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TRANSCRIPT
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
64
31 Plant Material Collection And Authentication
The flowers of Nerium oleander L were collected from Kota district of Rajasthan in
India and authenticated by routine pharmacognostic procedures by Dr SN Sharma
senior scientist botany division of Indian Institute of Integrative Medicine (IIIM)
Jammu India A voucher specimen was retained and deposited at the crude drug
repository of the herbarium of IIIM vide CDR accession no21869
32 Preparation Of Extracts
The flowers (2 kg) were dried in shade and coarsely powdered The powder was
extracted with petroleum ether (40-60 ordmC) for defatting and then macerated with ethanol
water (11) with constant stirring (1200 rpm 3h) The solvent containing extract was
filtered and the marc was completely drained this process was repeated thrice for the
complete extraction of the phytochemicals The extracts obtained during the three cycles
were independently combined and reduced to 18th of its original volume under rota
evaporator at 45 degC and then lyophilized (Vis Tis Advantage freeze Dryer 20 EL-85) to
obtained a yield of 11 (ww)
33 Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams as well as
male and female Swiss albino mice (8-12 weeks old) weighing 18-20 g were purchased
from Central Animal House Facility IMTECH Chandigarh 551999 CPCSEA The
protocol for CNS studies was approved by the Institution Animals Ethics Committee
(IAEC) Of ASBASJSM College of Pharmacy Bela (Ropar) Punjab (Registration No
72402aCPCSEA-29102002) The animals were housed under standard laboratory
conditions with temperature (23plusmn1degC) relative humidity (55plusmn10) 1212 h light dark
cycles and fed with standard pellet diet (Ms Ashirwad Industries Mohali) and purified
water ad libitum
34 Preparation of Drugs
Test doses of various fractions were freshly prepared as a homogenized suspension of N
oleander flowerrsquos extract in doses of 100 200 and 400 mgkg except chloroform extract
(25 50 and 100 mgkg) administered orally to Swiss albino mice once during the
experiment Chemicals reagents and testing kits etc were procured from the central
stores of the ASBASJS Memorial College of Pharmacy Bela (Rupnagar) Punjab
(India)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
65
35 Ascertaining Margin Safety by Acute Oral Toxicity Studies in Rats And Mice
(OECD Guideline 423)
Whenever an investigator administers a chemical substance to a biological system
different types of interactions can occur and a series of dose-related responses result In
most cases these responses are desired and useful but there are a number of other effects
which are not advantageous These may or may not be harmful to the patients The types
of toxicity tests which are routinely performed by pharmaceutical manufacturers in the
investigation of a new drug involve acute sub-acute and chronic toxicity Acute toxicity
is involved in estimation of LD50 (the dose which has proved to be lethal (causing death)
to 50 of the tested group of animals) Determination of acute oral toxicity is usually an
initial screening step in the assessment and evaluation of the toxic characteristics of all
compounds
Category 1
gt 0-5
Category 2
gt 5 ndash 50
Category 3
gt 50 - 300
Category 4 gt 300 - 2000
Category 5
gt 2000 - 5000
Category 5
Or unclassified
5 25 30 50 200 300 500 1000 2000 2500 5000 infin
5mgkg 3animals
2-3
300mgkg 3animals
50mgkg 3animals
50mgkg 3animals
5mgkg 3animals
2000mgkg 3animals
300mgkg 3animals
2000mgkg 3animals
2-32-32-3 2-3
2-3 2-3 2-3
0-1 0-1
0-1
0-1
0-1 0-1
0-1
0-1
0
0
3 2other
3(at 300)At 1st stepother
13 (at 50)At 1st step
0
Test Procedure with a Starting Dose of 300 mgkg Body weight
Acute toxicity study according to OECD guideline 423
Figure 5 Scheme of acute toxicity study according to OECD guideline 423
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
66
36 Preliminary Exploration of the CNS Activity Of 50 Hydroalcoholic Extract
(HyE) of N oleander Flowers in Mice
361 Spontaneous motor activity
The animals were divided into 4 groups with each group constituting 6 mice
Group-1 Control
Group-2 Standard (Diazepam) 4 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
Each Group of animal constitutes 6 mice In this experiment Group-I served as control
Group-II as standard (Diazepam 4 mgkg ip) while Group-III to Group-IV were
administered with drug (100 and 200 mgkg) po respectively For the spontaneous motor
activity individual mouse was introduced into actophotometer (INCO) and its spontaneous
locomotor activity was measured in 10 min duration
362 Pentobarbitone-induced sleeping time
The animals were divided into three new groups (n=6) Group-I was treated orally with
pentobarbital (50 mgkg ip) Group-II and Group-III received N oleander orally at the
dose of 100 and 200 mgkg respectively Pentobarbital (50 mgkg ip) was injected 30 min
before oral administration for all groups The time elapsed between loss and recovery of
the righting reflex was noted and taken as sleep time
Group-1 pentobarbital (50 mgkg ip) Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
363 Explored the muscle relaxant property of HyE in mice using Rota rod
apparatus (Motor coordination)
Before performing the experiment the fresh animals were trained to remain for 3 min on
rotarod apparatus the rod rotating at a speed of 25 rpm After training all mice were
divided into three groups (n=6) Group-I served as control Group-II and Group-III as N
oleander treated 100 and 200 mgkg respectively and the effect on motor coordination was
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
67
assessed by rotarod apparatus The fall-off time from the rotarod was noted for each
mouse
Group-1 Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
364 Determined the anti anxiety effect of HyE in mice using elevated plus maze
(EPM)
To conduct the EPM test 30 mice were taken and divided into 4 groups with each group
consisting 6 mice The plus maze consists of two open arms and two closed arms (50 x 10
x 50 cm each) elevated to a height Thirty minutes post treatment each mouse was placed
in turn in the centre of the maze facing one of the closed arms The cumulative times spent
by each mouse in the open and closed arms of the maze were recorded for 5 min
Group-1 Control
Group-2 Standard (Diazepam) 1 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
365 Explored the anticonvulsant property of HyE against Pentylenetetrazol (PTZ)
induced convulsions in mice
Three groups of mice (n=10) were used Group-I served as vehicle control Group-II and
Group-III as drug treated 100 and 200 mgkg po respectively After 2 h of treatment PTZ
was administered (60 mgkg ip) in all three groups Each group was observed for 30 min
and the onset of convulsion and duration of convulsion showed was counted
Group-1 Control + PTZ (60 mgkg ip)
Group-2 HyE of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-3 HyE of N oleander 200 mgkg + PTZ (60 mgkg ip)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
68
366 Explored the anticonvulsant property of HyE against maximal electroshock
(MES) induced convulsions in mice
Group-I served as vehicle control Group-II and III were treated orally with N oleander
dose 100 and 200mgkg po respectively After 2 h from treatment the above mention
groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by
using ear electrode The onset of convulsion duration of tonus was noted 100 protection
considered when hind limb tonic extension was completely abolished
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)
Group-3 HyE of N oleander 200 mgkg + MES (12 mA for 02 seconds)
37 Successive solvent extraction of the flowers of Nerium oleander L
The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely
powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using
petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete
extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC
and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel
All the five extracts were subjected to acute toxicity studies as per the OECD guidelines
Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
65
35 Ascertaining Margin Safety by Acute Oral Toxicity Studies in Rats And Mice
(OECD Guideline 423)
Whenever an investigator administers a chemical substance to a biological system
different types of interactions can occur and a series of dose-related responses result In
most cases these responses are desired and useful but there are a number of other effects
which are not advantageous These may or may not be harmful to the patients The types
of toxicity tests which are routinely performed by pharmaceutical manufacturers in the
investigation of a new drug involve acute sub-acute and chronic toxicity Acute toxicity
is involved in estimation of LD50 (the dose which has proved to be lethal (causing death)
to 50 of the tested group of animals) Determination of acute oral toxicity is usually an
initial screening step in the assessment and evaluation of the toxic characteristics of all
compounds
Category 1
gt 0-5
Category 2
gt 5 ndash 50
Category 3
gt 50 - 300
Category 4 gt 300 - 2000
Category 5
gt 2000 - 5000
Category 5
Or unclassified
5 25 30 50 200 300 500 1000 2000 2500 5000 infin
5mgkg 3animals
2-3
300mgkg 3animals
50mgkg 3animals
50mgkg 3animals
5mgkg 3animals
2000mgkg 3animals
300mgkg 3animals
2000mgkg 3animals
2-32-32-3 2-3
2-3 2-3 2-3
0-1 0-1
0-1
0-1
0-1 0-1
0-1
0-1
0
0
3 2other
3(at 300)At 1st stepother
13 (at 50)At 1st step
0
Test Procedure with a Starting Dose of 300 mgkg Body weight
Acute toxicity study according to OECD guideline 423
Figure 5 Scheme of acute toxicity study according to OECD guideline 423
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
66
36 Preliminary Exploration of the CNS Activity Of 50 Hydroalcoholic Extract
(HyE) of N oleander Flowers in Mice
361 Spontaneous motor activity
The animals were divided into 4 groups with each group constituting 6 mice
Group-1 Control
Group-2 Standard (Diazepam) 4 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
Each Group of animal constitutes 6 mice In this experiment Group-I served as control
Group-II as standard (Diazepam 4 mgkg ip) while Group-III to Group-IV were
administered with drug (100 and 200 mgkg) po respectively For the spontaneous motor
activity individual mouse was introduced into actophotometer (INCO) and its spontaneous
locomotor activity was measured in 10 min duration
362 Pentobarbitone-induced sleeping time
The animals were divided into three new groups (n=6) Group-I was treated orally with
pentobarbital (50 mgkg ip) Group-II and Group-III received N oleander orally at the
dose of 100 and 200 mgkg respectively Pentobarbital (50 mgkg ip) was injected 30 min
before oral administration for all groups The time elapsed between loss and recovery of
the righting reflex was noted and taken as sleep time
Group-1 pentobarbital (50 mgkg ip) Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
363 Explored the muscle relaxant property of HyE in mice using Rota rod
apparatus (Motor coordination)
Before performing the experiment the fresh animals were trained to remain for 3 min on
rotarod apparatus the rod rotating at a speed of 25 rpm After training all mice were
divided into three groups (n=6) Group-I served as control Group-II and Group-III as N
oleander treated 100 and 200 mgkg respectively and the effect on motor coordination was
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
67
assessed by rotarod apparatus The fall-off time from the rotarod was noted for each
mouse
Group-1 Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
364 Determined the anti anxiety effect of HyE in mice using elevated plus maze
(EPM)
To conduct the EPM test 30 mice were taken and divided into 4 groups with each group
consisting 6 mice The plus maze consists of two open arms and two closed arms (50 x 10
x 50 cm each) elevated to a height Thirty minutes post treatment each mouse was placed
in turn in the centre of the maze facing one of the closed arms The cumulative times spent
by each mouse in the open and closed arms of the maze were recorded for 5 min
Group-1 Control
Group-2 Standard (Diazepam) 1 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
365 Explored the anticonvulsant property of HyE against Pentylenetetrazol (PTZ)
induced convulsions in mice
Three groups of mice (n=10) were used Group-I served as vehicle control Group-II and
Group-III as drug treated 100 and 200 mgkg po respectively After 2 h of treatment PTZ
was administered (60 mgkg ip) in all three groups Each group was observed for 30 min
and the onset of convulsion and duration of convulsion showed was counted
Group-1 Control + PTZ (60 mgkg ip)
Group-2 HyE of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-3 HyE of N oleander 200 mgkg + PTZ (60 mgkg ip)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
68
366 Explored the anticonvulsant property of HyE against maximal electroshock
(MES) induced convulsions in mice
Group-I served as vehicle control Group-II and III were treated orally with N oleander
dose 100 and 200mgkg po respectively After 2 h from treatment the above mention
groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by
using ear electrode The onset of convulsion duration of tonus was noted 100 protection
considered when hind limb tonic extension was completely abolished
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)
Group-3 HyE of N oleander 200 mgkg + MES (12 mA for 02 seconds)
37 Successive solvent extraction of the flowers of Nerium oleander L
The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely
powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using
petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete
extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC
and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel
All the five extracts were subjected to acute toxicity studies as per the OECD guidelines
Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
66
36 Preliminary Exploration of the CNS Activity Of 50 Hydroalcoholic Extract
(HyE) of N oleander Flowers in Mice
361 Spontaneous motor activity
The animals were divided into 4 groups with each group constituting 6 mice
Group-1 Control
Group-2 Standard (Diazepam) 4 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
Each Group of animal constitutes 6 mice In this experiment Group-I served as control
Group-II as standard (Diazepam 4 mgkg ip) while Group-III to Group-IV were
administered with drug (100 and 200 mgkg) po respectively For the spontaneous motor
activity individual mouse was introduced into actophotometer (INCO) and its spontaneous
locomotor activity was measured in 10 min duration
362 Pentobarbitone-induced sleeping time
The animals were divided into three new groups (n=6) Group-I was treated orally with
pentobarbital (50 mgkg ip) Group-II and Group-III received N oleander orally at the
dose of 100 and 200 mgkg respectively Pentobarbital (50 mgkg ip) was injected 30 min
before oral administration for all groups The time elapsed between loss and recovery of
the righting reflex was noted and taken as sleep time
Group-1 pentobarbital (50 mgkg ip) Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
363 Explored the muscle relaxant property of HyE in mice using Rota rod
apparatus (Motor coordination)
Before performing the experiment the fresh animals were trained to remain for 3 min on
rotarod apparatus the rod rotating at a speed of 25 rpm After training all mice were
divided into three groups (n=6) Group-I served as control Group-II and Group-III as N
oleander treated 100 and 200 mgkg respectively and the effect on motor coordination was
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
67
assessed by rotarod apparatus The fall-off time from the rotarod was noted for each
mouse
Group-1 Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
364 Determined the anti anxiety effect of HyE in mice using elevated plus maze
(EPM)
To conduct the EPM test 30 mice were taken and divided into 4 groups with each group
consisting 6 mice The plus maze consists of two open arms and two closed arms (50 x 10
x 50 cm each) elevated to a height Thirty minutes post treatment each mouse was placed
in turn in the centre of the maze facing one of the closed arms The cumulative times spent
by each mouse in the open and closed arms of the maze were recorded for 5 min
Group-1 Control
Group-2 Standard (Diazepam) 1 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
365 Explored the anticonvulsant property of HyE against Pentylenetetrazol (PTZ)
induced convulsions in mice
Three groups of mice (n=10) were used Group-I served as vehicle control Group-II and
Group-III as drug treated 100 and 200 mgkg po respectively After 2 h of treatment PTZ
was administered (60 mgkg ip) in all three groups Each group was observed for 30 min
and the onset of convulsion and duration of convulsion showed was counted
Group-1 Control + PTZ (60 mgkg ip)
Group-2 HyE of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-3 HyE of N oleander 200 mgkg + PTZ (60 mgkg ip)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
68
366 Explored the anticonvulsant property of HyE against maximal electroshock
(MES) induced convulsions in mice
Group-I served as vehicle control Group-II and III were treated orally with N oleander
dose 100 and 200mgkg po respectively After 2 h from treatment the above mention
groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by
using ear electrode The onset of convulsion duration of tonus was noted 100 protection
considered when hind limb tonic extension was completely abolished
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)
Group-3 HyE of N oleander 200 mgkg + MES (12 mA for 02 seconds)
37 Successive solvent extraction of the flowers of Nerium oleander L
The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely
powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using
petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete
extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC
and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel
All the five extracts were subjected to acute toxicity studies as per the OECD guidelines
Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
67
assessed by rotarod apparatus The fall-off time from the rotarod was noted for each
mouse
Group-1 Control
Group-2 HyE of N oleander 100 mgkg
Group-3 HyE of N oleander 200 mgkg
364 Determined the anti anxiety effect of HyE in mice using elevated plus maze
(EPM)
To conduct the EPM test 30 mice were taken and divided into 4 groups with each group
consisting 6 mice The plus maze consists of two open arms and two closed arms (50 x 10
x 50 cm each) elevated to a height Thirty minutes post treatment each mouse was placed
in turn in the centre of the maze facing one of the closed arms The cumulative times spent
by each mouse in the open and closed arms of the maze were recorded for 5 min
Group-1 Control
Group-2 Standard (Diazepam) 1 mgkg
Group-3 HyE of N oleander 100 mgkg
Group-4 HyE of N oleander 200 mgkg
365 Explored the anticonvulsant property of HyE against Pentylenetetrazol (PTZ)
induced convulsions in mice
Three groups of mice (n=10) were used Group-I served as vehicle control Group-II and
Group-III as drug treated 100 and 200 mgkg po respectively After 2 h of treatment PTZ
was administered (60 mgkg ip) in all three groups Each group was observed for 30 min
and the onset of convulsion and duration of convulsion showed was counted
Group-1 Control + PTZ (60 mgkg ip)
Group-2 HyE of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-3 HyE of N oleander 200 mgkg + PTZ (60 mgkg ip)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
68
366 Explored the anticonvulsant property of HyE against maximal electroshock
(MES) induced convulsions in mice
Group-I served as vehicle control Group-II and III were treated orally with N oleander
dose 100 and 200mgkg po respectively After 2 h from treatment the above mention
groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by
using ear electrode The onset of convulsion duration of tonus was noted 100 protection
considered when hind limb tonic extension was completely abolished
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)
Group-3 HyE of N oleander 200 mgkg + MES (12 mA for 02 seconds)
37 Successive solvent extraction of the flowers of Nerium oleander L
The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely
powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using
petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete
extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC
and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel
All the five extracts were subjected to acute toxicity studies as per the OECD guidelines
Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
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Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
68
366 Explored the anticonvulsant property of HyE against maximal electroshock
(MES) induced convulsions in mice
Group-I served as vehicle control Group-II and III were treated orally with N oleander
dose 100 and 200mgkg po respectively After 2 h from treatment the above mention
groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by
using ear electrode The onset of convulsion duration of tonus was noted 100 protection
considered when hind limb tonic extension was completely abolished
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)
Group-3 HyE of N oleander 200 mgkg + MES (12 mA for 02 seconds)
37 Successive solvent extraction of the flowers of Nerium oleander L
The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely
powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using
petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete
extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC
and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel
All the five extracts were subjected to acute toxicity studies as per the OECD guidelines
Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
69
38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin
Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)
The maximum tolerable dose determination was performed using OECD (Organization for
Economic Corporation and Development) guideline 423 The study is performed as a
stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3
animals at each dose level Three female Swiss mice (18-22 g) were used for the study
and were not fed for 3-4 h prior to the experiment They were individually administered
various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal
was continuously monitored during first 30 min and then they were monitored on an
hourly basis for next 4 hrs Subsequently they were observed after a four hour interval
After this they were under observation for 14 days to monitor any abnormal signs and
symptoms depicting toxicity These animals were humanely killed for animal welfare
reasons after termination of the experiment The animals were observed for any change in
skin and fur eyes and mucous membranes respiratory circulatory and autonomic and
central nervous systems somatosensory activity and behavioral pattern Attention was
given to observation like tremors convulsions salivation diarrhoea lethargy sedation
hypnosis and coma
39 Preliminary Phytochemical Screening
The successive solvent extracts were subjected to preliminary phytochemicals screening
for the detection of various phytoconstituents such as alkaloids steroids flavonoids
glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats
The following tests were carried out to identify the various phytoconstituents present in the
extracts (Harborne 1998)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
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Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
70
Phyto
Chemical
Test Reagent Reaction Results
1
Alkaloids
A small quantity
sample + 5 ml of
15 vv HCl and
filtered
These filtrates (TFl) were then used
for testing alkaloids with following
reagents
Mayerrsquos
test
Mayerrsquos reagent
(MR) 136 gm
HgCl2 + 60 ml H2O
(dil upto 100 ml
H2O)
To a little of the TFl (taken in a
watch glass) + a few drops of the
MR
Cream coloured
precipitate
Dragen-dorffs
test
Sol A (17 gm of
bismuth subnitrate
+ 200 gm tartaric
acid + 800 ml H2O)
Sol B (160 gm
potassium iodide +
4 ml H2O)
Soln A + Sol B (11
vv proportion)
Working standard (WS) was
prepared by taking 50 ml of this
soln (A+B) + 100 gm of tartaric acid
( upto 500 ml H2O)
WS was sprayed on whatmann No
1 filter paper and the paper was
dried The TFl (basification) +
dilute ammonia was extracted with
chloroform and the CE was applied
on the filter paper impregnated
with WS with the help of a
capillary tube
Orange red colour
Wagnerrsquos test 127 g of I + 2 gm
of KI + 5 ml H2O
(dil upto 100 ml
H2O)
Few drops reagent + TFl Brown flocculent
precipitate
Hagerrsquos
reagent
A saturated aq Sol
of picric acid was
employed for this
test
When the test filtrate was treated
with this reagent
an orange yellow
ppt was obtained
(alkaloids)
2
Saponins
Froth
test
A few mg sample taken in a test
tube and shaken vigorously with a
small amount of NaHCO3 +H2O
Honeycomb like
froth is obtained
3
Sterols
Salkowask
i reaction
Few mg sample + 2 ml CHCl3 + 2
ml conc H2SO4 was added from the
side of the test-tube The test-tube
was shaken for few minutes
red colour in the
chloroform layer
(sterols)
Lieberman
nrsquos test
Few mg sample + few ml Ac2O
gently heated The contents of the
test-tube were cooled + Few drops
conc H2SO4 from the side of the
test-tube
A blue colour
(sterols)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
71
Lieberman
n
Burchardrsquos
reaction
Few mg sample + CHCl3 + few
drops of Ac2O + conc H2SO4 from
the side of the tube
Red blue
green
4
Carbohydr
ates
Molischrsquos
test
2-3 ml of extract + few drops 95
α-naphthol sol + alcohol shaken
and conc H2SO4 was added from
side of the test tubes
Red brown ring at
the junction of the
liquids (presence of
carbohydrates)
Fehlingrsquos
solution
test
1 ml of fehlingrsquos A
and B sol were
mixed and boiled
for one minute
Equal volume of extract was added
and heated on boiling water bath for
5-10 min
yellow brick red
(reducing sugars)
Benedictrsquos
solution
test
Equal volume of Benedictrsquos reagent
+ extract were mixed in test tube
and heated on boiling water bath for
5 min
red solution
(reducing sugars)
5
Tannins
The test residue of each extract was
taken separately in water warmed
and filtered Tests were carried out
with the filtrate using following
reagents
Ferric
chloride
reagent
A 5 wv sol of FeCl3 + 90
alcohol was prepared Few drops of
this sol + little of the above filtrate
Dark green or Deep
blue colour (tannins
present)
Lead
acetate
test
10 wv sol of basic lead acetate +
H2O + test filtrate
If ppt is obtained
tannins present
6
Proteins
and amino
acid
Ninhydrin
test
The Ninhy drin reagent is 01 wv
solution of ninhydrin in n-butanol
A little of this reagent was added to
the test extract
Violet or Purple
colour if amino
acids present
Millonrsquos
test
Millonrsquos reagent 3
ml of mercury in 27
ml of fuming nitric
acid
This sol was then diluted with
equal quantity H2O Aq Sol of the
residue was taken + 2 to 3 ml of
Millonrsquos reagent
The white
precipitate which
slowly turns to
pink is obtained if
proteins are present
7
Tests for
flavonoids
Shinoda
test
A small quantity to test residue was
dissolved in 5 ml ethanol (95 vv)
and reacted with few drops of
concentrated hydrochloric acid and
05 gm of magnesium metal
Pink crimson or
magenta colour is
developed within a
minute or two
(flavonoids)
Ammonia
test
Few mg sample in 1 ml of 95 vv
ethanol and apply a drop of this
solution on filter paper then expose
this paper to ammonia vapors
Yellow color
(flavonoids)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
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Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
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Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
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Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
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Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
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Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
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Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
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Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
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Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
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Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
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Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
72
Lead
acetate sol
test
To small quantity of extract + lead
acetate solution
Yellow coloured
ppt (flavonoids)
8
Triterpenoi
ds
Libermann
-Burchard
test
Extract treated with few drops of
acetic anhydride boil and cool
concentrated sulphuric acid is
added from the side of the test tube
A brown ring at the
junction of two
layers and the
(upper layer turns
green) sterols and
deep red colour
triterpenoids
Salkowskirsquo
s test
Treat extract in chloroform with
few drops of concentrated
Sulphuric acid shake well and
allow to stand for some time
lower layer red
colour (sterols) and
yellow coloured
(triterpenoids)
310 Preliminary In Vitro Screening of the Extracts
3101 Antioxidant activity
31011 DPPH radical scavenging activity of all the successive solvent extracts
Free radicals contribute in some severe chronic diseases in humans including
atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many
tissues central nervous system injury neurological disorders gastritis cancer and AIDS
ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are
generated under physiological and pathological stresses in human body Oxidation process
is one of the most important routs for producing free radicals in food drugs and even
living systems Antioxidants played an important role in lowering oxidative stresses
caused by ROS and protect against these complex diseases through scavenging free
radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like
butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated
hydroquinon and gallic acid esters have been suspected to cause or prompt negative health
effects Hence strong restrictions have been placed on their application and there is a trend
to substitute them with naturally occurring antioxidants Moreover these synthetic
antioxidants also show low solubility and moderate antioxidant activity An easy rapid
and sensitive method for the antioxidant screening of plant extracts is free radical
scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical
spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more
electron and the absorbance decreases We have also found the relationship of total
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
73
flavonoid and phenol contents with antioxidant activity In the longer term plant species
(or their active constituents) identified as having high levels of antioxidant activity in vitro
may be of value in the design of further studies to unravel novel treatment strategies for
disorders associated with free radicals induced tissue damage Therefore it increases
interest in natural antioxidant products for use as medicines and food additives Vitamin C
vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et
al 1992)
The free radical scavenging activity of extract was measured by DPPH radical using the
method described previously (Shimada et al 1992) A 01mM solution of DPPH radical
in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in
water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and
allowed to stand at room temperature for 30 min Then the absorbance was measured at
517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity The percent DPPH
scavenging effect was calculated using the following equation
DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the standard sample or extract All the tests were performed in triplicate and
graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant
compound
31012 Reducing power assay
The reducing power of extract was determined according to the method described
previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in
1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and
potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for
20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which
was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was
mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was
measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher
absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was
used as standard antioxidant compound
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
74
31013 Total antioxidant activity in linoleic acid emulsion system
The total antioxidant activity of the extract was determined according to the thiocyanate
method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different
concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium
phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in
potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175
g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On
the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml
potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC
in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value
was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601
Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals
during incubation During the linoleic acid oxidation peroxides are formed These
compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had
maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid
oxidation The solutions without extract or standards were used as blank samples All data
about total antioxidant activity are the average of triplicate analyses The inhibition of lipid
peroxidation in percentage was calculated by following equation
Inhibition () = (A0 ndash At) A0 times 100 (02)
Where A0 was the absorbance of the control reaction and At was the absorbance in the
presence of the sample All the tests were performed in triplicate and graph was plotted
with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound
31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical
decolorization assay
ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was
produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash
16 h before use For the test of samples the ABTS stock solution was diluted with
phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the
addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
75
5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS
radical scavenging that is calculated as follows
ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100
[Control absorbance]
31015 Superoxide anion scavenging activity
The assay was based on the capacity of the methanolic extract to inhibit blue formazon
formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue
tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant
mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate
buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample
solution Reaction was started by illuminating the reaction mixture with different
concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After
illumination the absorbance was measured at 590 nm The reaction assembly was
enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction
mixture served as blank The percentage inhibition of superoxide anion generation was
calculated using the equation 01 Ascorbic acid was used as standard compound
31016 Hydroxyl radical scavenging activity
The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in
phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)
were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)
trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The
supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA
reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)
31017 Metal chelating activity
The chelating of ferrous ions by the methanolic plant extract was measured by the method
described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)
Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2
(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The
reaction mixture was then shaken vigorously and allowed to stand at room temperature for
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
76
10 min The absorbance of the solution was then measured at 562 nm The percentage
inhibition of ferrozine ndashferrous complex formation was calculated by using the equation
01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound
3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays
The COX-1 assay was performed according to the method described elsewhere (Redl K et
al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M
Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02
units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)
was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at
37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the
concentration of PGE2
The COX-2 assay was performed according to the method describes earlier (Redl K et al
1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-
HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-
EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-
incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC
was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-
enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2
3103 Antibacterial activity
31031 Microorganisms
The microbial strains used for testing antimicrobial activities included the Gram positive
bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia
coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures
were maintained by subculturing periodically and were stored at 4 degC prior to use
31032 Screening for antibacterial activity
Antibacterial activity was tested by the agar-well diffusion method Different
concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the
extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the
medium by gently mixing 05 ml (For determination of antibacterial activity bacterial
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
77
cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures
with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After
hardening four 6 mm diameter wells were made using a sterile borer The wells were
filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates
were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served
as positive antibacterial control The diameter of the zones of inhibition around each of the
wells (well diameter included) was taken as measure of the antimicrobial activity Each
experiment was carried out in triplicate and the mean diameter of the inhibition zone was
recorded (Mukherjee PK et al 1995)
31033 Determination of Minimum Inhibitory Concentration (MIC)
The extracts which showed antibacterial activity in the agar-well diffusion method were
subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts
was determined for each of the test organisms in triplicates 05 ml of varying
concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005
mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)
Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0
mgml as a control) and then a loopful of the test organism previously adjusted to 05
McFarland turbidity standard for bacteria was introduced to the tubes The procedure was
repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A
tube containing nutrient broth only was seeded with the test organisms as described above
to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24
hr period After incubation the tubes were then examined for microbial growth by
observing the turbidity present in the tubes (Salama HMH et al 2009)
311 Neuropsychopharmacological Profiling of the Extracts
3111 Spontaneous motor activity
The animals were divided into 14 groups with each group constituting 6 mice For
spontaneous motor activity every mouse was introduced into the actophotometer (INCO)
and its score of locomotor was measured for 10 min duration
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
78
Group-1 Control
Group-2 Standard (Dia) 4 mgkg
Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)
Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)
Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)
Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)
In this study all extract were administrated orally at three selected dose level
3112 Elevated plus maze test
To conduct the EPM test 66 mice were taken and divided into 11 groups each group
contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x
40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in
the centre of the maze facing one of the closed arms The cumulative times spent by each
mouse in the open and closed arms of the maze were recorded upto 5 min
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) CE (25 50 and 100 mgkg po)
Group-(6-8) EA (100 200 and 400 mgkg po)
Group-(9-11) MENO-F (100 200 and 400 mgkg po)
3113 EPM model for Nootropic activity
The animals were placed individually on the EPM 30 min after extract administration the
animal was placed at the end of the open arms facing away from centre of the maze and
the time to move from the open arm to the closed arm was recorded as transfer latency
(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first
day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit
learning (Banji Otilia et al 2007)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
79
Experimental design
Group-1 Control
Group-2 Dia 2 mgkg
Group-(3-5) PE (100 200 and 400 mgkg po)
Group-(6-8) CE (25 50 and 100 mgkg po)
Group-(9-11) EA (100 200 and 400 mgkg po)
Group-(12-14) MENO-F (100 200 and 400 mgkg po)
3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced
convulsions in mice
Five groups of mice (n=10) were used Group-I served as positive control Group-II served
as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400
mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all
five groups Each group was observed for 30 min and the onset of convulsion and duration
of convulsion showed was counted
Experimental design
Group-1 Control + PTZ (60 mgkg ip)
Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)
Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)
Group-4 EA of N oleander 200 mgkg + PTZ (60 mgkg ip)
Group-5 EA of N oleander 400 mgkg + PTZ (60 mgkg ip)
3115 Anti-convulsant property of EA against MES induced convulsions in mice
Animals were divided into five groups containing 10 animals each Group-I served as
positive control Group-II served as standard Group-III Group-IV and Group-V were
treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
80
2 h from treatment the above mention groups were given current of 12 mA for 02 sec in
electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of
tonus was recorded 100 protection considered when hind limb tonic extension was
completely abolished
Experimental design
Group-1 Control + MES (12 mA for 02 seconds)
Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)
Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)
Group-4 EA of N oleander 200 mgkg + MES (12 mA for 02 sec)
Group-5 EA of N oleander 400 mgkg + MES (12 mA for 02 sec)
312 Exclusion and Inclusion Criteria for Further Biological Activity Selection
In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor
of lipid peroxidation as found by the results of total antioxidant activity This indicated the
possible effectiveness of the methanolic extract as a hepatoprotective In the following
hepatoprotective activity of the methanolic extract of the flowers of N oleander was
evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed
the study towards evaluating the anti-inflammatory potential of the extract
Neuropsychopharmacological profiling of all the successive solvent extracts revealed that
the methanolic extract consistently and dose dependently enhanced the cognition As the
methanolic extract proved to be a consistent cognition enhancer in elevated plus maze
model for Nootropic activity further the methanolic extract was evaluated for
neuroprotective effect in dementia related to AD
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
81
Figure 7 Flow chart representing schematically selection criteria for biological activities
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
82
313 Evaluation of Hepatoprotective Potential of the MENO-F
3131 Test Animals
Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM
College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals
were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2
degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals
were acclimatized to laboratory environment for a week prior to experiments Animals
were provided with a standard rodent pellet diet and clean drinking water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3132 Experimental design
A total of 36 rats were divided into 6 groups of 6 rats each
bull Group I served as normal control and received only the vehicle (1 mlkgday of 1
CMC po)
bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days
bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100
mgkg orally (po) for 7 days
bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body
weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)
for 7 days
All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before
sacrifice blood was collected from the retro-orbital sinus plexus under mild ether
anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for
15 min for carrying out further biochemical investigations One part of liver was dissected
out and used for biochemical and histopathological studies
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
83
3133 Measurement of serum biochemical parameters
The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)
alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912
clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)
3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)
In chilled normal saline excised livers were perfused to remove all the blood cells Then
they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and
homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus
obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an
Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The
final supernatant was used for the determination of malonaldehyde (MDA) as a lipid
peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also
assayed by the method described previously (Misra and Fridovich 1972)
3135 Histopathology
The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated
in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick
sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for
photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections
under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)
bull 0 = Normal liver histology
bull 1 = Tiny and short septa of connective tissue without influence on the structure of
hepatic lobules
bull 2 = Large septa of connective tissue flowing together and penetrating into the
parenchyma Tendency to develop nodules
bull 3 = Nodular transformation of the liver architecture with loss of structure of
hepatic lobules
bull 4 = Excessive formation and deposition of connective tissue with subdivision of
the regenerating lobules and with development of scars
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
84
3136 Statistical analysis
The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the
groups were analyzed by one-way ANOVA followed by Turkey as post hoc using
GraphPad Instat software package The IC50 values were calculated graphically by linear
regression analysis
314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD
3141 Test animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10
The animals were maintained on standard pellet diet and purified water ad libitum The
care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3142 Experimental design
A total of 30 rats were divided into 5 groups of 6 rats each
Group 1 Served as normal control and received normal drinking water ad libitum and
standard laboratory feed
Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad
libitum (PC)
Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum
plus vitamin E (100 mgkg)
Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (200 mgkg)
Group 5 Served as test and received (Al and Cu in micro mol) in water ad libitum plus
MENO-F (400 mgkg)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
85
Metals (M) in drinking water ad libitum to induce Alzheimer disease
Figure 8 A schematic diagram of the experimental setup depicted
3143 Radial 8 arm maze test
The maze consisted of eight arms extending radially from a central area (287 cm in
diameter) The doors 9 cm high will be placed between each arm and the central platform
The floor of arms and central area were painted black The apparatus was placed 40 cm
above the floor and surrounded by various extramaze cues such as a laboratory bench
posters and a clock The extramaze cues were placed in the same positions during the
study In order to investigate the spatial memory each arm was numbered from 1-8 outside
in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that
held a few mg of food pellet Prior to the performance of the maze task the animals were
kept on a restricted diet Before the actual training began the animals were shaped for 4
days to run to the end of the arms and consume the bait in groups of four The bait was
initially available throughout the maze but was gradually restricted to the food cup
Following this shaping period each animal was placed individually in the center of the
maze and subjected to the maze training The rats received five training trials every day for
5 days with a 5-min inter-trial interval The trial was continued until all four baits in the
food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned
with 70 ethanol and dried before each trial Each animal was placed individually in the
center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each
daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm
1-7
days
8-14
days
15-21
days
21-28
days 29-35 days
36-42 days
Day 1st to 35th metals (in micro mol) in water ad libitum)
Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)
Day 33rd to 35th training on radial eight arm maze
Body weight (weekly) Food and Water intake (average of every 7 days)
Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
86
entry was counted when all four limbs of the rat were within an arm Measurements were
made of the number of reference memory errors (entering an arm that does not contain
food) and working memory errors (entering an arm containing food) The number of entry
into the baited and non-baited arms that were previously visited was calculated as memory
errors The pattern in the arm entries until all four baits had been consumed was also
recorded After a 5-day training period (total 25 trials) the rats were maintained with one
trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or
less over three consecutive trials) were used for behavioral and pharmacological
experiment
Figure 9 Radial eight arm maze
3144 Biochemical estimations
31441 Determination of lipid profile
For determination of lipid profile blood was collected from the rats after 12 h of fasting
and 24 h after the last dose of drug The blood was collected from the retro orbital plexus
under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples
were allowed to clot for 45 min at room temperature Serum was separated from the
clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by
using Necpath triglyceride testing kit (GodPod method) according to manufacturer
instructions Serum total cholesterol was determined using Necpath Total cholesterol test
kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method
using Enzopak HDL- Cholesterol test kit
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
87
VLDL and LDL were calculated as
VLDL cholesterol = TG5
LDL cholesterol = TC ndash (VLDL + HDL cholesterol)
The values of lipid profile are expressed in mgdl
31442 Preparation of brain homogenate
The rats were sacrificed by decapitation and brains were collected rinsed in ice cold
normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv
of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of
lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of
homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for
estimation of glutathione by the method of The rest of the homogenate was centrifuged at
15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation
of superoxide dismutase (SOD) by the method described previously (Kakkar P et al
1984)
31443 Estimation of superoxide dismutase (SOD)
SOD activity of the brain tissue was analyzed by the method described previously
(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium
pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03
ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by
addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the
addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml
of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was
separated Color intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as unitsmg protein
31444 Protein estimation
The protein content was measured according to the method described previously (Lowry
OH et al 1951) using bovine serum albumin as standard
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
88
31445 Estimation of GSSG
To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock
solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG
assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range
of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for
both of samples and standards was adjusted to 74 by addition of triethanolamine solution
11 in water (TEAM solution) Samples and standards were kept on ice The reaction
mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM
5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of
glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm
Results were expressed in micromoles of GSSGmg protein
31446 Estimation of GSH
To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M
phosphate buffer pH 74) was taken The procedure was followed initially as described
previously (Ellman GL 1959) The homogenate was added with equal volume of 20
trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The
mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was
then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-
dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1
of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After
completion of the total reaction solutions were measured at 412 nm against blank
Absorbance values were compared with a standard curve generated from standard curve
from known GSH
31447 Nitrite estimation
Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was
measured in cell-free supernatants from brain homogenate by spectrophotometer assay
based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with
equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine
dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to
yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite
concentration was calculated from a standard curve and expressed as micromolar per
milliliter (Green LC et al 1982)
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
89
31448 Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by modified technique described
elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed
with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added
to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2
min Change in optical densityminute was calculated and results were expressed as
percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the
control group
31449 Estimation of acetylcholinesterase (AChE) activity
Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)
for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was
measured by the method described previously (Ellman GL et al 1961) Enzyme activity
was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture
consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as
substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)
(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme
containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained
005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced
yellow colour which was measured at 412 nm
3145 Histopathology
At the end of the experiment the animals were sacrificed by an overdose of diethyl ether
and brain from the animals were collected and placed in 40 vv neutral buffered
formalin Histopathology of the brain was performed by the pathologist at Guru Angad
Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and
eosin dyes were used for the staining of tissue The stained slides were observed by the
pathologist for the pathological changes and results were interpreted
3146 Statistical analysis
Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for one way analysis of variance
(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
90
315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of
Nerium Oleander L
3151 Test Animals
Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used
for the experiment Animals were procured from the central animal house facility
IMTECH Chandigarh The animals were housed under standard laboratory conditions
with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn
10 The animals were maintained on standard pellet diet and purified water ad libitum
The care and use of laboratory animals were strictly in accordance with the guidelines
prescribed by the Institutional Animal Ethical Committee constituted under the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) India
3152 Carrageenan induced rat paw oedema
The animals were divided into four groups (n=6) Group I served as control and received
vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received
Indomethacin (5 mgkg po) Group III group IV and group V served as test group which
received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally
Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan
induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et
al 2009) The animals were first treated with methanolic fraction (RP-M) or
Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in
1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the
paw was measured by using a volume displacement technique using a plethysmometer
immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume
as compared to the control group (vehicle only treated) was considered as an anti-
inflammatory response
3153 Exudative inflammation
A total of 24 rats were used in the experiment They were divided into four groups of six
rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
91
control vehicle (normal saline ip) were administered to different groups of animals
Thereafter peritoneal exudates were tested for total leukocyte count and total protein
content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N
acetic acid (Chaudhuri AKN et al 2005)
3154 Statistical analysis
Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad
prism software Statistical analysis was carried out for One way analysis of variance
(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package
Minimum significant value was set as P le 005
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
92
REFERENCES
Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I
Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J
Ethnopharmacol 2002 81 271-276
Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon
tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987
246 503-509
Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay
applicable to acrylamide gels Anal Biochem 1971 44 276-277
Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic
acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638
Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of
Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175
Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen
Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as
peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169
Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric
determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95
Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77
Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate
nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138
Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ
assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem
1987 165 215- 219
Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic
collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232
Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide
dismutase Ind J Biochem Biophys 1984 21 131-132
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
93
Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of
Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151
Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin
phenol reagent J Biol Chem 1951193 265-275
Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a
simple assay for superoxide dismutase J Biol Chem 1972 247 3170
Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the
autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214
Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of
Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276
Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated
levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058
Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by
thiobarbituric acid reaction Anal Biochem 1979 95 351-358
Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J
Nutr 1986 44 307-315
Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant
activity applying an improved ABTS radical cation decolorization assay Free Radical Biol
Med 1998 72 1231-1237
Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens
campylotheca Planta Medica 1994 60
Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of
Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and
Appl Sci 2009 3(3) 2008-2015
Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on
autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-
948
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287
Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
94
Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an
assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547
Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory
and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian
Stud 2009 2 280minus287