72

CHAPTER – 2

MATERIALS AND METHODS

Fig. 9: Soymida febrifuga Bark Sample

73

2.1 EXTRACTION AND PHYTOCHEMICAL SCREENING

The commonly used technique for isolation of active substances from crude drug

is called Extraction. It is the method where different solvents are used to separate

the active constituents.

2.1.1 Extraction

Materials: The leaves, bark, and root of Soymida febrifuga were collected from

the forest areas of Rajahmundry, East Godavari (Dist), Andhra Pradesh, India.

Immediately after collection they were authenticated by Dr. V.S. Raju,

Taxonomist, Department of Botany, Kakatiya University and a voucher specimen

was deposited at K.V.S.R. Siddhartha College of Pharmaceutical Sciences,

Vijayawada for future reference. All the required solvents and chemicals were of

analytical grade procured from local suppliers.

The plant material was dried and powdered in a mechanical grinder. Chloroform

and methanol extracts were prepared by maceration. The ratio of powder to

solvent was 1:3 with intermittent stirring, maceration was continued for 7 days.

They were filtered using vacuum filter, under reduced pressure. The mark left was

further macerated by adding solvent in the ratio 1:2 for 3 days. After filteration the

combined extracts were concentrated under reduced pressure and a dry powder

was obtained. Aqueous exract was prepared by soxhlet extraction. The extract

was concentrated under vacuum conditions. Finally all the dried extracts were

kept in a dessicator.They were stored in a refrigerator for future use.

The extracts were named as follows and the yield was calculated.

SFLC-Soymida febrifuga leaf chloroform extract.

SFLM-Soymida febrifuga leaf methanol extract.

SFLA-Soymida febrifuga leaf aqueous extract.

SFBC-Soymida febrifuga bark chloroform extract.

74

SFBM-Soymida febrifuga bark methanol extract.

SFBA-Soymida febrifuga bark aqueous extract.

SFRC-Soymida febrifuga root chloroform extract.

SFRM-Soymida febrifuga root methanol extract.

SFRA-Soymida febrifuga root aqueous extract.

Calculation of yield: The dried extracts obtained with each solvent were weighed

and yield was calculated with reference to air dried weight of the plant material.

Results are shown in Table 12.

Weight of the extract % Yield = _________________________ X 100 Weight of the plant material

2.1.2 Preliminary Phytochemical Analysis

Knowledge of the chemical constituents is useful to understand the herbal drugs

and their preparations.In addition it helps in isolation and characterization of

various chemical constituents present in the extracts. (Fransworth et al., 1966)

(Trease and Evans, 1989)

Systematic study of drug is required for understanding the primary and secondary

metabolites obtained as a result of plant metabolism. Therefore the plant material

should be tested for detecting various plant constituents. Preliminary

phytochemical screening was carried out for chloroform, methanol and aqueous

extracts of leaves,bark and root of Soymida febrifuga A. Juss Meliaceae;

following standard procedures(Kokate, 2008) (Harbone et al.,1983) (Sofowora et

al., 1993). The results are depicted in Table 13.

1. Test for Alkaloids: Take a small portion of solvent free chloroform, alcohol

and aqueous extracts separately with a few drops of dilute hydrochloric acid

and filter. The filterate is tested with various alkaloidal reagents such a

Mayer’s, Dragendorff’s, Hager’s and Wagner’s reagent.

75

a. Mayer’s Reagent: It consists of two solutions’ (i) Mercuric chloride

(1.36 gm) in distilled water (60 ml) (ii) Potassium Iodide (5g) in

distilled water (20ml). Alkaloids present in the sample give yellow (or)

buff coloured (or) cream coloured precipitate.

b. Dragendorff’s reagent: It consists of sodium Iodide (14g), Basic

bismuth carbonate (15.2gm) in 50 ml glacial acetic acid. They are

boiled for few minutes. Allow it to stand overnight, discard the

precipitate and take the filterate. To every 40ml of filterate, 160ml of

ethyl acetate and 1 ml water were added. Before conducting the test,

for every 10 ml of this stock solution add 20 ml of acetic acid and

make upto 100 ml with water. Alkaloids give orange brown precipitate.

c. Hager’s reagent: It consists of saturated solution of picric acid.

Alkaloids give yellow ppt.

d. Wagner’s reagent: It consists of Iodine (1.27gm) and Potassium

Iodide (2 gm) in 5ml distilled water and make the volume upto 100 ml

with distilled water. Alkaloids give Reddish brown ppt.

2. Detection of Glycosides:

a. Dissolve small quantities of extracts separately in 4 ml of distilled

water and filter. The filterate may be subjected to Molisch’s test to

detect the presence of carbohydrates.

b. Hydrolyse a small portion of extracts with dilute HCl in a water bath

and subject the hydrolysate to (i) Liebermann – Burchard’s test, to

detect steroidal glycosides (ii) Legal’s test to detect cardiac glycosides

/ sterol glycosides (pink to red colour) (iii) Borntrager’s test to detect

anthraquinone glycosides (Pink, red (or) violet colour).

76

3. Detection of saponins:

Extract

Dissolve in minimum quantity of water

And shaken in a graduated cylinder for 15min

A stable foam occupying 1cm

Presence of Saponins

4. Detection of Tannins:

Extract

Dissolve in water and filter

Filterate divided into 3 portions and taken in three test tubes

a. Treat with 10% aqueous potassium dichromate solution - development of yellow

brown precipitate, indicates positive test.

b. Treat with 10% aqueous lead acetate solution – development of yellow precipitate

is positive reaction.

c. Treat with 1 ml of 5% Ferric Chloride solution - formation of greenish black

colour is positive reaction.

These reactions are positive for Tannins.

77

5. Detection of Steroids and Triterpenoids

a. Libermann – Burchard Reaction :-

Extract

Dissolve in chloroform

1ml Acetic anhydride is added

2ml conc. Sulphuric acid was added along the sides of test tube

Reddish violet ring forms at the function of two liquid layers

Indicates the presence of Triterpenoids (or) Steroids

b. Noller’s Test :

Extract

2ml of 0.01% anhydrous Stannic chloride

was dissolved in pure Thionyl chloride

Initially purple colour develops which

Changes to deep red after few minutes

Indicates the presence of Triterpenoids

78

c. Salkowski Test :

Extract

Dissolved in chloroform

Conc. Sulphuric acid was added.

Chloroform layer acquires reddish – blue colour and acid layer shows green

fluorescence

Indicates the presence of steroids

6. Test for reducing sugar:

Extract

Dissolved in minimum amount of distilled

Water and filtered

To the filterate equal quantity of Fehling’s reagent A and B were

added and heated for few minutes

Brick red colour precipitate is formed

Reducing sugars.

79

7. Test for Flavoniod and theirs glycosides:

Extract

Dissolved in ethanol

Hydrolysed with 10% sulphuric acid and cooled

Extracted with diethyl ether, made into 3 parts and taken in three test tubes

a. Add 1ml dil. Sodium carbonate to first test tube

b. Add 1ml of 0.1 M Sodium hydroxide to second test tube

c. Add 1ml of dil. Ammonia solution to the third test tube.

Yellow color indicates presence of flavonoids

2.1.3 Treatment with different Chemical Reagents

The bark powder was treated with various chemical reagents. The results are

depicted in Table 14.

The powder after treatment with chemical reagents was analyzed for characteristic

study using UV and Visible light. The results are shown in Table15.

2.2 IN VITRO STUDIES ON BARK EXTRACTS OF SOYMIDA

FEBRIFUGA

Soymida febrifuga is traditionally used for the treatment of several ailments.

Based on this information, in vitro studies were carried out to evaluate Anti-

oxidant activity, 5-lipoxygenase inhibiting activity, Antihelmintic activity, and

anticancer activity.

80

2.2.1 Invitro models for assessement of antioxidant activity

There are a number of invitro methods for evaluating the antioxidant activity of

synthetic as well as plant drugs. Some important methods are described below.

a) DPPH method (1, 1- diphenyl -2- picryl hydrazyl):

This is one of the widely used methods for screening of antioxidant activity of

plant drugs (Vani et al., 1997) (Navarro, 1993). DPPH Assay method is based on

the reduction of methanolic solution of DPPH by free radical scavenger.

The procedure involves measurement of decrease in absorbance of DPPH at its

absorption maxima at 516nm which is proportional to concentration of free radical

scavenger added to DPPH reagent solution. Free radical scavengers and anti

oxidants bleach the colour from purple to yellow.

b) Super oxide radical scaveniging activity:

In this method, superoxide radical is generated by auto oxidation of riboflavin in

the presence of light. This causes reduction of NBT resulting in formation of blue

coloured formazan (Babu et al., 2001) that can be measured at 560nm.

c) Hydroxyl radical scavenging activity

In this method hydroxyl radicals are generated in vitro using Fe3+/

ascorbate/EDTA/H2O2 system in Fenton reaction. Hydroxyl radical is scavenged

in the presence of an antioxidant. The hydroxyl radicals formed by the oxidation is

allowed to react with DMSO (dimethyl sulphoxide) and yields formaldehyde.

Formaldehyde reacts with Nash reagent (2M ammonium acetate with 0.05M

acetic acid and 0.02M acetyl acetone in distilled water) producing intense yellow

color. The intensity of yellow color formed is measured at 412nm

spectrophotometrically against reagent blank (Babu et al., 2001).

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d) Nitric oxide radical inhibiting activity

Nitric oxide, has an unpaired electron, and is classified as a free radical and

displays important reactivities with certain types of proteins and other free

radicals. Anti oxidant activity can be measured by invitro inhibition of nitric oxide

radical. This method is based on the inhibition of nitric oxide radical generated

from sodium nitroprusside in buffer saline and measured by Griess reagent. In

presence of scavengers, the absorbance of the chromophore is evaluated at 546nm.

The activity is expressed as % reduction of nitric oxide (Babu et al., 2001).

e) Peroxynitrite radical scavenging activity

Peroxynitrite radical is involved in many toxic reactions. The scavenging activity

is measured by monitoring the oxidation of dihydro rhodamine 123 on a

microplate fluorescence spectrophotometer at 485nm (Hye Rhi Choi, 2002).

f) DMPD (N, N-dimethyl-p-phenylene diamine dihydrochloride) Method

This assay is based on the reduction of buffered solution of colored DMPD in

acetate buffer and ferric chloride. In the presence of free radical scavengers

absorbance of DMPD decreases at its absorption maxima at 505nm (Vinson et al.,

1995).

g) ABTS (2,2-azinobis(3-ethyl benzothiazoline-6-sulfonicacid) diammonium salt) Method

The assay is based on interaction between antioxidant and ABTS radical cation

which has a characteristic color showing maxima at 645,734 and 815nm (Vinson

et al., 1995).

82

h) Oxygen Radical Absorbance Capacity (ORAC)

Trolox (a water soluble analog of Vitamin E) serves as a standard to determine the

Trolox Equivalent (TE). The ORAC value is then calculated from the Trolox

Equivalent and is expressed as ORAC units or value. Compounds with high

ORAC value, have greater Antioxidant Power. This assay is based on of free

radicals generated using AAPH (2, 2-azobis 2-amido propane dihydrochloride)

and decrease in fluorescence in presence of free radical scavengers is measured

(Ronald et al., 1998).

i) β-Carotene Linoleate model

Linoleic acid, is an unsaturated fatty acid, it can be readily oxidized by Reactive

Oxygen Species (ROS) produced by oxygenated water. The products formed will

initiate the β-carotene oxidation, which leads to discoloration. Antioxidants

decrease the extent of discoloration, which is measured at 434nm (Joseph et al.,

1994).

j) Conjugated diene assay

During linoleic acid oxidation, the double bonds are converted into conjugated

double bonds, which are characterized by a strong UV absorption at 234nm. The

activity is expressed in terms of Inhibitory concentration (IC50) (Lingnert et al.,

1999).

k) Reducing Power Method

In this method increase in absorbance indicates increase in the antioxidant

activity. Antioxidants form a colored complex with potassium ferricyanide,

trichloroacetic acid and ferric chloride, which is measured at 700nm.Increase in

absorbance, indicates the reducing power of the samples (Jayaprakash et al.,

2001).

83

k) Phospho molybdenum Method

It is a spectroscopic method for the quantitative estimation of antioxidant activity,

through the formation of phosphomolybdenum complex. The principle involved is

reduction of Mo (VI) to Mo (V) by the sample analyte and subsequent formation

of a green phosphate-Mo (V) complex at acidic pH. (Kanner et al., 1994).

m) Xanthine oxidase Method

This is one of the recent methods for evaluation of anti oxidant activity (Opoku et

al., 2002). Presence of anti oxidant inhibits xanthine oxidase activity, and the %

inhibition is measured. Xanthine oxidase enzyme produces uric acid together with

super oxide radicals from xanthine and the amount of uric acid is measured at

292nm.

n) TRAP Method

TRAP stands for total radical trapping antioxidant parameter. The fluroscence of

R-Phycoerythrin is quenched by ABAP (2, 2′ -azo-bis (2-amidino-propane

hydrochloride) as a radical generator. This quenching reaction is measured in

presence of antioxidants. The antioxidative potential is evaluated by measuring

the delay in decoloration (Ghiselli et al., 1995).

o) FRAP Method

FRAP stands for Ferric Reducing Ability of Plasma. The antioxidant activity is a

measure of the increase in absorbance caused by the formation of ferrous ions

from FRAP reagent containing TPTZ (2, 4, 6 - tri (2 - pyridyl) - s - triazine) and

FeCl36H2O. The absorbance is measured spectrophotometrically at 595nm

(Benzie et al., 1996).

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2.2.2 Evaluation of In vitro Antioxidant activity

Materials : 1,1-diphenyl, 2-Picryl hydrazyl (DPPH) was obtained from Sigma

Aldrich, U.S.A., Gallic acid, Vit ‘C’ were procured from S.D.Fine Chemicals. All

the other reagents used were of analytical grade procred from local suppliers. O.D

values in invitro studiesvwere measured using Systronics UV-VIS (2201)

spectrophotometer.

Method

DPPH radical Scavenging activity

DPPH (1, 1-diphenyl –2-Picry1 hydrazy1) Scavenging activity was measured by

the method of Szabo (Szabo et al., 2007). The reaction mixture contained

1.5x10-7 M Methonolic solution of DPPH and various concentrations of the test

substances (SFBC, SFBM &SFBA) After mixing of reagent with 3ml of extract

(Different conc.), they were kept for 50min in dark. O.D was measured at 516nm

against blank. IC50 values were calculated using linear regression analysis. The

results are given in Table 16.

Superoxide free-radical Scavenging activity

Chloroform, methanol and aqueous extracts of bark of Soymida febrifuga were

evaluated for Superoxide free-radical Scavenging activity by Nitro Blue

Tetrazolium (NBT) riboflavin photo reduction method of Mc Cord and Fridovich

(Cord et al., 1969). The assay mixture contained EDTA solution (6.6mM), NaCn

(3µg), riboflavin (2µM), NBT (50µM), test substances and Phosphate Buffer

(67mM, PH 7.8) in a final volume of 3ml. The absorbance at 560nm was

measured before and 15min after illumination. All tests were run in triplicate and

mean values considered to calculate percentage scavenging ability. IC50 values

were calculated using linear regression analysis. Lower abs. indicates higher free

radical scavenging activity. Experiment was done in triplicate. Results are given

in Table 17.

85

2.2.3 Evaluation of 5-Lipoxygenase Inhibitory Activity

Products of 5-LOX pathway of arachidonic acid metabolism may mediate some

pathological events associated with acute inflammation and reversible airways

obstruction of asthma (Lewis et al., 1985) (Bray, 1986). Thus, activity of various

extracts barkof Soymida febrifuga on 5-LOX inhibition was studied.

Materials

Potato 5- LOX was purchased from Biosense Ltd., Norway, Linoleic acid and

other chemicals were procured from Merck Specialities Pvt. Ltd., Mumbai.

Method

5-LOX enzyme inhibitory activity of Soymida febrifuga barks extracts were

measured using the method of Reddanna et al (Reddanna et al.,1990) modified by

Ulusu et al (Ulusu et al.,2002). SFBC, SFBM& SFBA were tested. The assay

mixture contained 80mM linoleic acid, 10µl potato 5 – LOX in 50mM phosphate

buffer (pH 6.3). The reaction was initiated by the addition of enzyme buffer

mixture to linoleic acid and the enzyme activity was monitored as the increase in

absorbance at 234nm. The reaction was monitored for 120 Sec and the inhibitory

potential of extracts was measured by incubating various concentrations of test for

two minutes before addition of linoleic acid. All assays were performed in

Triplicate. The percentage inhibition was calculated by comparing slope of test

substance with that of enzyme activity. Results are depicted in Table 18.

86

2.2.4 Evaluation of Anti cancer activity

63% of drugs used in treatment of cancer are derived from natural products

(Cragg et al., 2009) (Newman et al., 2007) Invitro cytotoxicity is assessed by

microculture of cell lines and conducting assays of cell growth and viability

(Kishore Singh et al.,2009) (Yue et al.,2009).MTT method is most popular

method used (Lei Yuan et al.,2010) ( Paresh N Patel et al., 2010).

Materials

The cell lines used were

• MCF-7 Human breast tumor, Adeno carcinoma, Mammary gland (Negative)

• MDA-MB-231, Human breast tumor, Adeno carcinoma, Mammary gland

(Positive)

• A-431-Human Epidermoid carcinoma.Epidermis

• HT-1080-Fibrosarcoma, Human

All these cell lines were obtained from National Centre for Cell Sciences (NCCS),

Pune, India. Dulbecco’s Modified Eagle’s red medium (DMEM), MTT - (3-[4,5-

dimethyl thiazo1-2-y1]-2,5-diphenyl tetrazolium bromide) and EDTA were

purchased from Sigma life sciences, USA. Foetal bovine serum was purchased

from Arrow labs, culture plates from Tarson, Buffer from R&D Systems, USA.

All the chemicals and solvents employed were of analytical grade purchased from

local suppliers.

Method

MTT Cell Proliferation assay

The inhibitory activity of extracts were tested on proliferation of selected cell lines

using MTT (3-[4, 5-dimethylthiozol-2-yl]-2,5-diphenyl tetrazolium bromide)

assay.

87

• Cell lines were cultivated in DMEM containing 10% FBS and 4.5g/L, D-

Glucose.

• Cells (1.5X103 cells/well) were seeded in a 96-well microplate containing 100

µl of DMEM + 10% FBS + 4.5 g/L D-glucose medium per well and incubated

at 370C with 5% CO2 for overnight.

• The cells were treated with different concentrations of compounds (0-

100µg/ml) upto 72 h.

• Negative control was maintained with 0.5% DMSO.

• After 72 h treatment, 5 µl of MTT reagent, along with 45 µl of DMEM White

medium without FBS was added to each well and plates were incubated at

370C with 5% CO2 for 4h. Thereafter, 50 µl of solubilization buffer was added

to each well to dissolve formazan crystals produced by the reduction of MTT.

After 24h, the optical density was measured at 550 nm using microplate reader

(Bio- Rad USA).

The results of anti cancer activity are given inTable 19.

2.2.5 Evaluation of Antihelmintic activity Materials

Sodium Chloride, Gum Acacia and other chemicals were procured from Merck

Specialities Pvt. Ltd., Mumbai. Albendazole was purchased from local medical

store.

Method

The Antihelmintic activity was evaluated on adult Indian earth worm, Phertima

posthuma because of its anatomical and physiological resemblance with the

intestinal round worm parasites of human being (Bate Smith, 1962) (Thomson et

al., 1995). Healthy earth worms of 3-5 cm length and 0.1-0.2cm width were

collected form moist soil and authenticated in the department of zoology. They

88

were washed with normal saline to remove faecal matter, earthy matter and were

used for the evaluation of Antihelmintic activity.

The activity of SFBC, SFBM and SFBA of bark was determined by Mathew et al.

The samples were prepared by dissolving the dried extracts in gum acacia (1%)

solution prepared in normal saline. Different concentration ranges were prepared

from stock solution containing 100mg/ml of the respective extracts (2.5gm in

25ml gum acacia)

Equal sized earthworms were selected. They were made into six groups, 6 in each

group. Each group was placed in a petri dish and treated with one of the

following:

I group - Vehicle (1% Gum acacia in normal saline)

II group - 10mg/ml SFBC

III group - 25mg/ml SFBC

IV group - 50mg/ml SFBC

V group - 10mg/ml SFBM

VI group - 25mg/ml SFBM

VII group - 50mg/ml SFBM

VIII group - 10mg/ml SFBA

IX group - 25mg/ml SFBA

X group - 50mg/ml SFBA

XI group - 25mg/ml Albendazole

Observations were made for time taken to paralyze and/or death of individual

worms.paralysis was said to occur when worms do not revive even in normal

saline. Death was concluded when worms lost motility followed with fading away

of their body color (Martin, 1997). The results of the study are depicted in Table

20.

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2.3 FRACTIONATION AND INVITRO STUDY OF METHANOL EXTRACT OF BARK OF SOYMIDA FEBRIFUGA

2.3.1. Fractionation of Methanol extract

In in vitro studies SFBM exhibited potent antioxidant activity, 5-LOX inhibitory

activity and Antihelmintic activity.Fractionation of this extract was carried out to

isolate constituents responsible for the activity. The details of column

chromatographic separation are shown in Table 9.

About 20g SFBM was dissolved in minimum quantity of methanol and mixed

with silica gel.This mixture was taken in rotary evaporator for ensuring proper

adsorption of the extract.About 150-200gm silca gel was made into a slurry with

hexane and glass column was packed with the slurry, taking care to avoid

entrapment of air. Enough care was taken to prevent drying of packed column. On

top of this column,the dried adsorbed SFBM was packed without disturbing the

column.A cotton disc was placed on the top of the column.The column was eluted

with solvents of increasing polarity.Each fraction was collected,dried in flash

evaporator and preserved properly for further study.

Summary of Column Chromatography

Weight of Alcoholic extract = 20 g

Weight of silica gel 100-200 # = 40 g

Weight of silica gel (for packing) = 150-200 g

Volume of each fraction = 100 ml collected

The objective of column chromatographic separation was to facilitate further

purification of bioconstituents which have desired therapeutic activity. The

percentage yield of various fractions obtained is recorded in Table23. The

maximum yield could be obtained by elution with acetone: chloroform(80:20),

methanol : acetone (40:60) and methanol : acetone (80:20). And the fractions were

named as AFSF,MF1SF and MF2SF.

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Table 9 : Fractionation of Soymida febrifuga bark methanol extract

Eluant Fractions Compound Isolated

Hexane

8% Benzene in Hexane

20% Benzene in Hexane

40% Benzene in Hexane

60% Benzene in Hexane

80% Benzene in Hexane

Benzene

8% Chloroform in Benzene

20% Chloroform in Benzene

40% Chloroform in Benzene

60% Chloroform in Benzene

80% Chloroform in Benzene

Chloroform

8% Acetone in chloroform

20% Acetone in chloroform

40% Acetone in chloroform

60% Acetone in chloroform

80% Acetone in chloroform

Acetone

8% Methanol in Acetone

20% Methanol in Acetone

40% Methanol in Acetone

80% Methanol in Acetone

100% Methanol

1-5

6-10

11-15

16-20

21-26

27-31

32-35

36-40

41-45

46-50

51-55

56-60

61-66

67-74

75-78

79-80

81-82

83

84-100

101-102

103-104

105-106

107-108

109-112

-

Waxy

Waxy

Oily

Oily

Oily

-

Sticky

Sticky

Brown, sticky

Sticky

Brown, sticky

-

Dark brown

Dark brown

Brown band

Clear Band

AFSF

-

3 compounds

-

MF1SF

MF2SF

4 compounds

The yield and phytochemical evaluation of the fractions obtained from column

chromatography are shown in Table 21 and 22.

91

2.3.2 TLC of fractions Accurately weighed amount of sample SFBM was dissolved in methanol to yield

strength of 10µg/ml solution. This sample was loaded with calibrated capillary

tubes on activated silica get G coated ready made TLC sheets cut to required

size.Chromatograms were developed in saturated glass chambers using different

solvent systems.

The following solvent systems were used as mobile phases:

Chloroform:Methanol :Ethyl acetate (10:60:30)

Chloroform:Methanol(10:90)

Ethyl acetate:Methanol:water(100:13.5,10)

Ethyl acetate:Formic acid:Glacial acetic acid:water(150:11:11:26)

Developed plates were visualised under UV254 light and by spray reagents. The

spray reagents used were prepared using standard procedures (Wagner and Bladt,

1996)

i. Vanillin sulphuric acid reagent

a) Ethanolic solution of sulphuric acid (5%) and

b) Ethanolic solution of vanillin.

After spraying the plates vigorously with the vanillin sulphuric acid reagent it

was heated for 5-10 min at 1100c under observation. Steroids/triterpenoids and

their glycosides give blue, blue-violet or pink spots. The yellow color of

flavonoids and their glycosides gets intensified. Flavonoids are visualised as

yellow, lemon yellow, brick, orange or pink spots.

92

ii. Dragendorff’s Reagent

a. Dissolve 0.85 g of basic bismuth nitrate in 10ml glacial aceticacid and

40ml water under heating if necessary, filter.

b. Dissolve 8 g KI in 30ml water.

a + b in 1:1 ratio were taken as stock solution

For spraying: 1 ml stock + 2ml glacial aceticacid and 10 ml water.

iii. Liebermann – Burchard reagent (LB)

5 ml acetic anhydride and 5ml Conc. H2SO4 are added carefully to 50ml

absolute alcohol, while cooling in ice. The sprayed plate is warmed to 100o c

for 5-10min and then inspected in UV-365nm (Triterpenes and steroids).

iv. Potassium hydroxide reagent

5% (or) 10% ethanolic potassium hydroxide (Born Trager’s reaction). Plate is

sprayed with 10ml reagent and evaluated in Vis (or) UV – 356nm with (or)

without warming.

Anthraquinone (Red), Anthrones (Yellow)

2.3.3. DPPH Free radical scavenging activity of the selected bioactive fractions

DPPH (1, 1-diphenyl-2-Picry1hydrazy1) Scavenging activity was measured by the

method of Szabo et al. The reaction mixture contained 1.5x10-7M Methanolic

solution of DPPH and various concentrations of the test substances (AFSF,

MF1SF, & MF2SF). After mixing of reagent with 3ml of fractions (Different

conc.), they were kept for 50min in dark. O.D was measured at 516nm against

blank. IC50 values were calculated using linear regression analysis. The results are

given in Table 23.

93

2.4 TOXICITY STUDIES

Maintenance of animals

Wistar albino rats weighing 180-200 g were purchased from Mahaveer agencies,

Hyderabad, India and used for the studies after obtaining the permission from

institutional animal ethical committee (CPCSEA Reg. No. 146/1999). The animals

were housed in standard polypropylene cages, and maintained under standard

laboratory conditions (12:12 hour light and dark cycle; at an ambient temperature

of 25 ± 50C; 35%-60% of relative humidity). The animals were fed with standard

rat pellet diet and water ad libitum.

Acute toxicity study

Acute toxicity study was carried out according to the method described in the

literature (Glombitza et al., 1994) (Ghosh et al., 2002). Over night fasted Wistar

albino rats were divided into groups with each consisting of ‘6’ animals and were

orally fed separately with the fractions (AFSF, MF1SF and MF2SF) of Soymida

febrifuga in increasing dose levels of 100,500,1000 and 2000 mg/kg body weight.

The rats were observed continuously for 2h for behavioral, neurological and

autonomic profiles. After a period of 24h and 72h observations were made for

any lethality or death (Palanichamy et al., 1990).

2.5. EVALUATION OF ANTI DIABETIC ACTIVITY OF ISOLATED FRACTIONS

2.5.1 Screening methods for Diabetes mellitus

I. In vivo methods

a. Alloxan induced diabetes

Hyperglycemia and glycosuria after administration of Alloxan has been described

in several species, such as in dogs, in rats and in other species. Guinea pigs have

94

been found to be resistant. Alloxan is a pyrimidine, the uride of mesoxalic acid.

Alloxan produces a selective necrosis of beta cells in the islets of Langerhans of

the pancreas when injected to animals. This is followed by the development of

diabetes mellitus in 24-48 h. In its structure an intact pyrimidine ring seems to be

essential for diabetogenic action. In vitro Alloxan was found to inhibit the

conversion of Glucose-l-phosphate to Glucose-6-phosphate. It was believed that it

acts as an enzyme destroyer. The dose of Alloxan required to produce diabetes

varies with the animal. Alloxan is effective intraperitoneally in rats, but the degree

of liver damage tends to be greater, so subcutaneous or intravenous route of

administration is preferred. Preferable time period for the injection of Alloxan be

2-3 min, if too slow it is inactivated or neutralized on its way to pancreas (Glory,

2005).

Mechanism of induction of diabetes by Alloxan

Single dose of Alloxan induces diabetes in the experimental animals by selective

necrosis of β-cells resulting in insulin deficiency. This in turn leads to increased

blood glucose (Chude et al., 2001), increased triglycerides and cholesterol

(Venkateshwarlu et al., 2003) and decreased protein content (Sumana Ghosh,

2001).

It has several effects on β-cells of the pancreas and it is likely that a combination

of these effects results in the destruction of β-cells. Two different mechanisms

have been proposed. Alloxan is a highly reactive molecule that is readily reduced

to dialuric acid, which is then auto-oxidized back to Alloxan resulting in the

production of H2O2, O2 and hydroxyl radical. The H2O2 causes DNA

fragmentation, which in turn activates nuclear polyADP–ribose synthetase

resulting in depletion of cellular NAD levels. Two factors appears to make islet

especially sensitive to the effect of Alloxan,

(i) Alloxan is rapidly taken up into islet cells

(ii) Sensitivity of islet to peroxides.

95

A second mechanism involves the reaction of Alloxan with - SH group on

glucokinase, a signal recognition enzyme in the pancreatic β-cells, results in cell

necrosis. One of the effects of Alloxan on the β-cells is the inhibition of the

glucose stimulated insulin release. The major drawback of this model is the

difficulty in determining the dose of Alloxan that will produce sustained diabetes

without mortality. Mortality rate is higher than streptozotocin. There is a great

variation in the susceptibility of individual animals to Alloxan, and mortality may

occur because of the marked hypoglycemia, that follows about 6 h after the

injection of Alloxan, as well as hyperglycemia that occur in the week followed by

the injection of Alloxan. In addition to Alloxan, dialuric acid, alloxatin, β-

monomethylAlloxan, monopropylAlloxan, dimethylAlloxantin, diethylAlloxantin

and monoethyl dialuric acid are also diabetogenic in nature (Szkudelski, 2001).

b. Streptozotocin induced diabetes

Streptozotocin (STZ), 2-deoxy-2-(3-(methyl-3-nitroso uredo)-D-glucopyranose) is

synthesized by “Streptomycetes achromogenes” and is used to induce both IDDM

and NIDDM. The range of STZ dose is not as narrow as in the case of Alloxan.

The frequently used single i.v dose is adult rats to induce IDDM is between 40 and

60 mg/kg b.w may be ineffective. Intracellular action of STZ results in changes of

DNA in pancreatic β cells comprising its fragmentation. Recent experiments have

proved that the main reason for the STZ induced β cells death is alkylation of

DNA. The alkylating activity of STZ is related to its nitrosourea moiety,

especially at the O-6 position of guanine (Vogal et al., 1996).

c. Pancreatectomy

This technique is used in Beagle dogs. Total or 90% pancreas removal by surgical

method produces diabetes. The total removal of the pancreas results in insulin

dependent form of diabetes and insulin therapy is required to maintain

experimental animals.

96

d. Hormone induced diabetes

(i) Growth hormone induced diabetes

In intact adult dogs and cats the repeated administration of growth hormone

induces an intensively diabetic condition. Rats of any age subjected to a similar

treatment do not become diabetic but grow faster and show striking hypertrophy

of the islets of pancreas.

(ii) Corticosteroid induced diabetes

In the guinea pig and the rabbit, experimental corticoid diabetes could be obtained

without forced feeding. In the rat the adrenal cortex stimulated by corticotrophin

has the capacity to secrete amounts of steroids, which induce steroid diabetes

(Vogal et al., 1996).

e. Other diabetogenic compounds

Dithizone or gold thioglucose compounds have been found to induce symptoms of

diabetes and / or obesity.

f. Insulin deficiency due to insulin antibodies

A transient diabetic syndrome can be induced by injection of guinea pig anti–

insulin serum in various species (Vogal et al., 1996).

g. Virus induced diabetes

Type-1 diabetes mellitus may be due to virus infections and β-cell specific

autoimmunity. The D-variant of encephalomyocarditis virus (EMC-D) selectively

infects and destroys pancreatic β-cells in susceptible mouse strains similar to

human Type-1 diabetes.

97

h. Genetically diabetic animals

Several animal species, mostly rodents, were described to exhibit spontaneous

diabetes mellitus in a hereditary basis. Due to difficulties in breeding,severely

affected only a few species are used on a boarder scale, such as the Chinese

hamster, the obese hyperglycemic mice, the obese hyperglycemic zucker rat, and

various substrains of kk-mice. In the transgenic animals mostly mice also serve as

experimental model (Vogal et al., 1996).

i. The high-fat diet-fed mouse

It is a model for studying mechanisms and treatment of impaired glucose tolerance

and Type-2 Diabetes. There is a need for new modalities of Type-2 diabetes in

view of the progressive deterioration of metabolic control that occurs in spite of

intense treatment with existing modalities. New treatment should aim at

normalizing the basic defects in the disease, which are islet dysfunction in

combination with insulin resistance. These two needs require reliable and

clinically relevant experimental models. Most animals do not, however, fulfill

such requirements, since they are based on monogenic disorder of little relevance

for human diabetes or on chemical destruction of β-cells, which is also of less

clinical relevance. An important and relevant model, however, is the high-fat diet-

fed C57BL/6J mouse model. Surwit and co-workers originally introduced this

model in 1988. The model has shown to be accompanied by insulin resistance, as

determined by intravenous glucose tolerance tests and of insufficient islet

compensation to the insulin resistance. This model has, accordingly been used in

studies on pathophysiology of impaired glucose tolerance (IGT) and Type-2

diabetes (Verspohl, 2002) (So-young park et al., 2001) (Barbara, 2002) (Surwit et

al., 1988).

98

II. In vitro methods

a. Adipocytes - Adipogenesis - 3T3-L1 cell line

The first, and best characterized, model of adipogenesis in vitro is the 3T3-L1 cell

line, a sub strain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under

normal conditions have a fibroblastic phenotype. However, when treated with a

combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and

insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to

accumulate lipids intracellularly in the form of lipid droplets (Maria and Ahren,

1997). Another cell line, 3T3-F442A, is blocked from differentiating at a later

stage, and requires only insulin to differentiate.

b. Glucose uptake studies using rat diaphragm:

Diaphragm tissue was obtained from normal rats weighing 100-130 g

which had fasted for 18-20 h.

The rats were decapitated, the diaphragms quickly and gently excised,

divided into halves and allowed to soak for 15 min in buffer containing

250 mg of glucose/100 ml.

Each hemidiaphragm was then blotted and transferred to a beaker

containing 1-4 ml of the incubation medium (either buffer or the

previously prepared serum).

The tissues were incubated in a Dubnoff metabolic shaker for 30-90 min at

370C, with a gas phase of O2+ CO2 (95:5).

At the end of the incubation the hemidiaphragms were blotted and

weighed, and the glucose content of each beaker was estimated according

to the GOD-POD method.

The difference between the initial and final glucose concentrations was used to

calculate the glucose uptake of the diaphragm in terms of glucose utilized/g of

tissue/h (Rubin et al., 1978). In the present study, Diabetes is induced in

experimental animals by a single dose of Alloxan.

99

2.5.2 Assessment of hypoglycemic and anti hyperglycemic activity of fractions in euglycemic rats Materials

Glibenclamide was a generous gift from Dr.Reddy’s foundation; Hyderabad,

India. Alloxan monohydrate was purchased from Sigma-Aldrich Company,

Germany. While, assay kits [GOD-POD, serum glutamate pyruvate transaminase

(SGPT), serum glutamate oxaloacetate transaminase (SGOT)] were purchased

from Beacon Diagnostics Ltd., Navasari, India. All other chemicals and solvents

used were of analytical grade procured from local suppliers.

Method

The experiment was conducted according to the procedure described in the

literature (Turner, 1965). A total of 66normal rats fasted for 18 h were divided

into‘11’ equal groups (n=6). Initial fasting blood samples were taken from the

animals of all the groups and then different doses of fractions of methanolic

extract of bark (AFSF, MF1SF and MF2SF) of Soymida febrifuga and a reference

drug Glibenclamide (10 mg/kg b.w) suspended in 5% gum acacia were

administered orally to different groups of animals and their effect on fasting blood

glucose level was studied up to 24h. The details of untreated and treated groups

are as follows:

Group I - 5% gum acacia (Control group).

Group II - Glibenclamide 10 mg/kg b.w. (Standard group).

Group III - AFSF 100mg/kg b.w.

Group IV - AFSF 200 mg/kg b.w.

Group V - AFSF 400 mg/kg b.w.

Group VI - MF1SF 100 mg/kg b.w.

Group VII - MF1SF 200 mg/kg b.w.

Group VIII - MF1SF 400 mg/kg b.w.

100

Group IX - MF2SF 100 mg/kg b.w.

Group X - MF2SF 200 mg/kg b.w.

Group XI - MF2SF 400 mg/kg b.w.

Blood samples were drawn from the retro-orbital plexus of the rats at ‘0’ h (Initial

fasting blood sample) and 2, 4, 6,8,12 and 24h after the treatment. The samples

were analyzed on Autoanalyser for blood glucose content using glucose oxidase-

peroxidase method (Trinder, 1969). The results are depicted in Table 24 and Fig

10.

2.5.3 Assessment of glucose tolerance of fractions in normal healthy rats

The antihyperglycemic effect of the samples was assessed by improvement of

glucose tolerance. Oral glucose tolerance test is used to test the body’s ability to

manage high doses of glucose which is the main energy source (Khan et al.,

2010).

This was done as described in the literature (Abdel-Zaher et al., 2005) (Babu et

al., 2003) (Whittington et al., 1991) (Khan et al., 2010) Overnight fasted rats were

divided into 11 groups (I-XI) of each consisting of 6 rats and their fasting blood

glucose level was recorded. Group I served as control, received vehicle (5% gum

acacia),whereas the treatment groups II,III , IV , V, VI, VII,VIII, IX, X, and XI

received a reference drug, Glibenclamide (10 mg/kg b.w.), AFSF (100 mg/kg

b.w.), AFSF (200 mg/kg b.w.), AFSF (400 mg/kg b.w.), MF1SF (100 mg/kg b.w.),

MF1SF (200 mg/kg b.w.) and MF1SF (400 mg/kg b.w.), MF2SF (100mg/kg b.w),

MF2SF (200mg/kg b.w) and MF2SF (400mg/kg b.w) respectively in 5% gum

acacia(p.o). The rats of all the groups were loaded with glucose (2g/kg, p.o)

30min after the treatment. Blood samples were collected from the rats at 30min,

60min, 90min and 120 min after glucose loading for determination of blood

glucose levels. The results are discussed in Table 25 and Fig 11.

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2.5.4 Assessment of antihyperglycemic activity of fractions in Alloxan- induced diabetic rats

Rats were made diabetic by a single intraperitoneal injection of 125mg/kg b.w. of

Alloxan monohydrate. It was dissolved in saline and was injected to overnight fast

Wistar albino rats. Blood glucose level of the animals was checked after 72h.

Animals with blood glucose level >250 mg/dl were considered diabetic and were

used for the study. The diabetic rats were divided into ‘11’ groups of six animals

in each and treated orally in the following manner (Okokon et al., 2007).

Group I - 5% gum acacia (Control group).

Group II - Glibenclamide 10 mg/kg b.w. (Standard group).

Group III - AFSF 100mg/kg b.w.

Group IV - AFSF 200 mg/kg b.w.

Group V - AFSF 400 mg/kg b.w.

Group VI - MF1SF 100 mg/kg b.w.

Group VII - MF1SF 200 mg/kg b.w.

Group VIII - MF1SF 400 mg/kg b.w.

Group IX - MF2SF 100 mg/kg b.w.

Group X - MF2SF 200 mg/kg b.w.

Group XI - MF2SF 400 mg/kg b.w.

Blood samples were collected from all the animals at the time intervals of ‘0’

(Fasting blood sample), 2, 4, 6, 8, 12 and 24 h after the treatment to estimate the

blood glucose levels. The results are dicussed in Table 26 and Fig 12.

2.5.5 Effect of fractions on different biochemical parameters in Sub acute study (21days) of Alloxan induced Type-2 Diabetes in rats

Overnight fasted Alloxan induced Type-2 Diabetic rats were divided into 5

groups of each consisting of six rats and their body weight and fasting blood

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glucose level, serum insulin, serum glutamate oxaloacetate transaminase (SGOT)

and serum glutamate pyruvate transaminase (SGPT), serum cholesterol, serum

triglyceride, and serum total proteins were recorded. Then they were treated once

a day for 21days in the following manner (Chakrabarti S., 2005) (Syed Mansoor

A., 2005).

Group I - diabetic control (5% gum acacia)

Group II - Glibenclamide (10 mg/kg b.w.)

Group III - AFSF (200 mg/kg b.w.)

Group IV - MF1SF (200 mg/kg b.w.)

Group V - MF2SF (200 mg/kg b.w)

The results are depicted in Table 27 and 28, Fig. 13,14,15,16,17,18,19 and 20.

Estimation of Serum Biochemical Parameters

The principle, details of the kits and methodology used in the estimation of the

various bio-chemical parameters by Autoanalyser (Selectra Junior-Merck) in the

present investigation are as follows:

A. Blood glucose

Principle: Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen

peroxide. In a subsequent peroxidase catalyzed reaction, p-hydroxy benzoate and

4-amino antipyrine react with hydrogen peroxide to form red colored quinone

complex. Absorbance data measured at 510nm using Spectrophotometer are

directly proportional to glucose concentrations (Trinder,1969).

Glucose +H2O Glucose oxidase Gluconic acid + H2O2

H2O2+ 4 - amino antipyrine +p-Hydroxy benzoate Peroxidase Quinone dye

103

Glucose kit

Reagent 1: Phosphate buffer 100 mmol/l, phenol 10 mmol/l, pH 7.0

Reagent 2: Glucose oxidase ≥10000 U/L, peroxidase ≥ 600 U/L

4-amino antipyrine 270 µmol/l

Preparation and stability of Working Reagent

Dissolve reagent 2 in the suitable volume of reagent 1.

Stability: 1 month at 20-25°C, 3 months at 2-8°C.

B. Serum triglycerides

GPO reagent is used to measure triglyceride concentration by a timed end point

method. Triglycerides in the sample are hydrolyzed to glycerol and free fatty

acids, by action of lipase.

Principle: A sequence of 3 coupled enzymatic steps using glycerol kinase (GK),

glycerophosphate oxidase (GPO) and horse raddish peroxidase (HPO) causes the

oxidative coupling of 3, 5 di-chloro-2- hydroxy benzene sulphonic acid (DHBS)

with 4-amino anti-pyrine to form red quinoneimine dye.

The reaction used is 1 part sample to 100 parts reagent.

1. Triglycerides Lipase glycerol + free fatty acids.

2. Glycerol + ATP GK glycerol-3-phospate + ADP

Mg+2

3. Glycerol-3-phosphate + O2 GPO Di hydroxy acetone + H2O2

4. H202 + 4 amino antipyrine +DHBS HPO Quinoneimine dye + HCl + 2H2O.

104

C. Serum Cholesterol

Principle: Cholesterol and its esters are hydrolysed by cholesterol esterase

(CHE). In the subsequent oxidation by cholesterol oxidase (CHO), H2O2 is

liberated. The colorimetric indicator quinoneimine is generated from 4-

aminoantipyrine and phenol by H2O2 under the catalytic action of peroxidase

(Trinder, 1969).

Cholesterol ester +H2O CHE Cholesterol + Fatty acid

Cholesterol +O2 CHO Cholesterol-3-one + H2O2

2 H2O2 + 4-Aminoantipyrine +Phenol POD Quinoneimine + 4H2O

D. Serum glutamate oxaloacetate transaminase (SGOT)

Principle: SGOT catalyzes the transfer of the amino group from L-aspartate

(ASP) to - ketoglutarate (α -KG) resulting in the formation of oxaloacetate (OAA)

and L-Glutamate (L-Glu). The oxaloacetate so formed, is allowed to react with 2,

4-DNPH to form 2, 4-dinitro phenyl hydrazone derivative which is brown colored

in alkaline medium. The hydrazone derivative of oxaloacetate similar to pyruvate

is considerably more chromogenic than that of α -KG. The final color developed

does not obey Beer’s law, hence calibration curve is plotted using pyruvate

standard. SGOT activity of the specimen is directly read on the calibration curve

(Reitman et al., 1957).

L-Asp + α -KG SGOT OAA + L-Glu

pH 7.4

OAA + 2, 4-DNPH Alkaline Medium 2, 4-Dinitrophenyl hydrazone

Reagents

Reagent 1. Buffered aspartate - α -ketoglutarate substrate pH 7.4

Reagent 2. 2, 4-DNPH Reagent

Reagent 3. Pyruvate Standard

Reagent 4. 4N NaOH (dilute 1:10 when used)

Stability: All reagents are stable upto the given expiry date when stored at 2-8oC.

105

E. Serum glutamate pyruvate transaminase (SGPT)

Principle: SGPT catalyzes the transfer of the amino group from L-Alanine to α-

ketoglutarate (α -KG) resulting in the formation of pyruvate and L-glutamate. The

pyruvate so formed is allowed to react with 2, 4-DNPH to produce 2, 4-

dinitrophenyl hydrazone derivative which is brown colored in alkaline medium.

The hydrazone derivative of pyruvate is considerably more chromogenic than the

hydrazone derivative of α - KG. The final color developed does not obey Beer’s

law, hence a calibration curve is plotted using pyruvate standard. SGPT activity of

the specimen is directly read on the calibration curve (Reitman et al., 1957).

L-Ala + α -KG SGPT Pyr + L-Glu

pH 7.4

Pyr + 2, 4-DNPH Alkaline Medium 2, 4-Dinitrophenyl hydrazone

Reagents

Reagent 1. Buffered alanine- α -ketoglutarate substrate pH 7.4

Reagent 2. 2, 4-DNPH Reagent

Reagent 3. Pyruvate Standard

Reagent 4. 4N NaOH (Dilute 1:10 when used)

Stability: All reagents are stable up to expiry date given on the label, when stored

at 2-8oC.

F. Serum total proteins

Principle: Serum total protein content was estimated by the method of Lowry et

al (Lowry et al., 1951). Proteins form chromophoric complex with phenol

reagent, which was measured at 610 nm.

106

Reagents

1) Alkaline Reagent: 2g of sodium carbonate was added to 100 ml of 0.1N

sodium hydroxide solution.

2) Alkaline Mixture: To 100 ml of alkaline reagent 1 ml of 4% aqueous copper

sulphate solution was added, this was prepared freshly.

3) Phenol reagent (Folin and Ciocalteu’s Reagent): Diluted by dissolving 0.5 ml

of phenol reagent in 4 ml of distilled water before use and stored in

refrigerator.

Methodology

The blood samples collected from the animals of the study were subjected to

centrifugation at 3000 rpm for 10min to separate the serum. Then 0.5µl of serum

sample was used to estimate each bio-chemical parameter. For the estimation of

the above mentioned biochemical parameters, 0.5µl of serum sample was

transferred to each of the pediatric sample cups. Then, these cups and the working

reagent bottles (25ml) corresponding to the bio-chemical parameters were placed

at their respective positions in the rotor system of the Autoanalyzer. After 30min

of programming the test parameter (s), the corresponding parameter values of the

different serum samples displayed on the computer were recorded.

Serum insulin levels by chemiluminescence assay

(Petteri kallio et al., 2008) (Rajeswari et al., 2011)

i. Instrument description: Automated chemiluminescence System (ACS: 180,

Bayer Health Care).

ii. Intended Use: For in vitro diagnostic use in the determination of insulin in

serum using the ACS: 180 automated chemiluminescence system. This assay can

be used to aid in the diagnosis of diabetes mellitus and hypoglycemia.

107

iii. Assay Principle: The ACS: 180 insulin assays was a two-site sandwich

immunoassay using direct chemiluminescent technology, which uses constant

amounts of two antibodies. The first antibody in the lite reagent was a monoclonal

mouse antiinsulin antibody labeled with acridinium ester. The second antibody in

the solid phase was a monoclonal mouse antiinsulin antibody, which was

covalently coupled to paramagnetic particles. The system automatically performs

the following steps:

• 25 µl of sample was dispensed into the cuvette.

• 50 µl of lite reagent was dispensed and incubated for 5 minutes at 37oC.

• 250 µl of solid phase was dispensed and incubated for 2.5 minutes at 37oC.

• Sample was separated, aspirated and the cuvettes were washed with reagent

water.

• 300 µl of reagent 1 and reagent 2 were dispensed to initiate the

chemiluminescence.

• Results were reported according to the selected option, as described in the

system operating instructions.

A direct relationship exists between the amount of insulin present in the sample

and the amount of relative light units (RLUs) detected by the system.

The details of reagents supplied in the kit as described in the printed brochure are

given below in Table 10.

108

Table10. Reagents used in chemiluminescence assay

Reagent Volume Ingredients

ACS : 180 IRI LR

2.5ml/vial Monoclonal mouse anti-insulin antibody (~0.59µg/vial) labeled with acridinium ester in buffered saline with bovine serum albumin, sodium azide (<0.1%) and preservatives.

ACS : 180 IRI SP

12.5ml/vial Monocloanl mouse anti-insulin antibody (~75.0 µg/vial) covalently coupled to paramagnetic particles in buffered saline with bovine serum albumin, sodium azide (<0.1%) and preservatives

IRI DIL

20ml/vial Buffered saline with casein, potassium thiocyanate (3.89%) sodium azide (<0.1%) and preservatives.

iv. Storage: All the reagents and material were stored at 2-80C

Stability: Until the expiration date on the vial label or cumulative 40 hours at

room temperature.

Caution: Sodium azide can react with copper and lead plumbing to form

explosive metal azides. Hence during disposal, reagents were flushed with a large

volume of water to prevent the buildup of azides.

v. Preparation of the reagents: For best results, the solid phase was thoroughly

mixed by inverting the vial before each use. The bottom of the vial was inspected

visually to ensure that all particles were dispersed and suspended.

vi. Assay Procedure: The details as described in the instrument manual are

given below.

1. Calibrating the ACS: 180 Insulin assay:

a. Define the sample probe setting

Sample probe: 6 primes

b. Start the system. This procedure takes approximately 7.5 minutes.

2. Schedule the requested tests or profiles for each sample.

3. Prepare and load insulin calibrator, if required:

109

a. Prepare the low and high calibrators according to the instructions in the

insulin calibrator product instructions.

b. Dispense the low and high calibrators into sample cups labeled with the

appropriate barcode labels.

c. Load the sample cups on the sample tray.

4. Prepare and load the quality control samples:

a. Prepare the quality control material according to the instructions in the

quality control product instructions.

b. Dispense the quality control samples into labeled sample cups.

5. If dilution is required, dispense insulin diluents into a sample cup labeled with the

appropriate barcode label and load the sample cup on the sample tray.

6. Prepare the primary tubes or sample cups and load them on the sample tray.

7. Load the solid phase and lite Reagent in adjacent positions on the reagent tray.

8. Start the system.

Dilutions

Samples with insulin levels greater than 300 mU/L must be diluted and retested to

obtain accurate results.

Samples can be automatically diluted by the system or prepared manually.

For automatic dilutions, ensure that insulin diluent is loaded and set the system

parameters as follows:

Dilution set point: ≤ 300 mU/L

Dilution factor: 2.5

Manually dilute serum samples, when sample results exceed the linearity of

the assay using automatic dilution, or when laboratory protocol requires

manual dilution.

Use insulin diluent to manually dilute patient samples, and then load the

diluted sample on the sample tray, replacing the undiluted sample.

Ensure that results are mathematically corrected for dilution. If a predilution

factor is entered when scheduling the test, the system automatically calculates

the result.

110

Specificity

The cross reactivity of theACS: 100 insulin assay was determined by spiking

serum samples with the following compounds at the indicated levels. These

compounds did not have a significant effect on the insulin measurement.

Table11. Interference testing in the assay

Substance Amount added Mean % Recovery

Proinsulin 1 µg/ml 100.8

C-Peptide 500 ng/ml 95.1

Gastrin-1 1 µg/ml 96.6

Glucagon 1 µg/ml 100.2

Secretin 1 µg/ml 101.6

Interference testing was determined according to NCCLS Document EP7-P12

Sensitivity and Assay Range

The ACS: S100 insulin assay measures insulin concentrations 300 mU/L with a

minimum detectable concentration of 0.5 mU/L. Analytical sensitivity is defined

as the concentration of insulin that corresponds to the RLUs that are two standard

deviations greater than the mean RLUs of 20 replicate determinations of the

insulin zero standard.

Standardization

TheACS: 100 insulin assay is standardized against World Health Organization

(WHO) 1st IR 66/304. Assigned values of calibrators are traceable to this

standardization.

111

2.6. EVALUATION OF ALDOSE REDUCTASE INHIBITORY ACTIVITY OF ISOLATED FRACTIONS

Materials

Albumin, Sodium chloride, Disodium hydrogen phosphate, monhydrate, Sodium

dihydrogen phosphate, monohydrate, Dipotassium hydrogen phosphate anhydrous

were procured from Merck Specialities Pvt. Ltd., Mumbai, Comassie Brilliant

Blue G-250 (B0770-5G) and Quercetin were purchased from Sigma Aldrich,

Germany. Nicotinamide adenenine dinucleotide phosphate, reduced tetra sodium

salt, Dulicitol (galactitol), D+ Galactose, Sorbitol, Phenylisocyanate and DL-

Glyceraldehyde were procured from Himedia Lab., Mumbai, Ethanol (Absolute)

Phosphoric acid, Dimethyl Sulphoxide, Dextrose (anhydrous), Pyridine, Methanol

and Acetonitrile for HPLC and Spectroscopy were purchased from SD Fine

Chemical Ltd., Glucose kit was purchased from Beacon Diagnostics Ltd.,

Navasari, India.

Method

2.6.1 Assesment of aldose reductase inhibitory activity of fractions using rat lens homogenate

Preparation of Lens homogenate

Wistar-albino rats (male) weighing 180-200g), five in number, were selected and

were sacrificed by spinal nerve dislocation. The eye balls were collected and

washed with saline. From these eye balls, lenses were enucleated through

posterior approach, washed with saline. To the obtained lenses 3 volumes of 0.1M

(100mM) Sodium phosphate buffer, pH 6.2, was added. This buffer was prepared

by dissolving Disodium hydrogen phosphate monohydrate (Dibasic Sodium

Phosphate) 0.66g, and Sodium dihydrogen phosphate monohydrate (Monobasic

Sodium Phosphate) 1.27g, in small quantity of water and by making the final

volume to 100ml using double distilled water. Then the pH of the obtained buffer

112

was adjusted to 6.2. The lenses added with buffer were homogenized by using

tissue homogenizer. The obtained homogenate was centrifuged at10,000 rev/min

for 20 minutes at 4°C and the supernatant was collected carefully using

micropipette. This supernatant obtained served as enzyme preparation and it was

stored in freezer at -40°C (Kador et al., 1998)

(Jung et al., 2008).

Estimation of protein content

0.15 M NaCl was prepared by dissolving 0.8766 g of NaCl in small quantity of

double distilled water and by making the final volume to 100 ml with the same.

To plot a standard graph, albumin was taken as a standard protein. 10mg of

albumin was weighed and dissolved in 0.15M NaCl and the final volume was

made up to 100 ml with the same. The concentration of the obtained solution was

100µg/ml and different concentration of solutions like 10, 20, 40, 60, 80µg/ml

were prepared from the stock by using the 0.15M NaCl solution. Bradford reagent

was used as a colour developing agent. This reagent was prepared by dissolving

10mg of Coomassie Brilliant Blue in 4ml of ethanol and to this 8.5ml of

phosphoric acid was added and shaken well. Then small quantity of double

distilled water was added and mixed well. The final volume was made up to

100ml with double distilled water. The reagent was stored in dark. 0.5ml of

different concentration solutions of albumin was taken and 5ml of Bradford

reagent was added to each concentration individually. The absorbance of each was

measured at 595nm. 0.5ml of 100 times diluted lens homogenate was also mixed

up with the 5ml of the reagent and the absorbance was checked at 595nm in UV-

spectrophotometer. The readings obtained were noted down. The standard graph

was plotted by taking albumin concentration (µg/ml) on x-axis and absorbance (at

595nm) on y-axis (Fig. 21). By using the standard graph, the protein concentration

of the lens homogenate was determined.

113

Determination of enzyme activity

The activity of the lens homogenate was determined by measuring the amount of

NADP converted from NADPH per unit time at 37°C, spectrophotometrically.

One unit (U) of activity is defined as ‘the amount of the enzyme catalyzing the

oxidation of 1µmol of NADPH per minute under experimental conditions’. For

the determination of the activity, initially 0.15mM NADPH, 10mM DL-

glyceraldehyde and Sodium phosphate buffer of pH 6.2 were prepared. 0.15mM

NADPH was prepared by dissolving 5mg of NADPH in 4ml of double distilled

water and from which 1ml was taken and the final volume was made up to 10ml

with water. 10mM DL-glyceradehyde was prepared by dissolving 9mg of DL-

glyceraldehyde in small quantity of double distilled water and by making the final

volume to 100ml with the same. Sodium phosphate buffer of pH 6.2 was prepared

by the method described under preparation of lens homogenate. In a reference

cuvette, 200µl of 0.15mM NADPH, 200µl of water (instead of substrate), 200µl of

lens homogenate were added and the final volume was made up to 2ml with the

sodium phosphate buffer of pH 6.2. Then the absorbance was checked at 340nm

in a double-beam UV-spectrophotometer.and the reading was recorded as control.

In a sample cuvette, all the components of reference and 200µl of 10mM DL-

glyceraldehyde (substrate) in place of water of reference cuvette were taken and

absorbance determined immediately after the addition of substrate, as the enzyme

reaction was started by addition of substrate. The absorbance (OD) was recorded

in a double beam UV-Spectrophotometer at 340nm for at least 1min at 5s interval.

Enzyme activity was calculated using the following formula;

( ) ( )Enzyme activity =6.2 Volume of enzyme taken( ) Total reaction volume(ml)

Abs Enzyme units Abs control unitsX ml X

∆ − ∆

Where, 6.2= micro molar extinction coefficient of NADPH at 340 nm.

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Determination of enzyme specific activity

Specific activity of the rat lens homogenate (enzyme) was calculated by

using the formula:

Specific activity = Activity

Protein concentration

Uml

mgml

Enzyme Inhibitory Assay

All concentrations of Quercetin, Glibenclamide, AFSF, MF1SF and MF2SF were

prepared by dissolving them in Dimethyl Sulphoxide (DMSO), which was found

to have no effect on the enzymatic activity at less than 1%(Jung et al.,2008 ). For

the preparation of stock solutions (1000µg/ml), 10mg of each test compound was

weighed and dissolved in 10ml of DMSO, individually. By using these stock

solutions, different concentrations like 0.5, 1, 5, 10µg/ml of Quercetin and

Glibenclamide; 5, 10, 50, 100µg/ml of each fraction was prepared separately. In a

blank cuvette, reaction mixture was added which includes 200µl of NADPH

(0.15mM), 200µl of rat lens homogenate (enzyme), 200µl of double distilled water

instead of substrate (DL-glyceraldehyde), 200µl of DMSO instead of test sample

and the final volume was made up to 2ml with sodium phosphate buffer (pH6.2).

Absorbance was checked at 340nm and the reading was recorded as correction

factor. In a control cuvette, 200µl of substrate, NADPH, lens homogenate, DMSO

(instead of sample) were added. In sample cuvette, all the components of reaction

mixture, as of control cuvette, were added and 200µl of different concentration of

solutions of samples, instead of DMSO of control cuvette, were added separately.

These sample cuvettes were read against control cuvette. The reaction was

initiated by the addition of 200µl DL-glyceraldehyde and absorbance was

measured at 340 nm under UV-Spectrophotometer for at least 1min at 5s intervals.

All the concentrations employed are in triplicate and absorbance was recorded.

The ARI activity of each sample was calculated using the formula;

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/ min / min% 1 100

/ min / min

A sample ABlankARI X

A Control ABlank

∆ − ∆ = −

∆ − ∆

Where, ∆A Sample/min = decrease of absorbance for a min with a test sample.

∆A Blank/min = decrease of absorbance with DMSO and water instead of a

sample and a substrate respectively.

∆A Control/min = decrease of absorbance with DMSO in place of a sample

The results are shown in Table 29.

2.6.2 Assessment of Aldose reductase inhibitory activity of fractions using rat kidney homogenate

Preparation of Rat Kidney Homogenate

Wistar-albino rats (male) weighing 180-200 g, five in number, were selected and

were sacrificed by spinal nerve dislocation. Rats were dissected ventrally and pair

of kidneys from each rat were collected and washed with saline. Kidneys were cut

into small pieces and 3 volumes of 0.1M (100mM) Sodium phosphate buffer, pH

6.2, was added and homogenized by using tissue homogenizer. The obtained

homogenate was centrifuged at10,000 rev/min at 4°C for 30min and the

supernatant was collected carefully using micropipette. This supernatant obtained

served as enzyme preparation and it was stored in freezer at -40°C until use

(Kinoshita, 1974).

Estimation of protein content

The protein concentration of the rat kidney homogenate obtained was estimated

by using the standard graph of Albumin which was obtained by plotting different

albumin concentrations (µg/ml) on x-axis and absorbance at 595nm on y-axis

(Fig. 21).

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Determination of enzyme activity

The activity of the rat kidney homogenate was determined by measuring the

amount of NADP converted from NADPH per unit time at 37°C,

spectrophotometrically. The procedure is as described earlier.

Determination of enzyme specific activity

The method and formula were followed the same as described for rat lens

homogenate.

Enzyme Inhibitory Assay

This assay method is same as that of lens homogenate .But in kidney model,

instead of lens homogenate; kidney homogenate was taken in reaction mixture for

spectrophotometric analysis at 340nm. All the concentrations employed were in

triplicate and the absorbance was recorded. Then the ARI activity of each

concentration was calculated using the same formula. The results are depicted in

Table 30.

2.6.3 Assessment of in vivo Aldose reductase inhibitory activity of fractions in Glactosemia induced rats

The accumulation of polyols such as sorbitol, galactitol, etc., is thought to be

responsible for the development of cataracts (Kinoshita et al., 1974). In order to

decrease the accumulation of polyols there should be inhibition of aldose

reductase enzyme. Unless there is a sufficient activity of AR enzyme, then only it

is possible to detect AR enzyme in human body. In order to achieve these there

should an increase of blood sugar levels. There are two methods which are

feasible for experimentation.

i. Diabetes induction.

ii. Galactosemia induction by oral galactose feeding.

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In diabetic condition, there is an increased blood glucose level. This glucose will

convert into its alcoholic form, sorbitol, by AR enzyme. The sorbitol obtained is a

suitable substrate for the enzyme sorbitol dehydrogenase, second enzyme of

polyol pathway, and this enzyme oxidizes the sorbitol to fructose, which is an

energy source. The conversion of sorbitol to fructose is a very slow process, and

the quantity of sorbitol formed is difficult to detect under experimental conditions.

In galactosemia induction, the sugar galactose will convert to its alcohol,

galactitol, by AR enzyme. This galactitol is not a suitable substrate for

dehydrogenase enzyme. Hence most of the galactitol formed will accumulate in

the lens and it becomes comparatively easier to detect it under experimental

conditions. The onset and progression of retinal changes are more rapid in

galactosemia than other diabetes models (Kador, 1998). Hence, glactosemia

induction is considered to be more feasible method. In galactosemia induction,

galactose is given through oral route and any increment in the dose according to

their weights will not kill the animals. Hence the mortality rate will be very less

when compared to diabetes induced animals. Hence in this study, galactosemia

induction was preferred to diabetes induction.

Grouping of animals

Six week old 30 male Wistar-albino rats, weighing 180-200g were used in the

present study. They were grouped into 5, according to their weights. Each group

contains 6 animals. The initial weight of each animal was recorded. All six groups

of animals were labeled according to the treatment given. All the sample solutions

for dosing were prepared in double distilled water.

• Group I (Control): Rats were treated only with 2ml of galactose, 100mg/ml

(800mg/Kg body weight), and solution for 14 days, for inducing galactosemia.

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• Group II (Quercetin): The rats were treated with 2ml of 2.5mg/ml (10mg/Kg

body weight) of quercetin solution along with simultaneous galactose

treatment.

• Group III (AFSF): The rats were treated with 2ml of 12.5mg/ml solution of

AFSF (200mg/Kg body weight) solution along with simultaneous galactose

treatment.

• Group IV (MF1SF): The rats were treated with 2ml of 12.5mg/ml solution of

the MF1SF (200mg/Kg body weight) along with simultaneous galactose

treatment.

• Group V (MF2SF): The rats were treated with 2ml of 12.5mg/ml solution of

the MF2SF (200mg/Kg body weight) along with simultaneous galactose

treatment.

• Group VI (Glibenclamide): The rats were treated with 2ml of 2.5mg/ml

(10mg/Kg body weight) of Glibenclamide solution along with simultaneous

galactose treatment.

Collection of blood samples

The rats were fasted for 16 hours with water ad libitum before blood collection.

Blood samples were collected from tail vein for blood glucose estimation under

diethylether aneasthesia. The obtained blood samples were centrifuged and serum

samples stored at -20°C. This was done before the administration of the test

compounds.

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Dosing

The dosing was done as mentioned earlier for 14days. During the dosing, the rats

were fed with standard rat pellet and water ad libitum. 12hours dark and 12hours

light cycle was maintained and the temperature was controlled (25°C) by using air

conditioner in animal house.

Collection of blood samples

After dosing for 14 days, all the rats were fasted for 16 hours with water ad

libitum. On 15th day, the blood samples were collected and serum samples were

prepared as described. The serum samples were stored at -20ºC.

Biological sample preparation

After blood collection, on the 15th day, all the animals were sacrificed by spinal

nerve dislocation and pair of eye balls were collected from each rat and lenses

were dissected from the eyes using a posterior approach. The lenses obtained were

washed with saline and weight was recorded. Then pair of lenses of each rat was

homogenized with 2ml of ice cold water, individually. The proteins were

precipitated with ethanol (70% of the final volume) and they were removed by

centrifugation (15min at 12,000g). This centrifugation was done at 4°C. Then the

supernatant was collected carefully using micropipettes (Dethy et al., 1969).

Lyophilization

The supernatant obtained was taken into screw capped lyophilization bottles. To

every 3rd sample of each group 100µl of internal standard (sorbitol) 500mg/ml

solution was added. Then the bottles were screw capped and first kept in freezer

at-400C for freezing for 4 hours. Then the caps were removed and the bottles were

covered with aluminum foil and were kept in Lyodel freeze-drier for 8hours for

lyophilization.

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Derivatization

In order to obtain derivatives of sugars and polyols, 250µl of pyridine and 500µl

of Phenyl isocyanate were added to each lyophilized sample obtained from lenses.

Then the flask reactors were stoppered and derivatives were formed by incubating

the solutions at 55°C for 1h in water bath with mechanical shaking. After the

incubation period, the flask reactors were cooled and 250µl of methanol was

added to eliminate the excess phenyl isocyanate, which otherwise could react with

the water of the eluent. The clear solutions obtained were then diluted twice with

pyridine to decrease the interferences due to the absorption of the reagents (Dethy

et al., 1984). The free hydroxyl groups of sugar alcohols and neutral sugars react

with Phenylisocyanate. The resulting urethane bond is very stable and insensitive

to pH. Derivated samples and standards can therefore be stored at room

temperature for several days. This reaction seems to be catalyzed by tertiary

nitrogen atoms such as pyridine or dimethylformamide. Although

dimethylformamide has the same catalytic properties as Pyridine, the latter is

preferred because it is a better solvent for neutral sugars and polyols (Dethy et al.,

1969).

HPLC Analysis

A mixture of 60% acetonitrile and 40% 0.01M K₂HPO₄ buffer adjusted to pH 7.0

with H₃PO₄ in double distilled water was used as mobile phase for the separation

of the derivatives in HPLC analysis. Then the prepared mobile phase was

sonicated for 15min to remove entrapped air. 0.01M K₂HPO₄ buffer was prepared

by dissolving 1.74g of K₂HPO₄ in small quantity of double distilled water and by

making the volume to 1000ml. The flow rate of the mobile phase was adjusted to

2ml/min and the injection volume was 20µl. Detector wavelength was adjusted to

240nm. Derivated samples of each group were analyzed by HPLC (Dethy et al.,

1984). The chromatograms are shown in figs.23-29.

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For plotting the standard graph, galactose (pure) 1g was weighed and was

dissolved in 15ml of double distilled water. This was lyophilized. After

lyophilization, 20mg of galactose lyophilized powder was weighed and was

dissolved in 20ml of pyridine, which gives 1mg/ml concentration solution, which

serves as stock solution. By using this stock solution, 1, 3, 5, 10, 30, 50 µg/ml

solutions were prepared and analyzed by HPLC. Galactitol standard graph is

shown in fig 22. A comparasion of galactitol conc. in test groups is shown in

Table 33, fig. 30.

Glucose estimation

The blood glucose level of collected serum samples were estimated using GOD-

POD method (Trinder, 1969). From each sample 20µl of serum was taken into a

test tube and 2ml of working reagent (buffer reagent), which was given in glucose

kit was added. This solution was kept a side for 10 min for colour development.

Then the absorbance was detected at 505nm. 20µl of glucose standard solution,

which was given in glucose kit, was taken and 2ml of buffer reagent was added to

it. The colour developed after 10 min was detected under UV-Visible

Spectrophotometer at 505nm. The serum glucose level is calculated using the

formula; the resuts of the study are shown in Table 32.

Absorbance of testSerum glucose level = 100Absorbance of control

X

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2.7 EVALUATION OF HEPATOPROTECTIVE AND ANTI HEPATO TOXIC ACTIVITY OF ISOLATED FRACTIONS

2.7.1 Screening methods

Hepatoprotective activity is defined as the ability to control or reduce the impact

of hepatotoxic substances on liver in cases were pretreatment with the test drug is

done. It can be evaluated by well-established invivo and in vitro methods.

Antihepatotoxicity is defined as the capacity of drug to reduce the toxic effects

produced previously by the toxic substance and recovery of hepatocytes to the

normal function of the liver.

In-vivo evaluation

I. Carbon tetrachloride induced liver toxicity in rats:

The principle cause of carbon tetrachloride induced hepatic damage is lipid

peroxidation and decreased activities of antioxidants, enzymes and generation

of free radicals. The inhibition of the generation of free radicals is important in

providing protection against hepatic damage. Liver microsomal oxidizing

system connected with cytochrome P-450 produce reactive metabolites of CCl4

such as trichloromethyl radical (CCl3●) or trichloro peroxy radical (CCl3O2

●)

these radicals cause lipid peroxidation which produces hepatocellular damage

and enhanced production of fibrotic tissue.CCl4 is metabolized by the mixed

function oxidase system in the endoplasmic reticulum of the liver. Cleavage of

the carbon-chloride bond results in the formation of free trichloromethyl

radicals (CCl3●) which are highly unstable and immediately react with

membrane components. They form covalent bonds with unsaturated fatty acids

or take a hydrogen atom from the unsaturated fatty acids of membrane lipids,

resulting in the production of chloroform and lipid radicals (Reckengel et al.,

1973).

II. The lipid radicals react with molecular oxygen, which initiate peroxidative

decomposition of phospholipids in the endoplasmic reticulum. The

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peroxidation process results in the release of soluble products that may affect

cell membranes. Microsomal oxidation of chloroform was found to involve in

formation of phosgene. CCl4 induced necrosis is most severe in the

centrilobular hepatocytes (zone 3) as here the concentration of cytochrome P-

450 is highest (Brattin et al., 1985). It is difficult to reproduce all the features of

human liver disease and no single laboratory model was found satisfactory.

Hence, it is necessary to isolate specific components of the disease and to study

them individually. To simulate various liver diseases, carbon tetrachloride has

been used as a hepatotoxic substance due to simplicity in its structure and

ability to produce various experimental models for study.It is believed that it

acts directly on liver cells causing swelling, resulting in mechanical obstruction

of sinusoidal blood flow. These alterations may be manifested within 24 hr or

more after the injury. CCl4 induces a coagulative necrosis of the hepatocytes

that does not affect the sinusoidal lining cells permitting the retention of intact

vascular system. After intra-gastric administration of CCl4 in rats, hepatic lipid

metabolism is disturbed within the first 30 minutes. Change in appearance of

the endoplasmic reticulum, depression of microsomal enzyme activity and

reduced hepatic protein synthesis were observed within the first hour of CCl4

intoxication. Within 2-4hr, calcium content of the liver mitochondria is doubled

and rises rapidly thereafter, becoming 15 times normal by 40 hours. Associated

with this abnormal movement of calcium into liver cells, disturbance in

electrolyte distribution and swelling of liver cells was observed. Liver glycogen

was depleted due to CCl4 intoxication. Between 5th and10th hr the lysosomes

become disrupted and intracellular enzymes appear in the plasma.

Mitochondrial damage sets in about 10hr after intoxication. Focal necrosis is

evident as early as 6hr after CCl4 intoxication, which at first is mainly mid-

zonal. After 12hr, the centrilobular cells exhibit prenecrotic changes and

balloon cells are prominent in the mid-zonal region. Within 14 hr, marked

centrilobular necrosis affecting up to half of the lobule occurs. At least, two

separate series of changes appear to be taking place in the cells of mid zonal

region. One is entirely cytoplasmic characterized by increased osmophilia. The

other involves both cytoplasm and nucleus and is characterized by gross

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vacuolization of cytoplasm and increased osmophilia and pyknosis of

nucleus.The onset of mitochondrial damage could be expected to have most

serious of consequences. Abnormal entry of calcium ions into the liver cells

produce mitochondrial swelling, inhibit oxidation dependent on pyridine

nucleotides, and activate mitochondrial ATPase. Following carbon

tetrachloride intoxication, rapid accumulation of triglycerides in liver was

observed, which may be due to marked inhibition of normal transfer of

triglycerides from liver to plasma. Depressed hepatic protein synthesis results

in reduced amount of apoprotein required for combining with liver triglycerides

to form lipoproteins. These lipoproteins represent the major vehicle for transfer

of triglycerides away from the liver. A drop in low-density plasma proteins

results in accumulation of triglycerides in liver (Smuckler, 1976).

III. Paracetamol induced toxicity: Paracetamol is an antipyretic and analgesic

drug, when taken in large doses, it becomes a potent hepatotoxin, generating

fulminated hepatic and renal tubular necrosis which is lethal in humans and

experimental animals (Goldin et al., 1996).

Mechanism of Paracetamol induced liver damage

Hepatotoxicity induced by paracetamol resembles other kinds of acute

inflammatory liver disease with prominent increase of SGOT, SGPT and ALP

levels. Paracetamol at therapeutic dosage is primarily metabolized and

detoxified by glucuronidation and sulphation, followed by renal excretion

(Bessems et al.,2001) at toxic doses, the compound is converted to a toxic

form, N-acetyl-p-benzoquinoneimine (NAPQI), which is an electrophilic

intermediate, oxidized by cytochrome P-450 and gets converted to a highly

reactive and toxic metabolite .NAPQI, which is normally conjugated with

hepatocellular reduced glutathione (GSH) leads to 90% total hepatic GSH

depletion in cells and mitochondria and also covalently binds with cellular

proteins, including the Ca+2–ATPase of the endoplasmic reticulum, increases

intracellular free Ca+2, contribute to hepatocellular death and mitochondrial

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dysfunction(Mitchel et al.,1973). NAPQI can also increase the formation of

ROS and reactive nitrogen species (RNS) such as superoxide anion, hydroxyl

radical, and hydrogen peroxide, and peroxynitrite, respectively. Increased

levels of ROS and RNS can attack biological molecules such as DNA, protein,

phospholipids, which leads to lipid peroxidation, nitration of tyrosine, and

depletion of the antioxidant enzymes (SOD, CAT, and GPX), results in

oxidative stress (Michael et al., 1999).

IV. Thioacetamide induced hepatotoxicity: Thioacetamide is a potent

hepatotoxic that is metabolized by CYP450 enzymes present in the liver

microsomes and is converted to a toxic reactive intermediate called

thioacetamide oxide due to oxidative stress in the hepatic cells. It is responsible

for the changes in cell permeability, increased intracellular concentration of

Ca++, increase in nuclear volume and enlargement of nucleoli and also inhibits

mitochondrial activity which leads to cell death (Madani et al., 2008).

V. Alcohol induced hepatotoxicity: Alcohol consumption is known to cause fatty

infiltration, hepatitis and cirrhosis. Alcohol can induce invivo changes in

membrane lipid composition and fluidity, which may eventually affect cellular

functions.Mechanism responsible for effects of alcohol are increase in hepatic

lipid peroxidation which results in loss of membrane structure and integrity.

VI. D-galactosamine induced liver necrosis: Hepatotoxicity of galactosamine is

due to its metabolite which causes lowering of the levels of uracil nucleotides

(UTP, UDP- galactose) resulting in inhibition of RNA synthesis leading to

necrosis (Rao et al., 1998).

VII. Rifampicin induced hepatotoxicity: Rifampicin is a broad spectrum antibiotic

used in the treatment of tuberculosis. The metabolite desacetylrifampin which

reduces drug metabolizing enzymes and specifically bind to RNA polymerases

and there by inhibits the synthesis of nucleic acid and protein.\

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In vitro evaluation:

In vitro models employing primary cultures of hepatocytes (Nagagiri et al., 2003)

and using carbon tetrachloride or D-galactosamine, ethanol or phalloidin, as

hepatotoxic substance have been devised. Such methods have a number of

advantages over in vivo methods, including the ability to dispose numerous

samples at one time at a low cost, the requirement of small sample size, little

variation, and good reproducibility of results. It presents lot of disadvantages as

they require strict aseptic conditions and posses less in vivo correlation with

respect to the efficacy.

Assessment of the liver function:

Studying the morphology, histopathology of liver, serum biochemical changes and

funtional parameters assess the liver function.The following are the details of the

various parameters used in the assessment of the liver functions.

i. Biochemical changes in liver diseases: Liver is an important organ actively

involved in metabolic functions, is a frequent target of number of toxicants.

CCl4 is a widely used chemical to induce liver damage in experimental

studies, and its toxicity has been studied extensively. The resulting hepatic

injury was characterized by leakage of cellular enzymes into the blood stream

and by centrilobular necrosis (Zimmerman et al., 1970). Elevation of enzymes

in the body is most often associated with liver injury or disease. Elevation of

SGPT, SGOT often reflects hepatocellular damage. SGOT is found in

decreasing concentrations in liver, cardiac muscle, skeletal muscle, kidneys,

brain,pancreas, lungs, leukocytes and erythrocytes whereas ALT (SGPT) is

found primarily in liver and kidney. Thus an elevation of SGPT is more

specific for liver injury than an elevelation of AST (SGOT). Elevated SGOT

levels may be caused by disorder of other organs and tissues, particularly

striated muscle. The most common causes of elevated levels of

aminotransferases are: alcohol – related liver injury, chronic hepatitis B and C,

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autoimmune hepatitis, fatty infiltration of the liver (hepatic steatosis),

nonalcoholic steatohepatitis, hemochromatosis, Wilson’s disease, Alpha-1-

antitrypsin deficiency and celiac sprue. Specialized testing is required to

determine the specific diagnosis when elevated aminotransferases levels

occur.

Reference ranges for the aminotransferases are method dependent, which can

make comparison of elevated SGOT and SGPT levels difficult, but the SGOT:

SGPT ratio has been used in the differential diagnosis of liver disease. The SGOT:

SGPT ratio (with elevated levels of both enzymes) is approximately 1:1 for most

primary liver disease. The ratio is generally lowest in viral hepatitis (both acute

and chronic) and this ratio is typically less than 1:1. The levels of both SGOT and

SGPT are very high (greater than 10 times the highest normal level) in acute

hepatitis and are lower (often less than 4 times the highest normal level) in chronic

hepatitis. In patients with chronic hepatitis C, there is a subset of patients with

normal SGPT levels. In one study, almost half of these patients developed

persistently elevated SGPTlevels following alpha-interferon (INF) treatment and

this finding was used to suggest that INF treatment may be harmful to these

patients. Patients with nonalcoholic steatohepatitis usually also have a ratio of less

than 1:1. On the other hand, a high SGOT: SGPT ratio of 2:1 or 3:1 occurs in

patients with chronic alcohol-induced liver damage. In patients with alcohol

abuse, the SGOT level is rarely more than 8 times the normal range and the SGPT

is seldom more than 5 times the normal range and may, in fact, be normal. An

elevation of another liver function enzyme, GGT, is also helpful in the diagnosis

of alcohol abuse.

Bilirubin is found in urine in cholestatic jaundice wherein there is regurgitation of

conjugated bilirubin, which being water soluble and loosely attached to plasma

albumin passes into the urine. There is a low but variable threshold level for

conjugated bilirubin below which bilirubinuria does not occur. Bilirubinuria may

be found in early stages of viral hepatitis before clinical jaundice has developed.

The absence of urine bilirubin in the recovery phase of jaundice is due to non–

128

filterable protein bound S-bilirubin having long half–life at the glomerulus. The

urine urobilinogen is derived from urobilinogen reabsorbed from the intestine,

which is not excreted by the liver. The amount present thus depends both on the

amount of bilirubin entering the intestine and on the ability of the liver to excrete

the urobilinogen which is reabsorbed from the intestine. Urine urobilinogen tends

to be raised a little in hemolytic jaundice, since the liver is not able to excrete

completely the increased quantity absorbed from intestine (Limdi et al., 2003).

ii) Functional: The time of lost reflex induced by short acting barbiturate is

significantly prolonged in the event of any hepatic damage and this can be

used as a measure of the function of the liver drug metabolizing enzymes.

iii) Morphological: Changes in color, weight and volume of liver.

iv) Histopathological: Changes in liver architecture like hepatic lobules, swelling

of liver cells, fatty changes, focal necrosis, inflammatory cell in filtration

around portal areas, kupffer cell hyperplasia.

2.7.2 Assessment of Hepatoprotective activity of fractions in CCl4 induced

hepatotoxicity in rats

Material :

Silymarin and Paracetamol were the gift sample from Micro Labs Ltd., Hosur, Dr.

Reddys foundation, Hyderabad, India respectively. Thiopentone Sodium (Thiosol)

and olive oil were purchased from Neon Labs, Mumbai, Sevenships, and

Hyderabad, India respectively. The biochemical analytical kits for SGOT, SGPT,

ALP and TB were purchased from Beacon Diagnostics Ltd., Navasari, India. All

other chemicals and solvents used were of analytical grade purchased from local

suppliers.

129

Method :

The evaluation of the fractions of methanolic extract i.e AFSF, MF1SF and MF2SF

for hepatoprotective activity was done according to the procedure given in the

literature (Agarwal et al., 2006) with minor modifications. The animals were pre-

treated orally with the test material/standard drug suspended in 2% gum acacia

before inducing liver damage with CCl4 to evaluate the activity. Hepatic injury

was induced in rats by oral administration of a single dose of 1 ml/kg b.w. CCl4

mixed with equal volume of olive oil on the seventh day, 2 h after the last

treatment. The rats were divided into nine groups of six each, control, toxic,

standard, and two test groups of each fraction. The details of the treatment given

to these groups are as follows:

Group I (Control group): Treated with vehicle, (1 ml/kg b.w. of 2% gum acacia

in water) daily for seven days.

Group II (Toxic group): Treated with vehicle (1 ml/kg of 2% gum acacia in

water) daily for seven days followed by CCl4 on the seventh day.

Group III (Standard group): Treated with silymarin (50 mg/kg) daily for seven

days followed by CCl4 on the seventh day.

Group IV (AFSF 50): Treated with AFSF (50mg/kg.b.w.) for seven days

followed by CCl4 on the seventh day.

Group V (AFSF 100): Treated with AFSF (100 mg/kg.b.w.) for seven days

followed by CCl4 on the seventh day.

Group VI (MF1SF 50): Treated with MF1SF(50mg/kg.b.w) for seven days

followed by CCl4 on the seventh day

130

Group VII (MF1SF 100): Treated with MF1SF(100mg/kg.b.w) for seven days

followed by CCl4 on the seventh day

Group VIII (MF2SF 50): Treated with MF2SF(50mg/kg.b.w) for seven days

followed by CCl4 on the seventh day

Group IX (MF2SF 100): Treated with MF2SF(100mg/kg.b.w) for seven days

followed by CCl4 on the seventh day

The blood was collected from the retro orbital plexus of the rats of all groups 24 h

after administration of CCl4 under thiopentone sodium (35 mg/kg b.w.i.p)

anesthesia. The blood samples were allowed to stand for 30 min at room

temperature and then centrifuged at 3000 rpm for 30 min to separate the serum

using Remi Model: R8-C, centrifuge. The serum was analyzed for various

biochemical parameters such as SGOT, SGPT, ALP and TB .Their percentage

protection was calculated by using following formula.

Percentage protection = 1 100T V XC V

− − −

Where “T” is the mean value of test group (extract /standard), “C” is the mean

value of toxic group (CCl4) alone and “V” is the mean value of control group

(vehicle treated animals).

The animals were then dissected and the livers were carefully removed and

washed with 0.9% saline solution. A part of the liver sample was preserved in

formalin solution (10% formaldehyde) for histopathological studies. The results of

the study are shown in Table-34, 35 and Fig-31, 32, 33, 34 and 35.

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2.7.3 Assessment of Anti-hepatotoxic Activity of fractions in CCl4 induced

hepatotoxicity in rats:

The evaluation of the fractions of methanol extract of bark of Soymida febrifuga

i.e AFSF, MF1SF and MF2SF for antihepatotoxic activity was done according to

the procedure given in the literature with minor modification (Rasheeduz et al.,

1998). The rats were divided into nine groups of six rats each. The animals of

Group I served as control and was given orally a single daily dose of 2% gum

acacia (1 ml/kg.b.w.) for seven days starting from 4th day to 10th day of the study.

Group II served as toxic control and administered orally with a single dose of

CCl4 in olive oil (1ml/kg.b.w.) on 1st and 3rd day and a single dose of 2% gum

acacia from 4th day to 10th day. Group III, IV, V, VI, VII, VIII and IX treated with

standard and selected doses of test samples. The animals of group III to IX were

treated with CCl4 in a similar way as in group II i.e.; on 1st and 3rd day. From 4th

day to 10th day, group III- IXwere given orally a single daily dose of silymarin (50

mg/kg.b.w) and selected doses of test samples, suspended in 2% gum acacia

respectively.

After 24h of last treatment, blood and liver samples were collected from the

animals of all groups for estimation of various biochemical parameters (SGOT,

SGPT, ALP, and TB) and histopathological studies respectively in a similar way

as described in the previous section .The results of the study are summerised in

Tables- 36, 37 and Fig-36, 37, 38, 39 and 40.

2.7.4 Effect of fractions on Thiopentone induced sleeping time in CCl4 induced

hepatotoxicity in rats:

The effect of the fractions on thiopentone induced sleeping time in CCl4

intoxicated rats was determined according to the procedure described in literature

(Yadav et al., 2003). Rats were divided into six groups of six each. Group I was

kept as normal and normal sleeping time was determined after injecting sodium

thiopental (25 mg/kg b.w.i.p). Group II served as toxic control and was

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administered with CCl4 (1ml/kg b.w.i.p.) A single dose of silymarin (50mg/kg

b.w. p.o.) was administered to group III while AFSF (100 mg/kg b.w. p.o.) was

given to group IV, MF1SF (100 mg/kg b.w. p.o.) was given to group V and

MF2SF (100 mg/kg b.w. p.o.) was given to group VI. After 24 hours, a dose of

CCl4 (1ml/kg b.w.i.p.) was given to II-VI groups. Then sodium thiopental (25

mg/kg b.w.i.p.) was given to II, III, IV, V and VI groups after 2 h of CCl4

injection. The results of the study are given in Table 38 and Fig 41.

2.7.5 Assessment of Hepatoprotective activity of fractions in drug induced hepato

toxicity in rats:

The experiment was performed according to the method given in the literature

(Jafri et al., 1999). The rats were divided into nine groups comprising of six in

each. 2% gum acacia was used as vehicle for suspending the drugs and extract.

Group I was kept as control received a single daily dose of vehicle (2% gum

acacia 1 ml/kg.p.o.) for seven days. Groups II- IX were given orally a single daily

dose of vehicle, silymarin (50 mg/kg b.w.), AFSF 50 mg/kg, 100 mg/kg; MF1SF

50 mg/kg, and 100 mg/kg b.w.; MF2SF 50 mg/kg, and 100 mg/kg b.w for seven

days respectively. After 24 h of last treatment i.e., on 8th day a single dose of

Paracetamol (3 mg/kg.b.w) was administered to the animals of all groups leaving

group I. Then blood and liver samples were collected from the animals of all

groups after 24 h of administration of paracetamol for estimation of various

biochemical parameters (SGOT, SGPT, ALP, and TB) and for histopathological

studies respectively. The results of the study are given in Tables 39, 40 and

Fig 42-46.

2.7.6 Estimation of serum bio-chemical parameters:

The principle, details of the kits and methodology used in the estimation of the

various bio-chemical parameters by Autoanalyzer (Selectra Junior-Merck) in the

present investigation are as follows:

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A. Serum glutamate oxaloacetate transaminase (SGOT)

The principle and procedure is same as described in 2.5.7.

B. Serum glutamate pyruvate transaminase (SGPT)

The principle and procedure is same as described in 2.5.7

C. Alkaline Phosphatase

Principle: In an alkaline medium ALP hydrolyzes 4-nitrophenylphosphate to 4-

nitrophenol.

4-Nitrophenylphosphate + H2O Phosphate + 4-nitrophenolateALP

Reagents:

1117657/1 Reagent (1) Reagent solution 4×8 ml

1117675/2 Reagent (2) Start solution 1×9 ml

Reaction solution: Mix reagent (1) and reagent (2) at a ratio of 4:1, e.g. 4 ml

reagent solution plus 1 ml start reagent.

Test concentrations:

Diethanolamine HCl buffer, pH 9.8 1.0 mol/L

Magnesium chloride 0.5 mmol/L

4- Nitrophenylphosphate 10 mmol/L

Stability: All reagents are stable up to the expiry date given on the lable, when

stored at 2-80C.

D. Total Bilirubin

Principle:

Bilirubin reacts with diazotized sulphanilic acid after addition of caffeine, sodium

benzoate and sodium acetate. A blue azobilirubin is formed in alkaline Fehling

solution II. This blue compound can also be determined selectively in the presence

of yellow by products by photometry at 578 nm. The estimation of total bilirubin

is carried out according to the method (Jendrassik and Grof, 1938), in presence of

an accelerator.

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Total Bilirubin + Diazotized sulphanilic acid Azobilirubin compound

Bilirubin kit:

Reagents:

Reagent 1. Sulfanilic acid (29 mmol/L, 170 mmol/L HCl)

Reagent 2. Sodium nitrate (29 mmol/L)

Reagent 3. Accelerator (130 mmol/L caffeine, 156 mmol/L sodium benzoate,

460 mmol/ L sodium acetate).

Reagent 4. Fehling solution II (930 mmol/L potassium sodium tartrate,

1.9 mmol/L sodium hydroxide solution).

Stability : All reagents are stable up to the expire date stated when sealed and

stored at 150 to 250C.

Methadology:

The blood samples collected from the animals of the study were subjected to

centrifugation at 3000 rpm for 30 min to separate the serum. Then 0.5 µl of serum

sample was used to estimate each bio-chemical parameter. For the estimation of

above mentioned biochemical parameters, 0.5 µl of serum sample was transferred

to each of the pediatric sample cups. Then, these cups and the working reagent

bottles (25 ml) corresponding to the bio-chemical parameters were placed at their

respective positions in the rotor system of the autoanalyzer. After 30 min of

programming the test parameters, the corresponding parameter values of the

different serum samples displayed on the computer were recorded.

Histopathologial studies:

The liver is made up of hepatocytes and specialized cells called kupffer cells

interspersed with sinusoids. It is supplied with branches of bile duct. The normal

hepatocytes have intact plasma membrane. While in the presence of viral

infection/disease state or when drugs or chemicals affect liver cells there will be

changes in the permeability of plasma membrane. Disruption of cells is caused by

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excessive formation of fibrotic tissue eventually leading to necrosis.

Histopathological studies could be carried out to assess the degree of damage.

This is done by staining the fine section of liver isolates and examining under the

microscope. After the animals were sacrificed, livers were taken out and washed

with normal saline (0.9%). Then, 2-3 pieces of approximately 6cu.mm size were

cut and fixed in phosphate buffered 10% formaldehyde solution. After embedding

in paraffin wax, thin sections of 5µm thickness of liver tissue were cut and stained

with haemotoxylin-eosin stain.

Processing of liver Tissues:

Liver pieces were taken out from fixing solution and dehydrated for 30 min each in 30, 50, 70, 90 and 100% alcohol, successively. To remove the alcohol from the dehydrated tissues, they were kept for 30 minutes each in alcohol: xylene mixture (1:1) followed by pure xylene. The tissues were then kept in xylene: paraffin wax mixture (1:1) for 1 hr and then in molten paraffin wax at 620C, after which they were trimmed and mounted on wooden blocks for thin sectioning. Hand microtome (Yorco precision rotary microtome, model no YSI 114) was used to

cut thin sections of liver tissues of 5µm thickness. Staining and mounting of Liver tissues: Ribbons of thin sections of liver tissues were placed in rows on clean glass slides previously coated with albumin-glycerin mixture and few drops of water added to let the sections float. The slides were heated on hot plate to fix liver sections onto the slides. The slides were then placed for 5 minutes each in xylene to remove wax, then in absolute alcohol to remove xylene from the liver sections. Hydration of liver sections was attained by keeping them in descending series of alcohol and water mixtures (90%, 70%, 50%, 30% alcohol and in pure water) for three minutes each. Hydrated sections were stained with haemotoxylin stain for one minute and washed in running tap water to remove excess stain. Liver sections were dehydrated again by keeping in ascending proportions of alcohol-water mixtures (30%, 50%, 70%, and 90% alcohol) for one minute. After that, the sections were kept for 5 minutes each in absolute alcohol and then in xylene. Finally, the stained liver sections were

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mounted in DPX and viewed under optical microscope for histological examination. The photomicrographs (Motic, Germany) of the sections were also obtained.

2.8 STATISTICAL ANALYSIS OF DATA

All the values were expressed as Mean ± SD. The data was statistically evaluated

using one way analysis of variance (ANOVA) followed by post hoc Dunnet test in

Antidiabetic activity and Aldose reductase activity studies and using Tukeys

multiple comparison test for Hepatoprotective activity. The analysis was

performed using GraphPad Prism computer software version 5.0. (Saba et al.,

2010). Values corresponding to p<0.05 were considered significant.