50

METHODOLOGY AND

EXPERIMENTAL DETAILS

51

MATERIALS AND METHODS

Chemical used:

Methanol (SD Fine Chemical Ltd. Mumbai)

Anaesthetic ether (Sigma)

Carbon tetrachloride (SD Fine Chemical Ltd. Mumbai)

Paracetamol (Strides Arcolabs Ltd. Bangalore)

Thioacetamide (SD Fine Chemical Ltd. Mumbai)

Silymarin (Micro Labs Bangalore)

Liquid paraffin (CDH, Mumbai)

Nitroblue tetrazolium NBT (Loba Chemical, Mumbai)

EDTA (Nice Chemicals, Cochin)

Hydroxylamine hydrochloride (Nice Chemicals, Cochin)

Sodium carbonate (SD Fine Chemical Ltd. Mumbai)

Folin Cioalteau Reagent (Loba Chemical, Mumbai)

Copper sulphate (SD Fine Chemical Ltd. Mumbai)

Sodium hydroxide (SD Fine Chemical Ltd. Mumbai)

90% Alcohol (SD Fine Chemical Ltd. Mumbai)

Standard bovine albumin (SD Fine Chemical Ltd. Mumbai)

Pottasium dihydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)

Disodium hydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)

Hydrogen peroxide (SD Fine Chemical Ltd. Mumbai)

52

Kits used:

ASAT kit (Crest Biosystems, Coral Clinical Systems, Goa)

ALAT kit (Crest Biosystems, Coral Clinical Systems, Goa)

ALP kit9 (Crest Biosystems, Coral Clinical Systems, Goa)

Bilirubin kit (Crest Biosystems, Coral Clinical Systems, Goa)

Equipment used:

Analytical balance (Schimadzu/Japan)

Semi analyzer (Qualigen, AR 601 Mumbai)

53

4.1 STUDY SAMPLES

The leaves and bark of Buchanania lanzan were supplied by Yucca

Enterprises Mumbai. These were shade dried and used. Muppu and

Vidhakanachoornam were supplied by Dr. Jai Prakash Intergrated

Research Centre for Siddha Medicines Bangalore.

4.2 PLANT EXTRACTION

The leaves and bark of the plant were shade-dried and extracted using

various solvents in order of increasing polarity and extracts were

subjected for preliminary phytochemical investigation.

4.3 PHYTOCHEMICAL SCREENING 52

Standard phytochemical test were used to screen the extract for the

presence of different constituents. The various extracts of both the bark

and mature leaves of Buchanania lanzan were subjected to qualitative

analysis for the phytoconstituents like alkaloids, carbohydrates,

glycosides, steroids, tannins, proteins, amino acids and flavonoids as per

the standard procedures. The results are tabulated as shown in.

A. Tests for Alkaloids:

Hager’s test:- Extract treated with a few drops of Hager’s reagent

(saturated solution of picric acid) – yellow precipitates indicate presence

of alkaloids.

54

Mayer’s test:- Extract treated with Mayer’s reagent (potassium mercuric

iodide solution) – formation of cream precipitates indicate presence of

alkaloids.

Dragendorff’s test:- Extract treated with Dragendorff’s reagent

(potassium bismuth iodide solution) - orange precipitates.

Wagner’s test:- Extract treated with Wagner’s reagent (iodine,

potassium–iodine solution) –reddish brown precipitates.

B. Tests for carbohydrates:

Molish’s test:- Extract treated with Molish’s reagent (alpha naphthol in

95% ethanol) and a few drops of concentrated sulphuric acid were added

by the sides of the test tubes. A violet ring at the junction is formed.

Fehling’s test:- Extract treated with Fehling’s reagent (Fehling’s reagent

A-copper sulphate in water and Fehling’s reagent B-sodium potassium

tartarate) followed by heating. Red coloured precipitates indicate

presence of reducing sugars.

Barford’s test:- Extract treated with Barford’s reagent (copper acetate

in water and glacial acetic acid) - red colouration.

Benedict’s test:- Extract treated with Benedict’s reagent (copper

sulphate, sodium citrate and sodium carbonate in water)- red coloured

precipitates.

55

C. Tests for Steroids, Terpenoids and Cardiac Glycosides:

Liebermann-Burchard test:- 10 g of extract was dissolved in 1 ml of

chloroform, 1 ml of acetic anhydride was added followed by the addition

of 2 ml of concentrated sulphuric acid from the sides of the test tube.

Formation of reddish brown colour at the junction indicates the presence

of steroids/terpenoids/cardiac glycosides.

Salkowski test:- 1 ml of concentrated sulphuric acid was added to 10 g

of the extract dissolved in 1 ml of chloroform. A reddish brown layer

exhibited by the chloroform layer and green fluorescence by the acid

layer suggests the presence of steroids.

Noller’s test:- 5 g of the extract was dissolved in 2 ml of 0.01%

anhydrous stannic chloride in pure thionyl chloride .Yellow colour

indicates the presence of triterpenoids.

Legal’s test:- The extract was treated with sodium nitroprusside in

pyridine and methanolic alkali. The formation of pink colour indicates

the presence of cardiac glycosides.

Balget’s test:- 1 ml of the extract solution was treated with a few drops

of sodium picrate reagent, yellowish orange colour indicates the presence

of cardiac glycosides.

Killer Killani’s test:- 5 g of the extract was treated with 1 ml of glacial

acetic acid and a few drops of ferric chloride solution. 2 ml of

concentrated sulphuric acid were carefully added from the sides of the

test tube. Formation of a reddish brown colour at the junction of the two

56

layers and formation of bluish green colour in the upper layer indicates

the presence of deoxy sugar in the carbohydrates.

D. Tests for Saponins:

Diluted 1 ml of the extract with distilled water to 10 ml and shaken in

a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam

indicates the presence of saponins.

Haemolysis test:- 2 ml of 1.8% sodium chloride solution was taken in

two test tubes. To one test tube 2 ml of distilled water were added and to

another 2 ml of 1% extract were added. Blood is obtained by pricking the

thumb and 5 drops of blood were added to each tube, the contents were

gently mixed and observed under the microscope. Haemolysis indicates

the presence of saponins.

E. Tests for Tannins:

Ferric chloride test:- Extract treated with ferric chloride solution–blue

colour is formed.

Gelatin test:- Extract treated with gelatin solution–white precipitates

appear.

Lead acetate test:- Extract treated with lead acetate solution–yellow

precipitates appear.

57

F. Tests for Proteins and Amino Acids:

Millon’s test:- Extract treated with Millon’s reagent (mercuric nitrate in

nitric acid)-red colour is formed.

Biuret test:- Extract treated with sodium hydroxide and copper

sulphate solution drop wise –violet colour.

Ninhydrin test:- Extract treated with ninhydrin reagent and ammonia

and heated –violet colour is formed.

G. Tests for Flavonoids:

Ferric chloride test:- To the extract add a few drops of neutral ferric

chloride solution- blackish red colour is formed.

Lead acetate test:- To the extract add lead acetate solution-yellow

precipitates.

Magnesium ribbon test:- To the extract add few fragments of

magnesium ribbon and concentrated hydrochloric acid along the sides of

the test tube-magenta colour is formed is formed.

Zinc–hydrochloride test:- To the extract, a pinch of zinc dust was added

followed by addition of concentrated hydrochloric acid along the sides of

the test tubes-magenta colour is formed.

58

4.4 IN VITRO ANTIOXIDANT ACTIVITY

4.4.1 IN VITRO SCRENING OF BUCHANANIA LANZAN

The in vitro antioxidant screening was carried out using the following

models.

1. In vitro DPPH assay

2. Scavenging of ABTS

3. Scavenging of hydroxyl radical

2,2-Diphenyl-I-picryl hydrzyl (DPPH) Assay53

DPPH assay is based on the measurement of the scavenging ability of

antioxidant towards the stable DPPH radical. The free radical DPPH is

purple in colour in methanol and is reduced to the corresponding

hydrazine, which is yellow in colour, when it reacts with a hydrogen

donor.It is a discoloration assay, which is evaluated by the addition of

the antioxidant to 2,2-diphenyl-1-picryl hydrzyl (DPPH) solution in

ethanol or methanol and the decrease in the absorbance is measured .

The free radical-scavenging activity of the extracts was measured in

terms of hydrogen donating or radical-scavenging ability using the stable

radical DPPH.

NO2N N

Ph

Ph

NO2

NO2

+ R- OHO2N N

Ph

Ph

NO2

NO2

NH

+ R-O

DPPH radical Stable phenoxy radical

(Purple) (Yellow)

59

DPPH solution (100 μM): 22 mg of DPPH was accurately weighed and

dissolved in 100 ml of methanol. From this stock solution, 18 ml was

taken and diluted to 100 ml with methanol to obtain 100 μM DPPH

solutions.

Preparation of Test and Standard Solution

The extracts and the standards, ascorbic acid and rutin (21 mg each)

were separately dissolved in 5 ml of freshly distilled DMSO. These

solutions were serially diluted with freshly distilled DMSO to obtain the

lower dilutions.

Procedure

The assay was carried out in a 96 well microtitre plate. To 200 μl of

DPPH solution, 10 μl of various concentrations of the extract or the

standard solution was added separately in wells of the microtitre plats.

The plats were incubated at 370C for 30 min. Absorbance was measured

at 517 nm using ELISA reader. Thirty minutes later, the absorbance was

measured at 517 nm. Lower absorbance of the reaction mixture indicates

higher free radical-scavenging activity. Ascorbic acid and rutin were

used as a standard drugs.

Scavenging of 2, 2’-azino-bis (3-ethylbezothiazoline-6-sulfonic acid)

diammonium salt (ABTS) radical cation assay54

ABTS assay is relatively recent one, which involves a more drastic

radical, chemically produced and, is often used for screening complex

60

antioxidant mixture such as plant extracts, beverages and biological

fluids. The solubility in both the organic and aqueous media and the

stability in a wide pH range raised the interest in the use of ABTS radical

for the estimation of the antioxidant.

Preparation of test and standard solution:

13.5mg of each of the extracts and the standards, ascorbic acid and

rutin were accurately weighed and separately dissolved in 2 ml of DMSO.

These solutions were serially diluted with DMSO to obtain the lower

dilutions.

Procedure:

ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM

concentration and potassium persulphate (17 mM, 0.3 ml) was added.

The reaction mixture was left to stand at room temperature overnight in

dark before use. To 0.2 ml of various concentrations of the extracts or

standards, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution was

added to make a final volume of 1.36 ml. Absorbance was measured

spectrophotometrically, after 20 min at 734 nm.

Scavenging of Hydroxyl Radical by Deoxyribose Method55

The sugar deoxyribose (2-deoxy-D-ribose) is degraded on exposure to

hydroxyl radical generated by irradiation or by Fenton systems. If the

resulting complex mixture of products is heated under acid conditions,

malondialdehyde (MDA) is formed and may be detected by its ability to

react with thiobarbituric acid (TBA) to form a pink chromogen.

61

Preparation of test and standard solutions:

16 mg of each of the extracts and BHA were accurately weighed and

separately dissolved in 2 ml of DMSO. These solutions were serially

diluted with DMSO to obtain the lower dilutions.

Procedure:

To the reaction mixture containing deoxyribose (3 mM, 0.2 ml), ferric

chloride (0.1 mM, 0.2 ml), EDTA (0.1 mM, 0.2 ml), ascorbic acid (0.1 mM,

0.2 ml) and hydrogen peroxide (2 mM, 0.2 ml) in phosphate buffer (pH,

7.4, 20 mM), 0.2 ml of various concentrations of extracts or standards in

DMSO were added to give a total volume of 1.2 ml. The solutions were

then incubated for 30 min at 370C. After incubation, ice-cold trichloro

acetic acid (0.2 ml, 15% w/v) and thiobarbituric acid (0.2 ml, 1% w/v), in

0.25 N HCl were added. The reaction mixture was kept in a boiling water

bath for 30 min, cooled and the absorbance was measured at 532 nm.

4.4.2 IN VITRO ANTIOXIDANT ACTIVITY OF VIDAKANACHOORNAM

AND MUPPU

The formulations Muppu and Vidakanachoornam were evaluated by

Superoxide radical scavenging activity

Hydroxyl radical scavenging activity

Nitric oxide method

62

Superoxide Radical Scavenging Activity56

All the solutions were prepared in 100 mM phosphate buffer (pH 7.4).

1ml of nitroblue tetrazolium (NBT, 156 μM), 1 ml of reduced

nicotinamide adenine dinucleotide (NADH, 468 μM) 3 ml of HEPD

solution of different concentrations (50, 100, 200, 400, 800, 1000 1200

μg/ml) were mixed. The reaction was initiated by adding 100 μl of

phenazine methosulphate (PMS, 60 μM). The reaction mixture was

incubated at 25°C for 5 min, followed by measurement of absorbance at

560 nm.

Hydroxyl Radical Scavenging Activity57

Stock solutions of EDTA (1mM), FeCl3(10 mM), ascorbic acid (1 mM),

H2O2 (10 mM), deoxyribose (10 mM), were prepared in distilled deionized

water.The assay was performed by adding 0.1 ml FDTA, 0.01 ml of FeCl3,

0.1 ml H2O2, 0.36 ml of deoxyribose, 1.0 ml of the extract of different

concentration (200, 400. 800, 1000, 1200 mg/ml) dissolved in distilled

water, 0.33 ml of phosphate buffer (50 mM, PH-7.4), 0.1 ml of ascorbic

acid in sequence. The mixture was then incubated at 37°C for 1 hr. A 1.0

ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA

1.0 ml of 0.5% TBA (in 0.025 M NaOH containing 0.025% BHA) to

develop the pink chromogen measured at 532 nm. The hydroxyl radical

scavenging activity of the extract is reported as % inhibition of

deoxyribose degradation is calculated by using following equation.

63

Percentage inhibition = (1– absorbance of test/absorbance of control) ×

100

Nitric oxide Method58–60

In aqueous solution at physiological pH, sodium nitroprusside

generates nitric oxide, which interacts with oxygen to produce nitrite

ions. 1 ml of 10 mM sodium nitroprusside was mixed with 1 ml of test

solution of different concentrations (80, 160, 320, 500, 800, 1000 μg/ml)

in phosphate buffer (pH 7.4) and the mixture was incubated at 25°C for

150 min. From the incubated mixture, 1 ml was taken out 1 ml of Griess’

reagent (1% sulphanilamide, 2% o-phosphoric acid 0.1% naphthyl

ethylene diamine dihydrochloride was added to it. Absorbance of the

chromophore formed by the diazotization of nitrite with sulfanilamide

subsequent coupling with naphthyl ethylene diamine dihydrochloride

was read at 546 nm. Percentage inhibition was calculated by comparing

the results of the test with those of the control using following equation;

Percentage inhibition = (1– absorbance of test/absorbance of control) ×

100

64

4.4.3 COMPARATIVE IN VITRO ANTIOXIDANT ACTIVITY OF MUPPU,

BUCHANANIA LANZAN AND VIDAKANACHOORNAM

The comparative in vitro antioxidant activity of Vidakanachoornam ,

Muppu and plant Buchanania lanzan was carried out using DPPH

method.

4.5 IN VIVO ANTIOXIDANT ACTIVITY

4.5.1 Acute oral toxicity studies61,62

The acute oral toxicity study was performed according to the OPPTS

(Office of prevention of pesticides and toxic substance) Guidelines

following the up and down procedure62.Male albino mice (30-35 g) were

maintained under controlled standard animal house conditions with

access to food and water ad libitum.The mice were acclimatized for 5 days

and fasted overnight, with access for drinking water. Animals were

weighed; limit and main test were performed. The limit test is carried out

first at 5000 mg /kg body weight for one animal and if animal dies, main

test is performed. If the animal survives two more animals are dosed, if

both survive the test should be terminated. The main test is performed

with an initial dose of 175mg/kg body weight. The following sequence is

followed. 175, 550, 1750, and 5000 mg/kg body weight.First one animal

is dosed with 175 mg/kg body weight. If animal dies a much lower dose

is tested. If animal survives, then two more animals are dosed, after 48

65

hours observation of the first animal. If survives, then the main test

should be terminated .if animal dies, two more animals are dosed and

observed.

The dosing is stopped when one of the following stopping criteria is met.

Three consecutive animals survive at the upper bound.

5 reversals occur in any 6 consecutive animals tested

At least 4 animals have followed the first reversal and the specified

likelihood ratios exceed the critical value.

The control mice received vehicle (Tween 80 1%p.o) only.

4.5.2 IN VIVO ANTIOXIDANT STUDIES OF BUCHANANIA LANZAN

Test animals

Animal house was well maintained under standard hygienic

conditions. Rats were housed in groups of 6 in polypropylene cages

(32x24x16). They were provided with commercial food pellets [containing

(%w/w) protein 22.10, oil 4.13, fibre 3.15, ash 5.15, sand (silicia)1.12]

and water ad libitium. Cleaning and sanitation work was done on

alternate days. Paddy husk was provided as bedding material, which was

changed everyday. Wistar albino rats and mice of either sex were used

for the study of the crude extracts. The animals were housed at central

animal house under standard conditions of temperature (27 ± 2°C),

relative humidity (44-56%) and light and dark cycles of 10 and 14 h

respectively, for 1 week before and during the experiments. Animals were

66

provided with standard diet (Lipton, India) and water ad libitum. The food

was withdrawn at 18 to 24 h before the start of the experiment. All the

experiments were performed in the morning according to the ethical

guidelines for the care of the laboratory animals61.

In vivo antioxidant study of the Plant extract was carried out using

Biochemical estimation of the enzyme level in the liver homogenate.

1. S.O D estimation

2. Estimation of catalase

3. Estimation of total protein

4. Liver weight estimation

5. Histopathological studies

Experimental Design43

After seven days of acclimatization, the rats were divided into six

groups, each group consisting of six animals, (n=6). Treatment was done

for 8 days as follows:

Group I: Normal control (0.9% normal saline; 1 ml/kg p.o .) daily for 8

days and received liquid paraffin (2 mL/kg, s.c.) on last day. Group II

(CCl4) received (0.9% normal saline; 1 ml/kg p.o.) once daily for 8 days

and received CCl4: liquid paraffin (1:1, 2 mL/kg body weight, s.c.) on last

day. Group III received standard drug ascorbic acid (250 mg/kg, p.o.)

67

once daily for 8 days and received CCl4: liquid paraffin (1:1, 2 mL/kg

body weight, s.c.) on last day.Group IV received standard drug ascorbic

acid (500 mg/kg, p.o.) once daily for 8 days, and received CCl4: liquid

paraffin (1:1, 2 mL/kg body weight, s.c.) on last day. Group V received

methanolic extract of Buchanania lanzan (250 mg/kg, p.o.) once daily for

8 days and received CCl4: liquid paraffin (1:1, 2 mL/kg body weight, s.c.)

on last day . Group VI received the methanolic extract of Buchanania

lanzan (500 mg/kg, p.o.) once daily for 8 days and received CCl4: liquid

paraffin (1:1, 2 mL/kg body weight, s.c.) on last day. Animals were

sacrificed 24 h after the last treatment.

Blood was collected, allowed to clot and serum was separated at 2500

rpm for 15 min and biochemical investigations were carried out. Liver

was dissected out and used for histopathological studies.

Preparation of Liver Homogenate

The liver samples used for homogenate preparations were rinsed in

sterile ice cold saline and then homogenized in ice –cold 0.25 M sucrose

1:10 w/v solution using a mortar and pestle .Then, the obtained

homogenate was centrifuged at 5000 rpm for 15 minutes, the

supernatant was decanted and taken for estimation of antioxidants and

enzyme levels using kits.

Preparation of 0.25M Sucrose Solution

This solution was prepared by dissolving 342.30 gms of sucrose in

1000 ml of distilled water.

68

Estimation of Super Oxide Dismutase (SOD)6

Estimation of S.O.D is done by autooxidation of hydroxylamine

hydrochloride at pH 10.2 yielding nitrite, which was measured

colorimetrically. Ò2 can also be generated during auto oxidation of

hydroxylamine. Accompanying the autoxidation of hydroxylamine, NBT is

reduced and nitrite is produced in the presence of EDTA, which can be

detected colorimetrically at 560 nm. One enzymatic unit of S.O.D is the

amount in the form of proteins present in 100 µl of 5% tissue

homogenate required to inhibit the reduction of 24 mM NBT by 50% and

is expressed as units/mg of protein.

Reagents

Sodium carbonate solution: 1.06 gms of sodium carbonate was dissolved

in 100 ml of distilled water.

Nitroblue Tetrazolium (NBT) (Loba chemie, Mumbai, India ) 10 mg of NBT

was dissolved in 100 ml of distilled water .

EDTA solution (Nice chemicals, Cochin):37 mg of EDTA di-sodium salt

was dissolved in 100 ml of distilled water.

Hydroxyl amine hydrochloride (Nice chemicals, Cochin): 16.5 mg of

hydroxylamine hydrochloride was dissolved in 100 ml of distilled water.

Experimental Procedure

100 µl of 5% tissue homogenate in 0.2 M sucrose in phosphate

buffer (0.25 M pH 7.4) was taken in a test tube and to this a

mixture containing 1ml of sodium carbonate, 0.4 ml of NBT and

69

0.2 ml of EDTA were added and zero minute reading was taken at

560 nm.

The reaction was initiated by the addition of 0.4 ml of 1 mM

hydroxylamine hydrochloride to the test tube.

The reaction mixture was incubated at 25 C for 5 minutes; the

reduction of NBT was measured at 560 nm.

A parallel control without the tissue homogenate was also treated

in a similar manner as the test.

One enzymatic unit of S.O.D is the amount in the form of proteins

present in 100 µl of 5% tissue homogenate required to inhibit the

reduction of 24 mM NBT by 50% and is expressed as units/mg of

protein.

The concentration of enzyme is calculated using the formula

Units /mg=1000/ µgmof enzyme resulting in ½ max.inhibiton

Estimation of Catalase64

Estimation of catalase activity was done by determining the

decomposition of H2O2 at 240 nm in an assay mixture containing

phosphate buffer (0.25 M, pH 7).The estimation was done

spectrophotometrically following the decrease in absorbance at 240 nm.

The reaction mixture contained 0.01 M phosphate buffer (pH 7.0), 2 mM

H2O2 and the enzyme extract. The specific activity of catalase was

expressed in terms of units/gram of liver tissue. One international unit

70

of catalase utilized is that amount which catalyzes the decomposition of

1 micromole hydrogen peroxide per minute per mg of protein 370C.

Catalase activity was calculated using millimolar extinction coefficient of

0.07 and is expressed in terms of micromole per minute per milligram of

protein.

Reagents

Phosphate buffer (0.25M, pH7): 3.22gm potassium dihydrogen

orthophosphate and 7.268 gm of di-sodium hydrogen orthophosphate

was dissolved in distilled water and the volume was made upto 1000 ml.

Hydrogen peroxide: (0.042 ml=12.3 M) 0.34 ml of 30% hydrogen peroxide

was diluted to100 ml with distilled water.

Experimental Procedure:

100µl of 5% tissue homogenate in 0.15 M KCl buffer was added

to1.9ml of phosphate buffer (0.25 M, pH 7) and the absorbance was

measured at 240 nm. To the above reaction mixture 1 ml of hydrogen

peroxide was added and the absorbance measured after allowing it to

stand for 1 minute at 240 nm using phosphate buffer as blank solution.

One international unit of catalase utilized is that amount which catalyzes

the decomposition of 1 micromole of hydrogen peroxide per minute per

milligram of protein at 370C and is expressed in terms of units/mg of

protein.

The catalase concentration is calculated using the formula

71

Units/mg= Abs X1000/43.6 X mgof enzyme/ml reaction mixture.

Estimation of Proteins65

The method is based on the formation of a coloured complex of

proteins with Folin Ciocalteau reagent which can be measured

calorimetrically at 610 nm. Bovine serum was used as a standard.

Reagents

1. Alkaline reagent: 2 g of sodium carbonate was dissolved in 100 ml of

0.1 N NaOH solution.

2. Alkaline mixture: To 100 ml of alkaline reagent, 1 ml of 4% aqueous

copper sulphate solution was added. This was freshly prepared.

3. Phenol reagent (Folin Ciocalteau reagent) (Loba chemie, Mumbai)

commercially available reagent was diluted by adding equal volume of

distilled water before use and stored in the refrigerator.

4. 0.1 N NaOH solution.

5. 90% alcohol.

6. Preparation of standard bovine albumin solution: 8mg (0.08%)of

standard bovine albumin (S.D Fine chemicals, Mumbai) was dissolved

in 10 ml of distilled water.

72

Experimental Procedure

1. To 0.1 ml of the homogenate, 0.9 ml of 90% alchohol was added

and centrifuged at 3000 rpm for 10 minutes. The supernatant was

discarded and the precipitate was dissolved in 1ml of 0.1N NaOH.

2. 0.05 ml of the above solution was taken in a test tube, to which 4

ml of freshly prepared alkaline mixture was added and allowed to

stand for 10 minutes.

3. To the above reaction mixture 0.4 ml of phenol reagent was added

and allowed to stand for further 10 minutes for the reaction to

complete.

4. The absorbance was measured at 610 nm using distilled water as

blank.

5. 0.1 ml of standard was taken in a test tube and processed as

mentioned in step 2 and 3.

Histopathological Studies

The liver tissue was dissected out and fixed in 10% formalin,

dehydrated in gradual ethanol (50 to 100%), cleared in xylene and

embedded in paraffin. Sections were prepared and then stained with

hematoxylin and eosin (H to E) dye for photomicroscopic examination.

73

Estimation of Liver Weight

All the surviving animals were sacrificed after 48 hrs of CCl4

treatment; the liver was collected for weight determination and the mean

change in weight for each group was calculated and compared with CCl4

control.

4.5.3 IN VIVO ANTIOXIDANT ACTIVITY OF MUPPU AND

VIDAKANACHOORNAM

Experimental Design

After seven days of acclimatization, the rats were divided into six

groups (n=6). Treatment was done for 8 days as follows:Group I: Normal

control (0.9% normal saline; 1 ml/kg p.o), Group II: CCl4 control (CCl4:

liquid paraffin (1:2); 1ml/kg s.c.), Group III: CCl4 + Liquid Extract (LE-

100 mg/kg/day; p.o), Group IV: CCl4 + Liquid Extract (LE-200

mg/kg/day; p.o), Group V: CCl4 + Formulation (FR-100 mg/kg/day; p.o).

Group VI: CCl4 + Formulation (FR-200 mg/kg/day; p.o), Group II-IV:

CCl4 in liquid paraffin (1:2) (1.0 ml/kg s.c.) was given once every 72 h.

TISSUE SAMPLE PREPARATION FOR ESTIMATION OF MDA, GSH,

CAT

74

After 8 days dosing, animals were sacrificed by cervical dislocation.

Liver samples were dissected out and washed immediately with ice cold

saline to remove as much blood as possible. Liver homogenates (5% w/v)

were prepared in cold 50 mM potassium phosphate buffer (pH 7.4) using

a Remi homogenizer. The unbroken cells and cell debris were removed by

centrifugation at 1000 rpm for 10 minutes using a Remi refrigerated

centrifuge. The supernatant was used for the estimation of GSH66,

malondialdehyde MDA67, and catalase.

Reduced Glutathione (GSH)66

Reduced Glutathione was estimated spectrophotometrically by

determination of DTNB (Dithiobis-(2-nitrobenzoic acid) reduced by SH-

groups, expressed as μmol/mg wet tissue. The sulphahydryl groups

present in the glutathione forms a coloured complex with DTNB,which is

measured colorimetrically at 412nm.The amount of glutathione was

determined using its molar extinction coefficient of 13600/m/cm and is

expressed in terms of micro mol per mg of protein . To 0.1 ml of different

tissue samples, 2.4 ml of 0.02 M EDTA solution was added and kept on

ice bath for 10 min. Then 2 ml of distilled water and 0.5 ml of 50 %w/v

TCA were added. This mixture was kept on ice for 10-15 min, then

centrifuged at 3000 rpm for 15 min. To 1 ml of supernatant, 2.0 ml of

Tris buffer (0.4M) was added. Then 0.05 ml of DTNB solution (Ellman’s

reagent; 0.01M DTNB in methanol) was added and vortexed thoroughly.

75

Optical density (OD) was read (within 2-3 min after the addition of DTNB)

at 412 nm in spectrophotometer against a reagent blank.

Lipid Peroxidation67

To 1ml of tissue homogenate, 1ml of normal saline (0.9% w/v) and 2.0

ml of 10% TCA were added and mixed well. The mixture (3000 g) was

then centrifuged at room temperature for 10 min to separate proteins. 2

ml of supernatant was taken, 0.5 ml 1.0% TBA was added to it followed

by heating at 95°C for 60 min. to generate the pink coloured MDA. OD of

the samples was measured at 532 nm using Biotek Synergy 4

spectrophotometer. The levels of lipid peroxides were expressed as nM of

MDA/mg wet tissue using extinction co-efficient of 1.56 x105 M-1 cm-1.

Malondialdehyde is a secondary product of lipid peroxidation, reacts with

thiobarbituric acid at pH 3.5. The red pigment produced estimated by

measuring the absorbance at 532 nm.

4.5.4 STUDY OF COMPARATIVE IN VIVO ANTIOXIDANT ACTIVITY OF

VIDAKANACHOORNAM, MUPPU AND BUCHANANIA LANZAN 68

Experimental Design

Male albino mice 10–12 weeks old and weighing 18–22 g obtained, in

groups of six were employed for the study. These were maintained at a

room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access

76

to pellet food and water. Group I: Naïve Control animals received (saline,

p.o.),Group II:Stresc ontrol.,Group III: Animals received standard

ascorbic acid at the dose of 100 mg/kg, p.o.,Group IV: Animals received

Muppu at the dose of 100 mg/kg, p.o.,Group V: Animals received Muppu

at the dose of 200 mg/kg, p.o.,Group VI: Animals received Muppu at the

dose of 400 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam

at the dose of 100 mg/kg, p.o.,Group VIII: Animals received

Vidakanachoornam at the dose of 200 mg/kg, p.o.,Group IX: Animals

received Vidakanachoornam at the dose of 400 mg/kg p.o.,Group X

Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o. Group XI:

Animals received Buchanania lanzan leaf extract at the dose of 200

mg/kg, p.o.,Group XII: Animals received Buchanania lanzan leaf extract

at dose of 400mg/kg,p.o.Stress was induced in Mice by restraining

animals in well-ventilated horizontal 50-ml conical polypropylene tubes

for 12 h during the dark cycle (2000–0800 h) during experimental

periods of 14 days, One group of mice was taken as a non-treated naive

control group without any restrained conditions and was given full

access to food and water (naive-controll69. one group served as the

stress control to which drug was not administered but was subjected to

restrained stress (, treated groups were subjected to restrain stress as

well as drug administration for 14 consecutive days. Serum is separated

from whole blood which is taken from retro orbital plexus. After

microcentifugation for10min at 2000 rpm, RBCs and other cells are

77

settled down and supernatant straw coloured fluid is Serum. The serum

is separated by micropipettes in a separate micro-centrifuge tube and the

procedure is repeated thrice for complete removal

of any remaining RBCs and used blood of stressed animals for TBARS

and GSH Estimation.

Estimation of Blood Glutathione 70

0.2 mL fresh serum was collected from each animal and 1.8 mL

distilled water were added to it and 3 mL of precipitating solution were

added to the mixture. The mixture was then allowed to stand for

approximately 5 min and then filtered. 2.0 ml of filtrate were added to

8.0 ml of phosphate solution in cuvette and 1.0 mL DTNB reagent was

added to cuvette and the optical density (OD) was measured at 412 nm.

Estimation of Thiobarbituric Acid Reaction Substances (TBARS) 71

Mouse serum (0.2 ml) was pipetted out in a test tube, followed by

addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic

acid (pH 3.5), 1.5 ml of 0.8% of thiobarbituric acid and the volume was

made up to 4 ml with distilled water. The test tubes were incubated for 1

h at 950C and allowed to cool down at room temperature and 1 ml of

distilled water and 5 ml of n-butanol-pyridine mixture (15:1 v/v) were

78

added. The tubes were centrifuged at 4000 rpm for 10 min. The

absorbance of developed pink colour was measured

spectrophotometrically (Shimadzu 1700, Singapore) at 532 nm. A

standard calibration curve was prepared using 1-10 nM of 1,1,3,3-

tetramethoxypropane. The TBARS value was expressed as nanomoles per

mg of protein.

Histopathological Studies

For histopathology the following steps were involved:

Processing of the Isolated Liver

The animals were euthanized using anaesthic ether and their liver

was dissected out. The isolated organ was sliced and fixed in neutral

formalin (10%) solution for at least 3 days. Liver pieces were then washed

under running water for about 12 hours. This was followed by

dehydration with alcohol of increasing strength (70%, 80%, 90%) for 12

hours each. Final dehydration was carried out using absolute alcohol

with about three changes at 12 hours interval. Clearing was done with

xylene with 2 changes at 15 to 20 min interval. After cleansing, the organ

pieces were subjected to paraffin infiltration in automatic tissue

processing unit. The organ pieces were washed under running water to

remove the formalin completely. Alcohol of increasing strength was used

as a dehydrating agent. Alcohol was removed by using xylene, and the

xylene was removed by paraffin infiltration.

Embedding in Paraffin

79

Hard paraffin was melted and hot paraffin was poured into L–shaped

blocks. The liver pieces were then dropped into liquid paraffin quickly

and allowed to cool.

Sectioning

The blocks were cut using microtome to get sections of 5 microns

thickness. The sections were taken on micro slide on which egg albumin

(sticky substance) was applied. The sections were allowed to dry

completely before staining on a hot plate.

Staining

Eosin is an acid stain. It stains all the basic cytoplasm cells

constituents. Hematoxylin is a basic stain, which stains all the acidic cell

components blue (DNA, RNA, and nucleus).

Preparation of the Slide

The sections were deparaffinized by washing with xylene for about

15 minutes. The sections were dehydrated by washing in alcohol of

decreasing strengths (100%, 90%, 80%, and 70%).

The sections were finally washed with water.

The sections were stained with Hematoxylin for 15 minutes.

It was rinsed with tap water.

Acid alcohol was differentiated by 3 to 10 quick dips. The

differentiation with a microscope showed that the nuclei were

distinct light or colourless.

80

The slide was washed with tap water very briefly.

The section was dipped in ammonia water, lithium carbonate until

sections became bright blue by 3 to 5 dips.

The slide section was washed in running tap water for 10-20

minutes. If washing is inadequate, eosin will not stain evenly.

The slide was stained with eosin for 15 seconds to 2 minutes,

depending on the age of the eosin and the depth of the counter

stain desired. For even staining results, slides were dipped in

several times before allowing them to set in the eosin for the

desired time.

Slides were rehydrated in 95% alcohol and absolute alcohol until

excess eosin was removed. Two changes of 2 min interval were

performed of different treatments (95% alcohol, absolute alcohol

and xylene)

The section was then mounted in DPX (diphenylxylene) mountant.

Staining results showed blue coloured nucleus and cytoplasm with

various shades of pink with change in different tissue components.

Statistical Analysis

The data were expressed as mean ± S.E.M. The differences among

means were analyzed by one-way ANOVA. A value of P < 0.05 was

considered as statistically significant. The significance of difference

among groups was assessed using one way analysis of variance (ANOVA)

81

followed by Tukey-Karmer multiple comparison test between the data of

control and treated groups. The values were expressed as mean ± SEM,

where* p<0.05 was considered as significant, **p<0.01 was considered

very significant and ***p<0.001was considered extremely significant.

4.6 EVALUATION OF ANTISTRESS/ADAPTOGENIC ACTIVITY

4.6.1.FORCED SWIMMING TEST 72

Briefly, the animals were forced to swim individually in a glass jar (25

× 12 × 25 cm) containing water of 15 cm height at a temperature of 25 ±

3°C for a period of 15 min in a training session on day 1 and for 5 min on

day 2 of the experiment. Each animal made vigorous attempts to get out

of water bath during first couple of minutes and thereafter surrendered

to experimental conditions and assumed a typical immobile posture with

occasional escape attempts. This phenomenon is a measurement of

acute stress followed by depression. The total duration of immobility was

recorded. Animals were removed from water, dried and kept in their

respective cages.

Experimental Design

The animals were individually weighed and divided into eleven groups,

each group comprising of six animals. All the animals were fasted 3 h prior

to commencement of experiment but water was provided ad libitum. The

grouping of animals was done as follows:

82

Group I: Control animals received (saline, p.o.),Group II: Standard

Imipramine 15 mg/kg, p.o.,Group III: Animals received Muppu at the

dose of 100 mg/kg, p.o.,Group IV: Animals received Muppu at the dose

of 200 mg/kg, p.o.,Group V: Animals received Muppu at the dose of 400

mg/kg, p.o.,Group VI: Animals received Vidakanachoornam at the dose

of 100 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam at

the dose of 200 mg/kg, p.o.,Group VIII: Animals received

Vidakanachoornam at the dose of 400 mg/kg, p.o.,Group IX: Animals

received Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o.

Group X: Animals received Buchanania lanzan leaf extract at the dose of

200 mg/kg, p.o.,Group XI: Animals received Buchanania lanzan leaf

extract at the dose of 400 mg/kg, p.o. After 1 hr post-administration of

the drugs, the animals were placed in the jar one by one for despair swim

test (Forced Swim test). The immobility time in seconds was noted .

4.6.2 SWIMMING ENDURANCE TEST 73

The mice were divided into five groups of six animals each n=6. Group

I Normal control (0.9% normal saline; 1 ml/kg p.o.), Group II received

Impramine 15 mg/kg, p.o., Group III received Muppu at the dose of 250

mg/kg, p.o., Group IV received Vidakanachoornam 250 mg/kg, p.o., and

Group V received Buchanania lanzan leaf extract 250 mg/kg, p.o.The

drugs once daily for 14 days. On the fourteenth day all groups were

allowed to swim till exhaustion in separate cylindrical containers (bucket)

83

filled with water (maintained at 250C). Death due to drowning was taken

as the end point. Swimming time for individual animal was noted down

and the mean swim survival time for each group was calculated. Mean

swim time of drug treated groups were compared with that of the control

group.

4.6.3. ANOXIC STRESS TOLERANCE IN MICE 42

The mice were divided into five groups of six animals each n=6. Group

I Normal control received (0.9% normal saline; 1 ml/kg p.o.), Group II

received Impramine 15 mg/kg, p.o., Group III received Muppu at the

dose of 250 mg/kg, p.o., Group IV received Vidakanachoornam 250

mg/kg, p.o., and Group V with Buchanania lanzan leaf extract 250

mg/kg, p.o. Conical flasks of 250 ml capacity were used for the study.

The flasks were made airtight by using rubber cork before the beginning

of the experiment. Mice of equal weight were used in the experiments.

Each mouse served as its own control. The anoxic stress tolerance time

was measured as follows: Each animal was kept in the airtight vessel

and the time was noted by using a stop watch. The moment animal

showed its first convulsion, it was removed immediately from the vessel

and resuscitated if needed. The time duration from the entry of the

animal in a hermetic vessel to the appearance of the first convulsion was

taken as the time of anoxic stress tolerance. The appearance of

convulsion is a sharp end point, as delay of even one minute in removal

kills the animal. The anoxic stress tolerance time was determined for

84

each animal individually, before subjecting the animals to the drug

treatment and after one week, two weeks and third week of the drug

treatments.

4.6.4 RESTRAINT STRESS INDUCED CHANGES IN RATS 74

The mice were divided into six groups of six animals each n=6. Group

I Normal control (0.9% normal saline; 1 ml/kg i.p.), Group II Stress

control (0.9% normal saline; 1 ml/kg i.p.)and restraint stress, Group III

received 15 mg/kg, p.o., Impramine, Group IV received Muppu at the

dose of 250 mg/kg, p.o., Group V received Vidakanachoornam 250

mg/kg, p.o., and Group VI with Buchanania lanzan leaf extract 250

mg/kg, p.o.

Animals were pretreated with the drugs for eight days. They were

deprived of food 24 hrs before the commencement of experiment with

water ad libitum. After one hour of administration of the last dose, all

four limbs of the animal were tied and kept in separate chambers

(cylindrical vessels, 50 cm high and 40 cm wide) at 40C in the refrigerator

for two hours. Blood was collected by retro-orbital puncture method

using heparinised capillary tubes. The blood samples were analysed for

corticosterone estimation. Animals were sacrificed using cervical

dislocation. Adrenal gland was collected for assessing any changes in the

weight, when compared to the control group.

Plasma Corticosterone Estimation75

85

Plasma corticosterone was estimated by sulphuric acid induced

fluorescence measurement method.

Materials Required:

Flourescent reagent: Absolute alcohol and concentrated sulphuric

acid mixed in ratio of 3:7 and cooled.

Dichloromethane

Distilled water

Hydrocortisone standard (LOBA chemicals)

Two ml of plasma was taken in a separating funnel and 15 ml of

dichloromethane were added to it and shaken well, it was allowed to

stand, two layers are formed. The upper layer is separated out and is

discarded. 10 ml of the lower layer is treated with 5 ml of the Flourescent

reagent (as prepared by mixing 3 ml of absolute alcohol with 7 ml

concentrated of sulphuric acid, following by cooling) and shaken well.

Again the lower layer is separated and reading is taken at 530 nm using

photoflourimeter, as mentioned below:

(A) Blank; 2 ml of triple distilled water;

(B) Standard solution: 2 ml of hydrocortisone standard solution of

known strength (100mcg/100ml);

(C) Test solution: 2 ml of the lower layer obtained from the above

procedure.

The concentration of the plasma cortisol was calculated as follows:

86

Concentration of the plasma cortisol =reading of the test / reading of the

standard X100

4.6.5 ESTIMATION OF URINARY VMA AND ASCORBIC ACID LEVELS

76

Ascorbic Acid Estimation77, Urine VMA78

Experimental design

Rats of either sex weighing between 150-250 g were divided into six

groups (I, II, III, IV, V, and VI) each comprising of six animals. The 24 h

urine sample from each group was collected into two different beakers,

one containing 5 ml of 10% oxalic acid for spectrophotometric

determination of ascorbic acid at 550 nm and the other containing 0.5

ml of 6 N hydrochloric acid for the determination of vanillylmandellic

acid (VMA) spectrometrically at 360 nm. The experimental protocol was

divided into four phases. In the first phase of the experiment, 24 h urine

samples were collected in all the six groups and subjected to analysis for

both VMA and ascorbic acid and the normal values were recorded for

seven consecutive days. In the second phase, the animals in each group

were subjected to fresh water swimming stress individually. In this

method, rats were forced to swim until exhausted (three to four minutes)

in cylindrical vessel of 60 cm height and 45 cm diameter containing

water at room temperature (280C). Water depth was always maintained

at 40 cm. The 24 h urinary levels of VMA and ascorbic acid under

87

stressed conditions were determined again as described above daily for

seven consecutive days. Third phase of experiment consists of

administration of Methanolic Extract (ME) to same groups of animals

after having recovered completely to normal condition. Groups II, III, IV,

V and VI were administered orally with Methanolic Extract at daily doses

of 10, 20, 30, 40, and 50 mg/kg body weight, respectively for seven

consecutive days while group I serving as control. The 24 h urine

samples were collected and the levels of both VMA and ascorbic acid

were determined. The final phase of the experiment consisted of

administrated of Methanolic Extract (ME) to the same groups of animals

after a recovery period of one week. Groups II, III, IV, V, VI were

administered orally with Methanolic Extract at doses of 10, 20, 30, 40

and 50 mg/kg body weight, respectively, and hour prior to the daily

induction of stress for seven consecutive days while group I serving as

control. The 24 h urine sample were collected and analyzed for VMA and

ascorbic acid for seven consecutive days to study the influence of the

Methanolic Extract on the stress induced biochemical change.

4.6.6 ESTIMATION OF BLOOD GLUTATHIONE AND TBARS LEVELS

Male albino mice 10–12 weeks old and weighing 18–22 g obtained, in

groups of six were employed for the study. These were maintained at a

room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access

to pellet food and water. Group I: Naïve Control animals received (saline,

88

p.o.),Group II:Stresc control.,Group III: Animals received standard

ascorbic acid at the dose of 100 mg/kg, p.o.,Group IV: Animals received

Muppu at the dose of 100 mg/kg, p.o.,Group V: Animals received Muppu

at the dose of 200 mg/kg, p.o.,Group VI: Animals received Muppu at the

dose of 400 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam

at the dose of 100 mg/kg, p.o.,Group VIII: Animals received

Vidakanachoornam at the dose of 200 mg/kg, p.o.,Group IX: Animals

received Vidakanachoornam at the dose of 400 mg/kg p.o.,Group X

Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o. Group XI:

Animals received Buchanania lanzan leaf extract at the dose of 200

mg/kg, p.o.,Group XII: Animals received Buchanania lanzan leaf extract

at dose of 400mg/kg,p.o.Stress was induced in Mice by restraining

animals in well-ventilated horizontal 50-ml conical polypropylene tubes

for 12 h during the dark cycle (2000–0800 h) during experimental

periods of 14 days, One group of mice was taken as a non-treated naive

control group without any restrained conditions and was given full

access to food and water (naive-controll69. One group served as the

stress control to which drug was not administered but was subjected to

restrained stress , treated groups were subjected to restrain stress as

well as drug administration for 14 consecutive days. Serum is separated

from whole blood which is taken from retro orbital plexus. After

microcentifugation for10min at 2000 rpm, RBCs and other cells are

settled down and supernatant straw coloured fluid is Serum. The serum

89

is separated by micropipettes in a separate micro-centrifuge tube and the

procedure is repeated thrice for complete removal

of any remaining RBCs and used blood of stressed animals for TBARS

and GSH Estimation.

Estimation of Blood glutathione 70

In which 0.2 mL fresh blood was collected from each animal and1.8

mL distilled water was added to it and 3 mL of precipitating solution was

added to mixture. The mixture was then allowed to stand for

approximately 5 min and then filtered. 2.0 mL of filtrate was added to 8.0

mL of phosphate solution in cuvette and 1.0 mL DTNB reagent was

added to cuvette and the optical density (OD) was measured at 412 nm.

Estimation of TBARS71

The quantitative measurement of thiobarbituric acid reactive

substances (TBARS), an index of lipid peroxidation in mouse serum

.Mouse serum (0.2 ml) was pipetted out in a test tube, followed by

addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic

acid (pH 3.5), 1.5 ml of 0.8% of thiobarbituric acid and the volume was

made up to 4 ml with distilled water. The test tubes were incubated for 1

h at 95 0C and allowed to cool down at room temperature and 1 ml of

distilled water and 5 ml of n-butanol-pyridine mixture (15:1 v/v) were

added. The tubes were centrifuged at 4000 rpm for 10 min. The

absorbance of developed pink colour was measured

spectrophotometrically (Shimadzu 1700, Singapore) at 532 nm. A

90

standard calibration curve was prepared using 1-10 nM of 1, 1, 3, 3-

tetramethoxypropane. The TBARS value was expressed as nanomoles per

mg of protein.

4.7 HEPATOPROTECTIVE ACTIVITY 79–81

4.7.1 EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF MUPPU

Experimental design

Carbon Tetrachloride-induced Hepatotxicity in Rats

Rats were divided into four groups of six each: control, hepatotoxin,

positive control and test groups. The control group received oral vehicle

treatment at 0, 24 and 48 h. The animals in hepatotoxin-treated groups

received vehicle 0 h and at 24 h following by carbon tetrachloride diluted

in liquid paraffin (1:1, s.c.) at a dose of 1.25 ml/kg, while at 48 h these

animal received only vehicle. The test groups received the first dose of

extracts at 0 h, second dose of extracts at 24 h, which was followed by a

dose of extracts at 0 h, second dose of extracts at 24 h, which was

following by a dose of carbon tetrachloride and at 48 h the third dose of

extracts. The positive control group has received the first dose of LIV 52

(0.125 mg/kg body wt) 5 at 0 h, at 24 h the second dose of LIV 52

followed by a dose of carbon tetrachloride and at 48 h the third dose of

LIV 52. After 72 h blood was collected from all the groups, and allowed to

clot for the separation of serum. The serum was used for estimation of

biochemical parameters.

91

4.7.2 EVALUATION OF THE HEPATOPROTECTIVE ACTIVITY OF

VIDAKANACHOORNAM AND BUCHANANIA LANZAN

Biochemical Determinations

The biochemical parameters like serum enzymes: aspartate

aminotransferase (AST); alanine transaminase (ALT) alkaline

phosphatase (SALP); and total bilirubin were assayed using assay kits

supplied by Span Diagnostics, Surat.

4.7.3 EVALUATION OF THE HEPATOPROTECTIVE ACTIVITY OF

VIDAKANACHOORNAM AND BUCHANANIA LANZAN AND MUPPU

Drug-Induced Potentiation of Pentobarbitone Sleeping Time82

Drug treatment was continued for seven days.Group I:Vehicle control

received saline p.o;GroupII received groups received carbon tetrachloride

diluted in liquid paraffin (1:1, s.c.) at a dose of 1.25 ml/kg, Group III: N

animals received Liv -52 p.o,Group IV: Animals received

Vidakanachoornam at the dose of 250 mg/kg, p.o.,Group V: Animals

received Buchanania lanzan leaf extract at the dose of 250 mg/kg,

p.o.,Group VI: Animals received Muppu at the dose of 250 mg/kg, p.o.,

All the animals were deprived of food for 24 hrs before the experiment

with free access to water. The experiment was conducted in a quite and

temperature of room was maintained at 250C in the afternoon between

92

12 and 5 pm. Sleeping time was measured before and after the drug

treatment by injecting single dose of pentobarbitone 45 mg/kg body

weight i.p to all the mice. Sleeping time was taken using a stop watch

and the corresponding to the time elapsed between the loss of righting

reflex and the recovery of the same reflex. This time was expressed in

minutes. The mean narcosis time of the drug treatment was compared to

control group.

CARBON TETRACHLORIDE–INDUCED PROLONGATION OF

PENTOBARBITONE SLEEPING TIME AND MORTALITY IN ALBINO

MICE

After determining the effect of the drug treatment on narcosis time,

the treatment was continued till 14 days. On the fourteenth day, after

one hour of the last dose administration, all the mice were treated with

carbon tetrachloride 1ml/kg body weight of mice along with equal

volume of liquid paraffin.

After 30 minutes of carbon tetrachloride administration, sodium

pentobarbitone was injected at a dose of 45 mg/kg body weight by i.p

route. The mean sleeping time was calculated for each group. 48-hour

mortality was assessed and compared with the control group.

4.8 EVALUATION OF ANTIINFLAMMATORY OF BUCHANANIA

LANZAN 83,84

93

4.8.1 EVALUATION OF ANTIINFLAMMATORY USING CARRAGEENAN

INDUCED RAT PAW OEDEMA

Experimental Design

Rats weighing 150-250 g were used in the experiments. The animals

were housed at room temperature (20 ± 20C) in standard cages with

standard pellet food and kept under controlled environment following the

standard operating procedures of animal house with the approval of

institutional animal ethics committee.

Carrageenan (Sigma), acetylsalicylic acid, CMC (Carboxymethyl

cellulose) and DMSO (Dimethyl sulfoxide) were employed; all the

compounds were dissolved in mixture of CMC and DMSO (9:1).

In this method, rats were divided into seven groups of six animals

each. The animals were pretreated with drug and Methanolic Extracts

(ME) in concentration of (ME-10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg,

and 50 mg/kg) were given by i.p route 30 minutes before carrageenan

injection (phlogistic agent) of 0.1 ml dose. Carrageenan was injected into

the sub plantar tissue of left hind paw of each rat. Swellings of

carrageenan-injected foot were measured at 30 min, 60 min, 120 min,

180 min using plethysmometer. The right hind paw was injected with 0.1

ml of vehicle. The animals received the standard drug aspirin (20 mg/kg,

i.p.) which served as reference standard. The experimental groups were

as follows (n=6);,Group 1:Control (CMC+DMSO), i.p;,Group 2:

Acetylsalicylic acid (20 mg/kg), i.p;,Group 3: ME (10 mg/kg), i.p;,Group

94

4: ME (20 mg/kg), i.p;,Group 5: ME (30 mg/kg), i.p;,Group 6: ME (40

mg/kg), i.p;,Group 7: ME (50 mg/kg), i.p;

Statistical Analysis

Results were expressed as mean ± S.E.M.

Percentage of inhibition (Ι %) = [(1-(dt/dc)] x 100.

Where dt is the difference in paw volume in drug-treated group and dc

the difference in paw volume.

4.9 ISOLATION AND CHARACTERIZATION

4.9.1 HPTLC FINGER PRINTING OF BUCHANANIA LANZAN

4.9.2 ISOLATION OF PHYTOCONSTITUENTS FROM LEAVES OF

BUCHANANIA LANZAN

Column chromatography

Preparation of the extract

Shade-dried powdered leaves (5 kg) of B. Lanzan were extracted with

methanol in a Soxhlet apparatus. The extract was filtered, and

concentrated under reduced pressure to give dark brown mass. The yield

of the extract was 10%. The extract was then subjected to preliminary

phytochemical analysis using standard methods.

95

Chemicals: The chemicals for isolation were obtained from Merck, and

SD Fine Chemicals, Mumbai.

Spectral analysis: was done at II Sc Bangalore and Quest Lab.

Isolation: The methanolic extract (30 gm) was subjected to column

chromatography on silica gel using solvents of increasing polarities

starting from petroleum ether, chloroform, ethyl acetate and methanol to

yield several fractions (181 fractions in total Elution was monitored by

then layer chromatography (TLC) ).

Fractions 32-36 eluted with chloroform: petroleum ether (1:1 mixture)

were combined together due to similarities in TLC pattern. The solvent

system [toluene: ethyl acetate 1:1] showed a major spot at Rf 5.2. This

fraction was re-chromatographed on a column of silica gel, elution being

carried out using different ratios of petroleum ether and chloroform.

Fractions of 10 ml were collected. Fraction ninth showed a single spot on

TLC. This fraction was recrystalised and dried to get 0.43 mg of

compound which was coded as SKM-L-1.

Fractions 89-112, eluted with methanol: ethyl acetate (4:6 mixture)

when subjected to TLC, using the solvent system toluene: ethyl acetate

7:3, showed a two major spots at Rf value of 0.4 and 0.7. This combined

fraction was subjected rechromatography on a column of silica gel and

also eluting with ethyl acetate and methanol in different ratios. Thirty six

96

fraction of 5 ml each were collected and monitored by TLC. The fractions

(17-19) of the second column were mixed and recrystallise to get 0.40 mg

of the pure compound which was coded as SKM-L-2. Fractions 26 and

27 of the second column showed a single spot on TLC. These fractions

were mixed and left over night to obtain 0.51 mg of SKM-L3.

4.9.3 ISOLATION OF PHYTOCONSTITUENTS FROM BARK OF

BUCHANANIA LANZAN

5.9.3 CHEMICAL STUDIES ON THE BARK OF BUCHANANIA LANZAN

Bark of Buchanania lanzan was dried in shade and was rendered into

moderately coarse powder. The bark powder (1kg) was extracted with

methanol in a soxhlet apparatus till the last drops of the extract did not

show any residue on evaporation of the solvent. The extract was filtered

and the solvent removed under reduced pressure to obtain a semisolid

dark brown resinous residue (150 gm). The residue was refluxed for 1 hr

with a mixture of chloroform and ethanol (1:1), and filtered. This was

repeated one more time. The solvent from the combined extract was

removed under reduced pressure to give a brownish–yellow residue (40

g). The residue was dissolved in a mixture of chloroform and ethanol (1:1)

and silica gel for chromatography (100-200) mesh size (200 g) was slowly

added to the solution and stirred thoroughly directing hot air from a

blower to it. When the silica gel coated with the residue became free

97

flowing it was loaded on the column of silica gel (600 g) which served as a

stationary phase. Gradient elution with the solvents of increasing

polarity using petroleum ether (60-80): chloroform

(100:0 to 10:90); pure chloroform, chloroform: ethyl acetate (100:10 to

10:90); ethyl acetate (100:0 to 10:90); ethyl acetate pure; ethyl acetate:

methanol (95:5 to 50:50) was carried out. The eluate was collected in

250-ml fractions, solvent removed and the residue weighed after drying

the residue in vacuum desiccator.

Thin Layer chromatography:

The fractionation was monitored by thin layer chromatography on

TLC glass plates (520 cm) coated with silica gel G (0.25 mm thick).

The plates were activated at 1100C for 30 min before use. The

solvent system used were:

Toulene: Ethyl acetate (80:20)

Petroleum ether: Ethyl acetate : Methanol (5:3:2)

Chloroform: Ethyl acetate (5:5)

Detection of the spots was done by exposing the developed plate

under iodine vapors. Fractions with similar Rf values were pooled

together. The work up yielded residues which were crystallized

individually and were found homogenous on TLC, each showing a single

spot.