Chapter IV

MATERIALS AND METHODS

43

MATERIALS AND METHODS

4.1 Materials

4.1.1 Plant Material and drug.

Acalypha indica Linn (Kuppamani) of the family Euphorbiaceae, the

plant material selected for the present study, was collected from the suburbs

of Thiruvananthapuram and Kanyakumari districts and got identified by

comparing with the herbarium specimen kept in the Ayurveda Research

institute at Poojapura, Thiruvananthapuram.

Acalypha indica is an annual herb, up to about 75 em height. Leaves

are 3-8 ern long, ovate, thin, usually 3-nerved; margins of the leaves

toothed; leaf stalks longer than leaves. Flowers are in axilary erect spikes;

female flowers supported by conspicuous wedge-shaped bracts; male

flowers are minute, borne towards the top of the spike.225,253 Fruits are

small, hairy, concealed in the bracts.

After preliminary screening and pilot study to detect the most

effective extract, specific quantity of alcohol extract obtained by successive

solvent extraction of the dried leaves was suspended in 1% carboxy methyl

cellulose (CMC) and used as the test drug for the programmed studies.

The standard drug, Silymarin, used in this study was obtained from

Maneesh pharmaceuticals Pvt. Ltd, Mumbai.

44

4.1.2 Reagents and chemicals

The reagents and chemicals used in this study and their sources are

given below.

Reagents/ Chemicals Distributor/ Manufacturer

Nitroblue Tetrazolium (NBT) Sisco Research Lab Pvt. Ltd. Mumbai

Reduced glutathione (GSH) Do

Glutathione reductase (GR) Do

Nicotinamide adenine Do

dinucleotide - phosphate reduced

tetrasodium salt

5,5-dithiobis(2-nitro benzoic acid) Do

(DTNB)

2-deoxy- D- ribose Sigma chemicals

Calf thymus DNA Do

yeast RNA Do

Thiobarbituric acid E.Merck. India Ltd.,

Mumbai

Carbon tetrachloride (CCI4) Do

Paracetamol LP. Kerala State Drugs and

Pharmaceuticals, Alleppy

All other chemicals used were of analytical grade.

45

MASTER PLAN OF THE STUDY

p 1. Successive solvent extraction

L 2. Phytochemical screeningA

3. Ash valuesN

4. Extractive valuesT

5. Anti oxidant studiess

a. Lipid peroxide inhibitionT

ub. Super oxide scavenging effect

D c. Hydroxyl radical scavenging effect

y 6. Isolation and identification of compounds from ethanol extract

A I. Animal maintenance and CareN

2. Acute toxicity studies

3. Gross behavioural studiesM

4. Hepatoprotective study using CCl4A

5. Hepatoprotective study using paracetamolL

6. Curative study using CCl4

s 7. Curative study using paracetamolT 8. Histopathological studiesu

9. Choleretic studsyD

10. Liver regeneration studyy

11. Chronic toxicity study

IC N 12. Biochemical parameters of Blood (AST, ALT, ALKP, Total Protein,

v0 E Albumin and Total Bilirubin, Creatinin, Urea)

sM T

13. Anti oxidant enzymes like glutathione reductase,IM G

A glutathione peroxidase, superoxide dismutase and0 T

Imalondialdehyde, and glutathione contentN 0

Ns 14. Liver macromolecules such as RNA, DNA, Protein and

Cholesterol

46

4.2 Extraction and Preliminary Phytochemical Screening254.256

The plant may be considered a biosynthetic laboratory, not only for

the chemical compounds like carbohydrates, proteins and lipids that are

utilized as food by man, but also for a multitude of compounds like

glycosides, alkaloids, volatile oils, tannins etc. that exert a physiologic

effect. The compounds that are responsible for therapeutic effect are

usually the secondary metabolites. A systematic study of a crude drug

embraces thorough consideration of both primary and secondary

metabolites derived as a result of plant metabolism. The plant material may

be subjected to preliminary phytochemical screening for the detection of

various plant constituents on the following lines.

(A) Successive solvent extraction.

1. Thousand grams of the air-dried powdered plant material was

extracted successively with the following solvents in a Soxhlet

extractor.

(a) Petroleum ether (60-80°)

(b) Benzene

(c) Chloroform

(d) Acetone

(e) Ethanol (95%)

47

Each time before extracting with the next solvent, the powdered

material was dried in an air-oven below so'c.

2. Finally, the marc was macerated with chloroform water for 24 hours

to obtain the aqueous extract.

3. Each extract was concentrated by distilling off the solvent and then

evaporated to dryness on a water bath.

4. The extract obtained with each solvent was weighed and their

percentage calculated with reference to the air-dried material. The

colour and consistency was also noted.

Qualitative chemical examination

The extracts obtained as above were then subjected to qualitative

tests for the identification of various plant constituents.

1. Detection of alkaloids

A small portion of the solvent free chloroform, alcoholic and

water extracts were separately stirred with a few milliliters of dilute

hydrochloric acid and filtered. The filtrates were tested carefully with

various alkaloidal reagents.

a) Mayer's test: To 2 ml each of the acidified extracts added 0.5 ml of

Mayer's reagent and noted for the presence of a cream precipitate.

b) Dragendorff's test: The acidified extracts (2ml) were treated with a

few drops of Dragendorff's reagent and observed for the presence of

an orange brown precipitate.

48

c) Hager's test: To 2 ml each of the acidified extracts, added 0.5 ml of

Hager's reagent and observed for the presence of a yellow

precipitate.

d) Wagner's reagent: The acidified extracts were treated with a few

drops of Wagner's reagent and observed for the presence of

reddish brown precipitate.

Detection of carbohydrates and sugars

a. Small quantities of alcoholic and aqueous extracts were mixed

separately with 5ml (each) of water and filtered.

Molisch's test: 2ml each of the extracts were treated with few drops

of Molisch's reagent and 2 ml of cone. H2S04 was added through the sides

of the test tubes without shaking. Observed for the presence of a violet ring

at the junction of the two solutions.

b. A small portion of the extract was hydrolyzed with dilute

hydrochloric acid for a few hours on a water bath and the

hydrolysate was later subjected to Liebermann- Burchard's, Legal's

and Borntrager's tests to detect the presence of different glycosides.

(i) Liebermann-Burchard test: 5 ml each of the hydrolysate taken in test

tubes were evaporated, the residue taken in dry chloroform (lml) and then it

was mixed with 2 ml each of freshly distilled acetic anhydride followed by

a few drops of Conc.H2S04 through the sides of the test tubes. Observed

49

the development of a deep red colour in the lower portion and green colour

in the upper portion which changes to blue and violet.

(ii) Legal's test: The residue left after evaporation was dissolved in a few

ml of hydrolysate in 2 ml of pyridine. Added 2 ml each of sodium

nitroprusside solution to each test tube and then made alkaline with sodium

hydroxide solution; observed for pink to red colour.

(iii) Borntrager's test: A little of the residue obtained from the

hydrolysate was dissolved III water and shaken with equal volume of

chloroform. The chloroform layer was separated and added dilute ammonia

solution and shaken well. Noted whether any pink colour was present in the

ammoniac layer.

c. A small portion of the extract was dissolved in water and treated

with Fehling's, Barfoed's and Benedict's reagents for the presence

of sugars.

3. Detection of phytosterols:

Refluxed petroleum ether, acetone and alcoholic extracts separately

with solution of alcoholic potassium hydroxide till the completion of

saponification. Diluted the mixtures and extracted with solvent ether.

Evaporated the ethereal extract and subjected the residue to Liebermann's

and Liebermann-Burchard's test.

50

4. Detection of fixed oils and fats

(i) A small quantity of petroleum ether and benzene extracts were

pressed separately between two filter papers and observed for

the presence of oil stains.

(ii) Added a few drops of 0.5N alcoholic potassium hydroxide to a

small quantity of petroleum ether or benzene extract along with

a drop of phenolphthalein. Heated on a water bath and

observed for the presence of soap or partial neutralisation of

alkali.

5. Detection of saponins

(i) Foam test: Diluted 1 ml each of alcoholic and aqueous extracts

separately with distilled water to 20 ml in a graduated cylinder

and shaken for 15 minutes.

(ii) Haemolysis test: One drop each of alcoholic and aqueous

extracts in little warm water were added to blood samples on

glass slides and mixed well. Observed the presence of clear

haemolytic zones.

6. Detection of phenolic compounds and tannins.

Small quantities of alcoholic and aqueous extracts taken in water

were treated with dilute ferric chloride (5%), 1% solution of gelatin

containing 10% sodium chloride and 10% lead acetate solution. Observed

the presence of blue colour and white precipitate respectively.

51

7. Detection of proteins and free amino acids

Small quantities of alcoholic and aqueous extracts were subjected to

ninhydrin test and noted the presence of violet colouration.

8. Detection of gums and mucilage

10 ml of aqueous extract was added slowly to 25 ml of absolute

alcohol (with constant stirring) and filtered. The precipitate was then

observed for its swelling properties and for the presence of carbohydrates.

4.3 Ash values257

(i) Ash content: The residue remaining after incineration is the ash,

content of the drug, which simply represents inorganic salts,

naturally occurring in drug or adhering to it or deliberately added

to it, as a form of adulteration. Ash value is a criterion to judge

the identity or purity of crude drugs. Total ash usually consists of

carbonates, phosphates, silicates and silica. Acid insoluble ash,

which is a part of total ash insoluble in dilute Hel, is also

recommended for certain drugs. Adhering dirt and sand may be

determined by acid insoluble ash content.

4.3.1 Total ash value 258, 259

Procedure

2.5g of the ground drug (leaves) was accurately weighed and taken

In a tarred silica crucible previously ignited and weighed; scattered the

52

ground drug in a fine even layer in the bottom of the dish. Incinerated by

gradually increasing the heat until free from carbon. It was then cooled and

weighed to constant weight. Calculated the percentage of ash with

reference to the air-dried drug. The experiment was repeated thrice and the

average calculated.

4.3.2 Acid insoluble ash value.

Procedure

Boiled the ash obtained after total ash value determination for 5

minutes with 25 ml of dil. Hydrochloric acid. The insoluble matter was

collected in an ashless filter paper, washed with hot water, ignited and

weighed to constant weight. Calculated the percentage of acid insoluble ash

with reference to the air- dried drug. The procedure was repeated thrice and

the average calculated.

4.3.3 Sulphated ash

Procedure

About 2-3 g of drug was accurately weighed, moistened with

sulphuric acid and ignited gently. Again moistened and re-ignited, cooled

and weighed. Calculated the percentage of sulphated ash with reference to

the air dried drug.

53

4.3.4 Water-soluble ash

Procedure

Boiled the total ash with 25 ml of water for 5 minutes; insoluble

matter was collected in an ashless filter paper; wet with hot water, and

ignited to constant weight at a low temperature. The weight of insoluble

matter was subtracted from the weight of the ash. The percentage of water­

soluble ash was calculated with reference to the air-dried drug.

4.4 Extractive values 260

The extracts obtained by exhausting crude drugs are indicative of

approximate measures of their chemical constituents. Taking into

consideration the diversity in chemical nature and properties of contents of

drugs, various solvents are used for determination of extractives. The

solvent used for extraction is in a position to dissolve appreciable quantities

of substances desired.

4.4.1 Alcohol soluble extractive

Alcohol being an ideal solvent for extraction of various chemicals

like tannins, resins etc. this method is frequently employed to determine the

approximate resin content of drugs. Generally 95% ethyl alcohol is used for

determination of alcohol soluble extractive; dilute alcohol may also be used,

depending upon solubility of the constituents of crude drugs.

54

Procedure

Macerated Sg of dried coarse powder of the drug with 100ml of

alcohol 9S% in a closed flask for 24 hours, shaking frequently during 6

hours and allowed to stand for 18 hours. It was then filtered immediately

taking precautions against loss of alcohol. Twenty-five milliliter of the

filtrate was evaporated to dryness in a tarred flat-bottomed shallow dish.

Dried at 10SoC and weighed. Calculated the percentage of alcohol soluble

extractive with reference to the shade-dried drug.

4.4.2 Water-soluble extractive

This method is applied to drugs, which contain water-soluble active

constituents, such as tannins, plant sugars, mucilage, glycosides etc.

Procedure

Added Sg of coarse powder of the drug to SOml of water at 800e in a

stopper flask. Shaken well and allowed to stand for 10 minutes. Cooled to

lSoC and added 2g of kieselguhr; filtered and transferred Sml of the filtrate

to a tarred evaporating dish; evaporated the solvent on a water bath and

weighed the residue. Calculated the percentage of water-soluble extractive

with reference to the shade-dried drug. The values were tabulated.

4.5 Atomic Absorption Spectroscopy'?'

This technique is based on the fact that when atoms, IOns or IOn

complexes of an element are atomized at ground state in a flame, they

absorb light at the characteristic wavelength of that element. If the

55

absorption process takes place in the flame under reproducible conditions,

the absorbance is proportional to the number of absorbing atoms.

Procedure

19 of air-dried leaves of the plant was accurately weighed and

ashed at 800DC thoroughly. The ash was dissolved in concentrated

hydrochloric acid and made up to l Ouml in a standard flask. For the

analysis of zinc, since it is volatile, the sample was accurately weighed and

dissolved in hydrochloric acid - perchloric acid mixture, boiled for 5

minutes, filtered and made up to 100m!.

The instrument was standardized usmg known concentrations of

solutions and a graph of absorbance vs. concentration drawn by the

instrument itself (instrument used was VARIAN AUSTRALIA). The

sample solutions were fed into the instrument and the concentration of each

element detected using the calibration graph.

4.6 Anti oxidant studies (IN VITRO)

As pilot studies with extracts of successive solvent extraction did not

show significant results except for ethanol extract, further programmed

studies were concentrated on ethanol extract only.

4.6.1 Superoxide Scavenging Activity.

Superoxide scavenging activity of the ethanol extract was

deiermined by the method of McCord and Fridovich262 which depends on

56

the light induced superoxide generation by riboflavin and the corresponding

reduction of nitroblue tetrazolium(NBT).

The reaction mixture contained EDTA (6Ilm) containing 31lg NaCN,

riboflavin (2J.!m), NBT (50llm), various concentrations of the ethanol

extract and phosphate buffer (67mM, pH 7.8) in a final volume of 3ml. The

tubes containing the reaction mixture were uniformly illuminated with an

incandescent lamp for 15 minutes and the absorbance was measured at

530nm before and after the illumination. The percentage inhibition of super

oxide generation was evaluated by comparing the absorbance values of the

control and experimental tubes.

4.6.2 Hydroxyl radical scavenging activity263

Hydroxyl radical scavenging activity was measured by studying the

competition between deoxyribose and the plant extract for hydroxyl radical

generated from the Fe3+- ascorbate - EDTA - H20 2 system (Fenton

reaction). The hydroxyl radical attacks deoxyribose that eventually results

in the formation of thiobarbituric acid reactive substances (TBARS).

The reaction mixture containing deoxyribose (2.8mM),ferric chloride

(O.lmM), ~DTA (O.lmM), H20 2 (lmM), ascorbate (O.lmM), KH2P0 4­

KOH buffer (20mM; pH 7.4) and various concentrations of the extracts in a

final volume of 1ml was incubated for 1 hour at 37°C. Deoxyribose

degradation was measured as thiobarbituric acid reactive substance

57

(TBARS) by the method of Okhawa et ae64 and percentage of inhibition

was calculated from the control where no test extracts were added.

4.6.3 Effect on Lipid peroxidation

Effect on the inhibition of lipid peroxidation was determined by the

thiobarbituric acid method. Different concentrations of the plant extract

were incubated at 37°C with rat liver homogenate (25%) (O.lml) containing

30mM KCI, Tris-HCI buffer (O.04M; pH7), ascorbic acid (O.06mM) and

ferrous iron (O.16mM) (total volume was O.5m!) for 1 hour. At the end of 1

hour, thiobarbituric acid reactive substance (TBARS) was measured by the

method of Okhawa et ae64 and percentage of inhibition calculated from the

control where no test extract was added.

4.7 Phytochemical study

10 gm of ethanol extract obtained after successive solvent extraction

was dissolved in 50ml of ethanol. Then it was partitioned with ethyl acetate

and the ethyl acetate fraction was concentrated and dried under vacuum.

The ethanol fraction was also concentrated and dried.

The ethyl acetate fraction was subjected to TLC analysis usmg

hexane- ethyl acetate (1:1) solvent system and 3 spots were obtained. 10%

H2S04 in methanol was used as spray reagent. Later the ethyl acetate

extract was subjected to column chromatography using silica gel as the

adsorbent. Gradient elution using hexane and ethyl acetate, in different

58

proportions, was carried out. The fractions eluted using hexane : ethyl

acetate in the ratio 9:1 gave identical spots with same Rf in TLC analysis

(Rf = 0.92) and they were pooled and concentrated, crystallized and

recrystallized from 9: I hexane - ethyl acetate system. It was designated

as Compound A.

The fractions collected with the same solvents in the ratio (8:2) gave

another compound, crystallized in the same way, designated as compound

B (Rf = 0.86). Further elution was carried out with increasing concentration

of ethyl acetate. Finally at 50:50 ratio of hexane: ethyl acetate system, the

third compound was obtained as in the previous cases. This was designated

as compound C. (Rf = 0.49).

The ethanol fraction was subjected to TLC using n-butanol: acetic

acid: water (40:10:50) system. Mainly 3 spots were observed with the

spray reagent 10% H2S0 4 - methanol. During column chromatography

after gradient elution with chloroform-methanol, the first compound was

obtained at 50:50 ratio. This was designated as compound D.(Rf = 0.62)

Later the proportion of methanol was increased. Finally chloroform ­

methanol at 10:90 ratio gave another compound which was recrystallised

and later designated as compound E.

59

4.8 ANIMAL STUDIES

Studies related to hepatoprotection

4.8.1 Animal maintenance and care:

Wistar strain of rats (Rattus norvigicus) and Swiss albino mice (Mus

musculus), were the animals used for the study. They were bred in colony

and brought up in our own animal house. Animals were housed in well­

ventilated polypropylene cages and fed with a standard pellet diet (Gold

Mohur laboratory animal feed) and water ad libitum. They were kept in

standard environmental conditions. (Temperature- 25-28oC and 12 hours

light/dark cycle) Rats of both sexes weighing 150-250g and mice of average

20-30g were used for animal experiments. (All ethical formalities were

cleared for the conduct of animal experiments using albino rats and mice.)

4.8.2 Acute Toxicity Studies265, 266

Albino rats of either sex weighing 150 - 250g and Albino mice

weighing 20-30 g of either sex were used for carrying out acute toxicity

studies. Before the actual study, a pilot study was carried out in small group

of animals (2 each) giving them widely spaced doses to select the Jose

ranges for the actual acute toxicity studies. Finally the assay was done using

5-6 dose levels with larger number of animals in each group (10 animals in

one group). All animals were fasted overnight before the acute toxicity

studies. After oral administration of the drug, the animals were observed

60

continuously for 2 hours and then intermittently for another 4 hours. After

24 hours, the deaths if any, were noted to calculate the LDso.

4.8.3 Study of Gross behavioral changes

Along with the toxicity tests, the animals were observed for

behavioral, neurological and anatomical profiles and the changes observed

in animals were recorded.

4.8.4 Determination of the most effective extract and its dose

As the pilot study, with all the extracts, showed maximum effect for

the ethanol extractf", a dose response assay was carried out using ]15 th,

1I1Oth and 1I20th of the maximum dose given to the animals to determine the

minimum dose producing maximum hepatoprotective effect. This was used

for the subsequent programmed studies.

4.8.5 Induction of Hepatotoxicity

Hepatic injury was induced using Carbon tetrachloride (CC14) and

paracetamol. In the former case CCl4 was mixed with olive oil in the ratio

1:1 and given intraperitoneally at a dose of 0.5ml/kg body wt. for 5 days or

as needed. In the latter case an overdose of paracetamol (3g/kg body wt.) or

accordingly, was given orally, suspended in 1% carboxy methylcellulose

(CMC).

61

4.8.6 Grouping of animals and different treatment

Animals were grouped and given different treatments as follows.

Male Wistar albino rats weighing IOO-120g were used for

hepatoprotective studies. The animals were divided into four groups of six

each.

(a) Hepatoprotective activity of A.indica using Carbon tetrachloride

Group I

(control)

Group II

Group III

(CCI4 +

A. indica ext.)

Group IV

(CCI4+Silymarin)

Animals did not receive any treatment;

but only 1% CMC (Iml/lOOg)

Animals received CCl4 (O.5mllkg body wt.

intraperitoneallyand 1% CMC (lmVIOOg), orally.

Animals received CCl4 (O.5mllkg body wt.)

and extract (IOOmg / kg body wt) after

10 min, orally

Animals received CCl4 (O.5mllkg body wt.) and

Silymarin (1OOmg / kg body wt) after 10 minutes,

orally.

This procedure was repeated for 5 consecutive days and on the 6th

day animals were sacrificed. The blood was collected for determination of

biochemical parameters and liver for histopathological studies.

62

(b) Paracetamol - induced hepatotoxicity

Group I

(Control)

Group II

(Paracetamol)

Group III

(Paracetamol +

A.indica ext)

Group IV

(Paracetamol +

Silymarin)

Animals did not receive any drug but only 1%

CMC (lml / IOOg), orally; repeated for 4 days.

Animals received 1% CMC on i", 2nd and 4th day

in the normal case. On day 3, paracetamol suspension

in a dose of 3g/kg body wt, was given orally.

Ethanol extract in a dose of IOOmg / kg

body wt. orally for 4 days and paracetamol(3g/kg

body wt.) on the 3rd day.

Silymarin in a dose of IOOmg/kg body wt. orally for 4

days and paracetamol (3g/kg body wt.) on the 3rd

day.

Animals were sacrificed 48 hours after paracetamol intoxication.

Blood was taken for determination of biochemical parameters and liver for

histopathological studies.

(c) Curative effect of Acalypha indica extract after CCl4-induced

liver injury268.

Liver damage was induced by the administration of CCl4 mixed with

olive oil. The experimental protocol was as stated below.

63

Experimental protocol

Group Pretreatment Duration Day of Inferencein days withdrawal of

bloodI - - 0 Control value to

compare with the testII CC14 0-5 6111 To induce liver

damageIII CC14 0-5 11th To assess the

prophylactic effectIV A. indica + 1-5 To assess curative

CC14 6-10 11th effectV CC14 + 1-5 6111

"A. indica 1-5

VI CC14 + 1-5 11th"

A. indica 1-5VII CC14 + 1-10 11th

"A. indica 6-10

A.indica extract(lOOmglkg body wt) was administered orally and

CC14 (O.5mllkg body wt), intraperitoneally. A few animals from each group

were sacrificed on the day of withdrawal of blood from that group. Liver

was isolated, weighed and preserved in 10% formalin for histopathological

studies. With the blood all the liver function tests were done and with the

liver all the enzyme studies.

(d) Curative effect of Acalypha indica extract on paracetamol

induced liver injury122.

Liver damage was induced by the administration of paracetamol

(lg/Kg) orally, once daily. The experimental protocol was as stated below

64

Experimental protocolN =6

DurationDay of

Group Pretreatmentin days withdrawal Inference

of bloodI I%CMC - Control values to

0compare with the test

II Paracetamol 2 3HJ To induce liver damageIII Paracetamol 2 s" To assess the

spontaneous recoveryIV Paracetamol+ 1-5 s" To assess the

A. indica 6-7 preventive effectV Paracetamol + 1-5

3rd To assess the curativeA. indica 1-5 effect

VI Paracetamol + 1-56th To assess the curative

A. indica 1-5 effect ..VII Paracetamol + 1-7

8th To assess the curativeA. indica 3-7 effect

As in the previous case the drug was administered orally in a

dose of 100mg/kg body wt. The dose of paracetamol was 1g/kg body wt. A

few animals from each group were sacrificed on the day of withdrawal of

blood from that group: Liver was isolated, weighed and preserved in 10%

formalin for histopathological studies.

4.8.7 Effect of Acalypha leaf extract on Bile flow rate

(Choleretic Activity) 163,269

Male rats of 200-250 g weight were selected. They were divided

into two groups of 6 each. These rats were anesthetized with i.p injection of

Sodium pentobarbitone. The common bile duct was surgically exposed by

middle line laparotomy and cannulated with polyethylene tubing (No. 48).

A heating lamp maintained body temperatures of rats. Bile collected for

65

first 10 minutes was discarded and then collected in graduated tubes. Bile

was collected for 1 hour and then 1 ml of 1% CMC was administered

intraduodenally to control rats (group I). Group II rats received ethanol

extract of Acalypha leaf suspension (100 mg/kg body weight)

intraduodenally. Then the bile was collected for 4 hours. The volume of

bile was noted. An increase in the bile flow in the treated animals

compared to control was taken as the criterion for choleretic activity.

4.8.8 Effect of Acalypha leaf extract on liver regeneration205, 206.

Albino rats (male) of average weight (100 -llOg) were randomly

divided into four groups. Group I was normal sham operated, GroupII,

hepatectomised -untreated, Group III, hepatectomised - test drug treated,

while the group IV consisted of hepatectomised - silymarin treated animals.

Ethanol extract of A. indica and Silymarin, in 1% CMC suspension were

administered orally to rats for 7 consecutive days at a dose of 100mg./kg

body weight. On day 8, partial hepatectomy (about 70% removal of the

liver) of rats of group II, III and IV were done under light ether anaesthesia

according to the method of Higgins and Anderson/". Rats in sham­

operated group were laparotomised in the same way except that the partial

hepatectomy was not done.

On selected time intervals post surgery (from 12 to 120 hrs) animals

were sacrificed by decapitation after fasting overnight. The liver was

66

promptly excised, washed thoroughly and subjected to DNA, RNA, protein

and cholesterol estimations.

4.8.9 Chronic toxicity studies271

The alcoholic extract of A. indica was subjected to chronic toxicity

studies.

Albino rats of either sex weighing 150-200 g were selected. Three

dose levels of drugs were administered in the study viz, 200 mg, 400 mg

and 800mg/kg body weight.

A control group with 1% CMC (Iml/l00g) was also used to rule out

the effect of the vehicle if any.

Animals were divided into 4 groups of 12 each. Each group was

again divided into subgroups (a) and (b) of 6 males and 6 females.

Grouping of animals and dose of drug given are summarised below.

Groups of animals and dose of the drug in chronic toxicity studies

Grouping ofTreatment given (p.o) Purpose

animals

A a b 200 mg of extract/kg Chronic effect of low dose

body weight.

B a b 400 mg of ext/kg body Chronic effect of middle

weight. dose

C a b 800 mg of ext/kg body Chronic effect of high

weight. dose

D a b Control animals. 1% Control to compare with

CMC (lml/lOOg) other groups.

67

All animals were given measured amount of food and water daily.

Period of study was 3 months. During the study, body weight and food

intake were monitored and hematological parameters were determined at

regular intervals of every 14 days. At the end of three months, all animals

were sacrificed and autopsy performed. Vital organs (liver, kidney and

spleen) were removed, weighed and examined for gross changes, as well as

for any histological changes. Blood was also collected during sacrifice and

estimations of alkaline phosphatase, AST, ALT, blood urea, creatinine and

serum bilirubin were also done and compared with those of control animals.

4.9 Blood Chemistry

(Determination of various constituents and enzymes)

4.9.1 Collection of blood for various estimations

At the end of each experiment (or even in between, as specified)

blood was collected by retro-orbital bleeding. The blood was then allowed

to clot, and centrifuged at 3000 rpm for 10 minutes. Clear serum obtained

as supernatant was transferred to clean dry screw cap bottles. This was

used for various estimations.

4.9.2 Alkaline Phosphataset"

Reaction conditions

Temperature

pH

68

Final concentration of reagents.

PNPP

MAP

Mg2+

16 n mol/l

1.0 rnol/l

1.0 m mol/l

Principle

The method of Bowers and McComb273 is widely used today

because of its simplicity and sensitivity. In this, p-nitrophenyl phosphate

(PNPP) is the substrate and 2-methyl-2-amino-1-propranol (MAP), the

transphosphorylating buffer. The colourless substrate is hydrolysed to

yellow coloured P-nitro-phenol (PNP) which has an absorption maximum

at 405 nm. This method measures the rate of release of P-nitrophenoxide

in trans phosphorylating buffer.

Procedure

Pipetted out 1ml of substrate and 0.02ml of serum into test tubes.

Mixed well and absorbance was read at 30, 60, 90, and 120 seconds, at

405nm. The changes in absorbance were noted and calculated the results.

lUlL = fj, Almin x F (F=2713)

4.9.3 Alanine Aminotransferase (ALT)

Principle

In the method of Berg and Horder273 NADH is the reaction product

that is quantitated. Lactate dehydrogenase (LDH) and its co-factors are

69

added, with allowance of the enzymatic conversion of pyruvate to lactate

and with simultaneous oxidation of reduced Nicotinamide Adenine

Dinucleotide (NADH). The disappearance of NADH is followed

spectrophotometirically at 340 nm.

GPT

L-Alanine + a-Ketoglutarate ---+ Pyruvate + L-glutamate

LDH

Pyruvate + NADH + H+ ---+ L-Lactate + NAD+

Reaction condition

Temperature 30De

Fraction of serum volume - 0.083 ml

Final concentration of reagent.

L - alanine 500 mmolll

a - ketoglutarate 15 mmolll

pH 7.5

Tris buffer 100m molll

NADH 0.18m molll

Pyridoxal phosphate O.lm mol/l

Lactate dehydrogenase 1.21J molll

Type of analysis Kinetic

70

Procedure

Working reagent (1.0 ml) (Span Diagnostic kit) and sample (0.1 ml)

were mixed, incubated at 37°C for 1 minute. Started the stopwatch and read

the change in absorbance at 1, 2, 3 and 4 minutes.

Wave length, 340 nm

Blank Distilled water.

Determined the i1E/min for every reading and found the mean value

U/L = 1768 x (i1E/min.)

4.9.4 Aspartate Aminotransferase (AST)

Principle

In the method of Bergmeyer et ae74 first described by Karman,

oxaloacetate is the product quantified. Malate dehydrogenase (MDH) is

used to convert the oxaloacetate to malate. The decrease in absorbance at

340 nm caused by NADH (reduced nicotinamide adenine dinucleotide)

consumption is recorded spectrophotometrically.

GOTL - aspartate + a - ketoglutarate ~ Oxalo acetate + L-glutamate

MDHOxalo acetate + NADH + H + ~ Malate + NAD+

Reaction conditions:

Reaction temperature

Sample volume

71

Blank Distilled water

Wave length 340nm

Tris buffer 80mmoVI

pH 7.8

L -Aspartate 200mmoVI

a - ketoglutarate 12 m mol/l

ReducedNAD 0.18mmoVI

Pyridoxal P04 0.10mmoVl

Malate dehydrogenase 250 x 103U/I

Type of analysis Kinetic

Procedure

1 ml of working reagent (Span Diagnostic Kit) and 0.1 ml of sample

were mixed and incubated at 37°C, absorbance read after 1 minute, (change

in absorbance per min (~ OD/min) during 3 minutes. Calculated the

amount using the formula U/L = ~ OD/min x 1768

4.9.5 Estimation of Serum Bilirubirr'"

Principle

Sulphanilic acid is diazotised by the nitrous acid produced from the

reaction between sodium nitrite and hydrochloric acid. Bilirubin reacts with

the diazotized sulphanilic acid (diazo reagent) to form the purple compound

azobilirubin, the absorbance of which is measured at 540 nm.

72

Reagents Used:

1. Diazo reagent.

Sulphanilic acid solution 1% in 0.2N Hel

Sodium nitrite solution 0.5%

2. Methanol

- 10 ml

- 0.3 ml

3. Bilirubin standard - A solution containing 10mg/l00 ml

chloroform. Sulphanilic acid (0.5% solution)

Procedure

0.2 ml of serum was mixed with 5.4 ml water. Pipetted out 2.8 ml of

this into a second tube and used as blank. To the test added 0.7 ml of diazo

reagent and to the blank 0.7 ml of sulphanilic acid solution; mixed and

allowed to stand for 5 minutes. Then added 3.5 ml of methanol to each tube

and read the absorbance at 540 nm against the blank. For the standard

solution, added 0.2 ml bilirubin standard to 3.5 ml of methanol, and 0.7 ml

of diazo reagent, mixed well and added 2.6 ml of distilled water. Read

against a water blank after 5 minutes.

Total bilirubin = Reading of unknown X 20Reading of standard

4.9.6 Estimation of Serum Protein

Serum protein was estimated by the method of Huerga et at276

73

Reagents

1. Stock biuret reagent (45.0g sod.pot.tartarate dissolved in 400ml

of 200mM/l NaOH and added 15gm CUS04 . 2H20 Added 5gm

Potassium iodide and made up to llitre with 200m Mil NaOH.

2. Working biuret reagent (200ml stock reagent diluted to llitre

with 200m MIL NaOH containg 5gm pot.iodide llitre

3. Tartarate iodide solution (9gm sod.pot.tartarate in llitre of 200m

Mil NaOH containing 5gm KIf litre

4. Protein standard (80gm/litre)

Five test tubes marked as test (containing O.lml serum +5ml working

biuret reagent), serum blank (containing O.lml serum +5.0ml tartarate

iodide solution), standard containing O.lml standard +5ml working biuret

reagent), standard blank (containing O.lml standard +5ml tartarate iodide

solution) and reagent blank (O.lml water +5ml working biuret reagent),

were incubated at 37°C for Irnt. After cooling to room temperature

absorbance were measured at 555nm against reagent blank.

Procedure: 5 ml of Biuret reagent was taken to which 0.1 ml of serum was

added. Similarly 0.1 ml protein standard solution was added to 5 ml of

Biuret reagent I. 5 ml Biuret standard I was the blank. All the tubes were

kept at room temperature for 5 minutes. Then the absorbance of the

solutions-standard and test-were measured at 550 nm

74

4.9.7 Estimation of Serum Albumin 277

Serum albumin was estimated by the method of Drupt. Three test

tubes marked as blank (containing 4.5ml bromocresol green reagent),

standard (containing 4.5ml bromocresol green reagent +30 III (4gm%)

albumin standard) and test (containing 4.5ml bromocresol green reagent

+30 III serum were allowed to stand at room temperature for 1min.

Measured the absorbance of test and standard against blank at 600nm.

4.9.8 Urea Estimation 278

Diacetyl monoxime method

Principle

Serum proteins are precipitated with trichloro acetic acid and clear

supernatant is obtained after centrifugation. A portion of this supernatant is

heated with diacetyl monoxime in the presence of acid, oxidizing agent and

thiosemicarbazide and a red complex is formed with urea. The absorbance

of this complex is measured at 520 nm.

Reagents

1. Trichloro aectic acid solution, 10% (w/v). Dissolved 109 of trichloro

acetic acid in water and made up to 100 ml. This solution is stable

indefinitely at 20-25°C.

2. Ferric chloride solution: Dissolved 5.0g of ferric chloride in 100 ml

of water and added 1.0 ml of cone. Sulphuric acid. This solution

75

should be stored in a dark coloured bottle where it is stable for

several months at 20-25°C.

3. Acid Reagent: To 100 ml of water added 8.0 ml of cone. Sulphuric

acid, 1 ml of ortho phosphoric acid and 1 ml of iron III chloride

solution. This is stable at 20-250C indefinitely.

4. Diacetyl monoxime solution: Dissolved 25 g of diacetyl monoxime

in water and made up to 1 litre. This solution is stable for several

months at 20-25°C.

5. Thiosemicarbazide Solution: Dissolved 2.5 g of thiosemicarbazide

in water and made up to 1 litre. This solution is stable for several

months at 20-25°C.

6. Colour reagent: To 30 ml of acid reagent, added 20 ml of water, 1.0

ml of diacetylmonoxime solution and 0.25 ml of thiosemicarbazide

Solution. This should be prepared freshly each day.

7. Stock urea standard 10m mol/l: Dissolved 1.0 g of benzoic acid in

about 800 ml of warm water, allowed to cool, then transferred to a 1

litre volumetric flask and added 0.601 g of urea. When dissolution

was completed, made up to volume with water. This solution is

stable for several months at 20-25°C.

8. Working urea standards: Into 100 ml volumetric flasks, pipetted out

2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 ml of the stock urea standard and

76

made up to volume with benzoic acid solution (1.00 g/l.). The

working standards are equivalent to 2.5, 5.0, 7.5, 10.0, 15.0, 20.0 m

molll under the assay conditions. These standards are stable for

several months at 20-25°C.

Method

1. Pipetted into centrifuge tubes 0.8 ml of water, 0.2ml of sample and

mixed. Then added 1.0 ml of trichloro acetic acid solution, mixed

again and centrifuged.

2. To 0.2 ml of supernatant added 3 ml of colour reagent. For the

reagent blank 0.2 ml of water was used and for the standards 0.2 ml

of each working standard solution used.

3. After mixing, the tubes were closed and incubated at 100°C for 20

minutes.

4. Cooled the tubes in water for 5 minutes. Then read the absorbance of

the solutions at 520 nm, setting the spectrophotometer to zero with

the reagent blank.

4.9.9 Estimation of Creatinine

Principle

Creatinine is determined by quantitating the red pigment, alkaline

creatinine picrate (Jaffe reaction), as described by Giorgio279 (1974).

77

132 mg creatinine /lOml of 01 N NaOH.

Reagents

I.Picric acid

2. Tungstic acid

Polyvinyl alcohol

Sodium. tungstate dihydrate

Sulphuric acid

3.Sodium hydroxide

4. Standard-

0.36 molll.

1gil

11.1gil

O.lmlll

54 gil

Procedure: 0.5 ml of sample was deproteinised with 4 ml of tungstic acid,

centrifuged and 3 ml supernatant was mixed thoroughly with 1 ml of picric

acid followed by 0.5 ml sodium hydroxide. Absorbance was recorded after

15 minutes at 500 nm.

4.10 Estimation of Malondialdehyde (In Vivo)

Preparation of tissue homogenate

Weighed quantity of tissue was homogenised in O.IM Tris-HCl

buffer, pH 7.5, and allowed to stand for 5 minutes. The supernatant was

used for the determination of lipid peroxide level.

Thiobarbituric acid reactive substance (TBARS)

TBARS were estimated by the assay method of Nichans and

Samuelson.v"

78

Reagents

TCA-TBA-HCI reagent

0.25NHCl.

15%(w/v) TCA, 0.375%(w/v) TBA III

Procedure: 1ml of the tissue homogenate was combined with 2ml of TCA-

TBA-HCI reagent and mixed thoroughly. The solution was heated for 15

minutes in a boiling water bath. After cooling, the flocculent precipitate was

removed by centrifugation at 1000 rpm for 10minutes. The absorbance of

the sample was read at 535nm against a blank that contained no tissue

homogenate. The extinction co-efficient of malondialdehyde is 1.56 x 105

M -1 -Iem .

4.11 Activity of Scavenging Enzymes

4.11.1 Assay of Superoxide dismutase (SOD) [EC 1.15.1.1]

SOD activity was determined by the method of Kakkar et a1. 28 1 The

tissue was homogenized in 0.25M sucrose and differentially centrifuged at

10,000 rpm under cold conditions to get the cytosol fraction. The initial

purification was done by precipitating the protein from the supernatant with

90% ammonium sulphate and after dialysis against 0.0025M Tris-HCI

buffer, pH 7.4. The supernatant was used as the enzyme source.

The assay mixture contained 1.2m1 Sod.pyrophosphate buffer

(0.052M, pH8.3), O.lml 1861lM phenazine methosulphate (PMS), 0.3ml

300llm nitroblue tetrazolium (NBT) 0.2ml 780llM NADH, appropriately

79

diluted enzyme preparation and water in a total volume of 3ml. Reaction

was started by the addition of NADH. After incubation at 30°C for 90

seconds reaction was stopped by the addition of 1ml glacial acetic acid. The

reaction mixture was stirred vigorously and shaken with 4ml of n-butanol.

Mixture was then allowed to stand for ten minutes. Centrifuged and butanol

layer was taken out. Colour intensity of the chromogen in the butanol

fraction was measured at 560nm against butanol. A system devoid of

enzyme served as control.

One unit of enzyme activity is defined as the enzyme concentration

required to inhibit chromogen production (OD of 560 nm ) 50% in one

minute under the assay conditions. The specific activity is expressed in

units/ mg protein. Unit is defined as the velocity constant per second.

4.11.2 Assay of Catalase [EC 1.11.1.6]

( HzOz: Hydrogen peroxide oxido reductase)

The catalase activity was assayed by the method of Maehly and

Chancez8z, The tissue was homogenized with 0.91M-phosphate buffer (pH

7.0) at 1- 4°C and centrifuged at 5000 rpm. The estimation was done

spectrophotometrically following the decrease in absorbance at 240nm.

The reaction mixture contained 0.91 M phosphate buffer pH 7.0, 2mM

HzOz (diluted O.lml HzOz to 100ml using buffer) and 50~1 enzyme extract.

80

Specific activity is expressed in terms of units/ mg protein. Unit is defined

as the velocity constant per second.

4.11.3 Assay of Glutathione Peroxidase (EC.1.11.1.9)

(Glutathione: hydrogen peroxide, oxido reductase)

The activity of glutathione peroxidase was determined by the method

of Lawrence and Burk283 as modified by Agerguard and Jense284. Tissue

homogenate (10%) was prepared in 0.25M sucrose, centrifuged at 10,000

rpm for 30 minutes and the supernatant fraction was used for the assay.

Activity was determined in phosphate buffer (50mM pH7.0) containing

EDTA (1.5mM), sodium azide (1.0mM), reduced glutathione (1.0mM),

NADPH (O.lmM) and glutathione reductase (1.0flM/ml). Absorbance was

measured at 340nm at 20 seconds interval. Enzyme activity is defined as

flM ofNADPH oxidized/min /mg protein using 0.25mM H20 2 as substrate.

4.11.4 Assay of Glutathione reductase (EC.1.6.4.2)

(Reduced NAD (P): oxidized glutathione oxido reductase)

Glutathione reductase activity was determined by the method

described by Bergrneyer'f". Tissue homogenate (10%) was prepared in 0.25

M sucrose, centrifuged at 10,000 rpm for 30 minutes and supernatant

fraction was used for the enzyme assay. The assay system contained 1ml

phosphate buffer (0.12M, PH 7.2) O.lml EDTA, O.lml sodium azide

(10mM/I), O.lml oxidized glutathione (6.3mM) and 0.1 ml enzyme source.

81

It was kept for 3 minutes. Then 0.1 ml NADPH (9.6 mM/I) was added. The

absorbance at 340 nm was measured at an interval of 15 seconds for 2

minutes. The activity is expressed as JlM NADPH oxidized per minute I mg

protein.

4.12 Estimation of Antioxidants

4.12.1 Estimation of Glutathione Content

Glutathione content was estimated by the method of Benke et a1286•

20% tissue homogenate prepared in 5% TeA containing O.OOIM

EDTA, was centrifuged at 2000 rpm for 5 minutes; 0.2ml aliquot of each

supernatant fraction was transferred to another tube containing 4.75ml of

O.IM sodium phosphate buffer (pH 8) and to it 0.05ml of O.Olm 5,5

dithiobis-2-nitrobenzoic acid (DTNB) was added. The absorbance was read

at 412nm within 4 minutes.

4.13 Estimation of Liver Protein

The method of Lowry et ae87 was used for the assay.

Principle: The method relies on the formation of a protein- copper

complex and the reduction of the phosphomolybdate - phosphotungstate

reagent (Folin-ciocalteu phenol reagent) by the tyrosine and tryptophan

residues of protein to form coloured product.

82

Reagents

Solution A containing I ml CUS04 (1%), 1 ml sodium potassium

tartarate 2% and 98ml of Na2C03 (2% in O.IN NaOH); solution B ­

containing Folin-ciocalteu phenol reagent diluted (l: 1) with distilled water.

Procedure

O.lml of tissue homogenate (2.5%) was diluted to 1.2 ml with

distilled water and mixed with 6 ml of solution A. The mixture was

incubated at room temperature for 10 minutes. To this solution 0.3ml

solution B was added. Mixed well and kept at room temperature for 30

minutes. The absorbance was read at 660nm.

4.14 Estimation of Total Cholesterol

Extraction of Liver

Liver was homogenized and extracted with chloroform: Methanol

(2:1). It was filtered and the residue was washed with chloroform: methanol

(2:1) at least 3 times. The filtrate was combined and washed with 0.7% KCI

solution (20% of the total volume of the extract) The aqueous upper phase

was removed and the lower layer was washed 3 times with 5ml of

chloroform : methanol : KCI solution. (3:48:47v/v). The washed lower

layer of chloroform was evaporated to dryness and the residue re dissolved

in a known volume of chloroform.

83

Procedure

Cholesterol was estimated by Carr and Drekter288 method. An aliquot

was taken from the above solution and evaporated to dryness. It was

dissolved in 0.45ml of glacial acetic acid. An aliquot from standard (50mg

cholesterol/50ml glacial acetic acid) and a blank containing glacial acetic

acid was also taken. 2 ml of acetic anhydride was added followed by 0.05ml

water to enhance heat of formation. It was kept for five minutes. One drop

of dehydrating reagent (colour reagent: Acetic acid (1:1 v/v» was added

directly, mixed by gentle rotation, and kept in a water bath at 20De for 10

minutes. Added 0.5ml colour reagent (Acetic acid: H2S04 1:1 v/v) directly

and kept at room temperature exactly for 20 minutes. Absorbance was read

at 620nm.

4.15. Estimation of DNA289

2gm of tissue (liver) was homogenized with 10 ml of ice cold lO9'o

trichloro acetic acid. It was centrifuged at 3000 rpm for 10 minutes.

Supernatant was discarded and the precipitate was suspended in 5 ml ice

cold 10% TCA. Again centrifuged and discarded the supernatant.

Suspended the residue in 5ml of ethanol-ether mixture and once again

centrifuged for ten minutes. The precipitate was taken in 0.5 N NaOH.

Mixed well and kept for 18hours at 37°e and centrifuged. To the

precipitate was added 1 ml of perchloric acid and heated on a boiling water

84

bath for 1 hour, cooled and centrifuged. The supernatant was made up to a

known volume.

The deoxyribose in DNA in presence of acid forms hydroxylevulinic

aldehyde which reacts with diphenylamine to give a blue colour.

Pipetted out different volumes of the solution in test tubes and added

5 ml of diphenylamine reagent. Mixed well and heated on a boiling water

bath for 10 minute. After cooling the absorbance was measured at 595nm.

Calculated the amount of DNA present in the tissue.

4.16 Estimation of RNA289

The supernatant obtained after incubation with 0.5N NaOH(in the

extraction) was mixed with 10% TCA and centrifuged for l0 minutes. The

supernatant obtained was made up to a known volume.

Acid hydrolysis of RNA releases ribose and this in presence of

strong acid, dehydrates to furfural. Orcinol reacts with furfural in presence

of ferric chloride to give a green colour.

Set up different tubes with measured volume of the supernatant and

added 3.0ml of orcinol reagent to each tube and mixed well. Heated on a

boiling water bath for 15 minutes and cooled. The absorbance was read at

665nm. Estimated the amount of RNA in the known volume of solution.

85

4. 17 Histopathological Examinatiorr't"

Tissue specimens were fixed in 10% buffered formalin immediately

after sacrifice of the animals. Specimens were dehydrated by passing

through ascending grades of alcohol, cleared in xylene impregnated and

embedded in paraffin. Thin sections were cut (3 - 5~M) and were stained

using hematoxylin and eosin and studied microscopically. Liver of animals

in all groups was studied besides kidney and spleen after chronic study.

4.18 Statistical Analysis 291

Data obtained were described as mean ± SEM. One way Analysis of

Variance (ANOVA) was carried out to estimate the variation in the means

between groups. Multiple Analysis (MANOVA) of Variance and their F

ratio were also calculated using the SPSS window version 6.