chapter 4 4.1 hepatoprotective activity 4.1.1...

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CHAPTER 4

4.1 HEPATOPROTECTIVE ACTIVITY

4.1.1 Introduction

Liver is the most important organ of metabolism and excretion of

drugs and food. About 20,000 deaths occur every year due to liver diseases.

Hepatocellular carcinoma is one of the ten most common tumors in the world

with over 2, 50,000 new cases each year (Hikino and Kiso 1988). Although

viruses are the main cause of liver diseases, environmental pollutants,

xenobiotics, hepatotoxins, excessive drug therapy and chronic alcohol

ingestions can also cause severe liver injury (Subrata et al., 1993). Liver injury

induced by chemicals has been recognised as a toxicological problem for more

than 100 years. During the late 1800s, scientists were concerned about the

mechanisms involved in the hepatic deposition of lipids following exposure to

yellow phosphorus. Hepatic lesions produced by arsphenamine, carbon-

tetrachloride and chloroform were also studied in laboratory animals during the

first 40 years of the 20th century. During the same period the correlation

between hepatic cirrhosis and excessive ethanol consumption was recognized.

(Plaa and Charbonneau, 2001).

4.1.2 Experimental model

4.1.2.1 CCl4 induced hepatotoxicity

Carbontetrachloride is a potent hepatotoxin producing

centilobular necrosis which causes liver injury. CCl4 an extensively studied

liver toxicant and its metabolite such as trichloro methyl peroxy radical (•CCl3

O2) is involved in the pathogenesis of liver damage (Al-Shabanah et al., 2000).

The toxicity of CCl4 depends on the cleavage of a carbon-chlorine bond to

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generate a trichloromethyl free radical(•CCl3); this free radical reacts rapidly

with oxygen to form a trichloromethyl peroxy radical (•CCl3 O2), which may

contribute to the toxicity (Cheeseman et al., 1985, Mitchell et al., 1984,

Recknagel et al., 1973 & 1977), demonstrates that the cleavage occurs in the

endoplasmic reticulum and is mediated by the cytochrome P450 mixed function

oxidase system; the product of the cleavage can bind irreversersibly to hepatic

proteins and lipids ; and the CCl4 derived free radical(s) can initiate a process of

autocatalytic lipid peroxidation by attacking the methylene bridges of

unsaturated fatty acid side chains of microsomal lipids.

The peroxidative process initiated by the •CCl3 radical, for

example, is thought to result in early morphological alteration of the

endoplasmic reticulum, loss of activity of the cytochrome P450 xenobiotic

metabolizing system, loss of glucose-6-phosphatase activity, loss of protein

synthesis, loss of the capacity of the liver to form and excrete VLDL and

eventually, through as yet unidentified pathways, to cell death. Dingell and

Heimburg, (1968), Lal et al., (1970), Jaegar et al., (1973) and Anderson et al.,

(1979) have made use of barbiturate sleeping time to assess chemically induced

hepatotoxicity.

Normal cellular metabolism can result in the production of the

reactive oxygen species (superoxide, hydrogen peroxide, singlet oxygen,

hydroxyl radical) and all cells contain defense systems to prevent or limit

damage, glutathione is the major component of this system, but α-tocopherol

and Vitamin C play an important role (Liebler and Reed, 1997). The imbalance

between the prooxidants and antioxidants is known as oxidative stress (Reed,

1998). Toxicants are associated with the induction of lipid peroxidation or

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oxidative stress in liver cells (Comporti, 1998). The CCl4 biotransformed

reactive free radicals promote membrane lipid peroxidation directly.

CCl4 intoxication disrupts the Ca2+ homeostasis within 2 to 4 hrs

and the calcium content of liver, mitochondria is also doubled (Reynolds et al.,

1962). Associated with this abnormal movement of calcium into the liver cells,

there are other disturbances in electrolyte distribution and swelling of liver

cells (Mclean et al., 1965). The lysosomes become disrupted between 5th and

10th hrs (Dianzani, 1963). Intracellular enzymes appear in the plasma due to

leakage (Rees et al., 1962). Focal necrosis is evident as early as 6 hrs after

poisoning and at first is midzonal. By 12 hrs the centrilobular cells exhibit

prenecrotic changes and ballon cells are prominent in the midzonal region. By

24 hrs, there is marked centrilobular necrosis affecting upto half of the lobule

(Recknagel, 1967).

In the present study, CCl4 has been selected, as its immense use

in industry leads to severe exposure to mankind, resulting in acute liver

diseases.

4.1.3 Herbal drugs

In spite of the tremendous advances made in allopathic medicine,

no effective hepatoprotective medicine is available. Herbal drugs are known to

play a vital role in the management of liver disorders. There are numerous

plants and polyherbal formulations claimed to have hepatoprotective activities.

Nearly 150 phyto constituents from 101 plants have been claimed to possess

liver protecting activity (Doreswamy and Sharma, 1995, Handa et al., 1989).

Some of the plants possessing hepatoprotective activities (Subrata et al., 1993)

are Silybum marianum, Withania somnifera, Ocimum sanctum, Tinospora

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cordifolia, Picorrhiza kurroa, Andrographis paniculata, Phyllanthus embelica,

Phyllanthus amarus, Boerhaavia diffusa and Curcuma longa etc (Scott

Treadway, 1998).

4.2 Materials and Methods

4.2.1 Animals

Wistar albino mice of either sex and Wistar albino rats of either

sex were used for the experimental study as given in the section 3.1.2.1

4.2.2 Hepatoprotective evaluation

4.2.2.1 Pentobarbital induced sleeping time in mice

Pentobarbital induced sleeping time in mice model was evaluated

by the method Montilla et al., (1990).

4.2.2.1.1 Experimental protocol

The effect of plant extracts on pentobarbital induced sleeping

time and CCl4 induced prolongation of pentobarbital induced sleeping time was

studied in mice. The animals were divided into seven groups 10 in each group

and received the following regime of treatment.

Group I Animals received 1% carboxy methyl cellulose (10

ml/kg/po) and pentobarbital (45 mg/kg/ip).

Group II Animals received CCl4 mixture (1.5 ml/kg/po in 50%

v/v olive oil) followed by pentobarbital (45 mg/kg/ip).

Group III RCA (500 mg/kg/po) 4 doses of extract at 12 hr

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intervals and CCl4 mixture (1.5 ml/kg /po in 50% v/v

olive oil ) 1 hour post treatment of the last dose of

extract, followed by pentobarbital (45 mg/kg/ip).

Group IV RCM (500 mg/kg/po) 4 doses of extract at 12 hr




Group V CFA (500 mg/kg/po) 4 doses of extract at 12 hr




Group VI CFM (500 mg/kg/po) 4 doses of extract at 12 hr

intervals and CCl4 mixture (1.5 ml/kg/po in 50% v/v



Group VII Silymarin (25 mg/kg/po) 4 doses of extract at 12 hr




After the administration of pentobarbital injection the animals

were placed on their back on a table and sleeping time was noted. The time

between loss of righting reflex and its recovery was taken as duration of

pentobarbitone induced sleeping time.

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4.2.2.2 CCl4 induced liver injury model

CCl4 induced liver injury model was used to evaluate the

hepatoprotective activity by the method of Zhao et al., (2001).

4.2.2.2.1 Experimental protocol

The animals were divided into eleven groups of six animals each

Group I - Control animals received 1% CMC (10 ml/kg/po) for 7

days

Group II - CCl4 treated animal

Group III - RCA (250 mg/kg/po) for 7 days

Group IV - RCA (500 mg/kg/po) for 7 days

Group V - RCM (250 mg/kg/po) for 7 days

Group VI - RCM (500 mg/kg/po) for 7 days

Group VII - CFA (250 mg/kg/po) for 7 days

Group VIII - CFA (500 mg/kg/po) for 7 days

Group IX - CFM (250 mg/kg/po) for 7 days

Group X - CFM (500 mg/kg/po) for 7 days

Group XI - Silymarin (25 mg/kg/po) for 7 days

All the animal in Groups (II – XI) received single dose of equal

mixture of CCl4 and olive oil (50% v/v 5 ml/kg/po) on 7th day except normal

control animals (Group-I). All the animals were sacrificed by cervical

decapitation under light ether anesthesia on the eight day. Blood was collected

from jugular veins and centrifuged (3000 rpm for 10 min) to obtain serum. The

serum was used for marker enzyme estimation. Immediately after sacrifice, the

liver was dissected out, washed in the ice cold saline and the homogenate was

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prepared in 0.1 ml Tris –HCl buffer (pH 7.4).The homogenate was centrifuged

and the supernatant was used for the biochemical analysis. Small pieces of liver

tissue were collected and preserved in 10 % formalin solution for

histopathological studies.

4.2.3 Estimation of Tissue and Serum enzymes

4.2.3.1 Aspartate aminotransferase (EC: 2.6.1.1 AST)

Aspartate aminotransferase was estimated by the method of King

(1965b) under the section 3.1.4.3.7

4.2.3.2 Alanine aminotransferase (EC: 2.6.1.2 ALT)

Alanine aminotransferase was assayed by the method of King

(1965b) under the section 3.1.4.3.8

4.2.3.3 Alkaline phosphatase (EC 3.1.3.1 ALP)

Alkaline phosphatase was assayed by the method of King (1965 a)

under the section 3.1.4.3.9

4.2.3.4 Estimation of total bilirubin

Total bilirubin was estimated by the method of Malloy and

Evelyn (1937) under the section 3.1.4.3.6

4.2.3.5 Estimation of protein

Protein was estimated by the method of Lowry et al., (1951) under

the section 3.1.4.3.5

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4.2.4 Estimation of Enzymic Antioxidants

4.2.4.1 Superoxide dismutase (EC: 1.15.1.1, SOD)

The enzyme was assayed according to the method of Marklund and

Marklund (1974).

Reagents

1. Tris-HCl buffer 0.1 M, pH 8.2: 1.21 g of Tris was dissolved in 90

ml of distilled water, the pH was adjusted to 8.2 with 3 N HCl and

the volume was made up to 100 ml with distilled water.

2. Tris-HCl buffer 0.5 M, pH 7.4: 605 mg of Tris was dissolved in 90

ml of distilled water, the pH was adjusted with 3 N HCl and the

volume was made up to 100 ml with distilled water.

3. Pyrogallol solution 2 mM: 2.52 mg of pyrogallol was dissolved in

10 ml of 0.05 M Tris-HCl buffer in an aluminium foil wrapped

stoppered test tube.

4. Absolute ethanol (AR).

5. Chloroform (AR).

Procedure

To 1 ml of the sample, 0.25 ml of absolute ethanol and 0.15 ml of

chloroform were added. After 15 min of shaking in a mechanical shaker, the

suspension was centrifuged and the supernatant obtained constituted the

enzyme extract. The reaction mixture for autoxidation consisted of 2 ml of the

buffer (Tris-HCl, pH 8.2), 0.5 ml of 2 mM pyrogallol and 1.5 ml water.

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Initially, the rate of autoxidation of pyrogallol was noted at an interval of one

minute for 3 min.

The assay mixture for the enzyme contained 2 ml of 0.1 M

Tris-HCl buffer, 0.5 ml of pyrogallol, aliquots of the enzyme preparation and

water to give a final volume of 4 ml. The rate of inhibition of pyrogallol

autoxidation after the addition of the enzyme was noted.

The enzyme activity is expressed in terms of units/min/mg

protein in which one unit corresponds to the amount of enzyme required to

bring about 50% inhibition of pyrogallol autoxidation.

4.2.4.2 Catalase (EC: 1.11.1.6)

The catalase activity was assayed by the method of Sinha (1972).

Reagents

1. Dichromate–acetic acid reagent: 5% potassium dichromate in water

was mixed with acetic acid in the ratio 1:3 (v/v). The solution was

further diluted to 1:5 with distilled water.

2. Phosphate buffer 0.01 M, pH 7.0 : 173 mg of disodium hydrogen

phosphate and 122 mg of sodium dihydrogen phosphate were

dissolved in 61 ml and 39 ml of distilled water, respectively.

3. Hydrogen peroxide 0.2 M : 2.27 ml of hydrogen peroxide was

made up to 100 ml with distilled water.

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Procedure

0.1 ml of the homogenate was taken to which 1 ml of phosphate

buffer and 0.5 ml of hydrogen peroxide were added. The reaction was arrested

by the addition of 2 ml dichromate-acetic acid reagent. Standard hydrogen

peroxide in the range of 4 to 20 μmoles were taken and treated similarly. The

tubes were heated in a boiling water bath for 10 min. The green colour

developed was read at 570 nm.

Catalase activity in tissue homogenates is expressed as μmoles of

H2O2 consumed /min/mg protein at 37°C.

4.2.4.3 Glutathione peroxidase (EC: 1.11.1.9, GPx)

The activity of glutathione peroxidase was assayed by the method

of Rotruck et al., (1973).

Reagents

1. Sodium phosphate buffer 0.32 M, pH 7: 6.96 g of disodium

hydrogen phosphate and 3.89 g of sodium dihydrogen phosphate

were dissolved in 61 ml and 39 ml distilled water, respectively.

2. Ethylene diamine tetra acetic acid (EDTA) 0.8 mM : 233 mg of

EDTA was dissolved in 100 ml of distilled water.

3. Sodium azide 10 mM : 165 mg of sodium azide was dissolved in

100 ml of distilled water.

4. Reduced glutathione (GSH) 4 mM : 123 mg of GSH was dissolved

in 100 ml of distilled water.

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5. Hydrogen peroxide 2.5 mM: 0.03 ml of hydrogen peroxide solution

was made up to 100 ml with water.

6. 10% TCA

7. Disodium hydrogen phosphate 0.3 M : 5.33 g of disodium

hydrogen phosphate was dissolved in 100 ml of distilled water.

8. 5,5’-dithio bis (2-nitrobenzonic acid) (DTNB) : 40 mg of DTNB

was dissolved in 100 ml of 1% Tri sodium citrate.

9. Glutathione standard: 10 mg of reduced glutathione was dissolved

in 100 ml of distilled water.

Procedure

0.2 ml each of EDTA, sodium azide, glutathione (reduced),

hydrogen peroxide, and 0.4 ml of buffer and 0.1 ml of homogenate were mixed

and incubated at 37°C for 10 min. The reaction was arrested by the addition of

0.5 ml of TCA and the tubes were centrifuged. To 0.5 ml of supernatant, 4 ml

of disodium hydrogen phosphate and 0.5 ml of DTNB were added and the

colour developed was read at 420 nm immediately. Standards were also treated

similarly.

Glutathione peroxidase activity is expressed as μg of glutathione

utilized/minute/mg protein at 37°C.

4.2.4.4 Estimation of glutathione-S-transferases

Glutathione-S-transferase of liver homogenate was assayed

according to the method (Habig et al., 1974).

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Reagents

1. 0.3 mM Phosphate buffer pH 6.5

2. 30 mM 1-Chloro –2,4- dinitrobenzene (CDNB)

3. 30 mM Reduced Glutathione

Procedure

The reaction mixture (3 ml) contained 1.0 ml of 0.3 mM

phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro-2, 4-dinitrobenzene

(CDNB) and 1.7 ml of distilled water. After pre-incubating the reaction

mixture at 37°C for 5 min, the reaction was started by the addition of 0.1 ml of

tissue homogenate and 0.1 ml of glutathione as substrate. The absorbance was

followed for 5 min at 340 nm. Reaction mixture without the enzyme was used

as blank.

The activity of GST is expressed as μ moles of GSH-CDNB

conjugate formed/min/mg protein.

4.2.5 Estimation of Non-Enzymic Antioxidants

4.2.5.1 Reduced glutathione

Reduced glutathione was determined by the method of Moron

et al., (1979).

Reagents

1. Phosphate solution: 5.33 g of disodium hydrogen phosphate was

dissolved in 100 ml of distilled water.

2. 10% TCA

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3. DTNB 0.6 mM: 400 mg of DTNB was dissolved in 100 ml of 1%

trisodium citrate solution.

4. Standard: 10 mg of reduced glutathione was dissolved in 100 ml

of distilled water.

Procedure

One ml of tissue homogenate was precipitated with 1 ml of 10%

TCA. The precipitate was removed by centrifugation. To an aliquot of the

supernatant was added 4 ml of phosphate solution and 0.5 ml of DTNB

reagent. The colour developed was read at 420 nm.

The amount of glutathione in tissue is expressed as μg/mg

protein.

4.2.5.2 Vitamin C (Ascorbic acid)

Ascorbic acid was estimated by the method of Omaye et al.,

(1979).

Reagents

1. 5% TCA

2. 65% H2SO4

3. 2,4 dinitrophenyl hydrazine (DNPH), thiourea, copper sulphate

(CuSO4) reagent (DTC): 3 g DNPH, 0.4 g thiourea and 0.05 g

CuSO4 were dissolved in 9 N H2SO4 and made up to 100 ml.

4. Standard: Standards of ascorbic acid were made in 5% TCA in

the range of 0 to 20 µg/ml.

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Procedure

Aliquots of homogenate were precipitated with 5% ice-cold TCA

and centrifuged for 20 min at 3500 xg. 1ml of the supernatant was mixed with

0.2 ml of DTC and incubated for 3 hr at 37o C. Then 1.5 ml of ice cold 65%

H2SO4 was added, mixed well and the solutions were allowed to stand at room

temperature for an additional 30 min. Absorbance was determined at 520 nm.

Ascorbic acid values were expressed at µg/mg protein.

4.2.5.3 Vitamin E

Vitamin E was estimated by the method of Desai (1984).

Reagents

1. Absolute ethanol: Analytical grade ethanol redistilled in glass

apparatus after adding pellets of KOH and crystals of KMNO4.

2. Hexane: Analytical grade purified by glass distillation.

3. Bathophenanthroline reagent: 0.2% solution of 4,7-diphenyl-1,10

phenanthroline in purified absolute ethanol.

4. Ferric chloride reagent: 0.001 M ferric chloride solution in

purified absolute ethanol. This reagent was prepared fresh and

was kept in amber coloured bottle.

5. Orthophosphoric acid reagent: 0.001 M orthophosphoric acid

solution in purified absolute ethanol.

6. Vitamin E standard: α-tocopherol standards in the range of 1-10

µg/ml of purified absolute ethanol were prepared and treated in

the same manner as test samples.

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Procedure

a. Saponification and extraction

To 500 mg of the tissue, 5 ml of isotonic KCl was added and

homogenised. To 1.5 ml of homogenate, add 1 ml of ethanol, 0.5 ml of 25%

ascorbate and preincubate at 70oC for 5 min in glass-stoppered tubes. To this 1

ml of saturated KOH was added and mixed again. The mixture was further

incubated at 70oC for 30 min. The tubes were immediately cooled in an ice bath

and 1 ml of distilled water and 4 ml of purified hexane were added. The tubes

were shaken vigorously for 2 min and centrifuged at 1500 rpm for 10 min to

separate the phases.

b. Estimation

3 ml aliquots of hexane extract were pipetted out into suitable

reaction tubes and evaporated to dryness under nitrogen. The residue was then

carefully dissolved in 1 ml of purified ethanol. Tubes containing α-tocopherol

standards were treated in the same way as test samples. To all the tubes,

including a reagent blank, 0.2 ml of 0.2% bathophenanthroline reagent was

added and the contents of the tubes were thoroughly mixed. The assay

proceeded very rapidly from this point and care was taken to reduce

unnecessary exposure to direct sunlight. 0.2 ml of ferric chloride reagent was

added and the tubes were mixed by vortexing. After 1 min, 0.2 ml of

orthophosphoric acid was added and the tubes were thoroughly mixed again.

The absorbance was read at 536 nm. Vitamin E value is expressed as mg/g

tissue.

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4.2.6 Estimation of lipid peroxidation

4.2.6.1 Tissue lipid peroxidation

Lipid peroxidation was estimated by the method of Ohkawa et

al., (1979).

Reagents

1. 0.8% TBA

2. 8.5% Sodium dodecyl sulphate

3. 20% Glacial acetic acid

4. Distilled water

Procedure

1.5 ml of TBA, 0.2 ml of sodium dodecyl sulphate, 1.5 ml of

glacial acetic acid were added to test tubes containing 0.1 ml of samples. The

test tubes were heated in water bath for 1hr. The test tubes were then cooled

and 1 ml of distilled water was added. The optical density was determined at

532 nm using a reagent blank. Standard malondialdehyde was also processed in

a similar fashion.

4.3 Results and Discussion

4.3.1 Hepatoprotective activity

4.3.1.1 Pentobarbital induced sleeping time

Figure 1 shows sleeping time of CCl4 treated animals (Group II)

was significantly (P<0.001) increased when compared with control animals

(Group I). The extracts RCA, RCM, CFA and CFM at the dose level of 500

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mg/kg/po, significantly decreased (P<0.05, P<0.001) the sleeping time when

compared with CCl4 treated animals. Silymarin (25 mg/kg/po) treated animals

also showed significant decrease (P<0.001) in sleeping time when compared

with CCl4 treated animals.

Plaa et al.,(1958), demonstrated that prolongation of

pentobarbital sleeping time could be used to quantify the relative hepatotoxicity

of haloalkanes. Pentobarbital sleeping is directly dependent on the ability of the

liver to biotransform the barbiturate. Hepatocellular injury can lead to

decreased activity of hepatic drug metabolizing enzymes and therefore a

prolongation of pentobarbital hypnosis.

This method is used to screen anti - CCl4 toxicity of drugs in

animals (Burger, 1968). The extracts RCA, RCM, CFA, CFM were tested

against pentobarbital induced sleeping time. Hepatotoxic chemicals like CCl4

reduce the levels of drug metabolizing enzymes in liver. Therefore metabolism

of pentobarbitone is reduced resulting in prolongation of pentobarbitone

induced sleeping time. The extracts RCA, RCM, CFA, CFM reduced the CCl4

induced prolongation of sleeping time hence the extracts (RCA, RCM, CFA,

CFM) can be considered hepatoprotective against CCl4 toxicity.

4.3.1.2 CCl4 induced hepatotoxicity

4.3.1.2.1 Tissue and serum enzymes

Effects of RCA, RCM, CFA and CFM at dose levels (250, 500

mg/kg/po) on marker enzymes of serum, total bilirubin, total protein in CCl4

induced hepatotoxicity are shown in (Table 6 & 7). Liver damage induced by

CCl4 caused significant increase in marker enzymes AST, ALT, ALP in serum

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and liver homogenate. Oral administration of the extracts RCA, RCM, CFA,

and CFM significantly decreased the level of marker enzymes AST, ALT, ALP

in both serum and liver. The total bilirubin level was significantly increased in

CCl4 treated animals. The extracts treated animals showed a lower total

bilirubin level in serum. The total protein level in serum and liver was

considerably reduced in CCl4 toxicity. The extracts RCA, RCM, CFA, and

CFM treated animals showed significant increase in the total protein level in

both serum and liver.

Since the changes associated with CCl4 induced liver damage is

similar to that of acute viral hepatitis (Suja et al., 2004). CCl4 induced liver

toxicity was chosen as the experimental model. The ability of the liver

protective drugs to reduce the injurious effects or to preserve the normal

hepatic physiological mechanisms, which have been disturbed by a

hepatotoxin, is the index of its protective effect (Yadav and Dixit, 2003). The

liver damage induced by CCl4 is due to its metabolite •CCl3, a free radical that

alkylates cellular proteins and other macromolecules with a simultaneous

attack on polyunsaturated fatty acids, in the presence of oxygen, to produce

lipid peroxides, leading to liver damage (Bishayee et al., 1995). Hepatocellular

necrosis leads to elevation of the marker enzymes which are released from the

liver into blood (Ashok Shenoy et al., 2002). The increased levels of AST,

ALT, ALP and serum bilirubin are conventional indicators of liver injury

(Achliya et al., 2004). The present study revealed a significant increase in the

marker enzymes like AST, ALT, ALP and serum bilirubin levels,on exposure

to CCl4, indicating considerable hepatocellular injury, administration of RCA,

RCM, CFA, CFM at two different dose levels attenuated the increased levels of

the marker enzymes produced by CCl4 and caused a subsequent recovery

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towards normalization almost like that of standard silymarin treatment. The

decreased total protein level observed in the rats treated with CCl4 may be due

to the decrease in the number of hepatocytes which in turn may result in

decreased hepatic capacity to synthesis protein (Bhandarkhar and Khan, 2004).

On administration of extracts RCA, RCM, CFA, CFM showed significant

increase in total protein level, which indicates the increase in hepatocyte levels,

accounting for its hepatoprotective effect. The results show that the extracts

RCA, RCM, CFA, CFM at different dose levels offer hepatoprotection. But the

extracts (RCA, RCM, CFA, and CFM) at 500 mg/kg/po is more effective than

250 mg/kg/po treated groups.

4.3.1.2.2 Enzymic, non – enzymic antioxidants and tissue lipid

peroxidation

Decreased liver enzymic and nonenzymic antioxidant levels,

and enhanced activity of lipid peroxidation were seen in the CCl4 treated group

(Table 8), whereas the extracts (RCA, RCM, CFA, CFM) and standard drug

silymarin treated groups showed significant increase in antioxidant levels, with

reduction in lipid peroxidation level when compared with CCl4 treated animals.

It has been hypothesized that one of the principal causes of CCl4

induced liver injury is formation of lipid peroxides by free radical derivatives

of CCl4. Thus the antioxidant activity or the inhibition of the generation of free

radicals is important in the protection against CCl4 induced hepatotoxicity

(Castro et al., 1974). The body has an effective mechanism to prevent and

neutralize the free radicals induced damage. This is accomplished by a set of

endogenous antioxidant enzymes such as SOD, Catalase, GST and GPx. These

enzymes constitute a mutually supportive team of defense against ROS

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(Venukumar and Latha, 2002). In CCl4 induced liver toxicity, the balance

between ROS production and antioxidant defenses may be lost and leads to

“oxidative stress”, which deregulates the cellular functions through a series of

events leading to hepatic necrosis. The reduced activities of SOD, Catalase,

GST and GPx observed, pointed out the hepatic damage in the animals treated

with CCl4, but the drug treated animals showed significant increase in the

levels of these enzymes, which indicates the antioxidant activity of the extracts

RCA, RCM, CFA, and CFM.

Regarding non-enzymic antioxidants, GSH is a critical

determinant of tissue susceptibility to oxidative damage and the depletion of

hepatic GSH has been shown to be associated with an enhanced toxicity to

chemicals, including CCl4 (Hewawasam et al., 2003). In the present study, a

decrease in hepatic tissue GSH level was observed in the CCl4 treated groups.

The increase in the hepatic GSH level in rats treated with RCA, RCM, CFA,

CFM may be due to the denovo GSH synthesis or GSH regeneration. There

was also depletion of other nonenzymic antioxidants like Vitamin C and

Vitamin E in the hepatic tissues of CCl4 treated groups, when compared with

the control group. Vitamin C is reported to be associated with better

scavenging activities invivo than the antioxidant enzymes, because they are

present both in intracellular as well as extracellular fluid (Chatterjee and Nandi,

1991). The antioxidant effect of Vitamin E has ability to quench both singlet

oxygen and peroxides (Karthikeyan and Rani, 2003). Within the membrane

tocopherol is the only protective agent that can act against the toxic effects of

oxygen free radicals (Suntress and Shek, 1995). The significant increase in

these nonenzymic antioxidant levels in the hepatic tissues of extracts (RCA,

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RCM, CFA, CFM) treated groups indicates that the antioxidant effect of these

drugs exists both intracellularly and extracellularly.

LPO level is a measure of membrane damage and alterations in

structure and function of cellular membrane (Ilavarasan et al., 2003). In the

present study, elevation of lipid peroxidation level was observed in rats treated

with CCl4. Increase in MDA levels in liver suggests enhanced lipid

peroxidation leading to tissue damage and failure of antioxidant defense

mechanisms to prevent formation of excessive free radicals (Ashok Shenoy et

al., 2001). Administration of RCA, RCM, CFA, and CFM significantly

reversed these changes. Hence it is possible that the mechanisms of

hepatoprotection of RCA, RCM, CFA, and CFM may be due to its antioxidant

action. The results shows that RCA, RCM, CFA, CFM at the dose level of 500

mg/kg/po produced greater activity which is close to the values with standard

drug silymarin.

4.3.1.2.3 Histopathology of liver tissue (Plate 8)

Histopathology of the Group II (CCl4 treated) showed perivenular

inflammatory infiltration and hepatocytic fatty changes, diffused mild

hepatocellular vacuolation, where as extracts (RCA, RCM, CFA, CFM) treated

groups showed absence of cell necrosis, but minimal perivenular inflammation

and occasional hepatocytic fatty change. The extracts treated groups (RCA,

RCM, CFA, CFM) at 500 mg/kg/po dose level showed minimal inflammatory

conditions with near normal liver architecture possessing greater

hepatoprotective action.

From the above results, it can be concluded that the extracts

RCA, RCM, CFA and CFM at the dose level of 500 mg/kg/po, showed

significant protection against CCl4 induced liver injury in rats.

chapter 4 4.1 hepatoprotective activity 4.1.1...

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