PROTECTIVE EFFICACY OF THE EXTRACT OF VOLVARIELLA VOLVACEA (BULLIARD EX FRIES) SINGER. AGAINST CARBONTETRACHLORIDE INDUCED HEPATIC INJURY

PROTECTIVE EFFICACY OF THE EXTRACT OF VOLVARIELLA VOLVACEA (BULLIARD EX FRIES) SINGER. AGAINST CARBONTETRACHLORIDE INDUCED HEPATIC INJURY

S.V. Kalava* and S.G. Menon

Department of Biochemistry, Kongunadu Arts and Science College, Coimbatore-641029, Tamil Nadu, India

ABSTRACT

Carbon tetrachloride is a xenobiotic that produces hepatotoxicity. Aqueous extract Volvariella volvacea (500, 1000 mg/kg, p.o) showed significant hepatoprotective activity against carbontetrachloride induced hepatotoxicity in rats by normalizing the levels of serum AST, ALT, ALP, LDH and total bilirubin. The extract improved the activity of Catalase (CAT), Superoxide dismutase (SOD), and hepatic glutathione (GSH) content and depleted the lipid peroxidation levels in a dose dependent manner. Silymarin was used as the standard drug.

Hepatoprotective

INTRODUCTION: Liver, the largest organ in the vertebrate body, is the major site of intense metabolic activities. Liver injury induced by chemicals and drugs is a well-recognized toxicological problem. The hepatotoxicity possibly results from toxic intermediates that bind covalently to the hepatocytes, causing a centrilobular hepatic necrosis. Liver diseases are a serious health problem. In the absence of reliable liver-protective drugs in allopathic medical practices, herbs play a role in the management of various liver disorders ¹.

Carbon tetrachloride (CCl₄) is a xenobiotic producing hepatotoxicity in human beings and animals ². In fact, it has been shown that the trichloromethyl radical, formed in the metabolism of CCl₄ via the liver microsomal cytochrome P450 system, reacts rapidly with molecular oxygen to produce trichloromethyl peroxy radical. These radicals react with unsaturated fatty acids of phospholipids present in cell membranes, initiating lipid peroxidation (LPO) in liver cells ³. This leads to the formation of lipid peroxides, and a depression of protein synthesis ⁴ and elevated levels of serum marker enzymes such as AST, ALT and ALP⁵. The antioxidant activity or the inhibition of the generation of free radicals is important in the protection against CCl₄ induced hepatopathy ⁶.

CCl₄-induced liver injury is therefore considered as a model for the screening of hepatoprotective drugs ⁷.A number of investigators have previously demonstrated that antioxidants would prevent CCl₄ toxicity by inhibiting lipid peroxidation ^{8, 9}.

Many natural products of herbal origin are in use for the treatment of liver aliments.  Phytochemicals derived from plants are excellent antioxidants. Antioxidants appear to act against diseases by raising the levels of endogenous defense, by up-regulating gene expressions of the antioxidant enzymes ^{10, 11}.

Edible mushrooms are nutritionally endowed fungi. Mushrooms accumulate a variety of qualitatively good protein, crude fiber, minerals and vitamins but are poor sources of lipids and are rich in secondary metabolites ¹².

Various mushrooms viz. Ganoderma lucidum ^{13, 14}, P. ostreatus ¹⁵Antrodia camphorate ¹⁶,Phellinus linteus ¹⁷and Phellinus rimosus ¹⁸ have found to possess protective effects against liver diseases. Volvariella volvacea (Bulliard Ex Fries) Singer. is generally known as rice straw/ paddy straw  mushroom, is cultivated throughout East and Southeast Asia and used extensively in Asian cuisines The mushroom is found to possess antibacterial activity ^{19, 20}.

The present study was undertaken to evaluate the hepatoprotective potential of the aqueous extract of Volvariella volvacea (Bulliard ex Fries)Singer. against CCl₄-induced liver injury.

MATERIALS AND METHODS:

Preparation of the sample: Mushroom was obtained from Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. The fruiting bodies were shade dried and powdered. 10g of the powder was extracted with 100 ml of water at 100^oC for 4 hours, centrifuged at 5000rpm for 15 minutes and filtered through Whatman No. 1 filter paper. The residue was extracted twice with 100ml portions of water, as described above. The extracts were combined and vacuum evaporated. The extract obtained after vacuum evaporation was freeze dried and stored at 4⁰C until further use.

Drugs and chemicals:            Silymarin was obtained from Himedia, Bangalore, India. All other chemicals used in this study were obtained commercially and were of analytical grade.

Experimental Animals: Female Sprague Dawley rats, weighing, 160g-180g were purchased from, Small Animal Breeding Centre, College of Veterinary and Animal Science, Mannuthy, Kerala, India. The animals were maintained under standard conditions of humidity, temperature (25 ± 2ºC) and light (12 h light/dark). They were acclimatized to animal house conditions and were fed on a commercial pelleted rat chow (AVM Cattle Feeds, Coimbatore, Tamil Nadu) and water ad libitum. Experimental animals were handled according to the University and Institutional Legislation, regulated by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Experimental Design: The animals were divided into 5 groups of six animals each. Hepatotoxicity was induced by intraperitoneal injection of CCl₄   in paraffin oil ²¹.

1. Group I: Rats in this group served as a control.
2. Group II: Hepatotoxicity was induced with CCl₄ in paraffin oil (1:5, v/v) (1.5 ml/kg body weight, i.p) 3-times in a week for 5 weeks (total 15 doses)
3. Group III: Treated with 500mg/kg body weight/ day of aqueous extract of V. volvacea    (VVAE) orally, to the animals one hour prior to each CCl₄ injection.
4. Group IV: Treated with 1000mg/kg body weight/day of aqueous extract of V. volvacea (VVAE) orally, to the animals   one hour prior to each CCl₄ injection.
5. Group V: Treated with 25mg/kg body weight of silymarin administered orally, to the animals   one hour prior to each CCl₄ injection.

Biochemical Analysis: At the end of the last injection, the animals were subjected to fasting for a period of 12 hours. At the end of 12 hours fasting the animals were sacrificed, blood was collected and the liver, were excised and washed in saline. 10% homogenate of the liver tissues was prepared with 0.1 M Tri-HCl buffer, pH 7.4. Serum was prepared from whole blood. The homogenates were centrifuged at 3000 rpm for 15 min at 4^oC for cytosolic separation.

The levels of serum bilirubin were determined based on the method of   by the method of Malloy and Evelyn ²².  The activity of Aspartate Transaminase (AST) and Alanine Transaminase (ALT) by the method of Reitman and Frankel ²³ Alkaline phosphatase was determined by King and Armstrong ²⁴ and Lactate dehydrogenase by King ²⁵. The enzymatic activity of hepatic superoxide dismutase (SOD) was assessed according to the method of Das et al ²⁶ and Catalase (CAT) by the method of Sinha ²⁷, Glutathione (GSH) content of hepatic tissues were assessed using Ellman’s reagent according to the method described by Ellman ²⁸. Protein levels were determined   as described by Lowry ²⁹. Rat liver homogenate lipid peroxide (LPO) levels were determined by measuring MDA content according to the method of Niehus and Samuelsson ³⁰.

Histopathological Examination: The hepatic tissue of each animal were dissected out then fixed in buffered formalin for 12 hours and processed for histopathological examination. Four μm thick paraffin sections were stained with hematoxylin and eosin for light microscope examination using conventional protocol.

Statistical Analysis: The data are expressed as mean ± S.D. Statistical comparison was done at significance level, P<0.05 using SPSS package version 10.0. One way ANOVA followed by post hoc analysis of LSD was performed.

RESULTS: Table 1   represents the levels of   bilirubin and the activity of the liver marker enzymes- AST, ALT, ALP and LDH   in the serum of the experimental animals. There was observed a significant (p<0.05) raise in the activity of the   enzymes and levels of bilirubin in the CCl₄ intoxicated rats. The treatment with VVAE to the animals of group III and IV at a dose of 500mg/kg b wt and 1000 mg/kg b wt, respectively, resulted in a significant (p<0.05) decrease in the activity of the enzymes and serum bilirubin levels in a dose dependent manner. Silymarin administration to the group V animals, also resulted in a marked reduction (p<0.05) in   the serum bilirubin levels and the activity of the enzymes.

TABLE 1  EFFECTS OF AQUEOUS EXTRACT OF VOLVARIELLA VOLVACEA ON   SERUM BILIRUBIN, PROTEIN AND THE ACTIVITIES OF MARKER ENZYMES

Group I- Control;  Group II- CCl₄   (1.5 ml/kg b wt); Group III- VVAE (500mg/kg b wt) + CCl₄; Group IV- VVAE (1000mg/kg.b wt) + CCl₄; Group V- Silymarin (25mg/kg b wt) + CCl₄

Values are expressed as mean ± SD for six animals. Group comparison and statistical significance at p<0.05: ^a: Group I vs. II, III, IV, V     ^b: Group II vs. I, III, IV, V

The effect of VVAE on the activity of   the antioxidant enzymes (SOD and CAT), hepatic GSH content and lipid peroxidation levels are presented in Table 2. A significant reduction in the activity of SOD, CAT and GSH were observed in the group II animals that served as   CCl₄   control animals. Lipid peroxidation levels as MDA content was observed to be markedly (p<0.05) elevated in the group II animals. Treatment with VVAE at 500mg/kg b wt and 1000 mg/kg b wt to the animals of group III and IV respectively resulted in a marked improvement in the activity of SOD and CAT and a  significant (p<0.05) raise in the levels of  GSH and decline in MDA content. Group V animals that were supplemented with the standard, silymarin effectively   normalized (p<0.05) the activity of the enzymes- SOD and CAT, increased the hepatic GSH content and depressed the MDA levels.

TABLE 2: EFFECTS OF AQUEOUS EXTRACT OF VOLVARIELLA VOLVACEA ON THE ACTIVITY OF SOD, CAT AND THE LEVELS OF MDA AND GSH IN LIVER

Group I- Control;  Group II- CCl₄ (1.5 ml/kgb.w); Group III- VVAE (500mg/kg. b. wt) + CCl₄; Group IV- VVAE (1000mg/kgb.wt) + CCl₄; Group V- Silymarin (25 mg/kg b.wt) + CCl₄

Values are expressed as mean ± SD for six animals. Group comparison and statistical significance at p<0.05:   ^a: Group I vs. II, III, IV, V     ^b: Group II vs. I, III, IV, V

The results of the histopathological analysis of the liver tissue of the experimental animals are presented in figure 1 (a-e). Figure 1a represents the liver sectioning of the group I animals. Tissue presents normal architecture of the hepatocytes and the central vein. Figure 1b presents the hepatic tissue of the group II CCl₄ control animals. The sectioning depicts severe hemorrhage and necrosis of the hepatocytes. Figure 1c- The hepatic sectioning of the group III animals treated with 500mg/kg b.wt VVAE.

The tissue presents mild inflammation. Figure 1d- The slide represents the liver section of the animals treated with 1000mg/kg b.wt VVAE. The sectioning reveals negligible inflammation and near normal architecture. Figure 1e- The hepatic sectioning of the standard, silymarin treated group V animals. The section presents near normal architecture of the hepatocytes.

FIGURE 1A:  GROUP I   (CONTROL)

FIGURE 1B:  GROUP II   (CCl₄)

FIGURE 1C: GROUP III

(500mg/kg b. wt VVAE+ CCl₄)

FIGURE 1D: GROUP IV

(1000mg/ kg b.wt VVAE+ CCl₄)

FIGURE 1E: GROUP IV

(Silymarin 25mg/kg b.wt + CCl₄)

DISCUSSION: Serum bilirubin levels are indicators of hepatic damage. It is well known that necrotizing agents like CCl₄ produce sufficient injury to hepatic parenchyma to cause large increases in bilirubin content.

In the liver it has been shown that toxicity of CCl₄ is mediated by the Cyt P450-dependent mixed oxidase-mediated biotransformation product, trichloromethyl free radical (CCl₃) and subsequent derivative Cl₃COO ³¹. These free radical combines with the cellular lipid and proteins to produce lipid peroxidation, measured through its catabolite, malondyaldehyde (MDA), resulting in structural changes of endoplasmic reticulum and other biomembranes and loss of metabolic activity leading to liver damage ³².

A high concentration of bilirubin in serum is an indication for increased erythrocyte degeneration rate ³³. Due to the liver injury caused by the hepatotoxin, there is a defective excretion of bile by the liver which is reflected in their increased levels in serum ³⁴. The raised levels of bilirubin in the serum of the group II animals that served as CCl₄ control group, suggest the hepatocellular injury caused by CCl₄. Prior treatment with VVAE was observed to prevent severity of liver damage caused by CCl₄ as evidenced by the low level of bilirubin in the serum in a dose dependent manner.

Silymarin also reduced the serum bilirubin levels, thereby protecting the hepatocytes.

The results obtained were found to be in coordination with Shanmugasundaram and Venkataraman ³⁵. Hippophae rhanmoides L was found to decrease the raised bilirubin levels after induction with CCl₄ ³⁶   and Aerva lanata was exploited for its hepatoprotective activity also reduces the serum bilirubin levels upon CCl₄ induction ³⁷.

Liver injury induced by is the best-characterized system of the xenobiotic-induced hepatotoxicity and is a commonly used model for the screening the hepatoprotective activity of drugs ^{31, 38, 39}. Serum AST, ALT, ALP and bilirubin are the most sensitive markers employed in the diagnosis of hepatic damage, because these are cytoplasmic in location and are released into the circulation after cellular damage ⁴⁰.

In liver injury, the transport function of the hepatocytes is disturbed, resulting in the leakage of plasma membrane, thereby causing an increased enzyme level in serum, and soluble enzymes like AST will also be similarly released. The elevated activities of AST and ALT in serum are indicative of cellular leakage and loss of functional integrity of cell membranes in liver ^{3, 41}.

Thus, the increase in the serum levels of AST, ALT, ALP and LDH suggest the liver injury due to toxic insult of CCl₄. The treatment with VVAE was found reduce the serum activities of the enzymes in a dose dependent manner. The results suggest the capacity of the extract in counteracting against the damage caused by CCl₄.

The above results are similar to the earlier study reported on the effect of Ganoderma lucidum in CCl₄ induced hepatotoxic rats ¹⁴ and the effect of Aerva lanata on CCl₄ induction ³⁷.

Our findings also correlate with the study of Ajith and Janarthanan ⁴³ in Phellinus rimosus (Berk) and with the investigations of Zhou et al., ⁴⁴ BJ-JN, a Chinese formulation on CCl₄ induction.  The results are in agreement with those obtained by Jadon et al., ⁴⁵ who showed that gallic acid, at 50 mg/kg body weight, could decrease plasma AST and ALT activities elevated by acute hepatic damage.

This evidenced that the administration of VVAE and silymarin showed hepatoprotective effect under CCl₄-induced oxidative stress.

Samudram et al., ⁴⁶ investigated the effect of Bi-herbal formulation on CCl₄ induced hepatic damagein rats. The formulation decreased the elevated serum levels of the marker enzymes ALP, ACP, AST, ALT, LDH, 5’NT and γ-GT in the toxic control group animals.

Living tissues are endowed with innate antioxidant defence mechanisms, such as the presence of the enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx). A reduction in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes ^{47, 48}.

Administration of CCl₄ leads to generation of peroxy radical, O^2_; which is associated with inactivation of CAT and SOD enzymes. This probably explains the significantly reduced activities of CAT, SOD and Gpx observed in rats challenged with CCl₄ (group II). In rats receiving CCl₄ and VVAE (group III and IV ) the activities of CAT and  SOD were significantly higher than in Group II rats, and very similar to the values noted in normal (group I rats). This suggests a hepatoprotective effect by VVAE extract, which is a very encouraging finding. The extract possibly confers this protective effect by dampening the generation of free radicals that is induced by CCl₄.

Ohta et al., ⁴⁹ suggested that the reduced activities of these enzymes might reflect a feed-back inhibition or oxidative inactivation of protein caused by excess generation of ROS. So also, in the present study, significantly lower activities of these enzymes were noted in rats that had received CCl₄, when compared to the levels in normal rats.

GSH is the major non-enzymatic antioxidant and regulator of intracellular redox homeostasis, ubiquitously present in all cell types ⁵⁰. Studies with a number of models show that the hepatotoxicity of xenobiotics often is produced by GSH depletion ^{51, 52}. Significant increase in the hepatic GSH content suggests the protective role of VVAE.

Treatment of Z. mauritiana extract was found to modulate and increase the levels of vitamin E and glutathione in the CCl₄ induced rats ⁵³.

Grape seed extract was found to normalize the enzyme activities ⁵⁴. Yang et al., ⁵⁵ reported that Ganoderma lucidum when administered at a dose of 500mg exhibited hepatoprotective activity in rats with CCl₄ induced toxicity which is in agreement with our study. A significant restoration of antioxidant enzyme activities by treatment with Phellinus rimosus (Berk) Pilat ⁴³, A. camphorate ¹⁶and P. Ostreatus ¹⁴ in CCl₄ intoxicated animals also supports our study.  Hsu et al. ⁵⁷ reported that D.salina increased the activity of the antioxidant enzymes in CCl₄- induced hepatic damge in rats.

Lipid peroxidation has been implicated in the pathogenesis of increased membrane rigidity, osmotic fragility, reduced erythrocyte survival and perturbations in lipid fluidity. It has been hypothesized that one of the principal causes of CCl₄- induced hepatotoxicity is lipid peroxidation of hepatocyte membranes by free radical derivatives of CCl₄ ^{3, 58}.

The observation of elevated levels of hepatic MDA in Group II rats (administered CCl₄ alone) in the present study is consistent with this hypothesis. Thus, the maintenance of near normal levels of hepatic MDA in Group III and IV rats (administered with mushroom extracts) is of great interest since it provides additional evidence to suggest a hepatoprotective role for Volvariella volvacea extract.

The results of our investigations are in accordance with that of Hu et al., ⁴² who have reported the effect of Ganoderma lucidum against CCl₄ induced hepatotoxicity and Hwang et al., ⁵⁹ who have reported the activity of A.continentalis against CCl₄ induced hepatic damage.

Histoarchitectural improvement on treatment with VVAE suggests the protective effects of the extract in a dose dependent way against CCl₄ induced hepatic injury. Reduction in serum bilirubin and marker enzymes (AST, ALT, ALP, and LDH), and augmentation of endogenous antioxidants and suppression of MDA content supports the hepatoprotective and antioxidant activity.

This protective efficiency of VVAE may be due to its potent antioxidant activity/or by scavenging free radicals.

CONCLUSION: The results observed thus suggest the mushroom extract at both doses (500mg/kg b wt and 1000mg/kg b.wt) effectively ameliorated the toxic effect of CCl₄ in a dose dependent manner.

ACKNOWLEDGEMENT: The authors are thankful to the Management of Kongunadu Arts and Science College, Coimbatore, Tamilnadu, India.

REFERENCES:

1. Sethuraman MG, Lalitha KG, Rajkapoor B: Hepatoprotective activity of Sarcostemma brevistigma against carbon tetrachloride induced hepatic damage in rats. Current Science 2003; 84: 1186-1187.
2. Rudnicki M , Silveira, MM,  Pereira TV, Oliveira MR , Reginatto FH , Dal-Pizzol F, Moreira JCF: Protective effects of Passiflora alata extract pretreatment on carbon tetrachloride induced oxidative damage in rats, Food and  Chemical Toxicology 2007; 45: 656–661.
3. Recknagel RO, Glende Jr EA, Dolak JA. Waller RL:  Mechanisms of carbon tetrachloride toxicity. Pharmacology and Therapeutics 1989; 43: 139–154.
4. Faroon O, De Rosa CT, Smith L: Carbon tetrachloride: Health effects toxicokinetics, human exposure and environmental fate. Toxicology and  Industrial  Health 1994; 10: 04 –20
5. Zimmerman HJ and Seef LB: The functions and tests of liver. In: Diagnostic Enzymology, Philadelphia, Pergamon Press 1970; 1–4.
6. Castro JA , Ferrya GC, Castro CR , Sasame H, Fenos OM , Gillette JR. Prevention of Carbon tetrachloride-induced necrosis by inhibitors of drug metabolism. Further studies on the mechanism of their action. Biochemistry and   Pharmacology 1974; 23: 295-302.
7. Janbaz KH and Gilani AUH:  Evaluation of the protective potential of Artemisia maritima extract on acetaminophen- and CCl4- induced liver damage. Journal of Ethnopharmacology 1995; 47: 43–47.
8. Teselkin YO, Babenkova IV, Kolhir VK,  Baginskaya AI, Tjukavkina NA ,  Kolesnik, YA, Selivanova IA,  Eichholz AA: Dihydroquercetin as a means of antioxidative defence in rats with tetrachloromethane hepatitis. Phytotherapy Research2000; 14: 160–162.
9. Bansal  AK, Bansal M, Soni G, Bhatnagar D: N-nitrosodiethylamine induced oxidative stress in rat liver. Chemico Biological Interactions   2005; 156: 101–111.
10. Aruoma OI: Nutrition and health aspects of free radicals and antioxidants. Food Chemistry and   Toxicology 1994; 32: 671–683.
11. Kumaravelu P, Dakshinamoorthy DP, Subramaniam S. Devaraj H, Devaraj NS: Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver. Biochemistry and   Pharmacology 1995; 49: 1703–1707.
12. Frankel EN, Kanner J, German JB, Parks E,  Kinsella JE: Inhibition of human low density lipoprotein by phenolic substances in red wine. Lancet, 1993; 41: 454-457.
13. Yang XJ, Iu J, Ye LB, Yang F, Ye L, Gao JR, Wu ZH.: In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachloride induced liver injury. World Journal of   Gastroenterology2006; 12: 1379-1385.
14. He H, He JP, Sui YJ, Zhou SQ, Wang J: The hepatoprotective effects of Ganoderma lucidum peptides against carbon tetrachloride induced liver injury in mice Journal of  Food Biochemistry  2008; 32: 628-641.
15. Jayakumar T, Ramesh E , Geraldine P : Antioxidant activity of the oyster mushroom, Pleurotus ostreatus, on CCl4-induced liver injury in rats, Food Chemistry and  Toxicology 2006; 44: 1989–1996.
16. Hsiao G, Shen MY, Lin KH, Ian MH, Wu LU, Chou DS, Lin C.H, Su CH, Sheu JR: Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J. Agric. Food Chemistry 2003; 51: 3302-3308.
17. Jeon T, Hwang G, Lim BO, Park DK: Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats.Biotechnology Letters 2003;25: 2093–2096.
18. Ajith TA, Jose N, Janardhanan KK: Amelioration of cisplatin induced nephrotoxicity in mice by ethyl acetate extract of a polypore fungus, Phellinus rimosus. Journal of Experimental Clinical Cancer Research 2002; 21: 487–491.
19. Chihara G, Maeda Y, Hamuro J, Sasaki T, Fukuoka F: Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) Sing. Nature   1969; 222: 687–688.
20. Nanba H and   Kuroda H: Antitumor mechanisms of orally administered shiitake fruit bodies. Chemical and Pharmacological Bulletin 1987; 35: 2459–2464.
21. Ajith, TA, Sheena N, Janardhanan KK: Phellinus rimosus protects carbon tetrachloride induced chronic hepatotoxicity in rats Antioxidant defense mechanism. Pharma Biol 2006; 44: 467-474.
22. Malloy HT and Evelyn KA: The determination of bilirubin with the photoelectric colorimeter. Journal of   Biological Chemistry 1937; 119: 481-90.
23. Reitman S and   Frankel AS: A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. American Journal of   Clinical Pathology 1957; 28: 53-56.
24.  King EJ and Armstrong AR: A convenient method for determining serum and bile phosphatase activity. Canadian Medical Association Journal   1934; 31: 376- 9.
25. King J: The hydrolases-acid and alkaline phosphatases. In: Van, D. (Ed.), Pract. Clin. Enzymol. Norstand Co. Ltd, London 1965; 191-208.
26. Das S, Vasight S, Snehlata R, Das N, Srivastava LM: Correlation between total antioxidant status and lipid peroxidation in hypercholesterolemia. Current Science   2000; 78: 486-487.
27. Sinha KA: Colorimetric assay of catalase. Analytical biochemistry 1987; 47:389-394.
28. Ellman GL: Tissue sulfahydryl groups. Archives of Biochemistry and Biophysics 1959; 82:70-77.
29. Lowry OH, Roseobrough NJ, Farr AL, Randall RJ: Protein measurement with the folin’s phenol reagent. Journal of Biological Chemistry 1957; 193: 265-275.
30. Niehius WG and   Samuelsson D: Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation. European Journal of   Biochemistry 1968; 6: 126-130.
31. Brent JA   and Rumack BH: Role of free radicals in toxic hepatic injury. II. Are free radicals the cause of toxin-induced liver injury? Journal of Toxicology - Clinical Toxicology 1993; 31: 173–196.
32. Kadiska MB, Gladen BC, Baird DD, Dikalov AE, Sohal RS, Hatch GB, Jones DP, Mason RP, Barret, JC: Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl₄ poisoning? Journal of Free Radical Biology and Medicine 2000; 28: 838– 845.
33. Singh B, Saxena AK, Chandan BK, Anand KK, Suri OP, SuriSatti  KA, SuriSatti NK: Hepatoprotective activity of verbenalin on experimental liver damage in rodents. Fitoterapia 1998; 69: 134–140.
34. Rao RR: Mechanism of drug induced hepatotoxicity. Indian Journal of Pharmacology 1973; 5: 313–318.
35. Shanmugasundaram P and   Venkataraman S: Hepatoprotective and antioxidant effects of Hygrophila auriculata (K. Schum) Heine Acanthaceae root extract. Journal of Ethnopharmacology 2006; 104:124–128.
36. Suryakumar G, Purushothaman J, Pal K, Pandey S, Kumar R and Sawhney RC. Hepatoprotective effects of sea buckthorn (Hippophae rhamnoides L.) against carbon tetrachloride induced liver injury in rats. Journal of   Food Science and   Agriculture 2008; 88:1592-1597.
37. Nevin KG and Vijayammal PL: Effect of Aerva lanata against hepatotoxicity of carbon tetrachloride in rats.  Environmental Toxicology and Pharmacology 2005; 20:  471–477.
38. Brautbar N and Williams IInd, J: Industrial solvents and liver toxicity: risk assessment, risk factors and mechanisms. International   Journal of Hygiene and Environmental Health 2002; 205: 479–491.
39. Manibusan MK, Odin M, Eastmond DA:  Postulated carbon tetrachloride mode of action: a review. Journal of Environmental Science and Health- Part C Environmental Carcinogenesis and Ecotoxicology. Reviews 2007; 25: 185–209.
40. Sallie   R, Tredger JM, Willam R: Drugs and the liver. Biopharmaceutics and Drug Disposition 1991; 1: 251-259.
41. Rajesh MG and Latha MS: Protective effect of Glycyrrhiza glabra Linn. on carbon tetrachloride-induced peroxidative damage. Indian Journal of   Pharmacology   2004; 36: 284–286.
42. Hu CC, Lin JT, Lu FJ, Chou FP, Yang DJ:  Determination of carotenoids in Dunaliella salina cultivated in Taiwan and antioxidant capacity of the algal carotenoid extract. Food Chemistry 2008; 109: 439-446.
43. Ajith TA and Janardhanan KK: Antioxidant and antihepatotoxic activities of Phellinus rimosus (Berk) Pilat. Journal of Ethnopharmacolgy 2002; 81: 387-391.
44. Zhou YH, Yang Y, Li J , Wuc Q,  Li WP, Lua JT,  Roberts MS: Potential therapeutic effects of a traditional Chinese formulation, BJ-JN, on liver fibrosis induced by carbon tetrachloride in rats. Journal of Ethnopharmacology 2008; 120: 452–457.
45. Jadon A, Bhadauria M, Shukla, S: Protective effect of Terminalia belerica Roxb. and gallic acid against carbon tetrachloride induced damage in albino rats. Journal of Ethnopharmacology 2007; 109: 214–218.
46. Samudram P, Rajeshwari H, Vasuki R, Geetha A, Sathiya moorthi P: Hepatoprotective activity of Bi - herbal ethanolic extract on CCl4 induced hepatic damage in rats. African Journal of   Biochemistry and Research 2008; 2 (2): 61-65.
47. Krishnakantha TP and   Lokesh BR: Scavenging of super oxide anions by spice principles. Indian   Journal of Experimental Biology 1993; 30: 133–4.
48. Sheela CG and Angusti, K: Antiperoxide effects of S-allyl cysteine sulphoxide isolated from Allium sativum Linn and gugulipid in chleosterol diet fed rats. Indian   Journal of Experimental Biology1995; 33: 337–41.
49. Ohta Y, Kongo-Nishimura M, Matsura T, Yamada K,Kitagawa A, Kishikawa T: Melatonin prevents disruption of hepatic reactive oxygen speciesmetabolism in rats treatedwith carbon tetra chloride. Journal of   Pineal Research   2004; 36: 10–17.
50. Meister A and Anderson ME. 1983. Glutathione. Annual Review of Biochemistry; 52: 711–60.
51. Jollow DJ, Thogeirsson SS, Potter WZ, Hashimoto M, Mitchell JR: Acetaminophen-induced hepatic necrosis. IV Metabolic disposition of toxic and non-toxic doses of acetaminophen. Pharmacology 1974; 12: 251–271.
52. Rana S and Tyal MK: Influence of zinc, vit-B12 and glutathione on the liver of rats exposed to carbon tetrachloride. Industrial Health (Japan) 1981; 19: 65–69.
53. Dahiru   D, William   ET, Nadro MS: Protective effect of Ziziphus mauritiana leaf extract on carbon tetrachloride-induced liver injury. African   Journal of   Biotechnology 2005; 4 (10): 1177-1179.
54. Balu M, Sangeetha P, Haripriya D, Panneerselvam C: Rejuvenation of antioxidant system in central nervous system of aged rats by grape seed extract, Neuroscience Letters 2005; 383: 295–300.
55. Yang XJ, Iu, J, Ye, LB, Yang F, Ye L, Gao JR, Wu ZH: In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachloride induced liver injury. World Journal of   Gastroenterology 2006; 12: 1379-1385.
56. Chang WT, Shao ZH, Yin JJ, Mehendale S, Wang CZ, Qin Y, Li J, Chen WJ, Chien CT, Becker LB, Vanden Hoek, TL, Yuan CS: Comparative effects of flavonoids on oxidant scavenging and ischemia reperfusion injury in cardiomyocytes. European Journal of Pharmacology 2007; 566: 58-66.
57. Hsu YW, Tsai CF, Chang WH., Ho YC, Chen WK  Lu FL.Protective effects of Dunaliella salina – A carotenoids-rich alga, against carbon tetrachloride-induced hepatotoxicity in mice Food and Chemical Toxicology 2008;  46: 3311–3317.
58. Recknagel RO, Glende EA, Britton RS:  Free radical damage and lipid Peroxidation, in: R.G. Meeks (Ed.). Hepatotoxicology, CRC Press, Boca Raton, FL 1991; 401–436.
59. Hwang YP, Choi JH, Jeong GH: Protective effect of the Aralia continentalis root extract against carbontetrachloride-induced hepatotoxicity in mice. Food and Chemical Toxicology 2009; 47: 75–81.
    

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    How to cite this article:

    Kalava SV and Menon SG: Protective efficacy of the extract of Volvariella volvacea (Bulliard Ex Fries) Singer. against carbontetrachloride induced hepatic injury.Int J Pharm Sci Res 2012; Vol. 3(8): 2849-2856.

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