THE MODULATING EFFECT OF GARCINIA CAMBOGIA EXTRACT ON ETHANOL INDUCED PEROXIDATIVE DAMAGE IN RATS
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1 Indian Journal of Pharmacology 2001; 33: SHORT COMMUNICATION GARCINIA CAMBOGIA AND ETHANOL INDUCED PEROXIDATION THE MODULATING EFFECT OF GARCINIA CAMBOGIA EXTRACT ON ETHANOL INDUCED PEROXIDATIVE DAMAGE IN RATS P. MAHENDRAN, C.S. SHYAMALA DEVI Department of Biochemistry & Molecular Biology, University of Madras, Guindy Campus, Chennai Manuscript Received: Revised: Accepted: SUMMARY Objective: To determine the modulating effect of Garcinia cambogia fruit extract on ethanol induced peroxidative damage in rats. Method: Male albino rats weighing 125 to 150g were administered ethanol (7.11g per kg body weight / day) for 45 days. Ethanol administered rats were treated concomitantly with Garcinia cambogia fruit extract (1g/kg body weight / day) orally for 45 days. After the experimental period the antioxidant enzymes, LPO, conjugated diene in the liver tissue, serum AST, ALT and alkaline phosphatase and lipid levels in both serum and liver tissue were estimated. Results: Co-treatment of the rats with Garcinia cambogia significantly inhibited the rise in lipid levels and also the peroxidative damage caused by ethanol, which is evident from the improved antioxidant status. The levels of serum AST, ALT and alkaline phosphatase were maintained at near normalcy in Garcinia cambogia treated rats. Conclusion: The imbalance in lipid metabolism could be the reason for increase in lipid peroxidation. In our present study the treatment with Garcinia cambogia fruit extract resulted in reduction of both serum and liver lipid to near normalcy. This hypolipidemic property of Garcinia cambogia in turn reduces the peroxidative damage, enhanced by ethanol. KEY WORDS Ethanol Garcinia cambogia lipid peroxidation hyperlipidemia antioxidant enzymes INTRODUCTION Liver being the major site for detoxification is the primary target for environmental or occupational toxic exposure 1. The alcoholic liver injury appears to be generated by the effects of ethanol metabolism and the toxic effects of acetaldehyde which may be mediated by acetaldehyde altered proteins 2. Chronic alcohol intake is known to produce hypercholesterolemia, hyperlipidemia, hypertriglyceridemia 3,4. In chronic lipid accumulation the liver cells become fibrotic and leads to impaired liver function. Enhanced lipid peroxidation has been reported in hyperlipidemia 5, which is also induced by ethanol 3,4. Ethanol increases triglycerides and cholesterol levels thus inducing imbalance in lipid metabolism in liver, heart, kidney and other organs and this could explain the reason for the increase in lipid peroxidation in these organs. Recently free radical induced lipid peroxidation has gained much importance because of its involvement in several pathologies 6,7. Protection of cell membrane from lipid peroxidation becomes a necessity to prevent, cure or delay the aforesaid patho-logies. Rind of the fruits of Garcinia cambogia (Gaertn.) Desr. (Clusiaceae) is an astringent and is useful in the treatment of ulcers, haemorrhoids, diarrhoea and dysentry 8,9. Garcinia cambogia fruit extract, containing the principle organic acid, (-)-erythro-l s - hydroxycitric acid is a powerful anti-lipogenic agent 10. The present study is an attempt to elucidate the antiperoxidative property of Garcinia cambogia on ethanol induced peroxidative damage and its efficacy to inhibit lipid peroxidation. MATERIALS AND METHODS Animals: Male albino rats weighing 125 to 150 g were purchased from FIPPAT, Padappai, Chennai. The animals were housed in plastic bottom cages Correspondence: C.S. Shyamala Devi
2 88 P. MAHENDRAN AND C.S. SHYAMALA DEVI Table 1. Serum and hepatic levels of total cholesterol, triglycerides, free fatty acids, phospholipids, alkaline phosphatase, aspartate amino transferase (AST), alanine amino transferase (ALT) in experimental groups a. I II III IV ANOVA Parameters b Control Group 18% Ethanol treated Garcinia cambogia Garcinia cambogia F value* (n = 6) group (n = 6) treated group + 18% ethanol treated (n = 6) group (n = 6) Total cholesterol A 70.4 ± ± 2.19*** 64.3 ± 1.26*** 72.2 ± 1.81*** 4.24 B 4.36 ± ± 0. 51*** 3.72 ± 0.51* 4.76 ± 0.40*** 2.88 Triglycerides A ± ± 2.14*** 98.5 ± 2.09*** ± 2.11*** 3.97 B 4.23 ± ± 0.50*** 3.73 ± 0.46* 4.54 ± 0.40*** 2.94 FFA A 64.1 ± ± 2.28*** 56.6 ± 1.64*** 70.8 ± 2.07*** 3.68 B 11.5 ± ± 1.67*** 9.9 ± 0.81** 13.2 ± 1.24*** 3.24 Phospholipids A ± ± 2.17*** ± 1.98** ± 2.23*** 3.07 B 23.1 ± ± 1.83*** 20.6 ± 1.91* 30.4 ± 1.47*** 2.90 Alkaline Phosphatase A 0.76 ± ± 0.04*** 0.69 ± 0.04 NS 0.86 ± 0.03*** 2.78 B 1.41 ± ± 0.03*** 1.34 ± 0.05 NS 1.19 ± 0.04*** 2.91 AST A 0.48 ± ± 0.03*** 0.41 ± 0.04 NS 0.52 ± 0.04*** 2.83 B 0.72 ± ± 0.04*** 0.78 ± 0.05 NS 0.69 ± 0.05*** 2.79 ALT A 0.63 ± ± 0.04*** 0.61 ± 0.04 NS 0.58 ± 0.03*** 2.96 B 1.04 ± ± 0.02*** 1.07 ± 0.03 NS 0.93 ± 0.03*** 2.81 A : Serum B : Liver. a Values are expressed as mean ± SD for 6 animals in each group. Students t test: Group II vs Group I, Group III vs Group I, Group IV vs Group II. *p < 0.05; **p < 0.01; ***p < b The values of lipids in serum are expressed as mg/dl. The values of AST, ALT and ALP in serum are expressed as µkat/litre. The levels of AST and ALT in liver are expressed as µ moles of pyruvate liberated/sec/g protein. The level of ALP in liver is expressed as µ moles of phenol liberated/sec/g protein. F - test, * Significant at level of 5% (2.77). and allowed free access to standard laboratory chow (Hindustan Lever Foods, Bangalore, India) and water. Drug: Garcinia cambogia fruit extract was obtained from Siris Herbex, Vijayawada, India. Absolute alcohol (99%) was purchased from Anilax Chemicals, USA. All other chemicals used for the experiment were of analytical grade. Grouping Group I - Normal Control (n = 6). Group II - Group III - Rats given 18% ethanol (7.11g/kg body weight day) 5 ml/100 g body weight for 45 days (n = 6). Normal rats given Garcinia cambogia Group IV - 1 g/kg body weight/day orally for 45 days (n = 6). Rats given 18% ethanol (7.11g/kg/body weight/day) + Garcinia cambogia, 1 g/ kg body weight/day for 45 days (n = 6). After the experimental period the overnight fasted rats were sacrificed by cervical dislocation. Blood and tissues were collected in ice-cold containers for various estimations. AST, ALT 11 and alkaline phosphatase 11 were estimated in the serum. SOD 12, CAT 13, GSH 14, GSH-Px 15, GST 16, LPO 17 and conjugated dienes 18 were estimated in the liver tissue. Total cholesterol 19 phospholipids 20,21, triglycerides 22 FFA 23 and protein 24 were estimated both in serum and tissue.
3 GARCINIA CAMBOGIA AND ETHANOL INDUCED PEROXIDATION 89 Table 2. The level of lipid peroxide, conjugated diene and activities of antioxidant enzymes in the liver of experimental groups a. I II III IV ANOVA Parameters b Conrol group 18% Ethanol Garcinia cambogia Garcinia cambogia + F value* (n = 6) treated group treated group 18% ethanol treated (n = 6) (n = 6) group (n = 6) SOD 59.8 ± ± 0.98*** 62.2 ± 2.34 NS 54.4 ± 1.76*** 3.68 CAT 72.6 ± ± 3.52*** 70.8 ± 2.26 NS 66.7 ± 2.82*** 3.20 LPO 3.87 ± ± 0.10*** 3.70 ± 0.12 NS 4.06 ± 0.09*** 2.79 Conjugated diene 74.8 ± ± 1.14*** 73.6 ± 0.75 NS 81.2 ± 0.97*** 3.54 GST ± ± 2.85*** ± 3.71 NS ± 2.97*** 4.12 GSH-px ± ± 5.84*** ± 7.06 NS ± 5.44*** 3.96 GSH 5.11 ± ± 0.08*** 4.89 ± 0.09 NS 4.32 ± 0.04*** 2.84 a Values are expressed as mean ± SD for 6 animals in each group Students t test: Group II vs Group I, Group III vs Group I, Group IV vs Group II. ***p<0.001 b Lipid peroxide (LPO) = µmoles of TBA reactants/g protein. Conjugated diene = mm/100g wet tissue; Superoxide dismutase (SOD) = µkat/g protein. Catalase (CAT) = nmoles of H 2 O 2 decomposed/sec/g protein. Glutathione peroxidase (GSH - Px) = mmoles of GSH utilised/sec/g protein, Glutathione-S-transferase (GST) = µmoles of CDNB conjugated/sec/g protein. Glutathione (GSH) = nmoles of GSH/g tissue. F - test, * Significant at level of 5% (2.77). RESULTS The results are summarised in Table 1 and 2. The diet intake of rats in different groups were almost similar. After 45 days, the weight gain of ethanol fed rats was less than that of normal control rats. The activity of AST, ALT, and alkaline phosphatase increased significantly (p<0.001) in the serum after 45 days in ethanol treated rats (Table 1). The activity of superoxide dismutase catalase, GSH, GST, GSH-Px in the liver decreased significantly (p<0.001) in alcoholic rats (Table 2). The alcohol treated rats caused a significant increase (p<0.001) in the LPO content and conjugated diene in the liver when compared with that of normal rats. Concentration of cholesterol, triglyceride, FFA and phospholipids showed significant (P<0.001) increase in serum and liver tissue in alcohol treated rats. Normal rats treated with Garcinia cambogia (Group III) showed a decrease in lipid levels alone both in serum (p<0.001) and liver tissue (p<0.05). DISCUSSION Lipid peroxidation is a complex and natural deleterious process. The effect of free radicals on human beings have recently been considered as their close relation to toxicity and diseases 25. Chronic administration of ethanol was found to produce an accumulation 4 of cholesterol and triglycerides in the liver as well as in the blood. This imbalance in lipid metabolism could play a role in aggravating the lipid peroxidation 5. Varga et al, have proven that hyperlipidemia and elevated lipid peroxidation are inter-related 26. The possible explanation could be the hyperlipidemic condition would stimulate the catabolic pathway via oxidative breakdown 5. HMG CoA reductase catalyses the rate limiting step in cholesterol biosynthesis and its activity directly correlates with the extent of tissue cholesterol synthesis which is significantly activated during ethanol ingestion 27. Plasma LCAT is the enzyme involved in
4 90 P. MAHENDRAN AND C.S. SHYAMALA DEVI the esterification of cholesterol in the plasma. Increase in serum cholesterol, in the present study in alcohol treated rats may possibly be the result of decreased activity of this enzyme 27. Increase in serum triglycerides in alcohol treated rats may be due to decreased activity of lipoprotein lipase which is involved in the uptake of triglyceride rich lipoprotein by extra hepatic tissue. Increased synthesis or decreased lipid deposition or both resulted in simultaneous accumulation of lipids in the blood and in the liver 27. Ethanol induces hyperlipidemia 3,4 and hyperlipidemia enhances lipid peroxidation 5 causing hepatotoxicity by increasing the free radical formation which in turn increases the level of lipid peroxides in hepatic tissue. Garcinia cambogia containing the principal organic acid (-)-erythro-l s -hydroxycitric acid is an effective inhibitor of ATP-citrate lyase which cleaves citrate to produce acetyl CoA. 10 (-)-hydroxycitrate suppresses biosynthesis of both fatty acids and cholesterol in rat hepatocytes 28,29 and in rat liver, 30 hence cholesterol and triglyceride levels are drastically reduced in Garcinia cambogia treated experimental rats (Group IV). The reduction in lipids indirectly lowers the level of peroxides due to hyperlipidemia. The two antiperoxidative enzymes namely SOD and CAT decreased significantly in the hepatic tissues of alcohol administered rats suggesting the increased damage to this tissue as a result of uncontrolled generation of partially reduced oxygen species. The levels of AST, ALT and alkaline phosphatase in the serum have been elevated in ethanol treated rats suggesting hepatotoxicity as a result of high ethanol intake 31. Garcinia cambogia, an effective antilipogenic agent 32, prevents the hepatic cells to become fibrotic and the cellular damage due to hyperlipidemia. This is evident from the near normal activities of transferases and ALP in the liver of Garcinia cambogia treated group (Table 1). Glutathione protects the hepatocytes by combining with the reactive metabolites and thereby preventing their covalent binding to liver protein 31. Liver glutathione after alcohol administration was found to decrease due to increased utilization by the hepatocytes because GSH seems to act as scavengers for toxic chemical agents. The non availability of glutathione decreases the activity of glutathione peroxidase and glutathione transferase. Glutathione acts as the substrate for both GSH-Px and GST. Depletion of glutathione will render the enzymes (GSH-Px & GST) inactive and/or less active. It was established that the content of primary (acylhydroperoxide) and secondary (intermolecular seams in aminophospholipids) products of lipid peroxide oxidation in blood of patients with the background of hyperlipidemia and hypercholesterolemia increased intensively 33. Also in our present study the levels of LPO and conjugated dienes increased significantly in the ethanol administered rats suggesting its pathogenic role. Treatment of rats with Garcinia cambogia inhibited the deleterious process of lipid peroxidation and maintained the levels of glutathione to near normalcy. The observed abnormalities in the liver and serum may be in part due to hyperlipidemia leading to changes in the activity of antiperoxidative enzymes, glutathione and increased lipid peroxidation. Garcinia cambogia modulates its antiperoxidative role by inhibiting hyperlipidemia and peroxidation of biomembranes which is sufficient to cause cell death when uncontrolled. REFERENCES 1. Mehandale HM. Potentiation of halomethane hepatotoxicity by chlordecone - A Hypothesis for the mechanism. Med Hypotheses 1990;33: Ishak KG, Hyman, Zimmerman HJ. Alcoholic liver disease - pathologic, pathogenetic and clinical aspects. Alcoholism Clin Exp Res 1991;15: Baraona E, Sanolainen M, Karrenty C, Leo Maria A, Lieber CS. Pathogenesis of alcoholic hypertriglyceridemia. Trans Ass Amer Phys 1983;96: Baraona E, Lieber CS. Effects of ethanol on lipid metabolism. J Lipid Res 1979;20: Loeper J, Emerit J, Goy J, Bedu O, Loeper J. Lipid peroxidation during human atherosclerosis. IRCS Med Sci 1983;11: Rowley DA, Halliwell B. Formation of hydroxyl radicals from hydrogen peroxide and iron salts by superoxide and ascorbate-dependent mechanisms: relevance to the pathology of rheumatoid disease. Clin Sci 1983;64:
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