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Journal of Ethnopharmacology 94 (2004) 155-158
Effect of Garlic (Allium sativum) on Lipid Peroxidation in Experimental Myocardial Infarction in Rats
The present study was conducted to elucidate the antioxidant role of garlic oil in isoproterenol (IPL)-induced myocardial infarction in rats. During myocardial necrosis induced by isoproterenol, a significant increase in serum iron content with a significant decrease in plasma iron binding capacity, ceruloplasmin activity, and glutathione (GSH) levels were observed. There was also a significant increase in lipid peroxide levels on isoproterenol administration. Activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and glutathione reductase (GRD) were decreased significantly in the heart with isoproterenol-induced myocardial necrosis. Garlic oil produced a marked reversal of these metabolic changes related to myocardial infarction induced by isoproterenol. In conclusion, garlic oil exerts its effects by modulating lipid peroxidation and enhancing antioxidant and detoxifying enzyme systems.
Cardiovascular disease, including atherosclerosis and cardiac tissue injury after myocardial infarction, is associated with free radicals generated at the site of damage (Tappel, 1973). Free radicals may be formed by infiltration of white cells into ischemic myocardium or may be formed in the endothelial cells by the action of xanthine oxidase during the period of ischemia (McCord and Roy, 1982). It is generally accepted that oxygen-centered free radicals are key mediators associated with ischemia-reperfusion injury to the heart (David and Alvin, 1990). Isoproterenol (IPL), is a -adrenergic agonist and has been reported to increase lipid peroxidation through enhanced free radical formation (Sushamakumari and Menon, 1987). Isoproterenol-induced myocardial infarction has been used as a model for the evaluation of cardio-protective agents.
Consumption of garlic (Allium sativum, liliaceae) has been reported to have a variety of positive cardiovascular effects, including reduction in plasma cholesterol (Ali and Thomson, 1995). Bordia et al. (1975) reported that garlic oil can prevent fat-induced hyperlipidemia. It has been reported that ingestion of garlic has resulted in hypolipidemia and inhibition of atherogenesis (Jain, 1976, Jain and Konar, 1976). The present study was undertaken to evaluate the mechanism of myocardial damage in relation to lipid peroxides and antioxidant enzymes.
Materials and Methods
Male Wistar rats (weighing 200-210 g) were fed a commercial pellet diet, given water ad libitum, and maintained under standard environmental conditions.
Garlic pearls (as the source of garlic oil) were obtained from Ranbaxy Laboratories. Isoproterenol and bovine serum albumin were obtained from Sigma Chemical Company. All other chemicals used were of analytical grade.
Commercial garlic pearls (containing 0.25% garlic oil) were crushed and thoroughly mixed with diethyl ether (B.P., 40-60 ◦C) in a separatory funnel. The etherial fraction was then separated and ether was allowed to evaporate. The remaining oily material was used as garlic oil. (Jain and Konar, 1978).
The experimental animals were divided into four groups of six rats each. Group I served as the control group. Group II rats were administered isoproterenol (20 mg/100 g BW, subcutaneously twice at an interval of 24 h) in saline. Group III animals were orally treated with garlic oil (75 mg/kg per day, for a period of 60 days. Group IV animals were orally administered garlic oil at the above mentioned dosage for 60 days and isoproterenol (20 mg/100 g) administered subcutaneously twice at an interval of 24 h.
After the experimental period the animals were sacrificed under mild chloroform anaesthetic conditions by cervical decapitation. Blood was collected and kept in slanting position for serum separation. The heart was immediately removed, washed in ice-cold saline and homogenized in Tris-HCI buffer (0.1 M) pH 7.4. The homogenate was then centrifuged and the resulting supernatant was used for the assay of various
enzymes. Glutathione (GSH) was assayed by the method of Mornon et al. (1979), superoxide dismutase (SOD) was assayed according to the method of Misra and FridoVich (1972), based on the inhibition of epinephrine auto-oxidation by the enzyme, catalase (CAT) activity was measured by following decomposition of hydrogen peroxide according to the method of Beers and Sizer (1952), glutathione peroxidase (GPX) was assayed by the method of Rotruck et al. (1979) using hydrogen peroxide as substrate, glutathione-S-transferase (GST) activity was measured using 1,chloro 2,4 dinitro benzene as substrate according to Habig et al. (1975) and glutathione reductase (GRD) activity was measured by the method of Pinto and Bartley (1969). Serum iron content was estimated by the method of Ramsay (1969), plasma iron binding capacity was determined by Ramsay's dipyridyl method and ceruloplasmin activity was measured according to the method of Ravin (1961). Serum and heart lipid peroxides were estimated by the method of Okhawa et al. (1979) (The values were obtained from spectrophotometric measurements and confirmed by HPLC). Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
The data were statistically analyzed by employing a Student's t-test.
The effect of garlic oil administration on serum iron content, plasma iron binding capacity, and ceruloplasmin and glutathione content were determined. Isoproterenol administered rats showed a statistically significant increase in serum iron content (P < 0.001) with significant decrease in GSH level, ceruloplasmin activity, and iron binding capacity (P < 0.001) when compared to control rats. All the alterations except glutathione were prevented in rats pretreated with garlic oil (group IV). Levels of GSH, lipid peroxides, SOD, CAT, GPX, GST and GRD were also determined. The antioxidant enzyme activities were decreased significantly (P < 0.001) in isoproterenol treated rats when compared with those of control rats. There was a significant increase in lipid peroxidation (P < 0.001) with significant decrease in glutathione content in group II rats when their values are compared to those of control rats. The activities of antioxidant enzymes, glutathione, and lipid peroxide level were maintained at near normal (group IV) when compared with group II.
During myocardial infarction, reactive oxygen species like superoxide, hydrogen peroxide, and hydroxyl radicals are produced in enormous amounts (McCord, 1988) which contribute to myocardial tissue injury. Cardiovascular actions of isoproterenol may also lead to cardiac necrosis (Stanton and Schwart, 1967). The results of the present investigation support these studies.
The increased level of free iron is usually associated with decreased plasma iron binding capacity in isoproterenol treated rats. During ischemia, free iron is released from home dependent proteins like hemoglobin and myoglobin and a decreased iron binding capacity increases prostaglandin metabolism and in vivo lipid peroxidation (Halliwell and Gutteridge, 1986). There is increased mobilization of iron from ferritin in the heart by the enzyme xanthine oxidase and over production of free radicals results in myocardial damage (Biemond et al., 1986). Garlic oil pretreatment decreases the level of serum iron by increasing iron binding capacity and prevents hemolysis, thereby preventing iron catalyzed lipid peroxidation.
Ceruloplasmin activity is decreased significantly during isoproterenol administration (Altimani and Dormandy,
1977). Garlic oil pretreatment increases ceruloplasmin activity resulting in decreased production of free iron. Lipid peroxide is an important pathogenic event in myocardial infarction and the accumulated lipid peroxides reflects the various stages of the disease and its complications (Golikov et al., 1989). We are reporting enhanced serum and heart lipid peroxidation of isoproterenol treated rats when compared to control animals. Increased levels of lipid peroxides injure blood vessels, causing increased adherence and aggregation of platelets to the injured sites (Grylewski, 1980).
Garlic oil pretreated isoproterenol administered rats maintained the level of heart lipid peroxides to near normal when compared to control rats. Garlic has antioxidant activity (Banerjee et al., 2002). Garlic inhibits platelet aggregation and lowering of arterial blood pressure, which are the important events in myocardial infarction (Agarwal, 1996). Isoproterenol treated rats showed a significant decrease in glutathione levels, activities of glutathione-S-transferase, glutathione peroxidase, and glutathione reductase in the heart. Glutathione level and activities of glutathione-dependent enzymes were restored to near normal levels in rats pretreated with garlic oil (group IV). Glutathione reductase and glutathione peroxidase are essential for maintaining constant ratios of reduced glutathione to oxidized glutathione in the cell. Decreased glutathione levels in isoproterenol administration may be due to its increased utilization in protecting SH containing proteins from lipid peroxides. Reduced availability of glutathione also reduces the activity of glutathione peroxidase and glutathione-S-transferase on isoproterenol administration (Paritha Ithayarasi and Shyamala devi, 1997).
Inactivation of glutathione reductase in the heart leads to accumulation of oxidized glutathione (GSSG), the oxidized product of GSH (Ferrari et al., 1985). GSSG inactivates enzymes containing SH groups and inhibits protein synthesis (Lil et al., 1988). Garlic oil pretreatment restores glutathione levels and increases the
activities of glutathione peroxidase and glutathione-S-transferase. SOD and CAT activities were decreased on isoproterenol administration in accordance with the observation of Manjula and Shymaladevi (1994). During myocardial infarction, superoxide radicals generated at the site of damage modulate SOD and CAT, resulting in the loss of activity and accumulation of superoxide radicals, which damage myocardium. Garlic oil pretreatment increases the activity of SOD and CAT and their scavenging of superoxide radicals, reducing myocardial damage caused by free radicals.
In conclusion, garlic oil pretreatment offers significant protection to the myocardium by inhibiting lipid peroxidation and activating antioxidant defense enzymes in the system.
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