Antioxidant Activity of AO-8*, A Herbal Formulation In vitro and In vivo Experimental Models

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1 Antioxidant Activity of AO-8*, A Herbal Formulation In vitro and In vivo Experimental Models Mitra, S.K., Venkataranganna, M.V., Sundaram, R. and Gopumadhavan, S. R&D Centre, The Himalaya Drug Co., Makali, Bangalore, India. [Phytotherapy Research (1999): (13), ] ABSTRACT The effect of AO-8, a herbal formulation, was evaluated for free radical scavenging activity using in vitro and in vivo experimental models. AO-8 dose-dependently inhibited ferric ioninduced lipid peroxidation in vitro at µg. AO-8 was investigated at dose levels of 250, 500 and 750 mg/kg p.o. in isoproterenol-induced myocardial infarction and CCl 4 - induced hepatotoxicity in rats. The levels of serum GOT and LDH, cardiac glutathione, SOD and catalase were estimated following induction of myocardial infarction with isoproterenol. The estimation of parameters such as SGOT, SGPT, liver glycogen and lipid peroxidation were carried out in CCl 4 -induced hepatotoxicity. The treatment with AO-8 for 15 days at a dose of 500 and 750 mg/kg offered marked protection in both in vivo models. The reversal of increased serum enzymes in both the models may be due to the prevention of leakage of the intracellular enzymes by its membrane stabilizing activity. The inhibition of lipid peroxidation and enhancement of antioxidant enzymes by AO-8 may be due to the direct free radical scavenging activity which could be attributed to the antioxidant potential of various ingredients present in the formulation. Thus it can be concluded that AO-8 is an effective free radical scavenger and could prove beneficial in the treatment of various disorders associated with the involvement of free radicals. Keywords: Isoproterenol; antioxidant; CCl 4 ; lipid peroxidation; AO-8; free radical scavenging activity. INTRODUCTION Free radical reactions have been implicated in the pathology of many human diseases including atherosclerosis, ischemic heart disease, the aging process, inflammation, diabetes, immunodepression, the neurodegenerative condition and other disease conditions (Maxwell, 1995). Radicals and other reactive oxygen species are formed constantly in the human body and are removed by the enzymic and nonenzymic antioxidant defence system. Oxidative stress occurring when antioxidant defenses are inadequate can damage lipids, proteins, carbohydrates and DNA. Drugs with multiple mechanisms of protective action, including antioxidant properties, may be one way forward in minimizing tissue injury in human disease (Barry, 1991). A number of plants and plant isolates have been reported to protect free radical-induced damage in various experimental models. The essential oil of Syzygium aromaticum is a very good antioxidant in vivo and in vitro (Deans et al., 1995; Lemberkovies et al., 1992). The antioxidant properties of Vitis vinifera (Gabreilskee et al., 1997), Mangifera indica (Ghosal et al., 1996), Daucus carota (Kim and Lee, 1994) and Glycyrrhiza glabra *AO-08 is marketed as Oxitard 1

2 (Hatano et al., 1991) have been reported. Emblica officinalis is rich in vitamin C and is also a well known antioxidant (Jose and Kuttan, 1995). In the present study, AO-8, a herbal preparation, was tested for its antioxidant activity by in vitro and in vivo experimental models. MATERIALS AND METHODS Composition: Each gram of AO-8 contains Mangifera indica Linn. (Anacardiaceae; bark, 340 mg), Glycyrrhiza glabra Linn. (Papilionaceae; rhizome, 100 mg), Syzygium aromaticum Linn. Merr. and L.M. Perry (Myrtaceae; flower bud, 100 mg), Vitis vinifera Linn. (Vitaceae; fruit, 21 mg), Emblica officinalis Linn. (Euphorbiaceae; fruit, 200 mg) and Daucus carota Linn. D. Vulgaris (Umbelliferae; root, 200 mg). The constituent plants of the formulation were procured from authentic sources and were identified by Dr. S. Farooq, Botanist of The Himalaya Drug Co. A specimen of each plant was deposited in the herbarium of our R&D Centre. In vitro lipid peroxidation: The drug was prepared with a 1% suspension in 5% dimethyl sulphoxide (DMSO) using 0.005% of carboxy methyl cellulose. Lipid peroxidation was carried out by the following method. The liver of normal rat, after ether anesthesia, was perfused with ice-cold 0.9% sodium chloride and the tissue was homogenized at a concentration of 10% w/v in 1.15% of potassium chloride using a glass homogenizer. The homogenate was centrifuged at 800 g and the supernatant was used for the study. Various concentrations of extracts µg were taken with 0.5 ml of the homogenate and the lipid peroxidation was induced using ferric chloride (100 µl) of 2 mm solution. Tocopherol was used as a standard reference. The mixture was incubated at 37 o C for 20 minutes and the reaction was stopped by adding 2 ml of ice-cold 0.25 N hydrochloric acid containing 15% trichloroacetic acid, 0.38% thiobarbituric acid (TBA) and 0.05% butylated hydroxytoluene (BHT). Following heating at 80 o C for 15 minutes, absorbance of the supernatant was measured spectrophotometrically at 532 nm (Braughler et al., 1986). The percentage inhibition of lipid peroxidation was calculated with respect to the positive control. In vivo experiments: Albino rats of the Wistar strain weighing g were used for the study. The rats were fed commercial pelleted rat chow and given water ad libitum. Isoproterenol-induced myocardial infarction in rats: The rats were divided into five groups of eight animals each. Group I served as a control. Group II rats served as a positive control. Groups III, IV and V were administered AO-8 at a dose of 250, 500 and 750 mg/kg b. wt. p.o. respectively for 15 days. On day 15, Groups II, III, IV and V received isoproterenol (20 mg/100 g b. wt. s.c. twice at an interval of 24 h) in sterile saline. After 24 hours of the last dose of isoproterenol, the rats were killed by decapitation. Blood was collected and serum was separated for estimations of lactate dehydrogenase (LDH) (McQueen, 1972) and glutamic oxaloacetate transaminase (GOT) (Reitman and Frankel, *AO-08 is marketed as Oxitard 2

3 1957). The heart was dissected out, immediately washed in ice cold saline and a 5% homogenate prepared in 0.1 M Tris-HCl buffer (ph 7.4). The homogenate was centrifuged and the supernatant was used for the assay of glutathione (GSH) (Ellman, 1959), superoxide dismutase (SOD) (Kono, 1978), catalase and protein (Lowry et al., 1951). CCl 4 -induced hepatotoxicity in rats: The rats were divided into five groups of eight animals each. Group I served as a control. Group II rats served as CCl 4 group and Groups III, IV and V rats were administered AO-8 at a dose of 250, 500 and 750 mg/kg b. wt. p.o. respectively for 15 days. On day 15, Groups II, III, IV and V received CCl 4 1 ml/kg b. wt. in liquid paraffin (1:1). After 24 hours of the CCl 4 administration the rats were killed by decapitation. Blood was collected and serum separated for estimation of glutamic pyruvic transaminase (GPT) (Reitman and Frankel, 1957) and GOT (Reitman and Frankel, 1957). The liver was dissected out, immediately washed in ice-cold saline and a 5% homogenate prepared in 0.15M KCl. The homogenate was used for the assay of lipid peroxidation (Ohkawa et al., 1979). A small piece of liver was taken for the estimation of liver glycogen (Montgomery, 1957). Statistical analysis: The data of biochemical parameters were expressed as mean ± SEM. Results were analyzed statistically using ANOVA followed by Dunnet s t -test. The minimum level of significance was fixed at p<0.05. RESULTS In vitro lipid peroxidation: Compared with various stimulating agents such as tertiary butyl hydroperoxide and cumene hydroperoxide, ferric ions were the most effective in inducing lipid peroxidation. The effect of AO-8 and tocopherol on ferric ion-induced lipid peroxidation is given in Table 1. AO-8 formulation dose-dependently inhibited ferric chloride-induced lipid peroxidation which indicates free radical scavenging activity of the formulation. The protective effect of AO-8 at 1000 µg was comparable to that of 150 µg tocopherol. Table 1: Effect of AO-8 on in vitro lipid peroxidation Group Tocopherol Concentration (µg) Inhibition (%) ± ± ± ± 2.86 Values are mean ± SE (n=5) Isoproterenol-induced myocardial infarction: The treatment with isoproterenol resulted in a significant elevation in serum GOT and LDH activity compared with the normal control (Table 2). The administration of AO-8 at different doses resulted in reversal of these changes towards normalcy at 500 and 750 mg/kg b. wt. in the case of GOT, but it was significant only at 750 mg/kg b. wt. in the case of LDH. At the same time it was observed that there was a significant decrease in heart glutathione, SOD and catalase. AO-8 significantly increased the glutathione levels compared with Group II at 500 *AO-08 is marketed as Oxitard 3 AO ± ± ± ± ± 2.12

4 and 750 mg/kg b. wt. and catalase levels at all the doses. However, the SOD level was not significantly increased because of the high SEM. Table 2: Effect of AO-8 on isoproterenol-induced myocardial infarction Parameter Group I Group II Group III Group IV Group V SGOT (U/L) ± 6.26 a ± LDH (U/L) ± b ± Glutathione (mm/mg protein) ± a ± ± ± ± ± d ± a,e ± ± c,f ± d ± 0.39 c CCl 4 -induced SOD hepatotoxicity: Serum (U/mg protein) ± 0.77 b ± ± 1.02 ± 1.74 ± 1.29 GPT, GOT and liver lipid Catalase peroxidation were (U/mg protein) ± 2.63 a ± 0.79 ± 0.32 b ± 0.86 b ± 0.92 b significantly elevated after Values are mean ± SE (n=8) CCl 4 intoxication in rats a p<0.001, b p<0.01, c p<0.02, d p<0.05 compared with Group II e p<0.001 and f p<0.05 compared with Group III vs. V. compared with the control animals (Table 3). These parameters were reversed to normalcy after administration of AO-8 at 500 and 750 mg/kg b. wt. in the case of GOT and lipid peroxidation, but it was significant only at 750 mg/kg in the case of GPT. CCl 4 intoxication resulted in a significant depletion of liver glycogen and AO-8 significantly prevented the glycogen depletion at 500 and 750 mg/kg b. wt. SGPT (U/L) Table 3: Effect of AO-8 in CCl 4 -induced hepatotoxicity Parameter Group I Group II Group III Group IV Group V SGOT (U/L) Liver glycogen (mg%) Lipid peroxidation (nm/100 mg tissue) ± 3.86 a ± ± a ± ± 0.98 a ± ± 0.92 a ± ± ± ± ± ± ± c Values are mean ± SE (n=8); d p<0.001, b p<0.01, c p<0.02, d p<0.05 compared with Group II e p<0.02 and f p<0.05 compared with Group II vs. Groups III and IV ± d ± ± 0.62 b ± 0.68 b,f ± 2.62 a,f ± 2.92 a,e DISCUSSION The free radical scavenging activity of AO-8 formulation was studied using in vitro and in vivo experimental models. Ferric ions stimulate lipid peroxidation through various mechanisms, such as the generation of hydroxyl radical (Gutteridge et al., 1979), the decomposition of lipid peroxides (Braughler et al., 1987) or by forming perferryl species (Koppenol and Liebmarr, 1984). Ferric ions also undergo reduction by endogenous reducing substances, a process necessary for the initiation of lipid peroxidation. AO-8 dosedependently inhibited ferric ion-induced lipid peroxidation. Isoproterenol-induced myocardial infarction serves as a well-standardized model to study the beneficial effects of many drugs. Isoproterenol is a -adrenergic agonist and has been *AO-08 is marketed as Oxitard 4

5 reported to increase lipid peroxidation through free radical formation (Sushma Kumari and Menon, 1987). Activated lipid peroxidation is an important pathogenic element in myocardial infarction, with lipid peroxide levels reflecting the major stages of disease and its complications (Golikov et al., 1989). Increased levels of lipid peroxides indicate the excessive formation of free radicals and activation of free radicals resulting in irreversible damage to the heart and aorta in animals subjected to isoproterenol stress. The increased levels of serum enzymes in myocardial ischemia is due to the leakage of enzymes into blood (Kaul and Kapoor, 1991). AO-8 treatment significantly reduced the lipid peroxides in the heart by a significant increase in the catalase and glutathione levels and a mild increase in the SOD levels, which suggests its efficacy in preventing free radical-induced damage. Treatment with AO-8 also significantly decreased serum enzyme levels by preventing the release of lysosomal enzymes, which may be due to its membrane stabilizing activity. Carbon tetrachloride induces liver damage by producing free radical intermediates (Mitra et al., 1998). Carbon tetrachloride is converted to trichloromethyl radical (CCl 3 ) by the cytochrome P-450 system, which in turn is converted to peroxy radical (CCl 3 O 2 ) which causes lipid peroxidation (Butler, 1961). The inhibition of lipid peroxidation by AO-8 may be due to its free radical scavenging activity. The hepatic injury induced by CCl 4 resulted in increase in serum GOT and GPT levels due to the leakage of cellular enzymes into the blood stream. The reversal of these serum enzymes following treatment with AO-8 may be due to the reduction in cell membrane disturbances. CCl 4 -induced toxicity is known to deplete the glycogen levels in the liver because of the disturbance of the intracellular Ca +2 pool, which results in glycogenolysis leading to glycogen depletion (Zimmerman, 1978). AO-8 treatment increased the liver glycogen levels, which indicates that AO-8 protects the plasma membrane and prevents glycogen depletion. With all these findings it can be concluded that AO-8 is an effective free radical scavenger and can be used in the treatment of various disorders where free radicals are involved. REFERENCES: 1. Barry, H. (1991). Antioxidant effects a basis for drug selection. Drugs 42(2), Braughler, J.M., Chase, R.L. and Pregenzer, J.F. (1987). Oxidation of ferrous iron during peroxidation of various lipid substances. Biochem. Biophys. Acta 921, Braughler, J.M., Duncan, L.A. and Chase, R.L. (1986). The involvement of iron in lipid peroxidation. J. Biol. Chem. 261, Butler, T.C. (1961). Reduction of CCl 4 in vivo and reduction of CCl 4 and CHCl 3 in vitro by tissues and tissue constituents. J. Pharm. Exp. Ther. 134, Deans, S.G., Nobel, R.C., Hiltunen, R., Wuryani, W. and Penzes, L.G. (1995). Antimicrobial and antioxidant properties of Syzygium aromaticum. Flavour Fragr. J. 10(5), Ellman, G.L. (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys. 80, Gabreilskee, I., Oszmianski, J. and Lamer-zarawska, E. (1997). Protective effect of plant flavonoids on the oxidation of lecithin liposomes. Pharmazie 52(2), *AO-08 is marketed as Oxitard 5

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