Antioxidant and Hepatoprotective Effects of Anoectochilus formosanus and Gynostemma pentaphyllum

American Journal of Chinese Medicine,  Wntr, 2000  by Chun-Ching Lin,  Pei-Chen Huang,  Jer-Min Lin

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Abstract: Anoectochilus formosanus Hay. and Gynostemma pentaphyllum Makino are popular folk medicines that have been used for treating hepatitis, hypertension and cancer in Taiwan. Our previous studies showed that these crude drugs exert antiinflammatory activity and hepatoprotective activity against C[Cl.sub.4]-induced liver damage. In this study, the antioxidant effect of these crude drugs and their hepatoprotective activity on acetaminophen-induced liver injury in rat was evaluated. Our results suggest that A. formosanus and G. pentaphyllum do have antioxidant effects. On acetaminophenintoxicated model, the increased levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by acetaminophen administration were reduced by treatment with these two herbs. In histological observation, gross necrosis in the centribular area, sinusoidal congestion, infiltration of the lymphocytes and Kupffer cells around the hepatic central vein, and loss of cell boundaries and ballooning degeneration were reduced with herbal treatment. However, the effect of A. formosanus and G. pentaphyllum is biphasic. Methanol extract (100 and 300 mg/kg) and water extract (300 and 500 mg/kg) of A formosanus and water extract (100, 300 and 500 mg/kg) of G. pentaphyllum enhanced the recovery of liver injury while treatment with 500 mg/kg of A. formosanus methanol extract resulted in serious hepatic injury.

Anoectochilus formosanus Hay. and Gynostemma pentaphyllum Makino. are popular folk medicines in Taiwan. A. formosanus (Orchidaceae) is useful in treating hypertension, tuberculosis, impotence, underdeveloped children, liver and spleen disorders. Kan (1975, 1978) and Mak et al., (1990) reported that it inhibited the production of thromboxane and activated the production of prostacyclin. G. pentaphyllum (Cucurbitaceae) has been used as a diuretic, antipyretic and antiinflammatory agent and found to reduced serum levels of triglyceride, lipid peroxide, total cholesterol, phospholipids, ALT and AST in rats (Kimura et al., 1983; Lin et al., 1993). In this study, we evaluated the antioxidant activity and hepatoprotective activity on acetaminophen-induced liver damage in rats of these two herbal medicines.

Materials and Methods

Preparation of Plant Extracts

The whole plant of A. formosanus and G. pentaphyllum were shade-dried and decocted for 1 hour with 1 liter of methanol or boiling water three times. The decoction was mixed, filtered, concentrated and lyophilized. For evaluation of their antioxidant activity, the crude extract powder was dissolved in phosphate or Tris-HCl buffer solution to make up various concentrations (1, 3, 5, 8, 10, 15 mg/ml). Each concentration of test solution were tested three times, and the [IC.sub.50] values were calculated by the inhibition of difference concentration of test solutions. For evaluation of their hepatoprotective activity, crude extract powders were dissolved in normal saline (100, 300 or 500 mg/ml/kg in rats) prior to intragastric administration to the experimental animals. Plant materials were identified by C.C. Lin, and their voucher specimens were deposited at the Herbarium of the School of Pharmacy, Kaohsiung Medical College.

Chemicals

Chemicals were obtained from either E. Merck (Darmstadt, F.R.G.) or Sigma Chemical Co. (U.S.A.). Silymarin was obtained from Aldrich Chemical Co. (Milwaukee, WI). Xanthine oxidase (XOD) was purchased from Boehringer Mannheim. 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was supplied from Labtec (Tokyo, Japan). Superoxide dismutase (SOD) from bovine erythrocytes was from Toyobo (Osaka, Japan). All other chemicals were of reagent grade and were used without further purification.

Animals

Male Wistar albino rats (4-6 weeks, 120-150 g) were purchased through the animal center, College of Medicine, National Cheng Kung University and housed in an air conditioned room at 23 [+ or -] 1 [degrees] C, 55 [+ or -] 5% humidity, 12-hour light, and fed with a standard laboratory diet and tap water.

Preparation of Liver Homogenate

Liver tissues were removed after animals were sacrificed. Parts of the liver sections were cut into approximately 500 to 1000 mg portions on ice and stored separately at -70 [degrees] C in plastic tubes. The frozen liver samples were homogenized in Tris-HCl buffer to give a 20% homogenate. In measurement of lipid peroxidation levels, homogenates were centrifuged at 1700 rpm and 4 [degrees] C for 10 minutes. The protein of liver tissue also simultaneously assayed by the method of Lowry et al. (1951), which correlated with the A280 curve, a standard calibration curve.

Fe[Cl.sub.2]-Ascorbic acid Stimulated Lipid Peroxidation in Rat Liver Homogenate

The effect of anti-Fe[Cl.sub.2]-Ascorbic acid and stimulated lipid peroxidation was determined by the methods of Kimuya et al. (1981). The reaction mixture contains 0.5 ml of liver homogenate, 0.1 ml of Tris-HCl buffer (pH 7.2), 0.05 ml of 0.1 mM Ascorbic acid, 0.05 ml of 4 mM Fe[Cl.sub.2] and 0.05 mi of the various tested sample solution. After incubated for 1 hour at 37 [degrees] C, 0.9 ml of distilled water and 2 ml of 0.6% TBA were added to the incubation solution and shaken vigorously and then heated for 30 min in a boiling water bath. After cooling, 5 ml of n-butanol was added and the mixture was again shaken vigorously. The n-butanol layer, separated by centrifugation at 1000 xg for 10 min., was determined by a spectrophotometer (HITACHI U-2000) at A532. Rat liver homogenate was induced with Fe[Cl.sub.2]-Ascorbic acid to form nonenzymatic lipid peroxidation, the actions of the plant extracts on this system were determined by MDA-TBA adduct (complexion of malondialdehyde with thiobarbituric acid) at 532 nm (Wong et al., 1987).