Ginseng (the root of Panax ginseng C. A. MEYER, family Araliaceae) has been used as a herbal medicine in China, Korea, Japan and other Asian countries. Its major components are ginsenosides, which are glycosides with a dammarane skeleton.1,2) When ginseng is steamed at 98-100 ЊC, it is called Red Ginseng (RG). Their main components are different: the former is a ginsenoside Rb1, and the latter is a ginsenoside Rg3.3) These results suggest that the ginsenosides could be easily transformed under steaming. These ginsenosides have been reported to show various biological activities including anti-inflammatory, 4) antiallergic, 5,6) antitumor, 7-11) vascular relaxation, 12) and hepatoprotective activities. 13,14) To express these pharmacological actions, it is thought that ginseng saponins must be metabolized by human intestinal microflora after being taken orally. 7,15,16) For example, ginsenosides Rb1, Rb2 and Rc are metabolized to compound K by human intestinal microflora, and the ginsenoside Rg3 is metabolized to ginsenoside Rh2 by human intestinal microflora.17) The transformed compound K and ginsenoside Rh2 induces an anti-metastatic or anti-carcinogenic effect.8,11) However, the relationship between hepatoprotective effects of RG and the metabolism of its main constituent ginsenoside Rg3 have not been thoroughly studied.Therefore, we transformed 20(S)-ginsenoside Rg3 by human intestinal bacteria, isolated its metabolite 20(S)-ginsenoside Rh2, and investigated their hepatoprotective effects on HepG2 cells and mice injured by tert-butyl hydroperoxide (t-BHP).
MATERIALS AND METHODSMaterials Diagnostic kits for aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were purchased from Asan Pharmaceutical Co., Ltd. (Seoul, Korea). Silybin was purchased from Carl Roth (Karlsruhe, Germany). Minimum essential medium (MEM), fetal bovine serum (FBS) and antibiotics-antimycotics were obtained from Gibco BRL (Fig. 1) were isolated according to the previous method.
18)Culture of HepG2 Cells and Its Hepatoxicity Induction by t-BHP HepG2 cells (hepatocellular carcinoma cell line) donated from the Korean Cell Bank (Seoul, Korea) were cultured in MEM containing 10% FBS, 1% antibiotic-antimycotic solution, 1mM sodium pyruvate and 1.5 g/l sodium bicarbonate under 5% CO 2 at 37 ЊC. The protective effect of ginsenosides on HepG2 cells injured by t-BHP was measured using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. 19) Briefly, HepG2 cells were dispensed into 96 well plates at the concentration of 1ϫ10 4 cells per well. The test compounds were added into HepG2 cells, and preincubated for 2 h. Then the cultured media were replaced to the media containing t-BHP (100 mM), incubated for 3 h and then rinsed with phosphate-buffered saline. MTT reagent (0.25 mg/ml) was added into the cells, incubated for 1 h, and then added 100 ml of dimethylsulfoxide. Absorbance * To whom correspondence should be addressed. e-mail: dhkim@khu.ac.kr © 2005 Pharmaceutical Society of Japan
Hepatoprotective Ef...