Hepatoprotective Effect of Gallotannin-enriched Extract Isolated from Gall on Hydrogen Peroxide-induced Cytotoxicity in HepG2 Cells

Background: Gall (Galla Rhois [GRI) is known to have antibacterial, anti-inflammatory, antimetastatic, and anti-invasion activities and exert hepatoprotective effects. However, the hepatoprotective effects of gallotannin-enriched GR (GEGR) and their mechanisms have not yet been investigated. Objective: The potential protective effect of GEGR against hepatotoxicity induced by hydrogen peroxide (H2O2) was investigated. Materials and Methods: Changes in cell viability, apoptosis protein expression, and reactive oxygen species (ROS) generation were determined in HepG2 cells that were pretreated with four different concentrations of GEGR (6.25-50 mu g/ml) for 24 h before H2O2 exposure. Results: GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2-diphenyl-l-picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 mu g/ml). The lethal dose 50% and effective dose 50% values for the response of HepG2 cells to GEGR were determined to be 178 and 6.85. mu g/ml, respectively. Significant reductions in the immunofluorescence intensity indicating apoptosis were also detected in the nuclei of HepG2 cells stained with 4,6-diamidino-2-phenylindole and Annexin V after GEGR treatment. The Bax/Bcl-2 ratio and active caspase-3 level were higher in H2O2 + vehicle-treated cells. However, these levels gradually decreased to those of the No-treated group in the GEGR pretreated group even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of 2', 7-dichlorofluorescein diacetate stained cells, indicating ROS generation relative to the H2O2+ vehicle-treated group. Conclusion: The results of this study provide strong evidence that GEGR can prevent cell death induced by H2O2 in HepG2 cells through the induction of antioxidant conditions.