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

BACKGROUND: Gall (Galla Rhois [GR]) 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 (H(2)O(2)) 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 μg/ml) for 24 h before H(2)O(2) exposure. RESULTS: GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2-diphenyl-1-picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 μg/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 μg/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 H(2)O(2) + 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 H(2)O(2) + vehicle-treated group. CONCLUSION: The results of this study provide strong evidence that GEGR can prevent cell death induced by H(2)O(2) in HepG2 cells through the induction of antioxidant conditions. SUMMARY: The gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) are the main constituents of water extracts of GR. GEGR was more potent in DPPH scavenging, and gallotannin contributes to this extract activity. GEGR significantly reduced the increase of apoptosis, Bax/Bcl-2 ratio, and active caspase-3 level after H2O2 treatment. GEGR pretreatment showed protection against H(2)O(2)-induced ROS production in DCFH-DA staining analysis. Abbreviations used: COX: Cyclooxygenase; DAPI: 4’,6-diamidino-2-phenylindole; DMSO: Dimethyl sulfoxide; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GEGR: Gallotannin-enriched Galla Rhois; GR: Galla Rhois; HPLC: High-performance liquid chromatography; H(2)O(2): Hydrogen peroxide; MMP: Metallopeptidase; MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS: Reactive oxygen species; UV-Vis: Ultraviolet-visible.