| Objective:This thesis aims to explore the material basis of Polygonum multiflorum root which cause liver toxicity by using the precision-cut liver slice technique. Methods:The powder of P. multiflorum root was extracted with ethanol, and the extract solution was evaporated to no alcohol taste, then the extract was successively partitioned with ethyl acetate and n-butanol to obtain EtOAc, n-BuOH and water fractions. The three fractions were co-cultured with rat liver slices for 6 h respectively, and the liver tissue homogenate was prepared. Bicinchoninic acid(BCA) method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase(ALT), aspartate aminotransferase(AST), gamma-glutamine amino transpeptidase(GGT) and lactate dehydrogenase(LDH). The higher enzyme leakage rate, the more hepatoxicity it has.The chemical constituents of the toxic fraction were separated and purified by various chromatographic techniques including silica gel, ODS, MCI, Sephadex LH-20 column chromatrographies. Structures of compounds were elucidated based on the MS and NMR spectral analysis and comparison to the standard substance in TLC.MTT assay was used to investigate the toxicity of anthraquinones derivatives from P. multiflorum on HepG2 cell, then the determined cytotoxic ingredients were verified by precision-cut liver slices technique. Those toxic ingredients selected in the above mentioned experiments may be the material basis of P. multiflorum to cause hepatoxicity. Results:The precision-cut liver slices test results showed that EtOAC fraction of 0.4 g·m L-1 concentration significantly increased the leakage rate of ALT, AST, GGT and LDH(P<0.01或 P<0.05) and the group of 0.2 g·m L-1 concentration significantly increased the leakage rate of ALT, GGT and LDH(P<0.01 或 P<0.05). The hepatotoxicity of Et OAC fraction was more toxic compared to n-BuOH and water fractions at the same concentration. So, the EtOAC fraction was selected to study. Six compounds were isolated from EtOAC fraction and their structures are elucidated as emodin, citreorosein, physcion-8-O-(6′-O-acetyl)-β-D-glucopyranoside, gallic acid, emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside.MTT assay results showed rhein, emodin, physicion-8-O-β-D-glucopyranoside and physcion-8-O-(6′-O-acetyl)-β-D-glucopyranoside exhibited cytotoxic effects on HepG2 cell, and their IC50 values were 71.07, 125.62, 242.27, 402.32 μM, respectively. However, the other 7 compounds are basically nontoxic and their IC50 values can not be calculated. Next, rhein, emodin and physcion-8-O-β-D-glucopyranoside were selected to investigate the hepatoxicity by using precision-cut liver slices technique. The results showed that rhein group of 400 μM concentration significantly increased the leakage rate of ALT, AST and LDH(P<0.01), and the rhein group of 100 μM concentration only increased the leakage rate of LDH(P<0.05). With the increase of rhein concentration, the protein content in liver slices decreased significantly(P<0.05) within a certain range of does. Emodin group of 400 μM concentration significantly increased the leakage rate of ALT, GGT and LDH(P<0.01). Physcion-8-O-β-D-glucopyranoside group of 800 μM concentration also significantly increased the leakage rate of ALT, AST and LDH(P<0.01 or P<0.05), but the group of 200 μM concentration only significantly increased the LDH leakage(P<0.05). Along with the increase of the concentration of physcion-8-O-β-D-gluc-opyranoside, the leakage rate of ALT, AST and LDH showed a trend of increase, but the protein content in liver slices was in decline. Furthermore, MTT reduction ability of liver slices significantly decreased(P<0.01) in the physcion-8-O-β-D-glucopyranoside group of 800 μM concentration. Conclusion:Large doses of anthraquinones may cause hepatoxicity, but they are probably not the main material basis of the hepatoxicity of P. multiflorum. |
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