基于UGT1A1酶抑制探讨何首乌中顺(反)式二苯乙烯苷体外肝微粒体中潜在毒性作用

Study on Toxic Effects of cis(trans)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in Polygonum multiflorum Based on Inhibition of UGT1A1 Enzyme in Rat Liver Microsomes

目的:以胆红素代谢过程中UDP-葡萄糖醛酸转移酶1A1(UGT1A1酶)介导的胆红素葡萄糖醛酸结合环节为切入点,考察何首乌中含量高的二苯乙烯苷类(顺式、反式二苯乙烯苷)潜在肝毒性,探索何首乌致肝毒性物质基础。方法:以胆红素为UGT1A1酶底物,以表观抑制常数Ki为评价指标,采用体外肝微粒体孵育法,启动Ⅱ相代谢反应,考察顺式、反式二苯乙烯苷原型成分的抑制作用;启动Ⅰ相代谢反应,考察代谢产物及原型成分的抑制作用,推测待测物的潜在毒性作用。结果:当仅启动Ⅱ相反应时,顺式、反式二苯乙烯苷均以原型形式直接作用于UGT1A1酶,两个单体分别表现出中等抑制和弱抑制作用,抑制类型均为竞争型抑制;当同时启动Ⅰ、Ⅱ两相反应时,两个待测单体对UGT1A1酶的抑制作用均消失,提示两个单体存在Ⅰ相代谢过程,并且其Ⅰ相代谢产物对UGT1A1酶无抑制作用。结论:本实验初步证明何首乌中顺式、反式二苯乙烯苷原型成分对UGT1A1存在抑制作用,经由Ⅰ相代谢后,对Ⅱ相代谢酶UGT1A1抑制作用消失,肝毒性风险降低。

Objective:To investigate the potential hepatotoxicity of stilbene glycosides cis(trans)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in Polygonum multiflorum on the basic of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1 A1(UGT1A1 enzyme)in order to explore the material basis of hepatotoxicity caused by P.multiflorum.Methods:The monomers were added into the rat liver microsomes to test the hepatotoxicity using the bilirubin as UGT1A1 enzyme substrate,the inhibition of the UGT1A1 enzyme was investigated.In the microsomes incubation systems the apparent inhibition constant Ki was used to evaluate the hepatotoxicity.The phaseⅡmetabolic reaction was initiated to investigate the inhibitory effect of the cis(trans)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside;the phaseⅠandⅡmetabolic reaction were initiated to investigate the inhibition of metabolites and prototype components,and speculate the potential toxic effects of the analytes.Results:When only the phaseⅡreaction was initiated,both cis(trans)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside directly acted on the UGT1A1 enzyme in a prototype form,and the two monomers exhibited moderate inhibition and weak inhibition,respectively,and the inhibition types were competitive inhibition.When the two-phase reaction ofⅠandⅡwas started simultaneously,the inhibitory effects of the two monomers tested on UGT1A1 enzyme disappeared,suggesting that the two monomers had phase I metabolic processes,and the phaseⅠmetabolites had no inhibitory effect on UGT1A1 enzyme.Conclusion:This experiment preliminarily proved that the cis(trans)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside prototypic components of P.multiflorum inhibited UGT1A1.After phaseⅠmetabolism,the inhibitory effect of phaseⅡmetabolic enzyme UGT1A1 disappeared and the risk of hepatotoxicity decreased.