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目的以胆红素代谢过程中UGT1A1酶介导的胆红素葡糖醛酸结合环节为切入点,考察何首乌提取物体内、外肝毒性。方法以胆红素为UGT1A1酶底物,考察给药后大鼠肝微粒体体系中UGT1A1酶活性的变化(体内),以及何首乌提取物在人肝微粒体、大鼠肝微粒体,人重组UGT1A1酶中(体外)对胆红素葡糖醛酸结合的抑制作用,以表观抑制常数K_i及K_m,V_(max)变化为评价指标,预测其肝毒性有无及大小。结果何首乌提取物在三个体外体系中,对UGT1A1酶均有较强抑制作用,且抑制类型均为竞争型抑制。大鼠体内实验结果显示,何首乌提取物对UGT1A1酶有强抑制作用,抑制类型为反竞争型抑制。结论本实验所建立的体外研究方法为中药肝毒性药物的筛选提供了新思路和新方法,对中药安全性评价具有借鉴意义。
OBJECTIVE To investigate UGT1A1 enzyme-mediated bilirubin glucuronide binding in the process of bilirubin metabolism, and to investigate the hepatotoxicity of Polygonum multiflorum extract in vitro and in vivo. Methods The bilirubin was used as UGT1A1 enzyme substrate to investigate the changes of UGT1A1 enzyme activity in rat liver microsomes after administration (in vivo) and the effects of the extract of Polygonum multiflorum on human liver microsomes, rat liver microsomes, human recombinant UGT1A1 Inhibition of bilirubin glucuronidation by enzyme (in vitro), with apparent inhibition constants K_i and K_m, V_ (max) changes as the evaluation index to predict the presence and size of hepatotoxicity. Results Polygonum multiflorum Thunb extract had a strong inhibitory effect on UGT1A1 enzyme in three in vitro systems, and the inhibitory types were all competitive inhibition. The results of in vivo experiments in rats showed that the extract of Polygonum multiflorum has a strong inhibitory effect on UGT1A1 enzyme and the type of inhibition is anti-competitive inhibition. Conclusion The in vitro research methods established in this study provide new ideas and new methods for the screening of hepatotoxic drugs of traditional Chinese medicine, and have reference significance for the safety evaluation of traditional Chinese medicines.