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目的考察芫花醇提物对UGTs及UGT1A1活性的影响,为阐释芫花毒性机制提供依据。方法采用体外肝微粒体孵育模型,以4-硝基酚为底物检测UGTs活性,以β-雌二醇为底物检测UGT1A1活性,利用UV和HPLC测定底物及代谢物含量。结果芫花醇提物中3种黄酮类成分芹菜素、羟基芫花素、芫花素含量分别为6.34%、8.72%、6.06%,UV测得总二萜含量为31.40%。体外实验表明,在大鼠肝微粒体(RLM)和人肝微粒(HLM)孵育体系中,芫花醇提物均能显著抑制UGTs活性;对UGT1A1活性,在RLM、HLM和重组酶(rh UGT1A1)孵育体系中,芫花醇提物均表现为中等强度的抑制作用,以羟基芫花素计,半数抑制浓度分别为46.32、32.49、8.382μmol/L,抑制类型分别为竞争性抑制作用、反竞争性抑制作用和竞争性抑制作用。结论芫花醇提物对不同肝微粒体孵育体系中UGTs及UGT1A1活性均可产生抑制作用且存在种属差异性,这种抑制作用可能是芫花致肝损伤的机制之一。
Objective To investigate the effects of ethanol extract of genkwa on the activities of UGTs and UGT1A1 in order to provide basis for elucidating the toxic mechanism of genkwa. Methods The in vitro liver microsomal incubation model was established. The activities of UGTs were detected by using 4-nitrophenol as substrate, UGT1A1 activity by β-estradiol as substrate, and substrate and metabolite contents by UV and HPLC. Results The contents of apigenin, hydroxy-genkwanin and genkwanin in the three flavonoids from the ethanol extract of Daphne genkwa were 6.34%, 8.72% and 6.06%, respectively, and the total diterpene content was 31.40%. The results of in vitro experiments showed that the extract from Daphne genkwa both could significantly inhibit the activity of UGTs in the rat liver microsomes (RLM) and human liver microsomes (HLM) incubation system. The activities of UGT1A1 in RLM, HLM and rh UGT1A1 ) Incubation system, both of the ethanol extract of Daphne genkwa exhibited moderate inhibitory effects. The half-inhibitory concentrations of hydroxylabeled geranylol were 46.32,32.49 and 8.382μmol / L, respectively, and the inhibition types were competitive inhibition Competitive inhibition and competitive inhibition. Conclusions The ethanol extract of Daphne genkwaensis could inhibit the activities of UGTs and UGT1A1 in different liver microsomal incubation systems and there are some species differences. This inhibition may be one of the mechanisms of liver injury induced by Daphne genkwa.