芫花氯仿萃取物对大鼠及人肝微粒体UGTs及UGT1A1活性影响

In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1

考察芫花氯仿萃取物对尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及UGT1A1活性的影响,为预测芫花致肝损伤可能发生机制提供实验依据。采用体外肝微粒体孵育模型,分别以4-硝基酚和β-雌二醇为底物检测UGTs及UGT1A1活性;利用UV和HPLC测定底物或代谢物含量。结果表明,HPLC测得氯仿萃取物中3种黄酮类成分芹菜素、羟基芫花素和芫花素质量分数分别为1.00%,6.40%,18.38%;UV法测得总二萜质量分数为37.39%。与空白组相比,在大鼠肝微粒体(RLM)孵育体系,氯仿萃取物能显著抑制UGTs活性,而在人肝微粒体(HLM)孵育体系,抑制作用不明显;对UGT1A1,在RLM和HLM孵育体系中,氯仿萃取物均表现为中等强度的抑制作用(以芫花素计,IC50=8.76,10.36μmol·L-1);抑制类型分别为非竞争性抑制和反竞争性抑制。综上,氯仿萃取物对UGTs及UGT1A1活性均可产生抑制作用且存在种属差异性,推测这种抑制作用可能是芫花致肝损伤的机制之一。

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1 A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol （4-NP） and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18. 38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes （RLM） system, while the inhibitory effect was not obvious in human liver microsomes（HLM） system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes （based on genkwanin, IC50 = 8.76, 10. 36 μmol · L^-1 ）. The inhibition types were non-competitive inhibition（RLM） and uncompetitive inhibition（HLM）. In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.