聚乙二醇400对黄芩素在不同种属肝微粒体上代谢转化的影响

Effect of PEG400 on the metabolic transformation of baicalein in liver microsomes of different species

目的:建立一种UPLC-MS/MS的分析方法,考察PEG400对肝微粒体孵育黄芩素后其代谢转化的影响,并比较其在不同种属肝微粒体中的代谢特征。方法:基于体外肝微粒体孵育体系,分别将不同摩尔浓度(2.5,5,10,20μmol·L-1)的黄芩素于不同摩尔浓度(0,2,4,8μmol·L-1)的PEG400干预下的肝微粒体中进行37℃恒温孵育60 min,再加入含有内标(染料木素)的冰冷乙腈终止反应,以UPLC-MS/MS正离子模式监测孵育体系中黄芩素及其代谢产物的质量浓度,并根据质量浓度求算酶活性剩余百分含量及其参数。用于定量分析的离子对分别为m/z 447~271(黄芩苷和B6G);m/z 271~172(黄芩素);m/z 271~153(内标)。结果:基于肝微粒体体外孵育结果分析,在鼠、人和犬的肝微粒体孵育后B6G的vmax与黄芩苷的vmax都存在不同,表明黄芩素在不同种属肝微粒中的代谢转化存在差异;PEG400共孵育后,B6G与黄芩苷的vmax分别得到提高,表明了黄芩素的葡萄糖醛酸化程度加快,单位时间内催化生成的B6G与黄芩苷更多;Km降低,表明PEG400能够诱导UGT1A8与UGT1A9的酶活性,使药物与酶的亲和力加强;CLint的改变,表明药物在不同种属微粒体内的代谢稳定性不同。结论:黄芩素通过在不同种属肝微粒体的葡萄糖醛酸化后,其代谢产物存在着适度的差异,其中差异程度体现为:人>大鼠>犬。此外,PEG400能够通过诱导UGT1A8与UGT1A9的活性影响黄芩素的体外葡萄糖醛酸化水平,使单位时间内催化的代谢产物量增加。

OBJECTIVE To establish a analysis method of UPLC-MS/MS to investigate the effect of PEG400 on the metabolic transformation of baicalein in liver microsomes,and to compare its metabolic characteristics in different species of liver microsomes;METHODS Baicalein at different concentrations were dissolved in liver microsomes incubation system of rat,dog and human treated with different concentrations of PEG400;after incubation at 37℃for 60 min,the reaction was terminated with cold acetonitrile.Using genistein as the internal standard,the concentration of baicalein and its metabolites in different incubation systems were determined by UPLC-MS/MS,and the residual percentage and enzyme kinetic parameters in different incubation systems were calculated by mass concentration.The ion pairs for quantitative analysis were m/z 447-271 for baicalin and B6G,m/z 271-172 for baicalein and m/z 271-153 for the internal standard.RESULTS Based on the analysis results,the vmax of B6G and baicalin were different in rat,human and dog liver microsomes incubation systems,indicating the differences in the metabolic transformation of baicalein in different species of liver microsomes.With co-incubation with PEG400,the vmax of B6G and baicalin were both increased,indicating that the degree of glucuronidation of baicalein was accelerated,and more B6G and baicalin were generated in unit time;the decrease of Km indicated that PEG400 could induce the enzyme activity of UGT1A8 and UGT1A9,and enhance the affinity between drugs and enzymes.The changes of CLint indicated that the metabolic stability of drugs in different species of liver microsomes was different.CONCLUSION The metabolites of baicalein glucuronide in different species of liver microsomes are different,and the degree of difference is reflected in human>rat>dog.PEG400 is able to affect the glucuronidation level of baicalein in vitro by inducing the activity of UGT1A8 and UGT1A9,further increase the amount of catalyzed metabolite per unit time.