染料木苷在大鼠肝微粒体中的代谢及酶促反应动力学研究  

Analysis of the Metabolites of Genistin and Enzyme Kinetics of Genistin in Rat Liver Microsome

作　　者：贾佩佩[1] 张晓旭[1] 张智勇[1] 孙宇鹏[1] 张兰桐[1] 

机构地区：[1]河北医科大学药学院,石家庄050017

出　　处：《中国药学杂志》2015年第9期797-801,共5页Chinese Pharmaceutical Journal

摘　　要：目的建立HPLC-MS法,初步鉴定染料木苷在大鼠肝微粒体中的代谢产物,并研究其酶促反应动力学。方法建立大鼠肝微粒体体外孵育体系对染料木苷进行代谢研究,应用HPLC-MS鉴定其体外代谢产物。以磺胺甲唑为内标,采用代谢物生成法,对代谢产物染料木素进行定量测定。应用Graph Pad Prism 5.0软件分析数据,计算酶促反应动力学常数Vmax和Km。结果染料木苷在体外孵育体系中生成代谢产物,经鉴定其为苷元染料木素。染料木苷在大鼠肝微粒体中最佳温孵时间为40 min,最佳蛋白质量浓度为1 mg·m L-1,底物浓度为50μmol·L-1,代谢产物染料木素酶促反应的Vmax=(0.104 2±0.003 3)μmol·min-1·mg(pro)-1,Km=(28.96±2.80)μmol·L-1。结论染料木苷在大鼠肝微粒体中通过水解反应,生成相应苷元。代谢物生成法是一种可靠、简便测定肝微粒体酶促反应动力学参数的方法,所得到的酶促反应动力学参数为染料木苷进一步研究提供了重要参数。OBJECTIVE To characterize the metabolism of genistin and study its enzymatic kinetics in rat liver microsome by HPLC-MS. METHODS Genistin was incubated with rat liver microsomal incubation system. HPLC-MS method was used to charac- terize the metabolites. A metabolite generation method was established for quantitative analysis of genistein with sulfamethoxazole as in- temal standard. The enzyme kinetics parameters Vm ax and Km was calculated by the GraphPad Prism 5.0 software. RESULTS The me- tabolites in vitro incubation system was identified as genistein. The optimal time in rat liver microsomes incubation time of 40 min, the optimal protein concentration of 1 mg · mL- 1, a substrate concentration of 50 μmol · L-1. The enzyme kinetics parameters of genistein were as follows： Vmax = （0. 104 2 ±0.003 3） μmol· min-1 · min（pro） -1, K,n = （28.96 ±2. 80） μmol· L-1. CONCLUSION The results indicate that genistin can be metabolited as the form of hydroxylation in rat liver microsome. Metabolite generation method is a reliable and simple method for determination of kinetic parameters of hepatic microsomal enzymes, and enzyme kinetic parameters obtained of genistin provide important parameters for further study.

关 键 词：染料木苷 染料木素 肝微粒体 HPLC-MS 酶促反应动力学 

分 类 号：R969.1[医药卫生—药理学]