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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 establish a HPLC-MS method to identify the genomic products of genistin in rat liver microsomes and to study its enzymatic kinetics. Methods The rat liver microsome incubation system was established for the genomic analysis of genistin, and its metabolites were identified by HPLC-MS. To sulfamethoxazole as internal standard, the use of metabolite formation method, the quantitative determination of genistein metabolites. Data were analyzed using Graph Pad Prism 5.0 software and the enzymatic reaction kinetic constants Vmax and Km were calculated. Results Genistin produced a metabolite in an in vitro incubation system and was identified as aglycon genistein. Genistin in rat liver microsomes incubation time of 40 min, the best concentration of 1 mg · m L-1 protein concentration, the substrate concentration of 50μmol·L-1, genistein enzyme metabolites The Vmax of the reaction was (0.104 2 ± 0.003 3) μmol · min-1 · mg (pro) -1 and Km was (28.96 ± 2.80) μmol·L-1. Conclusion Genistin in rat liver microsomes through hydrolysis reaction, the corresponding glycoside. Metabolites generation method is a reliable and simple method for the determination of kinetic parameters of liver microsomal enzymatic reaction. The obtained kinetic parameters of enzymatic reaction provide important parameters for the further study of genistein.