白及有效成分Militarine在肝微粒体中的体外代谢途径及其酶促动力学特征

in vitro Metabolism Pathway of Effective Component of Bletilla striata as Militarine in Liver Microsomes and Kinetics Characteristics of Enzyme-catalyzed Reactions

目的:研究白及有效成分Militarine在肝微粒体中的体外代谢途径及其酶促反应动力学特征。方法:建立大鼠和人肝微粒体体外孵育体系,对Militarine进行体外孵育反应;采用超高效液相色谱-四极杆-飞行时间质谱联用技术,结合UNIFI数据库并参考文献对其代谢产物进行结构鉴定。同时,以葛根素为内标,采用超高效液相色谱-三重四极杆串联质谱联用技术对Militarine在大鼠肝微粒体中的代谢转化量进行定量分析,通过GraphPad Prism 5.0软件拟合代谢转化曲线,并计算在有/无还原性辅酶Ⅱ(NADPH)参与的反应条件下的Militarine酶促动力学参数[最大清除速率(v_(max))、米氏常数(k_m)、固有清除率(CL_(int))]。结果:Militarine在大鼠和人肝微粒体中孵育后,生成了1个化学式为C_(21)H_(29)O_(11)的化合物,推测其为Militarine的酯键水解代谢产物。酶促动力学研究显示,有/无NADPH参与的Militarine酶促反应的v_(max)分别为1.955、2.129 nmol/(h·mg),k_m分别为8.601、9.854 nmol/mL,CL_(int)分别为0.227 3、0.216 1 mL/(h·mg),两者无明显差异。结论:Militarine在肝微粒体中的主要代谢途径为C1或C4位酯键的水解;其代谢不依赖于由NADPH启动反应的细胞色素P_(450)酶代谢途径。

OBJECTIVE:To study in vitro metabolism pathway of effective component of Bletilla striata as Militarine in liver microsomes and kinetics characteristics of enzyme-catalyzed reactions.METHODS:The in vitro incubation system of rat and human liver microsomes was established,and incubation reaction of Militarine was performed.UPLC-QTOF-MS was used to identify the structure of its metabolites in combination with UNIFI database and references.Using puerarin as internal standard,UPLC-Triple Quad-MS was used to quantitatively analyze metabolic transformation of Militarine in rat liver microsomes.The kinetic parameters(vmax,km,CLint)of Militarine enzyme-catalyzed reactions with/without reducing coenzymeⅡ(NADPH)were calculated by fitting the curves with GraphPad Prism 5.0 software.RESULTS:After incubation in rat and human liver microsomes,Militarine produced a chemical formula C21H29O11,which was presumed to be a metabolite of Militarine ester bond hydrolysis.The kinetic study of enzyme-catalyzed reactions showed that vmax of Militarine enzyme-catalyzed reactions with/without NADPH were 1.955,2.129 nmol/(h·mg);km were 8.601,9.854 nmol/mL;CLint were 0.227 3,0.216 1 mL/(h·mg);there was no significant difference between with NADPH and without NADPH.CONCLUSIONS:The main metabolic pathway of Militarine in liver microsomes is the hydrolysis of C1 and C4 ester bonds.Its metabolism does not depend on the pathway of cytochrome P450 enzymes initiated by NADPH.