HPLC-MS/MS测定葛根芩连汤对大鼠肝脏细胞色素P450酶的影响  

Effect of Gegen Qinlian decoction on hepatic cytochrome CYP450 isozymes in rats by HPLC-MS / MS

作　　者：刘自华[1] 安叡[1] 张艺竹[1] 顾青青[1] 尤丽莎[1] 王新宏[1] 

机构地区：[1]上海中医药大学,上海201203

出　　处：《中国中药杂志》2015年第15期3072-3080,共9页China Journal of Chinese Materia Medica

基　　金：2012年度上海市自然科学基金面上项目(12ZR1431500);上海中医药大学预算内项目(2014YSN08)

摘　　要：研究葛根芩连汤及其主要药效成分对大鼠肝微粒体CYP450酶5种同工酶活性的影响。采用体外肝微粒体孵育技术将葛根芩连汤及其主要药效成分与各个探针底物共同孵育,利用LC-MS/MS建立CYP450 5种亚型探针底物的代谢产物的分析方法,考察微粒体蛋白浓度和孵育时间与代谢产物生成量之间的关系来确定蛋白浓度和孵育时间。运用HPLC-MS/MS检测CYP450酶5种同工酶特异性探针底物(非那西汀、甲苯磺丁脲、右美沙芬、氯唑沙宗、睾丸酮)相应代谢产物(对乙酰氨基酚、4-羟基氯唑沙宗、右菲烷、6-羟基氯唑沙宗、6β-羟基睾酮)的生成速率(V)来确定CYP450的各亚酶活性。结果表明,对乙酰氨基酚、4-羟基甲苯磺丁脲、右菲烷、6-羟基氯唑沙宗及6β-羟基睾酮线性关系良好,精密度、稳定性、平均回收率符合要求,所建方法可行;优化的肝微粒体体外孵育条件,符合药物相互作用指导原则的要求;葛根芩连汤对CYP450 5个亚型(CYP1A2,CYP2C11,CYP2D2,CYP2E1,CYP3A1/2)酶均有不同程度的抑制,其主要药效成分中小檗碱在高浓度下可对各亚型酶有一定的抑制(CYP1A2,CYP3A1/2除外)。To study the effect of Gegen Qinlian decoction and its major effective components on five hepatic microsomal CYP450 isozymes in rats. The in vitro hepatic microsomal incubation technique was used to co-culture Gegen Qinlian decoction and its major effective components together with each probe substrate. HPLC-MS / MS was used to establish the analytical method for metabolites of the five isoform probe substrates of CYP450 isozymes,detect the linearity among micoromal protein concentration,incubation time and metabolite formation amount. And HPLC-MS / MS was applied to determine the formation rate（ V） of corresponding metabolites（ acetaminophen,4-OH-chlorzoxazone,dextrophan,6-OH-chlorzoxazone and 6β-hydroxytestosterone） specific probe substrates of the five isoform probe substrates of CYP450 isozymes（ phenacetin,polbutamide,dextromethorphan,chlorzoxazone,testosterone）,in order to determine the activity of each isozyme. The result showed good linearity among acetaminophen,4-OH-tolbutamide,dextrophan,6-OHchlorzoxazone and 6β-hydroxytestosterone,satisfactory precision,stability and average recovery,suggesting the method was feasible. The optimized in vitro microsomal incubation conditions conformed to the requirements in the guideline of drug-drug interaction. Gegen Qinlian decoction showed different degrees of inhibitor effect on 5 CYP450 isoforms（ CYP1A2,CYP2C11,CYP2D2,CYP2E1,CYP3A1 /2）. Its major effective component berberine could inhibit each CYP450 isoform at high concentrations（ except for CYP1A2,CYP3A1 /2）.

关 键 词：葛根芩连汤 细胞色素P450 肝微粒体 葛根素 黄芩苷 

分 类 号：R285.5[医药卫生—中药学]