目的 建立测定肝微粒体孵育体系中诃子酸浓度的方法，并比较其在人、犬、猴、小鼠、大鼠肝微粒体中的Ⅰ相、Ⅱ相代谢稳定性及种属差异，确定其在人肝微粒中的代谢表型。方法 将诃子酸与不同种属肝微粒体共同孵育，应用UPLCMS/MS检测孵育液中诃子酸的含量，考察其代谢稳定性及体外动力学参数。采用化学抑制剂法确定其在人肝微粒中的代谢表型。将诃子酸与各CYP450同工酶CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1和CYP3A4的特异性抑制剂（α-萘黄酮、香豆素、磺胺苯吡唑、噻氯匹定、奎尼丁、二乙基二硫代氨基甲酸钠、酮康唑）共同孵育，确定其代谢酶表型。结果 诃子酸在Ⅰ相、Ⅱ相孵育体系中均可代谢，在Ⅰ相代谢中，犬肝微粒体孵育与人最为相似，半衰期（t_1/2）分别为115.50 min和121.58 min；Ⅱ相代谢中5个种属代谢稳定性均中等，其中猴与人肝微粒体代谢趋势最为相近。诃子酸在人肝微粒中的代谢是由多种CYP酶共同介导的，其中CYP2C9、CYP2E1和CYP3A4是主要的同工酶。结论 建立的UPLCMS/MS方法简便、快速、专属性强、灵敏性高，可用于肝微粒体孵育体系中诃子酸浓度的测定及体外代谢的研究。诃子酸在人、犬、猴、小鼠、大鼠肝微粒体中代谢存在一定种属差异，且其代谢过程与多种CYP酶相关。

Objective To establish a method for the concentration determination of chebulinic acid (CA) in liver microsomes, and to compare its phase I and phase II metabolic stability and species differences in human, dogs, monkeys, mice and rats liver microsomes, to predict the metabolic phenotypes of CA in human liver microsomes. Methods The remaining concentrations of CA incubated in various species of liver microsomes were determined to evaluate their metabolic stability and in vitro pharmacokinetic parameters. Selective chemical inhibition was used to predict the metabolic phenotypes of CA in human liver microsomes. CA was incubated with specific inhibitors of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 isoforms (α-naphthoflavone, coumarin, sulfaphenazolum, ticlopidine, quinidine, sodium diethyldithiocarbamatre, ketoconazole) to determine the phenotypes of metabolic enzymes. Results CA can be metabolized in phase I and phase II incubation systems. In phase I metabolism, the metabolic properties of dogs and human liver microsomes were the most resembles, and the half-lives are 115.50 min and 121.58 min, respectively. Metabolic stability in phase Ⅱ metabolism is moderate, and dogs, monkeys and human liver microsomes have the similar metabolic characteristic. CA is metabolized by a variety of enzymes in human liver microsomes, among which CYP2C9, CYP2E1 and CYP3A4 are the primary isoenzymes. Conclusion A UPLC-MS/MS method was established to determine the concentration of CA, and it is simple, rapid and specific. It can be used for the concentration determination of CA in liver microsomes incubation system. The metabolic characteristics of the compound are different among human, dogs, monkeys, mice and rats liver microsomes. It is metabolized by a variety of enzymes in human liver microsomes.

R285.5

中央高校基本科研业务费专项资助项目(3332021064);国家重点研发计划资助项目(2018YFC1708203);内蒙古自治区科技重大专项资助(2019ZD004)