虫草素体外大鼠肝微粒体代谢初步研究

目的：建立大鼠肝微粒体药物代谢酶体外转化模型，探讨虫草素的体外代谢情况，为规模化制备虫草素的代谢产物提供方法。方法：将虫草素与大鼠肝微粒体药物代谢酶共温孵，用液质联用的方法检测肝微粒体中虫草素代谢产物（2种生物碱类，2种葡萄糖醛酸结合产物，1种谷氨酰胺结合产物）。色谱条件：采用Diamonsil^TM ODS C₁₈（250 mm×4.6 mm，5 μm）色谱柱分离，以含0.1%甲酸的甲醇-含0.1%甲酸的水（15：85）为流动相，以0.5 mL·min^-1的流速进行洗脱，洗脱时间为32 min，进样量10 μL；质谱条件：电喷雾离子源（ESI源），正离子模式检测。采用虫草素代谢产物的MS和MS/MS以及MS-DIAL V1.57，MassLynx V4.1和CFM-ID等软件和本项目组建立的数据库来推测其化学结构式。结果：虫草素在大鼠肝微粒体药物代谢酶作用下可以被代谢，并且发现了8个代谢产物，其中鉴定7个代谢产物的分子式。在已鉴定的7个分子式中，确定了4个代谢物的结构式和1个与谷氨酰胺结合的代谢产物，确定的代谢产物分别是4-羧酸咪唑葡萄糖醛酸结合物，3，4，5-三甲氧基氧代四氢呋喃-2-羧酸葡萄糖醛酸结合物，C₆H₅O₂谷氨酰胺结合物，呋喃-2-甲醇葡萄糖醛酸结合物和氧化虫草素。结论：建立的肝微粒体药物代谢酶模型可靠有效，可用于虫草素的体外代谢研究。

Objective:To establish a transformation model of rat liver microsomes drug metabolic enzymes in vitro, to discuss the metabolism of the drug in vitro,and to provide methods for large-scale preparation of cordycepin metabolites.Methods:Cordycepin and rat liver microsomes drug metabolic enzymes were incubated together,and the metabolites were detected by the method of high performance liquid chromatography tandem mass spectrometry,including two kinds of alkaloids,two kinds of glucuronide conjugation compounds and one glutamine conjugation compound.Chromatographic separation was performed on a C₁₈ reversed-phase HPLC column(Diamonsil^TM ODS C₁₈ 250 mm×4.6 mm,5 μm).The ratio of the mobile phase consisting of methanol with 0.1% formic acid and an aqueous solution with 0.1% formic acid was 15:85.The flow rate was 0.5 mL·min^-1.Each analytical run was 32 minutes and the injection volume was 10 μL.For mass detection,the electrospray ionization source(ESI)and the positive ion electrospray mode were operated.The chemical structure of cordycepin metabolites were speculated by using the MS and MS/MS spectra,related softwares such as MS-DIAL V1.57,MassLynx V4.1 and CFM-ID and the database established by our project team.Results:Cordycepin in rat liver microsomes could be metabolized with drug metabolic enzymes.Eight metabolites were found wherein the molecular formula of seven metabolites were identified.Four metabolites and one glutamine binding product were identified among these seven metabolites.The identified metabolites were 4-carboxylic acid imidazole glucuronide,3,4,5-trimethoxytetrahydrofuran-2-carboxylic acid glucuronide,C₆H₅O₂ glutamine conjugation,furan-2-ylmethanol glucuronide and oxycordycepin.Conclusion:It is reliable and effective to establish the model of liver microsomes drug metabolic enzyme,which can be used in the metabolism of cordycepin in vitro.