HPLC法测定珊瑚菜内酯在大鼠肝微粒体生物转化的时程曲线  

Time course of phellopterin biotransformation in rat liver microsomes determined by HPLC

作　　者：赵爱红[1,2] 杨秀伟[1] ZHAO Ai-hong YANG Xiu-wei(State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China)

机构地区：[1]北京大学药学院天然药物及仿生药物国家重点实验室,北京100191 [2]兰州理工大学生命科学与工程学院,兰州730050

出　　处：《中国新药杂志》2017年第15期1837-1842,共6页Chinese Journal of New Drugs

基　　金：国家自然科学基金(81473321);"十二五"国家科技支撑专项(2011BAI07B08);北京市自然科学基金(7152086)

摘　　要：目的:建立高效液相色谱(HPLC)分析方法研究珊瑚菜内酯在大鼠肝微粒体生物转化的时程曲线,并对转化产物E-5-甲氧基紫罗绒苔素乙酸酯(M3)和Z-5-甲氧基紫罗绒苔素乙酸酯(M4)进行朔源。方法:应用HPLC分析法测定珊瑚菜内酯的大鼠肝微粒体生物转化中的原形及其转化产物白当归素的含量。色谱柱为DiamonsilTMODS C18(250 mm×4.6 mm,5μm);流动相为甲醇-水,梯度洗脱,流速为1.0 m L·min-1;检测波长为315 nm;柱温为25.0℃。通过转化产物不同萃取方法的比较,朔源M3和M4。结果:绘制了珊瑚菜内酯在大鼠肝微粒体体外的生物转化时程曲线,在60 min内下降较快,在60~240 min内基本稳定。其转化产物白当归素在60 min时达最大值,然后开始下降,到120 min时基本稳定。在大鼠肝微粒体温孵培养物中,珊瑚菜内酯和白当归素分别在600~1 800和0.2~2.4μg·m L-1的浓度范围内线性关系良好(r2>0.999),方法的精密度和稳定性RSD均小于15%;回收率分别为88.6%~92.9%和79.0%~89.5%,RSD均小于15%。结论:本研究建立的分析方法经方法学验证,可对大鼠肝微粒体温孵培养物中珊瑚菜内酯和白当归素含量进行测定,快速、准确、灵敏;珊瑚菜内酯在大鼠肝微粒体体外的生物转化缓慢;M3和M4是"人工"转化产物。Objective： To establish an HPLC method for determining time course of phellopterin biotransformation in rat liver microsomes in vitro,and to trace the source of the products E-5-methoxytrichoclin acetate（ M3） and Z-5-methoxytrichoclin acetate（ M4）. Methods： Phellopterin and its biotransformation product,byakangelicin,in rat liver microsomal incubation were measured by HPLC. The separation was performed on a DiamonsilTMODS C18column（ 250 mm × 4. 6 mm,5 μm） with mobile phase consisted of methanol and water in a gradient manner at a flow rate of 1. 0 m L·min-1. The detection wavelength was set at 315 nm and the column temperature was 25. 0 ℃. The products M3 and M4 were investigated for tracing the source by comparison of different extraction methods. Results： The time course of phellopterin biotransformation in rat liver microsomes in vitro showed a faster decline in less than 60 min,and then was basically stable between 60 min and 240 min. Theconcentration of its biotransformation product,byakangelicin,reached the maximum levels at 60 min of incubation,then slowly declined,and was basically stable at 120 min. Excellent linearity（ r2〉0. 999） was observed within their linear ranges,with 600 ~ 1 800 μg·m L-1for phellopterin and 0. 2 ~ 2. 4 μg·m L-1for byakangelicin. The RSDs of precision and stability of the developed method were less than 15%. The recoveries of the method were88. 6% ~ 92. 9% for phellopterin and 79. 0% ~ 89. 5% for byakangelicin,respectively,with RSDs less than 15%in all cases. Conclusion： The developed method was proved by methodological validation. It is rapid,reliable and sensitive,and can be used for content determination of phellopterin and byakangelicin in rat liver microsomal incubation. The biotransformation of phellopterin in rat liver microsomal incubation is slow. M3 and M4 are yielded during extraction process of rat liver microsomal incubation of phellopterin with solvent ethyl acetate,and may be artificial transformation products.

关 键 词：珊瑚菜内酯 白当归素 大鼠肝微粒体 生物转化 时程曲线 

分 类 号：R969.1[医药卫生—药理学]