基于HPLC-MS/MS法研究马兜铃酸Ⅰ和马兜铃酸Ⅱ的大鼠肝微粒体酶S9体外代谢

Research of Aristolochic AcidⅠ,Aristolochic Acid Ⅱ and Their Metabolites in Rat Liver Microsomes Using HPLC-MS/MS

马兜铃酸Ⅰ和马兜铃酸Ⅱ广泛存在于马兜铃属植物中,具有肾损伤毒性和致癌致突变毒性。为了研究马兜铃酸类化合物的毒性产生机制,建立了马兜铃酸Ⅰ和马兜铃酸Ⅱ及其大鼠肝微粒体酶S9代谢物分离与鉴定的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。采用体外代谢法分别研究了有氧和厌氧孵育条件下,马兜铃酸Ⅰ和马兜铃酸Ⅱ在大鼠肝微粒体酶S9作用下的代谢行为,并对各代谢物进行结构表征。研究结果显示:马兜铃酸Ⅰ在有氧条件下生成的代谢产物为马兜铃酸Ⅰa,在厌氧条件下生成的代谢产物为马兜铃内酰胺Ⅰ;马兜铃酸Ⅱ只在厌氧条件下生成了马兜铃内酰胺Ⅱ。马兜铃内酰胺类化合物的生成是马兜铃酸Ⅰ和马兜铃酸Ⅱ在肝微粒体酶S9代谢过程中毒性产生的主要原因。该结果对于更好地理解马兜铃酸Ⅰ和马兜铃酸Ⅱ的毒性作用机制,控制毒副反应的发生具有一定意义。

Aristolochic acids（AAs）,derived from aristolochia plant species,are a mixture of structural-related compounds.Aristolochic acid Ⅰ and aristolochic acid Ⅱ are reported to be the major components that are associated with kidney damage toxicity and carcinogenic mutagenicity toxicity.Since the toxicity mechanism of action of Aristolochic acids are still unclear,it is necessary to develop a sensitive analytical method to facilitate these studies.In this work,a rapid and sensitive method based on high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）was applied for the determination of aristolochic acidⅠ,aristolochic acid Ⅱ and their major metabolites in samples from in vitro metabolism studies.Aristolochic acid Ⅰ and aristolochic acid Ⅱwere incubated with rat liver microsome S9 fractions under aerobic and anaerobic conditions.The incubation samples were analyzed by HPLC-MS/MS.The obtained results showed that the in vitro metabolic pathways of aristolochic acid Ⅰ and aristolochic acidⅡ are different.The metabolites of aristolochic acid Ⅰ was detected and characterized as aristolochic acid Ⅰa under aerobic condition and aristololactam Ⅰ under anaerobic condition,respectively;however,in the case of aristolochic acidⅡ,the only metabolite of aristolochic acid Ⅱ was identified as aristololactam Ⅱ under the anaerobic condition.Besides,the fragmentation behaviors of aristolochic acid Ⅰ,aristolochic acid Ⅱ and their metabolites in rat liver microsomes S9 fractions were characterized and summarized.The results indicated that the production of aristololactams is the main cause of the toxicity of aristolochic acidⅠ and aristolochic acidⅡin the metabolism of rat liver microsome S9.This study have a certain significance for better understanding of the toxicity mechanism of action of aristolochic acid Ⅰ and aristolochic acid Ⅱ,as well as for toxicity monitoring of aristolochia plants.