5,7-二甲氧基香豆素和6,7-二甲氧基香豆素在大鼠体内的代谢产物鉴定与代谢途径分析

Metabolites and metabolic pathway analysis of 5,7-dimethoxycoumarin and 6,7-dimethoxycoumarin in rats

目的采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap HRMS)技术对5,7-二甲氧基香豆素和6,7-二甲氧基香豆素在大鼠血浆,组织(心、肝、脾、肺、肾、胃、小肠),尿液和粪便中的代谢产物进行鉴定分析,并推测其在大鼠体内的代谢途径。方法雄性SD大鼠ig给予5,7-二甲氧基香豆素和6,7-二甲氧基香豆素(100 mg/kg),收集血浆、组织、尿液以及粪便样品。样品处理后,采用Thermo Scientific AccucoreTM C18色谱柱(100 mm×3 mm,2.6μm),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相梯度洗脱,在电喷雾电离源正离子模式下采集生物样品的质谱数据。采用Xcalibur采集数据,利用Compound Discoverer 3.1软件的化合物代谢产物预测模板进行分析,结合相关文献以及Mass Frontier 7.0软件对代谢产物进行鉴定。结果在大鼠的生物样本中检测到33个5,7-二甲氧基香豆素代谢产物,21个6,7-二甲氧基香豆素代谢产物。在血浆、尿液、粪便、心、肝、脾、肺、肾、胃、小肠中分别鉴定出8、28、23、2、10、3、10、13、7、7个5,7-二甲氧基香豆素代谢产物和7、19、14、7、7、4、7、13、3、8个6,7-二甲氧基香豆素代谢产物。结论通过液质联用技术推测出了33个5,7-二甲氧基香豆素代谢产物和21个6,7-二甲氧基香豆素代谢产物,阐明了2种香豆素在大鼠体内通过去甲基化、羟基化、水解、还原、甲基化、磺酸化、葡萄糖醛酸化等途径进行代谢,对2种香豆素的代谢特点进行了比较分析,为进一步研究中药中2种香豆素的作用机制和作用途径奠定了基础,同时也为药物代谢研究提供一种综合研究方法。

Objective To identify the metabolites of 5,7-dimethoxycoumarin and 6,7-dimethoxycoumarin in plasma,tissues(heart,liver,spleen,lung,kidney,stomach and small intestine),urine and feces in rats by using hybrid quadrupole-orbitrap high resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS).Methods SD male rats were ig 5,7-dimethoxycoumarin and 6,7-dimethoxycoumarin(100 mg/kg),the plasma,tissue,urine,and fecal samples were collected.After sample pretreatment,a Thermo Scientific AccucoreTM C18 column(100 mm×3 mm,2.6μm)was used,the gradient elution was performed with 0.1%formic acid-0.1%formic acid acetonitrileas the mobile phase.The mass spectrometry data of biological samples were acquired in positive ion mode with an electrospray ionization source(ESI source).Xcalibur was used to collect data,and the compound metabolite prediction template of Compound Discoverer 3.1 software was used for analysis,and the metabolites were identified in combination with relevant literature and Mass Frontier 7.0 software.Results A total of 335,7-dimethoxycoumarin metabolites and 216,7-dimethoxycoumarin metabolites were detected in biological samples from rats.A total of 8,28,23,2,10,3,10,13,7,7 metabolites of 5,7-dimethoxycoumarin and 7,19,14,7,7,4,7,13,3,8 metabolites of 6,7-dimethoxycoumarin were identified in plasma,urine,feces,heart,liver,spleen,lung,kidney,stomach and small intestine,respectively.Conclusion A total of 33 metabolites of 5,7-dimethoxycoumarin and 21 metabolites of 6,7-dimethoxycoumarin were identified by liquid chromatography-mass spectrometry.The metabolism of the two coumarins in rats was elucidated through demethylation,hydroxylation,hydrolysis,reduction,methylation,sulfation and glucuronidation.The metabolic characteristics of the two coumarins were compared and analyzed.This study laid a foundation for further study on the mechanism and pathway of action of the two coumarin in traditional Chinese medicine,and also provided a comprehensive research method for drug metabolism identification.