Liao Man, Diao Xinpeng, Cheng Xiaoye, Sun Yupeng, Zhang Lantong
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University 361 East Zhongshan Road Shijiazhuang Hebei 050017 P. R. China
RSC Adv. 2018 Apr 19;8(27):14925-14935. doi: 10.1039/c8ra01221k. eCollection 2018 Apr 18.
Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, anti-apoptosis and neuroprotection. In this study, a rapid and reliable method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and MetabolitePilot2.0™ software with principal component variable grouping (PCVG) filtering was developed to observe probable metabolites of OST firstly. The high resolution mass data were acquired by data-independent acquisition mode (DIA), , sequential window acquisition of all theoretical fragmentation spectra (SWATH), which could significantly improved the hit rate of low-level and trace metabolites. A novel data processing method 'key product ions (KPIs)' were employed for metabolites rapid hunting and identification as an assistant tool. A total of 72 metabolites of OST were detected and , including 39 metabolites in rat liver microsomes (RLMs), 20 metabolites in plasma, 32 metabolites in bile, 32 metabolites in urine and 37 metabolites in feces. The results showed that mono-oxidation, demethylation, dehydrogenation, sulfate conjugation and glucuronide conjugation were major metabolic reactions of OST. More significant, oxydrolysis, 3,4-epoxide-aldehylation, phosphorylation, -cysteine conjugation and -acetylcysteine conjugation were considered as unique metabolic pathways of OST, and phosphorylation, -cysteine conjugation and -acetylcysteine conjugation reactions were characterized in rat biological samples for the first time. Preparation of active metabolites will be greatly helpful in elucidating the potential biological mechanism of OST, and the proposed metabolic pathways of it might provide further understanding of the safety and efficacy of simple coumarins.
蛇床子素（OST），即7-甲氧基-8-异戊烯氧基香豆素，是蛇床（学名：Cnidium monnieri (L.) Cuss.）中的特征性成分，具有出色的药理活性，包括抗癌、抗凋亡和神经保护作用。在本研究中，首先开发了一种基于超高效液相色谱-四极杆飞行时间质谱联用（UHPLC-Q-TOF-MS）以及带有主成分变量分组（PCVG）过滤功能的MetabolitePilot2.0™软件的快速可靠方法，以观察OST可能的代谢产物。高分辨率质谱数据通过数据非依赖采集模式（DIA），即所有理论碎片谱的顺序窗口采集（SWATH）获得，这可以显著提高低水平和痕量代谢产物的命中率。一种新颖的数据处理方法“关键产物离子（KPI）”被用作辅助工具，用于代谢产物的快速搜寻和鉴定。共检测到72种OST的代谢产物，其中包括大鼠肝微粒体中的39种代谢产物、血浆中的20种代谢产物、胆汁中的32种代谢产物、尿液中的32种代谢产物和粪便中的37种代谢产物。结果表明，单氧化、去甲基化、脱氢、硫酸酯结合和葡萄糖醛酸结合是OST的主要代谢反应。更重要的是，氧水解、3,4-环氧化物-醛基化、磷酸化、半胱氨酸结合和乙酰半胱氨酸结合被认为是OST独特的代谢途径，并且磷酸化、半胱氨酸结合和乙酰半胱氨酸结合反应首次在大鼠生物样品中得到表征。活性代谢产物的制备将极大地有助于阐明OST的潜在生物学机制，其提出的代谢途径可能为进一步理解简单香豆素的安全性和有效性提供帮助。
