Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (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), i.e., 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 in vitro and in vivo, 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, S-cysteine conjugation and N-acetylcysteine conjugation were considered as unique metabolic pathways of OST, and phosphorylation, S-cysteine conjugation and N-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.

中文翻译：

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使用与四极杆飞行时间质谱联用的超高效液相色谱法鉴定大鼠蛇床子素代谢物的非靶向 SWATH 采集模式†

蛇床子素 (OST)，7-methoxy-8-isopentenoxycoumarin，是在蛇床子(L.) Cuss 中发现的特征成分。并具有优异的药理活性，包括抗癌、抗凋亡和神经保护。在本研究中，一种基于超高效液相色谱耦合四极杆飞行时间质谱 (UHPLC-Q-TOF-MS) 和具有主成分变量分组 (PCVG) 的 MetabolitePilot2.0™ 软件的快速可靠方法) 过滤首先用于观察 OST 的可能代谢物。高分辨率海量数据采用数据无关采集模式（DIA）采集，即，所有理论碎片光谱（SWATH）的顺序窗口采集，可以显着提高低水平和痕量代谢物的命中率。一种新的数据处理方法“关键产物离子 (KPI)”被用作代谢物快速搜索和鉴定的辅助工具。体外和体内共检测到72种OST代谢物，其中大鼠肝微粒体（RLMs）39种，血浆20种，胆汁32种，尿液32种，粪便37种。结果表明，单氧化、去甲基化、脱氢、硫酸盐结合和葡糖苷酸结合是OST的主要代谢反应。更显着的是，氧化分解、3,4-环氧化物-醛基化、磷酸化、S-半胱氨酸结合和N-乙酰半胱氨酸结合被认为是OST的独特代谢途径，首次在大鼠生物样品中表征磷酸化、 S-半胱氨酸结合和N-乙酰半胱氨酸结合反应。活性代谢物的制备将有助于阐明 OST 的潜在生物学机制，所提出的代谢途径可能有助于进一步了解简单香豆素的安全性和有效性。