Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.
Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N-methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo.
Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, antiapoptosis and neuroprotection. In this study, a rapid and reliable method based on ultra-highperformance 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. Fig. 1 Chemical structure of osthole.
The metabolic pathways of pectolinarin, linarin and pectolinarigenin were summarized. This study not only proposed a practical strategy for rapidly screening and identifying metabolites but also provided useful information for further pharmacological studies and the design of new drugs based on Cirsium japonicum DC.
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