Dihydromethysticin, a natural component from Piper methysticum Forst, has been reported to display pharmacological effects in mental disorders and some malignant tumors. However, the metabolism of this component remained unknown. The goal of this work was conducted to discover the metabolic profiles of dihydromethysticin. The in vitro incubation was performed by incubating dihydromethysticin with rat, monkey, and human liver microsomes and hepatocytes. An analytical assay of ultra-high performance liquid chromatography combined with Orbitrap high-resolution mass spectrometry was utilized to detect and identify the metabolites. With high resolution mass spectrometric determination, the accurate mass, elemental composition, and product ions of the metabolites were determined, which enabled structural characterization to become easy. Under the present conditions, four phase-I metabolites, as well as six phase-II metabolites, were detected and their tentative structures were characterized by mass spectra. M4 was found as the most abundant metabolite both in liver microsomes and hepatocytes. Cytochrome P450 1A2, 2C9, and 3A4 contributed to the formation of this metabolite by using human recombinant P450 enzymes. M4 can be oxidized into reactive ortho-quinone intermediate followed by conjugating with glutathione. M4 was also subject to glucuronidation (M1 and M2) and methylation (M5). Demethylenation, oxidation, hydroxylation, glucuronidation, glutathionylation, and methylation were the primary metabolic pathways of dihydromethysticin. This study provides in vitro metabolism data of dihydromethysticin, which is indispensable for understanding the disposition of this compound.
Keywords: Dihydromethysticin; cytochrome P450; metabolic pathway; metabolite characterization.
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