Profiling and identification of the metabolites of calycosin in rat hepatic 9000 × g supernatant incubation system and the metabolites of calycosin-7-O-β-d-glucoside in rat urine by HPLC-DAD-ESI-IT-TOF-MSn technique

Calycosin and calycosin-7-O-β-d-glucoside are two main bioactive isoflavonoids in Astragali Radix. To profile the metabolites of calycosin in rat hepatic 9000 × g supernatant incubation system and the metabolites of calycosin-7-O-β-d-glucoside in rat urine, high performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-DAD-ESI-IT-TOF-MSn) technique was used. Totally, 24 new in vitro metabolites of calycosin and 33 new in vivo metabolites of calycosin-7-O-β-d-glucoside were identified. Monoglucosylation, monopentosylation, demethylation, dehydroxylation, dimerization, and trimerization were found to be new in vitro metabolic reactions of calycosin; hydroxylation and hydrogenation were new metabolic reactions of calycosin-7-O-β-d-glucoside in vivo. The major metabolic reactions of calycosin in rat hepatic 9000 × g supernatant incubation system were monohydroxylation on A-ring, dimerization (CO coupling), dimerization (CC coupling) and dehydroxylation; the major phase I metabolic reactions of calycosin-7-O-β-d-glucoside in rats were deglycosylation, hydroxylation, demethylation and dehydroxylation. Hydroxylation, dehydroxylation, and demethylation were common metabolic pathways to calycosin and calycosin-7-O-β-d-glucoside, and some of their metabolites formed through these reactions, such as 8-hydroxycalycosin (S10, M10), pratensein (5-hydroxycalycosin, S19, M27) and formononetin (S22, M28), daidzein (M22), 7,3′,4′-trihydroxyisoflavone (S13, aglycon of M3 and M8), equol (aglycon of M19 and M20) had been reported to have many bioactivities related to the pharmacological effects of calycosin and calycosin-7-O-β-d-glucoside. These findings would enhance understanding of the metabolism and real active forms of calycosin and calycosin-7-O-β-d-glucoside.