Systematic screening and characterization of prototype constituents and metabolites of total astragalosides using HPLC-ESI-IT-TOF-MS after oral administration to rats.

Astragalosides (AGs) are the main bioactive constituents in Astragali Radix (AR), and have a wide range of pharmacological properties, including immunoregulatory, cardioprotective, neuroprotective, antioxidative, antidiabetic, and antinociceptive effects. However, the metabolism of total AGs remains unclear. To clarify the metabolic fate of AGs after oral administration to rats, total AGs were isolated from AR extracts using AB-8 macroporous resin chromatography and preparative HPLC, and then analyzed using HPLC-DAD-ELSD and LC-MS. HPLC-ESI-IT-TOF-MS was used to systematically screen and characterize prototype constituents and metabolites of total AGs in rat feces, urine, and plasma samples. As a result, 123 AG-related compounds from feces were detected and structurally characterized. Among the 123 compounds, 107 were phase I metabolites, of which 91 were new metabolites, and 73 were new compounds. In addition, six prototype constituents in urine, and one in plasma were detected. The main metabolic sites in the structure of cycloastragenol (CAG), the aglycone of AGs, were found to be the 9, 19-cyclopropane ring (E ring) and the 20, 24-furan ring (F ring). The cleavage mode of CAG derivatives in negative ion mode was identified, and was found to be highly dependent on the integrity of the E ring. Mono- to tetra-hydroxylated and carboxyl substituted metabolites were tentatively identified. Deglycosylation, hydroxylation, dehydrogenation, isomerization, ring cleavage, and carboxyl substitution were considered to be the major metabolic reactions involved in the formation of the metabolites, among which carboxyl substitution was a novel metabolic reaction. In summary, after total AGs were orally administered to rats, their constituents were extensively metabolized in a phase I manner, and the metabolites were excreted mainly into feces. To our knowledge, this is the first systematic study on the metabolism of total AGs. The results give us insight into the metabolic profiles of total AGs in vivo, and provide a foundation for identifying effective forms of AGs and exploring their mechanism in future studies.