Gentiopicroside (GPS), the main bioactive component in Gentiana scabra Bge., has attracted our attention owing to its high bioactivity, especially the treatment of hepatobiliary disorders. The aglycone form of GPS, a typical secoiridoid glycoside, is considered to be more readily absorbed than its parent drug. This study aimed to identify and characterize the metabolites after GPS incubated with β-glucosidase in buffer solution at 37°C. Samples of biotransformed solution were collected and analyzed by ultraperformance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (Q-TOF MS). A total of four metabolites were detected: two were isolated and elucidated by preparative-HPLC and NMR techniques, and one of those four is reported for the first time. The mass spectral fragmentation pattern and accurate masses of metabolites were established on the basis of UPLC/Q-TOF MS analysis. Structure elucidation of metabolites was achieved by comparing their fragmentation pattern with that of the parent drug. A fairly possible metabolic pathway of GPS by β-glucosidase was proposed. The hepatoprotective activities of metabolites M1 and M2 were investigated and the results showed that their hepatoprotective activities were higher than that of parent drug. Our results provided a meaningful basis for discovering lead compounds from biotransformation related to G. scabra Bge. in traditional Chinese medicine.

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half-life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato-protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone-related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N-heterocyclization and N-acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by (1)H, (13)C and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q-TOF-MS analysis has provided an important analytical platform to gather metabolic profile of sweroside.

To test the metabolic mechanism, the ␤-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with -nitrophenyl-␤-D-glucopyranoside as a probe substrate, and the pathway of GPS biotransformation by strain 2T01Y01 is proposed. In addition, the hepatoprotective activities of GPS and metabolite compounds 2, 5, and 6 against human hepatocyte line HL-7702 injury induced by hydrogen peroxide were evaluated.