Abstract

Isoneochamaejasmin A (INCA), a biflavonoid, is one of main active ingredients in the dried root of Stellera chamaejasme L., a widely used traditional Chinese medicine. In the present study, we identified the glucuronidation metabolite of INCA and characterized the UGTs responsible for INCA glucuronidation. 7-O-Glucuronide (M1) and 4'-O-glucuronide (M2) were identified by incubation of INCA with human liver microsomes (HLMs) in the presence of UDP-glucuronic acid, and their structures were confirmed by high-resolution mass spectrometry and nuclear magnetic resonance analyses. Although INCA is a single enantiomer molecule, its M1 metabolite showed two equal-size peaks on a πNAP stationary phase but only one peak on a C18 stationary phase, indicating that the 7-/7"- and 4'"-hydroxyl groups of INCA were in different spatial configurations relative to each other. Among the twelve recombinant human UGT isoforms test and correlation analysis, UGT1A1, 1A3, and 1A9 were found to mediate M1 formation while only UGT1A3 mediated M2 formation. Kinetic studies showed obvious species differences between human, mouse, rat, dog, and pig liver microsomes. HLMs, human intestinal microsomes, and UGT1A1 exhibited substrate inhibition for the formation of M1. UGT1A1-mediated formation of M1 showed a 6- and 11-fold higher V_max than did UGT1A3- and 1A9-mediated formation of M1, respectively. The results of relative activity factor assay showed that UGT1A1 contributed approximately 75% in the formation of M1. These findings collectively indicate that UGT1A1 is the major enzyme in the formation of M1, whereas UGT1A3 is the major enzyme in the formation of M2.