Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (L.Y., M.Z., X.Z., Y.L., H.H., H.J., S.Z.); Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (J.P., Y.C., S.C., J.C.); and Department of Pharmacy, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China (Q.Z.).
Drug Metab Dispos. 2014 Apr;42(4):735-43. doi: 10.1124/dmd.113.055962. Epub 2014 Jan 22.
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 UDP glucuronosyltransferases (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 C(18) stationary phase, indicating that the 7-/7''- and 4'-/4'''-hydroxyl groups of INCA were in different spatial configurations relative to each other. Among the recombinant human UGT isoform test and correlation analysis, UGT1A1, UGT1A3, and UGT1A9 were found to mediate M1 formation, whereas only UGT1A3 mediated M2 formation. Kinetic studies showed obvious species differences between human, mouse, rat, dog, and pig liver microsomes. UGT1A1, HLMs, and human intestinal microsomes, but not human kidney microsomes, 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 UGT1A9-mediated formation of M1, respectively. The results of the 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.
异牡荆黄素 A（INCA）是瑞香科狼毒属植物狼毒干燥根中的主要活性成分之一，是一种广泛使用的传统中药。在本研究中，我们鉴定了 INCA 的葡萄糖醛酸化代谢物，并鉴定了负责 INCA 葡萄糖醛酸化的 UDP 葡萄糖醛酸基转移酶（UGTs）。通过在存在 UDP 葡萄糖醛酸的情况下将 INCA 与人肝微粒体（HLMs）孵育，鉴定了 7-O-葡萄糖醛酸苷（M1）和 4'-O-葡萄糖醛酸苷（M2），其结构通过高分辨率质谱和核磁共振分析得到确认。尽管 INCA 是单一的对映异构体分子，但它的 M1 代谢物在 πNAP 固定相上显示出两个等大小的峰，但在 C（18）固定相上仅显示一个峰，表明 INCA 的 7-/7''-和 4'-/4'''-羟基相对于彼此具有不同的空间构型。在重组人 UGT 同工型测试和相关性分析中，发现 UGT1A1、UGT1A3 和 UGT1A9 介导 M1 的形成，而只有 UGT1A3 介导 M2 的形成。动力学研究表明，人、鼠、大鼠、狗和猪肝微粒体之间存在明显的种属差异。UGT1A1、HLMs 和人肠微粒体，但不是人肾微粒体，对 M1 的形成表现出底物抑制。UGT1A1 介导的 M1 形成的 Vmax 比 UGT1A3 和 UGT1A9 介导的 M1 形成分别高出 6 倍和 11 倍。相对活性因子测定的结果表明，UGT1A1 对 M1 的形成贡献约为 75%。这些发现共同表明，UGT1A1 是 M1 形成的主要酶，而 UGT1A3 是 M2 形成的主要酶。
