山姜素在人肝微粒体的葡萄糖醛酸化反应研究

Study on the glucuronidation of alpinetin in human liver microsomes

目的研究体外肝微粒体孵育体系中山姜素的葡萄糖醛酸化代谢情况,鉴定参与山姜素葡萄糖醛酸化代谢的UGT亚型。方法用体外肝微粒体孵育体系,用HPLC-UV检测方法,检测山姜素的葡萄糖醛酸化代谢情况。将代谢产物进行纯化后,用质谱(MS)和核磁共振(NMR)法进一步鉴定其结构。用商业化重组表达的UGT单酶,鉴定代谢产物的结构和归属可能参与山姜素葡萄糖醛酸化代谢反应的葡萄糖醛酸转移酶(UGTs)亚型。结果山姜素葡萄糖醛酸代谢产生一个代谢产物,经结构鉴定为山姜素-氧-单葡萄糖醛酸化产物。人肝微粒体代谢山姜素的动力学行为,符合米方程且动力学参数:Vmax=(101.9±3.0)nmol·min-1·mg-1·pro,Km=(40.6±3.6)μmol·L-1。UGT1A1、UGT1A3、UGT1A9和UGT2B15均参与了山姜素的葡萄糖醛酸化反应。结论山姜素在人肝微粒体孵育体系中会被代谢成为一个单葡萄糖醛酸化产物,且归属了参与的UGT酶。

Objective To investigate the glucuronidation of alpinetin in human liver microsomes,including elucidation of the structure of alpinetin glucuronide and identification of UDP-glucuronosyltransferases（UGT） isoforms involved in alpinetin′s glucuronidation.Methods After incubation of alpinetin using in vitro human liver microsomal system,HPLC-UV was utilized to determine the glucuronides of alpinetin.MS and NMR were employed to elucidate the structures of metabolite.Screening assays with recombinant human UGTs were used to identify the UGT isoforms involved in the glucuronidation of alpinetin.Results One metabolite was identified and elucidated to be alpinetin-O-monoglucuronide.The metabolic behaviour of alpinetin was demonstrated to obey the typical monophasic Michaelis-Menten kinetics,and kinetic parameters were calculated to be（101.9±3.0） nmol·min-1·mg-1·pro and（40.6±3.6） μmol·L-1 for Vmax and Km,respectively.UGT1A1,1A3,1A9 and 2B15 were determined to participate in the glucuronidation of alpinetin.Conclusion In the present experiment,the glucuronidation behaviour of alpinetin was clearly investigated,which is beneficial to deeply understanding of alpinetin′s metabolism in human and its pharmacodynamic basis.