菊花黄酮类化合物与黄嘌呤氧化酶的药靶结合动力学研究

Study on drug-target binding kinetics profiles of flavonoids in Chrysanthemum morifolium and xanthine oxidase

基于靶点占有率数学模型,评估菊花中潜在降尿酸有效成分与黄嘌呤氧化酶的结合动力学过程。采用UPLC-Q-Exactive MS技术,对照品指认方法和体外酶促动力学实验鉴定菊花中潜在降尿酸有效成分;通过表面等离子共振(surface plasmon resonance,SPR)技术测定菊花中潜在抑制剂与黄嘌呤氧化酶的结合动力学参数;最后通过经验证的黄嘌呤氧化酶靶点占有率数学模型,评估抑制剂与黄嘌呤氧化酶在体内的结合动力学过程。该研究鉴定了菊花提取物中39个潜在降尿酸有效成分,测定了其中23个化合物的抑制活性,筛选潜在黄嘌呤氧化酶抑制剂3个,分别为染料木素、木犀草素、芹菜素。3个化合物的IC50分别为1.23,1.47,1.59μmol·L^(-1),结合速率常数(Kon)分别为1.26×10^(6),5.23×10^(5),6.36×10^(5) mol·L^(-1)·s-1,解离速率常数(Koff)分别为10.93×10^(-2),1.59×10^(-2),5.3×10^(-2) s^(-1)。经不同给药方法评估后,所选化合物在联合给药情况下能对体内黄嘌呤氧化酶产生快速且持续的抑制。该研究采用UPLC-MS技术、浓度-响应方法、SPR技术和黄嘌呤氧化酶靶点占有率模型综合评估菊花中3个有效成分在体内不同给药方式的体内靶点占有率过程,可为中药有效成分活性筛选提供一种新的研究思路和方法。

Based on the target occupancy mathematical model,the binding kinetic process of potential active ingredients of lowering uric acid in Chrysanthemum morifolium with xanthine oxidase(XOD)was evaluated.The potential active ingredients of lowering uric acid in Ch.morifolium were screened by UPLC-Q-Exactivems MS technology,reference substance identification and in vitro enzymatic kinetics experiments.The binding kinetic parameters of xanthine oxidase and potential inhibitor in Ch.morifolium were determined by surface plasma resonance(SPR).The verified mathematical model of the XOD target occupancy evaluated the kinetic binding process of inhibitors and xanthine oxidase in vivo.According to UPLC-Q-Exactive MS and reference substance identification,39 potential uric acid-lowering active ingredients in Ch.morifolium extracts were identified and the inhibitory activities of 23 compounds were determined.Three potential xanthine oxidase inhibitors were screened,namely genistein,luteolin,and apigenin.whose IC50 were 1.23,1.47 and 1.59μmol·L^(-1),respectively.And the binding rate constants(Kon)were 1.26×10^(6),5.23×10^(5) and 6.36×10^(5) mol·L^(-1)·s^(-1),respectively.The dissociation rate constants(Koff)were 10.93×10^(-2),1.59×10^(-2),and 5.3×10^(-2) s^(-1),respectively.After evaluation by different administration methods,the three selected compounds can perform rapid and sustained inhibition of xanthine oxidase in vivo under combined administration.This study comprehensively evaluated the target occupancy process of three effective components in different ways of administration in vivo by UPLC-MS,concentration-response method,SPR technology and xanthine oxidase target occupancy model,which would provide a new research idea and method for screening active ingredients in traditional Chinese medicine.