RP-HPLC法测定人尿苷二磷酸葡醛酸转移酶UGT1A3转基因细胞中槲皮素  

Determination of quercetin metabolism in UGT1A3 cDNA-expressing cells by RP-HPLC

作　　者：姚艳[1] 张霞[1] 刘瑶[1] 余露山[1] 蒋惠娣[1] 曾苏[1] 

机构地区：[1]浙江大学药学院药物分析与药物代谢研究室,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2011年第1期7-11,共5页Journal of Zhejiang University(Medical Sciences)

基　　金：国家"十一五"重大科技专项资助(2009ZX09304-003)

摘　　要：目的:建立反相高效液相色谱法测定人重组UGT1A3转基因细胞中槲皮素,并研究该酶对槲皮素的相关代谢及动力学参数测定。方法:Bac-to-Bac系统表达的人UGT1A3重组酶细胞破碎液与槲皮素共孵育之后,以乙腈沉淀蛋白,采用木犀草素为内标,以Phenomenex Luna C18柱为分析柱,甲醇(A)-0.1%甲酸溶液(B)为流动相梯度洗脱:0~25 min(30∶70-80∶20,A∶B),>25~25.5min(80∶20),>25.5~27 min(80∶20-30∶70),>27~30 min(30∶70);流速1.0 ml/min,紫外检测波长为368 nm。结果:槲皮素在5~200μmol/L内线性关系良好(r=0.9999),检测限为1.25μmol/L(S/N≥3),定量限为5μmol/L(S/N>10,RSD=6.99%),方法回收率达99.1%~103.5%,日内、日间RSD分别<2.5%和<8%。另测得UGT1A3催化槲皮素的Km为(62.95±13.16)μmol/L,Vmax为(284.50±24.35)nmol.min-1.g-1,清除率Vmax/Km为4.52 ml.min-1.g...Objective: To develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells.Methods: The lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin,the reaction was terminated with acetonitrile,and luteolin was used as internal standard.The determination was performed on a C18 reversed phase column with a mobile phase of methanol-0.1% formic acid(V/V) at a flow rate of 1.0 ml/min.The gradient el...

关 键 词：槲皮素/分析 色谱法 高压液相 葡糖醛酸基转移酶/生物合成 重组酶类 

分 类 号：R285.5[医药卫生—中药学]