LC-MS/MS法研究黄酮类化合物对人肝微粒体细胞色素P450酶6种亚型的体外抑制作用

Flavonoids inhibit six cytochrome P450 enzymes from human liver microsomes as analyzed by LC-MS/MS method

目的:建立液相色谱-串联质谱法(LC-MS/MS)研究黄酮类化合物对人肝微粒体细胞色素P450酶6种亚型的体外抑制作用。方法:采用ESI正离子和负离子选择反应检测同时测定扑热息痛、4-羟基甲苯磺丁脲、5-羟基奥美拉唑、氧去甲基右美沙芬、6-羟基氯唑沙宗和氧化硝苯地平,分别代表CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1,CYP3A4的活性;黄酮类化合物和6种探针底物在人肝微粒体中共同孵育,并计算其IC_(50)值表示对CYP450酶的抑制程度。结果:黄酮苷类(水飞蓟宾、金丝桃苷、儿茶素、表儿茶素、葛根素、芦丁、甘草苷以及柚皮苷)的IC_(50)值均大于50μmol·L_1,对6种亚型没有抑制作用。而黄酮苷元如槲皮素等均能不同程度地抑制CYP1A2,IC_(50)值从0.054μmol·L_1到3r.91μmol·L_1不等;有些对其他亚型如CYP2C9,CYP2C19,CYP2E1,CYP3A4也有一定的抑制作用。所有的黄酮类化合物对CYP2D6无抑制活性。黄酮对CYP3A4的活性也有激活作用。结论:建立的LC-MS/MS方法准确、灵敏、可靠。33种黄酮类化合物对CYP450酶6种亚型有不同的抑制作用,这些信息有助于中药-药物相互作用的预测。

Objective：To determine the inhibitory effect of flavonoids on six cytochrome P450 enzymes from human liver microsomes as analyzed by LC-MS/MS method. Methods：Six cytoehrome P450 probe substrate metabolites （including paracetamol for CYP1A2, 4-hydroxytolbutamide for CYP2C9, 5-hydroxyomeprazole for CYP2CI9, dextrorphan for CYP2D6, 6-hydroxychlorzoxazone for CYP2E1 and dehydronifedipine for CYP3A4） were measured by LC-MS/MS with positive and negative ion SRM detection. Flavonoids were incubated with human liver microsomes in the presence of six probe substrates of CYP450 isozymes, and the inhibitory effect were evaluated with IC50 values. Results： Flavonoids were classified as the potent, marginal,or weak inhibitors to the CYP450 isozymes. The IC50 values of flavonoids for glycosides substitutions （ silybin, hyperoside, catechin, epicatechin, puerarin, rutin, liquiritin and naringin） were above 50 μmol·L^-1,but did not show any inhibitory effect on the six CYP450 isoforms. All flavonoid aglycones such as quercetin showed inhibitory effects on CYP1A2 （IC50 values ranged from 0. 054 μmol·L^-1 to 31.91μmol·L^-1 ） ; some of them were able to inhibit other CYP450 isoforms such as CYP2C9, CYP2C19, CYP2E1 ,and CYP3A4. All flavonoids did not inhibit CYP2D6. Otherwise, flavone also stimulated the CYP3A4 activity. Conclusion：The presented method has acceptable accuracy, precision and sensitivity, and it is suitable for in vitro inhibition study. Flavonoids show different inhibitory effects on the activity of CYP450 isozymes;the information is useful for the prediction of flavonoids-drug interactions.