毛蕊花糖苷对大鼠肝微粒体中6种CYP酶的体外抑制作用

In vitro inhibitory effects of acteoside on 6 kinds of CYP enzymes in liver microsomes of rats

目的考察毛蕊花糖苷对大鼠肝微粒体细胞色素P450(CYP)酶的体外抑制作用。方法采用探针底物法,在大鼠肝微粒体中加入0.1、0.3、1、3、10、30μmol/L的毛蕊花糖苷,与探针底物非那西丁、美芬妥因、双氯芬酸、香豆素、右美沙芬、睾酮(分别为CYP1A2、CYP2C19、CYP2C9、CYP2A6、CYP2D6、CYP3A4酶的底物)共同孵育。在另设空白对照组和阳性抑制剂组[α-萘黄酮、噻氯匹定、磺胺苯吡唑、毛果芸香碱、奎尼丁、酮康唑(分别为CYP1A2、CYP2C19、CYP2C9、CYP2A6、CYP2D6、CYP3A4酶的抑制剂)]的基础上,以吲达帕胺为内标,采用超高效液相色谱-串联质谱法检测相应代谢产物(对乙酰氨基酚、4-羟基美芬妥因、4-羟基双氯芬酸、7-羟基香豆素、右啡烷、6β-羟基睾酮)的含量,运用GraphPad v8.0软件计算半数抑制浓度(IC_(50));采用计算机分子对接技术,将毛蕊花糖苷和阳性抑制剂分别与CYP酶进行分子对接,分析二者分子结合方式及结合能力强弱。结果毛蕊花糖苷对CYP1A2、CYP2A6酶的IC_(50)>30μmol/L,对CYP2D6、CYP2C19、CYP2C9、CYP3A4酶的IC_(50)分别为24.87、21.52、12.56、7.55μmol/L。毛蕊花糖苷与CYP3A4酶之间可形成氢键并产生疏水作用力,酮康唑与CYP3A4酶之间可形成氢键并产生静电相互作用;毛蕊花糖苷和酮康唑与CYP3A4酶的结合自由能分别为-10.2、-12.4 kcal/mol(1 kcal/mol=4.19 kJ)。结论毛蕊花糖苷对大鼠肝微粒体中CYP3A4酶有中等强度的抑制作用,且亲和力与阳性抑制剂相当;该化合物对其他5种CYP酶的抑制作用弱。

OBJECTIVE To investigate the in vitro inhibitory effects of acteoside on cytochrome P450(CYP)enzymes in liver microsomes of rats.METHODS Using probe substrates method,acteoside(0.1,0.3,1,3,10,30μmol/L)was incubated with probe substrates phenacetin,mephentoin,diclofenac,coumarin,dextromethorphan and testosterone(substrates of CYP1A2,CYP2C19,CYP2C9,CYP2A6,CYP2D6 and CYP3A4 enzymes,respectively)in liver microsomes of rats.Another blank control group and positive inhibitor group[α-naphthoflavone,ticlopidine,sulfabendazole,pilocarpine,quinidine and ketoconazole(inhibitors of CYP1A2,CYP2C19,CYP2C9,CYP2A6,CYP2D6 and CYP3A4 enzymes,respectively)]were set up.Using indapamide as the internal standard,the contents of corresponding metabolites(acetaminophen,4-hydroxymephenytoin,4-hydroxydiclofenac,7-hydroxycoumarin,dextran,6β-hydroxytestosterone)were detected by ultra high performance liquid chromatography-tandem mass spectrometry.The IC_(50)values were calculated by GraphPad v8.0 software.By computer molecular docking technology,acteoside and positive inhibitors were molecularly docked with the CYP enzyme,and the binding mode and strength of the two molecules were analyzed.RESULTS The IC_(50)values of acteoside to CYP1A2 and CYP2A6 enzymes were more than 30μmol/L,and those of acteoside to CYP2D6,CYP2C19,CYP2C9 and CYP3A4 enzymes were 24.87,21.52,12.56 and7.55μmol/L,respectively.The hydrogen bond and hydrophobic force could form between acteoside and CYP3A4 enzyme,and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP3A4 enzyme.The binding free energy of acteoside and ketoconazole to CYP3A4 enzyme were-10.2 and-12.4 kcal/mol(1 kcal/mol=4.19 k J),respectively.CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP3A4 enzyme in liver microsomes of rats,and its affinity is equivalent to that of positive inhibitor;the compound shows weak inhibitory effect on other 5 CYP enzymes.