基于Cocktail探针药物法研究木犀草素在大鼠体内对CYP450酶活性影响  

Investigation on effect of luteolin on CYP450 enzymes in rats using cocktail method

作　　者：宋可心 衣洁 高琳 任纪平 李倩 SONG Ke-xin;YI Jie;GAO Lin;REN Ji-ping;LI Qian(Department of Pharmaceutical Analysis and Analytical Chemistry,College of Pharmacy,Harbin Medical University,Harbin 150000,China;Department of Pharmaceutics,College of Pharmacy,Harbin Medical University,Harbin 150000,China)

机构地区：[1]哈尔滨医科大学药学院药物分析与分析化学教研室,哈尔滨150000 [2]哈尔滨医科大学药学院药剂学教研室,哈尔滨150000

出　　处：《药物分析杂志》2022年第2期279-286,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：国家自然科学基金项目(81970055)。

摘　　要：目的:应用Cocktail探针药物法评价木犀草素对大鼠体内CYP450酶亚型活性的影响。方法:首先将大鼠随机分为空白组和木犀草素组,2组分别灌胃羧甲基纤维素钠(2.0 mL·kg^(-1))和木犀草素(30.0 mg·kg^(-1))1 d,并于第7 d腹腔注射混合探针药物溶液(含10.0 mg·kg^(-1)的非那西汀和睾酮,2.5 mg·kg^(-1)的甲苯磺丁脲)。之后在不同时间点对大鼠进行眼眶取血,血浆样本经异丙醇沉淀后进入液质测定。UHPLC-MS/MS色谱部分采用ZORBAX SB-C_(18)(2.1 mm×30 mm,3.5μm)色谱柱进行分离,以乙腈-0.1%甲酸水为流动相,梯度洗脱;质谱部分使用电喷雾离子源(ESI),分别在正、负离子条件下采用MRM模式下进行监测,将所采集数据输入DAS软件计算药动学参数。另外,采用Western blot法观察木犀草素对CYP450蛋白表达的影响,对体内结果做进一步验证。结果:非那西汀、睾酮、甲苯磺丁脲分别在2.0~2000.0、0.3~300.0及1.7~1700.0 ng·mL^(-1)浓度范围内线性关系良好,定量限分别为2.0、0.3、1.7 ng·mL^(-1)。所建立UHPLC-MS/MS法专属性良好,各待测药物准确度在96.9%~108.0%范围内,日间和日内精密度RSD均小于8.9%,提取回收率、基质效应以及稳定性考察项目均符合测定要求。药动学结果显示,与空白组相比,木犀草素组非那西汀AUC值明显降低,甲苯磺丁脲和睾酮AUC值均呈现升高趋势,但睾酮变化非常轻微。Western blot结果表明,木犀草素组大鼠CYP1A2蛋白表达增加,而CYP2C9和CYP3A4蛋白表达减少。结论:根据FDA CYP450酶诱导剂和抑制剂标准,木犀草素可作为CYP1A2的中度诱导剂,CYP2C9的弱抑制剂,但对CYP3A4无显著影响。Objective:An in vivo cocktail probe drug method was conducted to evaluate the effect of luteolin onCYP450 enzyme activity.Methods:The rats were randomly divided into two groups,the control and the luteolin groups were respectively given gavage administration of CMC-Na(2.0 mL·kg^(-1))and luteolin(30.0 mg·kg^(-1))for 7 successive days.On the 7^(th) day,all rats were injected intraperitoneally with the mixed solution of 3 probe drugs(10.0 mg·kg^(-1) of phenacetin and testosterone and 2.5 mg·kg^(-1) of tolbutamide),the plasma samples were collected at different time points and precipitated with isopropanol,then determined by LC-MS/MS.The chromatographic separation was achieved on a ZORBAX SB-C_(18)(2.1 mm×30 mm,3.5μm)column by gradient elution of acetonitrile-0.1%formic acid in water,and the MS instrument was operated in multiple reaction monitoring(MRM)with positive and negative electrospray ionization(ESI)modes.The UHPLC-MS/MS method was established for the determination of the concentrations of three probe drugs in plasma,the acquired data were calculated for pharmacokinetic parameters by using DAS software subsequently.Further,the protein expression levels of the three CYP450 enzymes were tested by western blot.Results:Methodological results suggested that the linear range of phenacetin,testosterone,and tolbutamide were in the concentration of 2.0-2000.0 ng·mL^(-1),0.3-300.0 ng·mL^(-1),1.7-1700.0 ng·mL^(-1),and the lower limit of quantification(LLOQ)were 2.0 ng·mL^(-1),0.3 ng·mL^(-1),and 1.7 ng·mL^(-1),respectively.For all analytes,the accuracy was in the range of 96.9%-108.0%,and the RSD of intra-day and inter-day precision were within 8.9%.The extraction recovery,matrix effect and stability all met the criteria.The pharmacokinetic results showed that luteolin could significantly reduce the AUC value of phenacetin,and increase the AUC value of tolbutamide.But there was few significant variation in pharmacokinetic parameters of testosterone.The western blot results showed that the expression of CYP1 A

关 键 词：木犀草素 液质联用法 药代动力学 探针药物 CYP450酶 免疫印迹法 

分 类 号：R917[医药卫生—药物分析学]