探针药物法评价厚朴酚、和厚朴酚对大鼠肝微粒体中CYP450酶的抑制作用

Probe Drug Assay for the Inhibitory Effect of Magnolol and Honokiol on CYP450 Enzymes in Rat Liver Microsomes

关键词:  细胞色素P450酶  厚朴  代谢  抑制作用

Key words:  cytochrome P450 enzyme  Magnoliae Officinalis Cortex  metabolism  inhibition

摘要:

目的 研究厚朴中的主要成分厚朴酚、和厚朴酚对CYP450酶的7种亚型酶活性的影响，预测可能的药物间相互作用，为中药的临床应用提供理论依据。方法 采用Cocktail探针方法，将厚朴酚、和厚朴酚和CYP450酶7种亚型的特异性探针底物：甲苯磺丁脲（CYP2C）、香豆素（CYP2A6）、右美沙芬（CYP2D6）、氯唑沙宗（CYP2E1）、睾酮（CYP3A）、非那西丁（CYP1A2）、安非他酮（CYP 2B6）与大鼠肝微粒体进行孵化反应，结合UPLC-MS/MS的多反应监测技术，测定对应的7种代谢产物（4-羟基甲苯磺丁脲、7-羟基香豆素、右啡烷、6-羟基氯唑沙宗、6β-羟基睾酮、对乙酰氨基酚和羟基丙酮）的峰面积，通过与对照组比较，确定厚朴酚、和厚朴酚对以上7种酶活性的影响，计算相应的IC₅₀，评价是否有抑制作用。结果 厚朴酚、和厚朴酚对大鼠肝微粒体中的4种亚型酶（CYP2C、CYP2D6、CYP2E1和CYP2B6）活性均有抑制作用，且随化合物浓度和预温育时间增加而增加机械抑制；另外3种亚型酶（CYP2A6、CYP3A4和CYP1A2）则不呈现此抑制规律。结论 厚朴在与以上4种酶（CYP2C、CYP2D6、CYP2E1、CYP2B6）代谢的药物联合用药时，易产生药物相互作用，抑制药物代谢。本研究也为中药厚朴的临床应用以及新药研发提供重要的数据支持。

Abstract:

OBJECTIVE To study the effects of magnolol and honokiol on the 7 subtypes of CYP450 enzymes in Magnoliae Officinalis Cortex, and to predict the possible drug-drug interactions. To provide theoretical basis for the clinical application of traditional Chinese medicine. METHODS Used the Cocktail probe method, magnolol, honokiol and CYP450 enzyme specific substrates for 7 subtypes:Tolbutamide (CYP2C), Coumarin (CYP2A6), Dextromethorphan (CYP2D6), Chlorzoxazone (CYP2E1), Testosterone (CYP3A), Phenacetin (CYP1A2), Bupropion (CYP2B6) incubation reaction with rat liver microsomes, combined with UPLC-MS/MS multiple reaction monitoring technology, Determination of the peaks of the corresponding 7 metabolites (4-hydroxytoluidine, 7-hydroxycoumarin, dextrorphan, 6-hydroxy chlorzoxazone, 6-hydroxytestosterone, acetaminophen, and hydroxyacetone) area. By comparing with the control group, the effect of magnolol and honokiol on the activity of the above 7 enzymes was determined, and the corresponding IC₅₀ was calculated to evaluate whether there is an inhibitory effect. RESULTS Magnolol and honokiol had inhibitory effects on the activity of 7 isoforms (CYP2C, CYP2D6, CYP2E1 and CYP2B6) in rat liver microsomes. And that was an increase in mechanical inhibition with increasing compound concentration and pre-incubation time. However the subtypes of enzymes (CYP2A6, CYP3A4 and CYP1A2) did not exhibit this inhibition pattern. CONCLUSION It is likely to inhibit drug metabolism and produce drug-drug interactions, during the combination of magnolia and drugs metabolized by 4 enzymes (CYP2C, CYP2D6, CYP2E1 and CYP2B6). This study also provides very important data support for the clinical application of Chinese herbal medicine Magnoliae Officinalis Cortex and the development of new drugs.