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树豆酮酸A对人肝微粒体中5种常见细胞色素P450酶的体外抑制作用研究
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| 篇名： | 树豆酮酸A对人肝微粒体中5种常见细胞色素P450酶的体外抑制作用研究 |
| TITLE： | Inhibitory Effects of Cajanonic Acid A on 5 Kinds of Common CYP 450 Enzyme in Human Liver Microsomes in vitro |
| 摘要： | 目的：研究树豆酮酸A对人肝微粒体中的5种细胞色素P450（CYP）酶的体外抑制作用。方法：采用Cocktail探针药物法，在人肝微粒体中加入50.0、15.0、5.0、1.5、0.5、0.15、0.05μmol/L的树豆酮酸A，与混合探针药物[包含非那西丁、右美沙芬、奥美拉唑、睾酮、甲苯磺丁脲（分别为CYP1A2、CYP2D6、CYP2C19、CYP3A4、CYP2C9的探针药物）]共同孵育60min。在另设空白组和阳性对照组[α-萘黄铜、奎尼丁、（+）-N-3-苄基香酚、酮康唑、磺胺苯吡唑（分别为CYP1A2、CYP2D6、CYP2C19、CYP3A4、CYP2C9的特异性抑制剂）]的基础上，以葛根素为内标，采用超高效液相色谱-质谱联用法（UPLC-MS/MS）分析相应代谢产物（对乙酰氨基酚、右啡烷、5-羟基奥美拉唑、6β-羟基睾酮、羟基甲苯磺丁脲）的含量。以ACQUITYUPLC®BEHC18为色谱柱，以0.01％甲酸水溶液-0.01％甲酸乙腈为流动相（梯度洗脱），柱温为40℃，流速为0.4mL/min，进样量为2μL；采用电喷雾电离源，以多反应监测模式进行正负离子扫描，数据采集范围为m/z100～1200，碰撞气为氩气，雾化气为氮气，锥孔气流量为50L/h，脱溶剂气流量为800L/h，正、负离子模式下毛细管电压分别为2.0、1.5kV，离子源温度分别为120、110℃，脱溶剂的温度分别为400、450℃。使用Graph-padPrism5.0软件进行非线性回归分析并计算半数抑制浓度（IC50）。结果：上述各代谢产物检测浓度的线性范围分别为0.26～8.35、0.36～34.56、0.10～3.09、3.67～117.37、0.15～4.88μmol/L（R2＞0.99），定量下限分别为0.26、0.36、0.10、3.67、0.15μmol/L。阳性对照组特异性抑制剂对人肝微粒体中CYP1A2、CYP2D6、CYP2C19、CYP3A4、CYP2C9酶的IC50均在文献报道的可接受范围内。树豆酮酸A对人肝微粒体中CYP1A2、CYP2D6、CYP3A4酶的IC50值均大于50μmol/L，对CYP2C9、CYP2C19酶的IC50值分别为4.94、18.00μmol/L。结论：树豆酮酸A对人肝微粒体中CYP1A2、CYP2D6、CYP3A4酶没有抑制作用，对CYP2C9、CYP2C19酶有一定的抑制作用。 |
| ABSTRACT： | OBJECTIVE：To study the inhibitor y effects of cajanonic acid A on 5 kinds of cytochrome P 450（CYP）enzyme，in human liver microsomes in vitro . METHODS ：By Cocktail probe substrate method ，50.0，15.0，5.0，1.5，0.5，0.15，0.05 μmol/L cajanonic acid A were added into liver microsomes ， and incubated with mixed probe substrates [including phenacetin ， dextromethorphan，omeprazole，testosterone and toluenesulfonbutylurea （probe substrates of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]. On the basis of setting up blank group and positive control group [ α-naphthalene brass ， quinidine，（+）-N-3-benzyl vanillin ，ketoconazole and sulfabendazole （specific inhibitors of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]，using puerarin as internal standard ，UPLC-MS/MS method was adopted to determine the contents of corresponding metabolites （acetaminophen， dextrophane， 5-hydroxy omeprazole ， 6 β-hydroxytestosterone， hydroxytolbutamide）. The determination was performed on ACQUITY UPLC ® BEH C 18 column，with mobile phase consisted of 0.01％ formic acid aqueous solution- 0.01％ acetonitrile formic acid （gradient elution ）at the flow rate of 0.4 mL/min. The column temperature was 40 ℃，and the sample size was 2 μL. An electrospray ionization source was used to conduct positive and negative ion scanning in the multiple reaction monitoring mode. The data acquisition range was m/z 100-1 200，the collision gas was argon ， the atomized gas was nitrogen ，the gas flow rate of the cone hole was 50 L/h，the desorption gas flow rate was 800 L/h，the capillary voltage under positive and negative mode was 2.0， 1.5 kV，and the ion source temperature was 120 ℃，110 ℃， respectively. The desolvent temperature were 400 ℃ and 450 ℃ ， respectively. Non linear regression analysis was performed by using Graphpad Prism 5.0 software and IC 50 wascalculated. RESULTS ：The linear ranges of above metabolifes were 0.26-8.35，0.36-34.56，0.10-3.09, 3.67-117.37，0.15-4.88 μmol/L（R2＞0.99）. The limits of quantitation were 0.26，0.36， 0.10，3.67，0.15 μmol/L，respectively. The IC 50 values of specific inhibitors in positive control group to CYP 1A2，CYP2D6， CYP2C19，CYP3A4 and CYP 2C9 in human liver microsomes were all within the acceptable range reported in the literature. The IC50 values of cajanonic acid A to CYP 1A2，CYP2D6 and CYP 3A4 in human liver microsomes were all more than 50 μmol/L，and the IC 50 values of CYP 2C9 and CYP 2C19 were 4.94 and 18.00 μmol/L，respectively. CONCLUSIONS ：Cajanonic acid A has no inhibitory effect on CYP 1A2，CYP2D6 and CYP 3A4，but has a certain inhibitory effect on CYP 2C9 and CYP 2C19. |
| 期刊： | 2021年第32卷第02期 |
| 作者： | 陈瑞，周维，张丽，朱高峰，黄静，汤磊 |
| AUTHORS： | CHEN Rui，ZHOU Wei，ZHANG Li，ZHU Gaofeng ，HUANG Jing，TANG Lei |
| 关键字： | 树豆酮酸A；人肝微粒体；细胞色素P450酶；Cocktail探针药物法；超高效液相色谱-质谱联用法；特异性抑制剂；抑制作用 |
| KEYWORDS： | Cajanonic acid A ；Human liver microsome ；Cytochrome P 450 entyme；Cocktail probe substrate method ；UPLC-MS/ |
| 阅读数： | 290 次 |
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