Inhibitory Effects of Cajanonic Acid A on 5 Kinds of Common CYP 450 Enzyme in Human Liver Microsomes in vitro / 中国药房

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.