没食子酸及其氧化产物对大鼠肝微粒体CYP3A的抑制效应

Inhibitory effect of gallic acid and its oxidation products on CYP3A in rat liver microsomes

目的体外研究没食子酸对大鼠肝微粒体CYP3A的抑制效应。方法将不同浓度没食子酸与鼠肝微粒体及探针底物在37℃下孵育,用高效液相色谱-紫外法测定探针底物的代谢产物生成率,计算IC50值。在NADPH存在或不存在时,将没食子酸与鼠肝微粒体在37℃下分别预孵育0、15、30 min后继续孵育15 min,测定探针底物的代谢产物生成率,观察没食子酸对CYP3A抑制的时间依赖性。在无NADPH存在时,将不同浓度没食子酸与鼠肝微粒体在37℃下分别预孵育0、30 min后继续孵育15 min,测定探针底物的代谢产物生成率,计算出两实验组IC50平移值(IC50shift)。观察2.5 h内没食子酸在紫外-可见光光谱下的变化。在无NADPH存在时,将抗氧剂与没食子酸及鼠肝微粒体在37℃下共同预孵育,观察抗氧剂谷胱甘肽和维生素C的加入对没食子酸抑制强度的影响。结果没食子酸对CYP3A表现出浓度依赖性抑制(P<0.05),IC50为130.2μg/mL。预孵育无NADPH存在时,没食子酸对CYP3A表现出时间依赖性抑制(P<0.05),且抑制强度远大于NADPH存在的实验组。在无NADPH存在时,没食子酸与鼠肝微粒体在37℃下分别预孵育0 min和30 min,两实验组间的IC50平移值达11.9。紫外-可见光全波长扫描观察显示,没食子酸在孵育过程中生成了2种反应产物。抗氧剂的加入能显著降低没食子酸对CYP3A的抑制能力(P<0.05)。结论没食子酸对CYP3A的活性表现出较强的不可逆性抑制,具有诱导药物-药物相互作用的风险。

Objective To investigate the inhibitory effect and mechanism of gallic acid on CYP3A in rat liver microsomes in vitro. Methods Rat liver microsomes were incubated with different concentrations of gallic acid and probe substrates at 37℃. The formation of metabolites was detected by high performance liquid chromatography-UV and ICs0 was calculated according to the formation rates of metabolites at various concentra- tions of gallic acid. Gallic acid was used to pre-incubate with rat liver microsomes in the presence or absence of NADPH at 37℃for 0, 15 and 30 min separately. Probe substrate metabolite generation rate was measured to observe the time-dependent inhibition of CYP3A after 15 rain incubation. Different concentrations of gallic acid and rat liver microsomes were pre-incubated without NADPH at 37 ℃ for 0 and 30 rain separately and IC50 shift value was calculated according to the formation rates of metabolites after 15 rain incubation. Time-dependent changes in UV-visible spectra of gallic acid within 2.5 h were observed. Antioxidants such as glutathione and vitamin C were used to incubate with gallic acid and rat liver microsomes without NADPH at 37 μ and the effects of antioxidants on inhibitory potency of gallic acid were observed. Results Gallic acid exhibited con- centration-dependent inhibitory effects on CYP3A （P 〈0. 05）, with ICs0 value of 130.2 μg/mL. Pre-incuba- tion with rat liver microsomes in the absence of NADPH, gallic acid exhibited time-dependent inhibitory effects on CYP3A （P 〈 0. 05 ） and the inhibitory potency was much greater than the experimental group with NADPHin pre-incubation. Between 0 min and 30 min pre-incubation experimental groups in the absence of NADPH, the ICs0 shift value was 11.9. Two reaction products of gallic acid were observed with UV-visible spectra. Adding antioxidants could significantly reduce the inhibitory potency of gallic acid on CYP3A （P 〈 0. 05 ）. Conclusion Gallic acid shows irreversible inhibition on CYP3A which may induce drug-drug interaction.