白头翁皂苷B4对小鼠肝药酶CYP1A2表达及酶活性的影响  

Effect of anemoside B4 on expression and activity of CYP1A2

作　　者：龚琴 王木兰 李军茂 黄文平 冯育林[1,2] 付爱东 杨世林 李俊[1,2] GONG Qin;WANG Mu-lan;LI Jun-mao;HUANG Wen-ping;FENG Yu-lin;FU Ai-dong;YANG Shi-lin;LI Jun(Jiangxi University of Traditional Chinese Medicine,Nanchang 330006,China;State Key Laboratory of Innovative Drugs and Efficient Energysaving Pharmaceutical Equipment,Nanchang 330006,China;Jiangxi Muentang Biological Technology Co.,Ltd.,Nanchang 330006,China)

机构地区：[1]江西中医药大学,南昌330006 [2]创新药物与高效节能降耗制药设备国家重点实验室,南昌330006 [3]江西沐恩堂生物科技有限公司,南昌330006

出　　处：《药物分析杂志》2021年第6期970-978,共9页Chinese Journal of Pharmaceutical Analysis

基　　金：江西省中药学一流学科专项基金项目(JXSYLXK-ZHYAO012);国家重点研发计划资助(2019YFC1712300)。

摘　　要：目的:观察白头翁皂苷B4体内、体外对小鼠肝药酶CYP1A2酶活性及表达的影响,为白头翁B4的药物相互作用提供实验依据。方法:采用UHPLC XB-C18色谱柱(2.1 mm×50 mm,1.8μm)分离样品,以0.1%甲酸水和乙腈为流动相,流速0.3 m L·min^(-1),采用电喷雾离子源(ESI)正离子模式,多反应离子(MRM)扫描方式,定量离子对为m/z 180.1~109.9,建立检测非那西丁的UPLC-MS/MS检测方法。制备正常小鼠肝粗酶液并建立体外酶孵育体系,将粗酶液与探针非那西丁(4μg·m L^(-1))、B4溶液(50μg·m L^(-1))进行孵育,UPLC-MS/MS检测孵育5、15、30、60、120 min体系中非那西丁含量,绘制时间与含量的线性方程,对方程中a值进行统计分析以反映非那西丁的代谢速率。小鼠静脉注射给药3 d后腹腔注射探针非那西丁(2 mg·kg^(-1)),UPLC-MS/MS测定注射后5、15、30、60 min小鼠血浆中非那西丁含量,q PCR和WB分别检测肝脏中m RNA、蛋白表达。结果:非那西丁浓度在4~2000 ng·m L^(-1)范围内与峰面积线性关系良好(r2=0.9936),检测下限为0.1 ng·m L^(-1)(S/N≥3),定量下限为0.2 ng·m L^(-1)(S/N≥10),日内精密度RSD小于5%,日间精密度RSD小于10%,回收率为88.5%~92.9%。B4体外对非那西丁的代谢速率无明显影响(P>0.05);B4可明显加快小鼠血浆中非那西丁的代谢速率(P<0.01),且一定程度上调小鼠肝组织中CYP1A2 m RNA和蛋白表达。结论:白头翁皂苷B4体外对CYP1A2酶活性无明显影响,体内对该酶活性有缓和的促进作用,其促进作用与上调该酶的基因转录和蛋白表达有关。Objective:To study the effect of anemoside B4 on expression and activity of CYP1 A2 in vivo and in vitro,to provide experimental data for possible interaction between aemoside B4 and other drugs.Methods:The chromatographic separation was carried out using a UHPLC XB-C18 column(2.1 mm×50 mm,1.8μm)with mobile phase comprising of 0.1%fomic acid-acetonitrile at a flow rate of 0.3 mL·min^(-1),the quantitation was performed using multiple reaction monitoring of the transitions of m/z 180.1→109.9 in positive electrospray ionization.The liver micrisomes of normal mice was prepared.The incubation system was established.Liver micrisomes was incubated with probe phenacetin(4μg·mL^(-1))and B4(50μg·mL^(-1)),UPLC-MS/MS was adopted to detect the content of phenacetin after incubation for 5,15,30,60 and 120 min.Incubation time and content were used to draw a linear equation,the a value in the equation reflecting the metabolic rate of phenacetin was analyzed.Mice were intravenous administrated with Anemoside B4 for 3 days,then the probe phenacetin(2 mg·kg^(-1))was injected intraperitoneally,plasma was separated for the detection of phenacetin(UPLC-MS/MS)after 5,15,30 and 60 min of probe injection,the liver was removed for mRNA expression by qPCR assay.Protein expression was achieved with Western blot assay.Results:The linearity range was 4-2000 ng·m L^(-1)(r2=0.9936),the limit of detection was 0.1 ng·m L^(-1)(S/N≥3)and limit of quantification was 0.2 ng·m L^(-1)(S/N≥10),intraday and interday relative standard deviations were less than 5%and 10%respectively,the recoveries were between 88.5%-92.9%.Anemoside B4 has no effect on metabolic rate of phenacetin in vitro(P>0.05),but significantly promote the metabolic rate in plasma of mice(P<0.01)and upregulate the mRNA and protein expression of CYP1 A2.Conclusions:In vitro,anemoside B4 has no effect on CYP1 A2 activity,it has a mild stimulative effect on CYP1 A2 in vivo,which is related to the up-regulation of gene transcription and protein expression of CYP1 A2.

关 键 词：白头翁皂苷B4 UPLC-MS/MS 小鼠 非那西丁 CYP1A2 探针 体内 体外 

分 类 号：R917[医药卫生—药物分析学]