The Effect Of Yinzhihuang Injection On The Activity Of Rat Hepatic Cyp2e1

ObjectiveThe study was designed to study the active component of Yinzhihuang injection on the activity of CYP2E1 in rat liver microsome. We also studied the effect of Yinzhihuang injection on the pharmacokinetics of the CYP2E1 subtrate chlorzoxazone. The aim was to evaluate the effect of Yinzhihuang injection on the activity of CYP2E1 and guide reasonable clinical medication.Method1.The effect of Yinzhihuang injection on the acticity of CYP2E11.1 The inhibition of baicalin and geniposide on the acticity of CYP2E1 in rat microsome1.1.1 Preparation of liver microsome Liver microsome was prepared by using calcium precipitation approach.1.1.2 Determination of protein concentration Protein concentration was determinated by Bradfrod approach. 1.1.3 Incubation reaction The total volume of incubation system was 500μL, which contained NADPH, phosphate buffer, EDTA, MgCl2, and liver microsome. The reaction was preincubated for 5 min at 37℃, and was started since NADPH was added. The study was divided into control group and experiment group. The experiment group contained the active component Yinzhihuang of injection, the control group contained the same volume of phosphate buffer.1.1.4 Measurement of CYP2E1 enzyme activity HPLC method was used to determinate the turnover of midazolam.1.2 The inhibition of CYP2E1 activity after multiple administration of Yinzhihuang injection Male SD rats were randomly were divided into experiment group and control group. The experiment group were intravenously given Yinzhihuang injection(40ml·kg-1) and the control group were given glucose injection for 7 d. Immediately after the last treatment, rats were killed by decapitation to prepare the liver microsome, and then the turnover of chlorzoxazone of the two group were determinated.1.3 Effect of Yinzhihuang injection on the pharmacokinetics of chlorzoxazone The study was conducted by self-control design.14 male SD rats were given intravenously 10% glucose injection (40ml·kg-1), then chlorzoxazone at a dose of 10mg·kg-1 was infused via the tail vein to rats immediately after the last injection. A blood sample was collected at 2,8,20,40,70,100,150min the plasma was stored for analysis.3 days after the first blood collection, the 14 rats were given Yinzhihuang injection(40ml·kg-1) for 7 days. The blood sample was collected at designed time point.The concentration of chlorzoxazone was determinated by HPLC method.2. Statistical analysis The pharmacokinetic parameters of chlorzoxazone were calculated by software 3P97. The analyses were performed by SPSS 10.0 statistical software. The data of different groups was analyzed with independent t test. The differences of the concentration and pharmacokinetic parameters of chlorzoxazone single and its combination of Yinzhihuang injection were compared with paired t test. A value of P<0.05 was considered to be statistically significant.Result1. The effect of Yinzhihuang injection on the acticity of CYP2E11.1 The inhibition of baicalin and geniposide on the acticity of CYP2E1 in rat microsome Yinzhihuang injection(P<0.001), baicalin(P=0.002) and baicalin combine geniposide group(P=0.001) had inhibition on the activity of CYP2E1, the inhibition ratio was 66.79%,46.57%,52.46%, respectively; while geniposide (P=0. 628) did not.1.2 The inhibition of CYP2E1 activity after multiple administration of Yinzhihuang injection There were significant differences in the activities of CYP2E1 between control group and experiment group, the activities of CYP2E1 were inhibited obviously by administration Yinzhihuang injection(P<0.001). It decreased the metabolism of chlorzoxazone by 53.62%。1.3 Effect of Yinzhihuang injection on the pharmacokinetics of chlorzoxazoneThe main pharmacokinetic parameters of single administration of chlorzoxazone group and coadministration of Yinzhihuang injection group:the values of AUC0�'t(P=0.009) were 1202.3±157.34,926.93±169.7 (μg·mr-1)-min, CL(P=0.016) were 0.014±0.002,0.018±0.004 ml-kg-1·-min-1, both of which had significance statistically, while the values of T1/2(P=0.001) were 19.98±3.7,29.74±14.98 min, which also had significance statistically. Conclusion1. Yinzhihuang injection can inhibit CYP2E1 activity in rat microsome in vitro, and baicalin plays an important part in it.2. Yinzhihuang injection inhibits the activity of CYP2E1 after multiple administration.3. Continuous administration of Yinzhihuang injection inhibits the pharmacokinetics of chlorzoxazone in rats.