论文部分内容阅读
目的：研究葛根素对人细胞色素P450（ CYP）1D6酶活性的影响及其与CYP1D6基因型的关系。方法（1）体外试验：取肝内胆管结石患者手术切除标本中正常肝组织制备人肝微粒体孵育体系，加入不同浓度葛根素（0、0.015、0.050、0.100、0.100、0.400、0.800 mmol/L）及美托洛尔，以高效液相色谱法检测美托洛尔代谢产物α-羟基美托洛尔产率，以此反映CYP1D6酶活性。（1）体内试验：以18名健康男性[年龄19~16（11±4）岁，体重61~75（69±7）kg]为对象，其中CYP1D6基因型为*1/*1、*1/*10、*10/*10者各6名。试验分3个阶段进行，采用两阶段交叉试验设计。将受试者按照简单随机化分组方法随机分为1组。第1阶段（第1~11天）：1组连续10 d静脉滴注葛根素注射液400 mg/d，另1组不给药，第11天清晨1组受试者均口服美托洛尔100 mg；第1阶段（第11~17天）：1组均不采取任何措施；第3阶段（第18~18天）：第1阶段未服药组连续10 d静脉滴注葛根素注射液400 mg/d，另1组不给药，第18天清晨1组受试者均口服美托洛尔100 mg。第11和18天口服美托洛尔100 mg后收集受试者连续8 h尿液，采用高效液相色谱法测定尿液中美托洛尔及α-羟基美托洛尔浓度，以二者之比反映CYP1D6酶活性。结果体外试验显示经0、0.015、0.050、0.100、0.100、0.400、0.800 mmol/L葛根素处理的人肝微粒体反应体系α-羟基美托洛尔产率分别为（0.018±0.001）、（0.015±0.001）、（0.015±0.001）、（0.011±0.001）、（0.011±0.001）、（0.010±0.001）、（0.005±0.001）mmol/（mg·h），α-羟基美托洛尔产率随着葛根素浓度的升高而逐渐降低，总体均数间差异有统计学意义（ F =30.750，P =0.000）。两两比较显示，0.100、0.100、0.400和0.800 mmol/L葛根素组α-羟基美托洛尔产率明显低于0、0.015、0.050 mmol/L葛根素组（ P﹤0.05），0.100、0.400、0.800 mmol/L 葛根素组明显低于0.015、0.050 mmol/L 葛根素组（ P ﹤0.05），而0.800 mmol/L葛根素组明显低于0.100、0.100、0.400 mmol/L葛根素组（ P﹤0.05）。体内试验显示18名受试者应用葛根素前后尿样中美托洛尔与α-羟基美托洛尔浓度比值分别为1.11±0.55和1.73±0.94，差异有统计学意义（ P﹤0.01）。不同CYP1D6基因型的3组受试者应用葛根素前后尿样中CYP 1D6活性变化程度组间比较，差异无统计学意义。结论葛根素对CYP1D6活性有明显的抑制作用，浓度越高抑制作用越明显，但与CYP1D6基因型无明显相关性。“,”Objective To investigate the effect of puerarin on cytochrome P450( CYP)1D6 activity and its relationship with CYP1D6 genotypes in human. Methods (1)In vitro experiment:human liver microsomal incubation mixtures was prepared with normal liver tissues from a surgical excision of patients with introhepatic cholelithiasis;different concentrations of puerarin(0,0. 015,0. 050,0. 100, 0. 100,0. 400,and 0. 800 mmol/L)and metoprolol were added into the incubation mixtures and the productive rate of metoprolol metabolite(α-hydroxymetoprolol )was detected using HPLC,which was used to access the activity of CYP1D6.(1)In vivo test:18 male healthy volunteers with age of 19-16(11 ± 4)years and weight of 61~75(69 ± 7)kg were selected as the subjects. Of them,6 patients were genotype of CYP450 1D6 *1/ *1,6 were genotype of *1/ *10,and 6 were genotype of *10/ *10. The test comprised 3 stages and was carried out using a two-phase crossover design. The subjects were divided into 1 groups by simple randomization method. The first stage( the 1st day to the 11th day):the subjects in one group received an IV infusion of puerarin 400 mg once daily for 10 days and the subjects in another group received nothing. All subjects received metoprolol 100 mg orally early in the morning on the 11th day. The second stage(the 11th to the 17th day):all subjects received nothing. The third stage(the 18th to the 18th day):the subjects,who received nothing in the first stage,received an IV infusion of puerarin 400 mg once daily for 10 days and the subjects in another group received nothing. All subjects received metoprolol 100 mg orally early in the morning on the 18th day. Urine was collected from 0 to 8 h after metoprolol administration on the 11th and 18th days. The concentration of metoprolol andα-hydroxymetoprolol was detected by HPLC and its ratio was used to assess CYP1D6 activity. Results The results of in vitro experiment showed thatα-hydroxymetoprolol levels in human liver microsomal incubation mixtures after treatment with puerarin 0, 0. 015,0. 050,0. 100,0. 100,0. 400,and 0. 800 mmol/L were(0. 018 ± 0. 001),(0. 015 ± 0. 001), (0. 015 ± 0. 001),(0. 011 ± 0. 001),(0. 011 ± 0. 001),(0. 010 ± 0. 001),and(0. 005 ± 0. 001)mmol/(mg·h),respectively. The productive rate of α-hydroxymetoprolol decreased with increase of puerarin concentrations and the difference in population mean was statistically significant(F=30. 750,P=0. 000). The results of pairwise comparision showed that the productive rate of α-hydroxymetoprolol in the puerarin 0. 100,0. 100,0. 400,and 0. 800 mmol/L groups were markedly lower than those in the puerarin 0, 0. 015,0. 050,0. 100,0. 100,0. 400,and 0. 800 mmol/L groups(P﹤0. 05);the productive rate of α-hydroxymetoprolol in the puerarin 0. 100,0. 400,and 0. 800 mmol/L groups were markedly lower than those in the puerarin 0. 015 and 0. 050 mmol/L groups(P﹤0. 05);the productive rate ofα-hydroxymetoprolol in the puerarin 0. 800 mmol/L group was markedly lower than those in the puerarin 0. 100,0. 100,and 0. 400 mmol/L groups( P ﹤ 0. 05 ). The results of in vivo test showed that the ratios of metoprolol and α-hydroxymetoprolol in urine in the 18 subjects before and after puerarin treatment were 1. 11 ± 0. 55 and 1. 73 ± 0. 94,respectively. The difference was statistically significant(P﹤0. 01). The changes of CYP1D6 activity in urine in all subjects with different CYP1D6 genotypes in the 3 groups were compared and the differences were not statistically significant. Conclusion Puerarin could inhibit the activity of CYP1D6 in a dose-dependent manner,but it is not related to the CYP1D6 genotypes.