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目的研究葛根素对人细胞色素P450(CYP)2D6酶活性的影响及其与CYP2D6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、0.025、0.050、0.100、0.200、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CYP2D6酶活性。(2)体内试验:以18名健康男性[年龄19~26(22±4)岁,体重62~75(69±7)kg]为对象,其中CYP2D6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2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2组受试者均口服美托洛尔100 mg;第2阶段(第12~17天):2组均不采取任何措施;第3阶段(第18~28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28天清晨2组受试者均口服美托洛尔100 mg。第11和28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CYP2D6酶活性。结果体外试验显示经0、0.025、0.050、0.100、0.200、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)、(0.012±0.001)、(0.011±0.001)、(0.010±0.001)、(0.005±0.002)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、0.200、0.400和0.800mmol/L葛根素组α-羟基美托洛尔产率明显低于0、0.025、0.050 mmol/L葛根素组(P<0.05),0.200、0.400、0.800 mmol/L葛根素组明显低于0.025、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、0.200、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CYP2D6基因型的3组受试者应用葛根素前后尿样中CYP 2D6活性变化程度组间比较,差异无统计学意义。结论葛根素对CYP2D6活性有明显的抑制作用,浓度越高抑制作用越明显,但与CYP2D6基因型无明显相关性。
Objective To study the effect of puerarin on the activity of human cytochrome P450 (CYP) 2D6 and its relationship with CYP2D6 genotype. Methods (1) In vitro experiments: The liver tissues of patients with intrahepatic bile duct stones were removed from the normal liver tissue to prepare human liver microsome incubation system. Different concentrations of puerarin (0,0.025,0.050,0.100,0.200,0.400,0.800 mmol / L ) And metoprolol to detect the metoprolol metabolite a-hydroxy metoprolol productivity by HPLC to reflect the CYP2D6 enzyme activity. (2) In vivo test: 18 healthy men [age range 19 ~ 26 (22 ± 4) years and weight 62 ~ 75 (69 ± 7) kg] were enrolled. The CYP2D6 genotype was * 1 / * 1, * 1 / * 10, * 10 / * 10 each of six. The experiment was conducted in three stages, using a two-stage crossover design. Subjects were randomly divided into 2 groups according to a simple randomized grouping method. In the first phase (day 1 to day 11), 1 group received intravenous puerarin 400 mg once daily for 10 days, and the other group did not receive administration. On the 11th day morning, both groups received oral metoprolol 100 mg; Stage 2 (Days 12-17): No measures were taken in both groups; Stage 3 (Days 18-28): Group 1 received intravenous puerarin 400 mg / d, and the other one was not given. On the 28th day, two groups of subjects took metoprolol 100 mg orally. On days 11 and 28, metoprolol 100 mg was administered to the subjects for 8 h urine, and metoprolol and alpha-hydroxymetoprolol concentrations in urine were determined by high performance liquid chromatography The ratio reflects the CYP2D6 enzyme activity. Results The in vitro experiments showed that the yields of α-hydroxy metoprolol in human liver microsomes treated with 0, 0.025, 0.050, 0.100, 0.200, 0.400 and 0.800 mmol / L puerarin were (0.018 ± 0.001) (0.015 ± 0.001), (0.012 ± 0.001), (0.011 ± 0.001), (0.010 ± 0.001), (0.005 ± 0.002) mmol / (mg · h) With the concentration of Puerarin gradually decreased, the overall mean difference was statistically significant (F = 30.750, P = 0.000). The pairwise comparisons showed that the yields of α-hydroxy metoprolol in 0.100, 0.200, 0.400 and 0.800 mmol / L puerarin groups were significantly lower than those in 0, 0.025 and 0.050 mmol / L puerarin groups (P <0.05) , Puerarin group (0.800 mmol / L) was lower than that of puerarin group (0.025,0.050 mmol / L), while puerarin group (0.800 mmol / L) <0.05). The in vivo tests showed that the concentrations of metoprolol and a-metoprolol in urine of 18 subjects before and after the application of puerarin were 1.11 ± 0.55 and 1.73 ± 0.94, respectively, with statistical significance (P <0.01). There was no significant difference between the three groups of subjects with different CYP2D6 genotypes in the changes of CYP 2D6 activity before and after the application of puerarin. Conclusion Puerarin significantly inhibited the activity of CYP2D6. The higher the concentration of puerarin, the more obvious the inhibitory effect was, but no significant correlation with CYP2D6 genotype.