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Evaluation of Effect of Scrophularia Ningpoensis on Cytochrome P450 Enzymes Using Cocktail Probe Drug Method
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摘要: 采用Cocktail探针药物法研究玄参对大鼠4种细胞色素P450（CYP3A4、CYP2D6、CYP2C9和CYP1A2）活性的影响. 24只健康雄性SD大鼠（Sprague-Dawley rat）随机分为三组：A组为多剂量组，连续7 d给予150 mg/kg玄参. B组为单剂量组，第7 d给予150 mg/kg玄参. C组为对照组. 第7 d，各组在给予玄参30 min后，再给予4种探针药物的混合溶液，其中咪达唑仑3 mg/kg、美托洛尔20 mg/kg、氯沙坦5 mg/kg、非那西汀5 mg/kg，分别用以评价CYP3A4、CYP2D6、CYP2C9和CYP1A2的活性. 根据设定的时间点收集大鼠血浆样品，处理后的样品采用超高效液相色谱-串联质谱（UHPLC-MS/MS）法测定样品中4种探针药物的浓度. 结果可见，玄参对大鼠体内氯沙坦和非那西汀的药代动力学参数无显著影响. 单剂量的玄参导致咪达唑仑代谢减慢，多剂量给予的玄参显著诱导美托洛尔代谢. 玄参对CYP2C9和CYP1A2的酶活性无明显影响，可以抑制CYP3A4或诱导CYP2D6的酶活性.
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关键词:
- 玄参 /
- 鸡尾酒疗法 /
- 细胞色素P450 /
- 中药-西药相互作用 /
- 超高效液相色谱-串联质谱
Abstract: The effects of Scrophularia ningpoensis on the activities of 4 Cytochrome P450 enzymes (CYP3A4, CYP2D6, CYP2C9 and CYP1A2) in rats was investigated using the Cocktail probe drug method. Twenty-four healthy male Sprague-Dawley rats were randomly divided into three groups: group A (multiple-doses of 150 mg/kg Scrophularia ningpoensis for 7 d), group B (single-dose of 150 mg/kg Scrophularia ningpoensis), and group C (control group). To each group after administering Scrophularia ningpoensis for 30 min, the mixture solution of probe drugs midazolam (3 mg/kg, evaluation of CYP3A4 activity), metoprolol (20 mg/kg, evaluation of CYP2D6 activity), losartan (5 mg/kg, evaluation of CYP2C9 activity) and phenacetin (5 mg/kg, evaluation of CYP1A2 activity) were administered orally on the 7th d. Rat plasma samples were collected at the arranged time points, and the plasma concentrations of 4 probe drugs in the samples were simultaneously determined using the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method after processing. The results showed that Scrophularia ningpoensis had almost no significant effect on the pharmacokinetic parameters of losartan and phenacetin. However, a single-dose of Scrophularia ningpoensis induced a slow metabolism of midazolam. In addition, the pharmacokinetics of metoprolol was affected significantly by the multiple doses of Scrophularia ningpoensis, which induced rapid metabolism. The results suggested that Scrophularia ningpoensis has no significant effect on the enzymatic activities of CYP2C9 and CYP1A2, but it might inhibit enzymatic activities of CYP3A4 or induce CYP2D6.-
Key words:
- Scrophularia ningpoensis /
- cocktail /
- CYP450 /
- herb-drug interactions /
- UHPLC-MS/MS
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图 1 玄参多剂量组、单剂量组和对照组（n=8）中（a）咪达唑仑、（b）美托洛尔、（c）氯沙坦和（d）非那西汀的血药浓度-时间曲线图
Figure 1. Mean plasma concentration-time curves of (a) midazolam, (b) metoprolol, (c) losartan and (d) phenacetin after multiple doses or single dose of Scrophularia ningpoensis and control group (n=8)
表 1 4种探针药和地西泮的质谱参数信息
Table 1. Mass spectrometric parameters of four probe drugs and diazepam
化合物 母离子/(m/z) 子离子/(m/z) 碰撞能/eV 碎裂电压/V 咪达唑仑 326.1 290.8 28 170 美托洛尔 268.2 115.9 17 130 氯沙坦 423.2 206.8 22 130 非那西汀 180.1 109.9 24 122 地西泮 285.0 192.9 34 164 
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表 2 咪达唑仑、美托洛尔、氯沙坦和非那西汀在血浆中的精密度、准确度、回收率和基质效应
Table 2. Precision, accuracy, recovery and matrix effects of metoprolol, phenacetin, midazolam and losartan in rat plasma
分析物 加入质量浓度/
(ng/mL)日内精密度 日间精密度 回收率/% 基质效应/% Mean±SD/
(ng/mL)RSD/% RE/% Mean±SD/
(ng/mL)RSD/% RE/% 咪达唑仑 0.25 0.26±0.02 7.72 2.51 0.25±0.02 6.52 0.70 93.23 88.79 2.5 2.41±0.11 4.53 −3.73 2.62±0.18 7.03 4.70 91.97 99.57 25 24.98±1.62 6.48 −0.10 25.59±0.58 2.29 2.36 90.80 92.59 美托洛尔 5 5.14±0.19 3.60 2.75 5.02±0.11 2.16 0.34 93.92 93.16 50 54.47±3.67 6.73 8.93 51.50±2.71 5.27 2.99 89.49 94.32 500 539.34±28.64 5.31 7.87 532.06±15.41 2.90 6.41 91.28 98.14 氯沙坦 2.5 2.46±0.17 6.77 −1.72 2.43±0.11 4.52 −2.69 99.89 97.72 25 23.97±1.15 4.81 −4.12 24.12±0.79 3.26 −3.52 90.95 98.11 250 248.35±7.12 2.87 −0.66 251.63±3.79 1.51 0.65 96.31 100.38 非那西汀 5 5.12±0.15 2.84 2.31 4.98±0.13 2.54 −0.47 98.04 86.89 50 55.09±2.45 4.45 10.19 53.90±1.18 2.19 7.79 95.86 93.57 500 524.67±28.01 5.34 4.93 525.57±6.00 1.14 5.11 97.59 92.38
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表 3 大鼠血浆中咪达唑仑的主要药代动力学参数（Mean±SD, n=8）
Table 3. Main pharmacokinetic parameters of midazolam in rat plasma (Mean±SD, n=8)
参数 单位 A B C AUC0-t μg/ (L·h) 29.47+7.47 40.13+5.15** 24.70+8.03 AUC0-∞ μg/ (L·h) 30.35+7.86 41.95+5.55** 25.77+8.12 t1/2z h 1.04+0.19 1.25+0.33 1.19+0.40 Tmax h 0.66+0.19** 0.75+0.00** 0.50+0.00 Vz/F L/kg 154.94+40.55 131.93+45.08* 211.88+80.31 CLz/F L/h/kg 104.35+24.76 72.67+10.24** 125.63+35.25 Cmax μg/L 13.80+1.44 19.95+6.36 18.18+4.91 A:多剂量组，B:单剂量组，C:对照组. *：p<0.05，**：p<0.01代表与对照组相比存在显著性差异
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表 4 大鼠血浆中美托洛尔的主要药代动力学参数（Mean±SD, n=8）
Table 4. Main pharmacokinetic parameters of metoprolol in rat plasma (Mean±SD, n=8)
参数 单位 A B C AUC0-t μg/ (L·h) 4 632.88±608.22** 5 084.97±604.10 5 807.72±694.60 AUC0-∞ μg/ (L·h) 5 886.34±832.51 5 816.49±774.52 6 804.05±1 069.87 t1/2z h 3.96±1.25 2.84±0.70 3.03±0.54 Tmax h 0.50±0.13 0.54±0.09 0.56±0.18 Vz/F L/kg 19.42±5.63* 14.20±3.28 12.90±1.80 CLz/F L/h/kg 3.46±0.53 3.50±0.51 3.00±0.46 Cmax μg/L 1 532.22±153.54** 1 707.39±176.29** 2 140.76±220.66 A：多剂量组，B：单剂量组，C：对照组. *：p<0.05，**：p<0.01代表与对照组相比存在显著性差异
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表 5 大鼠血浆中氯沙坦的主要药代动力学参数（Mean±SD, n=8）
Table 5. Main pharmacokinetic parameters of losartan in rat plasma (Mean±SD, n=8)
参数 单位 A B C AUC0-t μg/ (L·h) 1 750.73±353.28 1 380.02±245.57 1 735.48±416.06 AUC0-∞ μg/ (L·h) 1 917.43±436.75 1 591.20±334.33 1 973.85±433.96 t1/2z h 2.94±0.83 4.14±2.09 3.53±0.67 Tmax h 2.00±0.00 2.00±0.58 2.25±0.46 Vz/F L/kg 11.36±3.09 18.48±6.45 13.73±4.70 CLz/F L/h/kg 2.73±0.61 3.25±0.63 2.64±0.58 Cmax μg/L 290.92±40.33 268.25±73.28 271.89±73.62 A：多剂量组，B：单剂量组，C：对照组
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表 6 大鼠血浆中非那西汀的主要药代动力学参数（Mean±SD, n=8）
Table 6. Main pharmacokinetic parameters of phenacetin in rat plasma (Mean±SD, n=8)
参数 单位 A B C AUC(0-t) μg/ (L·h) 1 454.88±452.60 1 149.00±360.03 1 580.96±446.16 AUC(0-∞) μg/ (L·h) 1 455.83±453.30 1 161.96±360.64 1 619.49±431.26 t1/2z h 0.52±0.08* 0.67±0.18 1.14±0.65 Tmax h 0.24±0.03 0.25±0.00 0.27±0.10 Vz/F L/kg 2.82±1.05 4.59±2.08 5.85±4.07 CLz/F L/h/kg 3.77±1.29 4.67±1.37 3.37±1.29 Cmax μg/L 1 460.41±323.01 1 404.60±323.73 1 822.10±558.58 A：多剂量组，B：单剂量组，C：对照组. *：p<0.05代表与对照组相比存在显著性差异
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