Microsoft Word - 韩现芹_news_.doc

Size: px

Start display at page:

Download "Microsoft Word - 韩现芹_news_.doc"

仔韦
7 years ago
Views:

1 CYP3A 1,2, 1,2, 1 (1,, ; 2., ) 摘要 : 研究甘草 (Glycyrrhiza uralensis) 和连翘 (Forsythia suspensa) 对牙鲆 (Paralichthys olivaceus) 肝细胞色素 P4503A(CYP3A) 活性的影响将牙鲆随机分为 3 组, 即对照组甘草组和连翘组, 对照组每日口灌 0.9% 的生理盐水, 试验组每日 1 次口灌甘草 30 mg/kg 和连翘 100 mg/kg, 连续口灌 6 d, 第 7 天时, 采用直接测定法和间接测定法 ( 探针药物 ) 进行 CYP3A 酶活性的测定结果显示 : 在肝微粒体水平上, 甘草和连翘均可明显提高红霉素 -N- 脱甲基酶活性, 分别提高了 1.79 倍 4.87 倍与对照组相比, 甘草组和连翘组氨苯砜的半衰期分别降低了 4.51%(P>0.05) 和 36.8%(P<0.01); 药时曲线下面积分别减小了 8.28%(P>0.05) 和 32.3%(P<0.01); 总清除率分别增加了 5.63%(P>0.05) 和 99.3%(P<0.01) 说明连翘对牙鲆 CYP3A 的活性具有极显著诱导作用, 甘草诱导作用不显著提示其他药物与连翘合用时, 其有效性和安全性可能会受到影响关键词 : CYP3A 活性 ; 甘草 (Glycyrrhiza uralensis); 连翘 (Forsythia suspensa); 氨苯砜 ; 牙鲆 (Paralichthys olivaceus) 中图分类号 : S948 文献标识码 : A 文章编号 : (2010) P450(CYP450) (mixed function oxidase system, MFOS),,, () [1], CYP3A CYP450, 50% [2],, CYP3A, CYP3A -N- (erythromycin N-demethylase, ERND) CYP3A, ERND, CYP3A, CYP450,, (Glycyrrhiza uralensis ) (Forsythia suspensa),, [3], CYP450 ERND, CYP3A, 1 材料与方法 1.1 实验动物 (Paralichthys olivaceus), ( )g, ( )cm, 4~5 /, (16±2), ( ),,, 2, (), 1 d, 1.2 药品试剂与仪器 : Sigma, , : ; : : (nyhyzx07-046); ( ) : (1981-),,,, : , xianqinhan@yahoo.com.cn;, : : , Lijian@ysfri.ac.cn Marine Sciences / Vol. 34, No. 12 /

9% 的生理盐水, 试验组每日 1 次口灌甘草 30 mg/kg 和连翘 100 mg/kg, 连续口灌 6 d, 第 7 天时, 采用直接测定法和间接测定法 ( 探针药物 ) 进行 CYP3A 酶活性的测定结果显示 : 在肝微粒体水平上, 甘草和连翘均可明显提高红霉素

2 99 %; :, ; >99.8%, Amresco, ; : 85%; Agilent 1100 ( ); 96 : 100 g 500 ml, 0.5~1 h,, 0.5 h, 4,,, 0.29 g ml [4], 0.24 g ml 1.3 给药及样品采集 3, : (1) (0.9%); (2) 30 mg/kg; (3) 100 mg/kg 1, 6 d, 7, 3 10,, 3 5 mg/kg, 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 h ml,, r/min 5 min,, 血浆中氨苯砜的测定 1.4.1,, 0.2 ml 1.5 ml, 0.3 ml,, r min 10 min, 20 µl HPLC [5] Agilent Tc-C 18 (250 mm 4.6 mm, 5µm), 30, - (ph=3.55)(40 : 60, V/V), 1.0 ml/min, 260 nm, 20 µl ml 1.5 ml, 0.25 ml, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10 mg/l, 1.4.1, 5,, ,, 0.25, 2.5, 10 mg/l 3,, 5,,, : 样品实测药物浓度回收率 = 100% 样品加入药物浓度 , 2.5, 10 mg L, 1.4.1, 5 5, 5, 1.5 ERND 活性测定 1.5.1,, 0.9% 3,,,, Potter 1 : 5(W/V), 3 ml/g, 8,,, 2 ml,, g, 2 30 min,,, (MS), S9 [6],, (BCA ) BCA A B, A:B=50:1 BCA 0.5 g/l 0, 2, 4, 8, 16, 20, 24, 32, 40 μl, 40 μl 200 μl BCA ; min,, 562 nm, OD,, , 40 μl 200 μl BCA,, OD, 20 / 2010 / 34 / 12

5 ml, 0.3 ml,, 12 000 r min 10 min, 20 µl HPLC [5] 1.4.2 Agilent Tc-C 18 (250 mm 4.6 mm, 5µm), 30, - (ph=3.55)(40 : 60, V/V), 1.0 ml/min, 260 nm, 20 µl 1.4.3 1.4.3.1 0.25 ml 1.5 ml, 0.25 ml, 0.01, 0.

3 , ERND , 0.1, 0.2, 0.4, 0.6, 0.8 ml,, 2 ml( : 0, 0.1, 0.2, 0.4, 0.6, 0.8 nmol/ml Nash 2 ml, min, 5 min, 420 nm OD, OD ERND 0.1 mol/l PBS(pH7.4)1.7 ml, 0.1 ml, 0.1 ml, 25 2 min, 10 mmol/l (nicotinamide adenine dinucleotide 2'-phosphate, NADPH) 0.1 ml, 0.1 ml, min ZnSO ml,, 5 min, Ba(OH) m L,, 5 min, r/min, 10 min, 2 ml, Nash 2 ml, min, 5 min, : (HCHO) nmol/(mg min)=(od OD ) K 4/10 min(k OD, HCHO nmol/l ) 1.6 数据分析与处理 (Mean SD), - DAS2.1.1,,,, SPSS 结果 2.1 探针药物氨苯砜间接测定 2.1.1,,,, min, ,, HPLC, (A) (C), A= C (n=5, r= ) : 0.01~10 mg L, 0.01 mg L, (Signal/Noise, S/N)>3 (0.25, 2.5, 10 mg L)3 94.0%~99.2%, 5%, 2.1.3, 2, DAS2.1.1,,, 1/C/C, 1, 1 (A) (B)(C) Fig. 1 Representative HPLC chromatograms of (A) blank fish plasma, (B) plasma spiked with dapsone standard, and (C) plasma sample after injections of dapsone Marine Sciences / Vol. 34, No. 12 /

35m L,, 5 min, 11 000 r/min, 10 min, 2 ml, Nash 2 ml, 60 10 min, 5 min, 1.5.3.3 : (HCHO) nmol/(mg min)=(od OD ) K 4/10 min(k OD, HCHO nmol/l ) 1.6 数据分析与处理 (Mean SD), - DAS2.1.1,,,, SPSS13.

4 表 1 甘草和连翘对牙鲆单次腹腔注射氨苯砜 (10mg/kg) 后主要药代动力学参数的影响 (n=5) Tab. 1 Effects of Glycyrrhiza uralensis or Forsythia suspense on main pharmacokinetic parameters of dapsone after a single intraperitoneal administration at 5mg/kg to Paralichthys olivaceus (n=5) CL(kg/(L h)) ** t 1/2β (h) ** AUC(mg/(L h)) ** : *(P 0.05); ** (P 0.01), ± (mean±sd), t (Student s t-test), ERND 2 2 (5mg/kg) (n=5) Fig. 2 Plasma concentrations of Glycyrrhiza uralensis or Forsythia suspensa after single intraperitoneal administration in dapsone at 5mg/kg(n=5) (t 1/2β ) 4.51%(P 0.05) 36.8%(P 0.01); (area under the concentrationtime curve, AUC) 8.28%(P 0.05) 32.3%(P 0.01)(the total body clearance, CL) 5.63%(P 0.05) 99.3%(P 0.01) 2.2 红霉素 -N- 脱甲基酶活性 BCA, Y (OD ), Y X , r ~0.05 mg/l, 0.01 mg /L 2.2.2, OD, y x , r ~0.80 μmol/l OD, 0.10μmol/L ERND Microsoft Excel 2003 表 2 两种药物对牙鲆红霉素 -N- 脱甲基酶活性的影响 Tab. 2 Effect of the two drugs on erythromycin N-demethylase activity in Paralichthys olivaceus (n=10) (mg/g ) -N- nmol/(mg min) 7.076± ± ±0.441* 0.141±0.0087** 6.935± ±0.0108** : ± ; **P 0.01, * P 讨论 3.1 氨苯砜色谱条件的优化,,, 0.2 mol/l,,,, ,,,,,, ph, 2 5 ph : 3.5, 4, 4.5, 5, 5.5; : 45%, 40%, 35%, 30%, 25%, ph, ph3.5,, 22 / 2010 / 34 / 12

h)) 0.142 0.010 0.150 0.014 0.283 0.015 ** t 1/2β (h) 13.301 1.115 12.701 0.580 8.401 0.202 ** AUC(mg/(L h)) 17.576 1.130 16.121 0.690 11.900 0.526 ** : *(P 0.05); ** (P 0.

5 25%~45%, 5%, min 5 min, 5 min, ph3.5, 40% 3.2 甘草和连翘对牙鲆肝微粒体中 ERND 活性的影响, CYP450, CYP450 CYP450 [7,8], CYP450 CYP450, (Pueraria lobata) (Gardenia jasminoides) (Glycyrrhiza uralensis) (Panax notoginseng) (Scutellaria baicalensis) P450 CYP3A, CYP450, [9], CYP3A [10], N- CYP3A 7.076±0.47 mg/g, [7] 5.238mg/g±0.397 mg/g -N nmol/(mg min)± nmol/(mg min), Vaccaro [11] (Dicentrarchus labrax) nmol/ (mg min)±0.200 nmol/(mg min),, CYP3A P450, CYP3A CYP450,, CYP3A 2,, ERND, ,, CYP3A [12] P 探针药物法间接评价甘草和连翘对牙鲆 CYP3A 活性的影响 CYP3A, CYP3A CYP3A : [13] [14] [15],, CYP3A : t 1/2β h, [5] t 1/2β h, t 1/2β, CYP3A P450,, CYP3A ERND, 6 d, t 1/2β CL AUC h kg /(L h) mg/(l h),, t 1/2β 4.51%, CL 5.63%, AUC 8.28%,,, CYP3A [12] (490 mg/kg), [16] [17], CYP1A2 CYP450, CYP,, t 1/2β CL AUC h kg /(L h) mg/(l h),, t 1/2β 36.8%; CL 99.3%; AUC 32.3%,, CYP3A [18] CYP3A [19] CYP3A Marine Sciences / Vol. 34, No. 12 /

P450 CYP3A, CYP450, [9], CYP3A [10], N- CYP3A 7.076±0.47 mg/g, [7] 5.238mg/g±0.397 mg/g -N- 0.0567 nmol/(mg min)±0.0038 nmol/(mg min), Vaccaro [11] (Dicentrarchus labrax) 1.162 nmol/ (mg min)±0.

6 1, [20], CYP3A (pregnane X receptor, PXR) CYP3A, CYP3A, CYP3A PXR [21,22] CYP3A PXR,, CYP3A PXR [23,24],,, [25], P450, [26],,, [27],,,,,,,, CYP, CYP450, CYP3A,,, : [1] Slaughter R L, Edwards D J. Recent advances: The cytochrome P450 enzymes[j]. Ann Pharmacother, 1995, 29: 619. [2] Rendic S, Di Carlo F J. Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates inducers and inhibitors[j]. Drug Metab Rev, 1997, 29(1-2): [3],,. [J]., 2007, 31(5): [4]. [M]. :, [5],,. Flunixin P450 [J]., 2006, 36(12): [6],,. M 2 :, [7],.. CYP2E1 [J]., 2008, 17(1): [8] Moutou K A, Burke M D, Houlihan D F. Hepatic P450 monooxygenase response in rainbow trout administered aquaculture antibiotics[j]. Fish Physiol Biochem, 1998, 18(1): [9] Goksoyr A, Forlin L. The cytochrome P-450 system in fish: Aquatic toxicology and environmental monitoring [J]. Aquat Toxicol, 1992, 22: [10] Celander M, Stegeman J J. Isolation of a cytochrome P450 3A cdna sequence (CYP3A30) from a marine teleost Fundulus heteroclitus and phylogenetic analyses of CYP3A genes [J]. Biochem Biophys Res Commun, 1997, 236: [11] Vaccaro E, Giorgi M, Longo V, et al. Inhibition of cytochrome P450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax) [J]. Aquatic Toxicology, 2003, 62: [12],,, [J]., 1986, 11(10): 55. [13] Thummel K E, Shen D D, Podoll T D, et al.use of midazolam as a human cytochrome P450 3A probe: in vivo and in vitro correlation in liver transplantation patients[j]. J Pharmaco Exper Ther, 1994, 271: 549. [14],,,. CYP3A4 CYP1A2 [J]., 2002, 18(3): [15],,,. P450 3A [J]., 2002, 5: [16],, [J], 1994, 16(3): 46. [17],,, P4501A2 [J]., 2007, 7(4): [18],,,. Cocktail P450 [J]., 2003, 38(10): [19] Bray B J, Perry N B, Menkes D B, et al. St. John s wort extract induces CYP3A and CYP2E1 in the Swiss Webster mouse [J]. Toxicol Sci, 2002, 66: / 2010 / 34 / 12

Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates inducers and inhibitors[j]. Drug Metab Rev, 1997, 29(1-2): 413-580. [3],,. [J]., 2007, 31(5): 655-660. [4]. [M].

7 [20] Wang LS, Zhou G, Zhu B, et al. St John swort induces both cytochrome P450 3A4-catalyzed sulfoxidation and 2C19-dependent hydroxylation of omep razole [J]. Clin Pharmacol Ther, 2004, 75: [21] Masuyama H, Hiramatsu Y, Mizutani Y, et al. The expression of pregnane X receptor and its target gene, cytochrome P4503A1, inperinatal mouse[j]. Mol Cell Endocrinol, 2001, 172(1-2): [22] Le Culyse E L. Pregnane X receptor: molecular basis for species differences in CYP3A induction by xenobiotics[j]. Chem Biol Interact, 2001, 134(3): [23] Kliewer S A, Goodwin B, Willson T M. The nuclear pregnane X receptor: a key regulator of xenobiotic me- tabolism. Endocr[J]. The Endocrine Reviews, 2002, 23: [24] Masuyama H, Hiramatsu Y. Endocrine disrupting chemicals, phthalic acid and nonylphenol, activate Pregnane X receptor-mediated transcription[j]. Mol Endocrinol, 2000, 14: [25],,,. CYP450 [J]., 2002, 6(37): [26],. [J]., 2007, 11: [27],. [J]., 1998, 38(5): 56. Effects of Glycyrrhiza uralensis and Forsythia suspensa on cytochrome CYP3A in Paralichthys olivaceus HAN Xian-qin 1,2, LI Jian 1,2, LI Ji-tao 1 (1. Key Laboratory of Sustainable Utilization of Marine Fishery Resources of the Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science, Qingdao , China; 2. National Center for Quality Supervision and Test of Aquatic Products, Qingdao , China) Received: Dec., 24, 2009 Key words: CYP3A activity; Glycyrrhiza uralensis; Forsythia suspensa; Dapsone; Paralichthys olivaceus Abstract: The effects of Glycyrrhiza uralensis (GU) and Forsythia suspense (FS) on activities of cytochrome CYP3A in Paralichthys olivaceus were studied. Fishes were divided into three groups as follows: GU and FS group were administered orally for six days at 30mg/kg bw and 100mg/kg bw, respectively; fish in the control group were given an equal volume of normal saline (0.9%). Seven day after the injection, the activities of CYP3A were measured directly and indirectly. We found that on the level of liver microsomes, GU and FS both increased erythromycin N-demethylase activity significantly by 1.79 times and 4.87 times respectively. Compared with the normal saline-treated control group, metabolism of dapsone in FS and GU group were higher than that in the control group, the elimination half -lives were shortened by 4.51% (P>0.05) and 36.8% (P<0.01), respectively. Area under the concentration-time curve were shortened by 8.28% (P>0.05) and 32.3% (P<0.01), respectively; and the total body clearances were increased by 5.63% (P>0.05) and 99.3% (P<0.01), respectively. The results showed that GU and FS can significantly induced the activity of CYP3A; and the induction effect of FS is higher than GU. A drug s efficacy and safety may be affected by other drugs used concomitantly. ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 34, No. 12 /