白及有效部位中militarine在模式生物斑马鱼中的代谢研究
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摘要
目的:采用模式生物斑马鱼研究白及有效部位中双[4-(β-D-吡喃葡萄糖氧)苄基]-2-异丁基苹果酸酯(militarine)的代谢产物及转化形式,探索斑马鱼用于研究药物Ⅰ,Ⅱ相代谢的可行性及合理性。方法:将斑马鱼置于militarine溶液中24 h,按时取药液和鱼体,采用UPLC-ESI-Q-TOF/MS~E技术,并结合UNIFI、Masslynx V4. 1及Single Mass Analysis等代谢产物分析技术,推测代谢产物。结果:militarine经斑马鱼作用后,主要以酯键水解、硫酸化反应为主。在斑马鱼体内外检测到militarine原形成分和1个酯键水解产物及1个硫酸化产物。结论:白及有效部位中militarine经斑马鱼代谢后存在Ⅰ,Ⅱ相代谢产物,这与大鼠体内的代谢机制高度一致,提示斑马鱼对白及有效部位中militarine的代谢具有合理性,为建立简单、快速、高效预测药物在体内代谢的斑马鱼新模型提供理论依据。
Objective: To investigate the metabolism of militarine components of the effective fractions of Bletilla striata in zebrafish and their metabolites and discuss the feasibility and rationality of using zebrafish as a metabolic model for phase Ⅰ and Ⅱ metabolism study of drugs. Methods: Zebrafish was exposed to militarine solution 24 h,and the samples of solution and zebrafish body were extracted on time and analyzed by using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry technology (UHPLC-QTOF MS) method with UNIFI,Masslynx V4. 1 and the data was treated with data mining software Single Mass Analysis. Results: After militarine is treated by zebrafish,the major metabolites were sulfate conjugate,ester bond hydrolysis of militarine. The militarine components were detected and three metabolites were identified as militarine original fraction,a ester bond hydrolysate and a sulfated product in the zebrafish in vivo and in vitro. Conclusion:Metabolic products of phase Ⅰ and Ⅱ in militarine of the effective fractions of Bletilla striata,and that were highly consistent with those in rats,which indicates that this method is rational. To provide a theoretical basis for the establishment of a new model of zebrafish that is simple,rapid and efficient in predicting the metabolism of drugs in vivo.
Objective: To investigate the metabolism of militarine components of the effective fractions of Bletilla striata in zebrafish and their metabolites and discuss the feasibility and rationality of using zebrafish as a metabolic model for phase Ⅰ and Ⅱ metabolism study of drugs. Methods: Zebrafish was exposed to militarine solution 24 h,and the samples of solution and zebrafish body were extracted on time and analyzed by using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry technology (UHPLC-QTOF MS) method with UNIFI,Masslynx V4. 1 and the data was treated with data mining software Single Mass Analysis. Results: After militarine is treated by zebrafish,the major metabolites were sulfate conjugate,ester bond hydrolysis of militarine. The militarine components were detected and three metabolites were identified as militarine original fraction,a ester bond hydrolysate and a sulfated product in the zebrafish in vivo and in vitro. Conclusion:Metabolic products of phase Ⅰ and Ⅱ in militarine of the effective fractions of Bletilla striata,and that were highly consistent with those in rats,which indicates that this method is rational. To provide a theoretical basis for the establishment of a new model of zebrafish that is simple,rapid and efficient in predicting the metabolism of drugs in vivo.
引文
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[3]FISHMAN MC.Zebrafish-the canonical vertebrate[J].Science,2001,294(5545):1290-1291.
[4]LANGHEINRICH U.Zebrafish:a new model on the pharmaceutical catwalk[J].Bioessays,2003,25(9):904-912.
[5]MILAN DJ,PETERSON TA,RUSKIN JN,et al.Drugs that induce repolarization abnormalities cause bradycardia in zebrafish[J].Circulation,2003,107(10):1355-1358.
[6]THOMPSON ED,BURWINKEL KE,CHAVA AK,et al.Activity of PhaseⅠand PhaseⅡenzymes of the benzopyrene transformation pathway in zebrafish following waterborne exxposure to arsenite[J].Comp Biochem Physiol C Toxicol Pharmacol,2010,152(3):371-378.
[7]ALMEIDA DV,NORNBERG BFDS,GERACITANO LA,et al.Induction of PhaseⅡenzymmes and hsp70 genes by copper sulfate through the electrophile-responsive element(EpRE):insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin[J].Fish Physiol Biochem,2010,36(3):347-353.
[8]BRESOLIN T,REBELO MDF,BAINY ACD.Expression of PXR,CYP3A and MDR1 genes in liver of zebrafish[J].Comp Biochem Physiol C Toxicol Pharmacol,2005,140(3-4):403-407.
[9]吕爱军,杨正行,刘欢,等.斑马鱼肠道细菌的分离与生理生化特性[J].中国农学通报,2010,26(24):412-415.
[10]陈秋霞,曾苏.斑马鱼在药物代谢中的研究进展[J].药学学报,2011,46(9):1026-1031.
[11]THOMPSON ED,BURWINKEL KE,CHAVA AK,et al.Activity of PhaseⅠand PhaseⅡenzymes of the benzo[a]pyrene transformation pathway in zebrafish[J].Comp Biochem C Toxicol Pharmacol,2010,152(3):371-378.
[12]国家药典委员会.中华人民共和国药典[S].北京:中国医药科技出版社,2015.
[13]国家中医药管理局《中华本草》编委会.中华本草苗药卷[M].贵阳:贵州科技出版社,2005:197-199.
[14]俞林花,史海霞,聂绪强,等.芦荟和白及创面愈合的药理作用比较[J].时珍国医国药,2010,21(6):1450-1453.
[15]TAKAGI S,YAMAKI M,INOUE K.Antimicrobial agents fromBletilla striata[J].Phytochemistry,1983,22(4):1011-1015.
[16]赵菲菲.白及BS-80EE组分的止血作用及其药效物质基础与作用机制研究[D].贵州:贵州医科大学,2017.
[17]韦英杰,宁青,贾晓斌,等.基于斑马鱼模型的中药代谢研究思路与方法[J].中草药,2009,40(7):1009-1011.