骨刺片对斑马鱼胚胎发育的急性毒性研究
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摘要
目的研究两批骨刺片样品对斑马鱼胚胎发育的急性毒性作用。方法以斑马鱼胚胎为试验对象,观察骨刺片样品A(0.01、0.05、0.10、0.20、0.30、0.50、1.00 mg/mL)、样品B(0.01、0.05、0.10、0.50、1.00、2.00、10.00 mg/mL)对斑马鱼胚胎发育的影响,包括胚胎致畸、致死检测;同时采用超高效液相色谱法-四极杆飞行时间-质谱(UPLC-QTOF-MS)对两批样品所含化学成分进行定性分析。结果两批样品对斑马鱼胚胎发育的影响相似,主要表现为高浓度下的致死作用,低浓度下主要以发育滞后表型为主;但二者作用浓度存在一定差异,样品A的半数致畸剂量(TD_(50))为225.4 mg/mL,半数致死剂量(LD_(50))为292.0 mg/mL;样品B的TD_(50)为60.3 mg/mL,LD_(50)为1.382 mg/mL。UPLC-QTOF-MS分析结果显示,两批样品所含主要化学成分基本一致,但某些成分含量存在一定差异;初步分析显示既是有效成分又是毒性成分的士的宁含量与斑马鱼胚胎试验结果呈正相关。结论斑马鱼胚胎模型可用于评价骨刺片样品的急性毒性,两批骨刺片样品对斑马鱼胚胎发育影响均表现为高浓度下的致死作用,低浓度下,特别是样品B表现为轻度非正常表型,且均可逆。
Objective To study the acute toxic effect of Guci Tablets on zebrafish embryo development.Methods Zebrafish embryo was used to investigate the development situation under different drug concentrations of two batches of Guci Tablets samples(sample A:0.01,0.05,0.10,0.20,0.30,0.50 and 1.00 mg/mL,sample B:0.01,0.05,0.10,0.50,1.00,2.00,and 10.00 mg/mL)including embryo teratogenic and lethal tests.Additionally,an UPLC-QTOF-MS was applied to analyze the chemical constituents of both samples.Results The result showed that the effects of both samples on development of zebrafish embryos were similar.The main manifestation was lethal effect under high concentrations and lag phenotypes of development under low concentrations.However,there was a certain difference in the action concentration between both samples,The half of teratogenic dose(TD_(50))and the half of lethal dose(LD_(50))of sample A were 0.225 4 mg/mL and 0.292 mg/mL,respectively;the TD_(50)and the LD_(50)of sample B were 0.060 3 mg/mL and 1.382 mg/mL,respectively.According to the results of UPLC-QTOF-MS,the main chemical constituents contained in both samples were basically in agreement.But there were some differences in the content of some constituents.And the preliminary analysis showed there was a positive correlation between the content of strychnine and the toxicity evaluation result by zebrafish embryo experiment.Conclusion The result indicated that the zebrafish embryo model was suitable for the toxicity evaluation of Guci Tablets.The effects of two batches of Guci Tablets on zebrafish embryonic development were lethal at high concentrations,and slightly abnormal phenotypes at low concentrations,especially sample B,all of which were reversible.
Objective To study the acute toxic effect of Guci Tablets on zebrafish embryo development.Methods Zebrafish embryo was used to investigate the development situation under different drug concentrations of two batches of Guci Tablets samples(sample A:0.01,0.05,0.10,0.20,0.30,0.50 and 1.00 mg/mL,sample B:0.01,0.05,0.10,0.50,1.00,2.00,and 10.00 mg/mL)including embryo teratogenic and lethal tests.Additionally,an UPLC-QTOF-MS was applied to analyze the chemical constituents of both samples.Results The result showed that the effects of both samples on development of zebrafish embryos were similar.The main manifestation was lethal effect under high concentrations and lag phenotypes of development under low concentrations.However,there was a certain difference in the action concentration between both samples,The half of teratogenic dose(TD_(50))and the half of lethal dose(LD_(50))of sample A were 0.225 4 mg/mL and 0.292 mg/mL,respectively;the TD_(50)and the LD_(50)of sample B were 0.060 3 mg/mL and 1.382 mg/mL,respectively.According to the results of UPLC-QTOF-MS,the main chemical constituents contained in both samples were basically in agreement.But there were some differences in the content of some constituents.And the preliminary analysis showed there was a positive correlation between the content of strychnine and the toxicity evaluation result by zebrafish embryo experiment.Conclusion The result indicated that the zebrafish embryo model was suitable for the toxicity evaluation of Guci Tablets.The effects of two batches of Guci Tablets on zebrafish embryonic development were lethal at high concentrations,and slightly abnormal phenotypes at low concentrations,especially sample B,all of which were reversible.
引文
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[11]佟军威,张靖溥,孟杰.17α-乙炔雌二醇对斑马鱼胚胎发育的致畸作用及其基因靶位[J].药学学报, 2011, 46(1):50-55.
[12]胡占英,张靖溥.长春新碱对斑马鱼神经发育和行为的影响[J].毒理学杂志, 2014, 28(12):98-103.
[13] Rinkwitz S, Mourrain P, Becker T S. Zebrafish:an integrative system for neurogenomics and neurosciences[J]. Prog Neurobiol, 2011, 93(2):231-243.
[14] De Esch C, Slieker R, Wolterbeek A, et al. Zebrafish as potential model for developmental neurotoxicity testing:a mini review[J]. Neurotoxicol Teratol, 2012, 34(6):545-553.
[15] Mittelstadt S W, Hemenway C L, Craig M P, et al.Evaluation of zebrafish embryos as a model for assessing inhibition of hERG[J]. J Pharmacol Toxicol Methods,2008, 57(2):100-105.
[16] Zhu J J, Xu Y Q, He J H, et al. Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish[J]. Appltoxicol, 2014,34(2):139-148.
[17]张勇,郭胜亚,邓中平,等.斑马鱼在中药毒性靶器官研究中的进展[J].中国新药杂志, 2016, 25(12):1343-1347.
[18]许冰洁,张立将,李春启,等.斑马鱼胚胎评价5种药物的发育毒性与模型验证[J].中国药理学通报, 2016, 32(1):74-79.
[19]汪艳平,戴德雄,谢媛媛,等.骨刺胶囊HPLC指纹图谱[J].中成药, 2016, 38(1):98-103.
[20]赵静,秦振娴,彭冰,等.基于UPLC-Q-TOF MS技术的三七中皂苷类成分质谱裂解规律研究[J].质谱学报,2017, 38(1):97-108.
[21]赵何峰,王永林,郑林,等.UPLC-PDA-ESI-MS分析杜仲中化学成分[J].中国实验方剂学杂志, 2014, 20(3):59-62.
[22] Yang Y, Yin X J, Guo H M, et al. Identification and comparative analysis of the major chemical constituents in the extracts of single Fuzi herb and Fuzi-Gancaoheb-pair by UFLC-IT-TOF/MS[J]. Chin J Nat Med, 2014, 12(7):542-553.
[2]国家食品药品监督管理总局标准WS3-B-3290-2002[S],2002.
[3]国家食品药品监督管理总局标准WS3-B-3290-98-1[S],2014.
[4]国家食品药品监督管理总局标准WS3-B-3290-98-2[S],2014.
[5]国家食品药品监督管理总局标准WS3-B-3290-98-3[S],2014.
[6]张舒,袁晓芳,张艳.HPLC法测定骨刺片中柚皮苷、芍药苷的含量[J].广州化工, 2015, 43(16):140-142.
[7]曹玲,冯有龙,王亚超,等.骨刺片的质量标准研究[J].中国药品标准, 2011, 12(3):191-195.
[8]袁晓芳,张舒.骨刺片制剂的HPLC指纹图谱[J].中国医院药学杂志, 2015, 35(4):314-317.
[9]梁爱华.斑马鱼-一种可用于中药药效和毒性筛选的鱼类模型[J].中国中药杂志, 2009, 34(22):2839.
[10]郭隽,耿兴超,汪巨峰.药物发育毒性体外替代方法研究进展及组合策略[J].药物评价研究, 2015, 38(4):345-349.
[11]佟军威,张靖溥,孟杰.17α-乙炔雌二醇对斑马鱼胚胎发育的致畸作用及其基因靶位[J].药学学报, 2011, 46(1):50-55.
[12]胡占英,张靖溥.长春新碱对斑马鱼神经发育和行为的影响[J].毒理学杂志, 2014, 28(12):98-103.
[13] Rinkwitz S, Mourrain P, Becker T S. Zebrafish:an integrative system for neurogenomics and neurosciences[J]. Prog Neurobiol, 2011, 93(2):231-243.
[14] De Esch C, Slieker R, Wolterbeek A, et al. Zebrafish as potential model for developmental neurotoxicity testing:a mini review[J]. Neurotoxicol Teratol, 2012, 34(6):545-553.
[15] Mittelstadt S W, Hemenway C L, Craig M P, et al.Evaluation of zebrafish embryos as a model for assessing inhibition of hERG[J]. J Pharmacol Toxicol Methods,2008, 57(2):100-105.
[16] Zhu J J, Xu Y Q, He J H, et al. Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish[J]. Appltoxicol, 2014,34(2):139-148.
[17]张勇,郭胜亚,邓中平,等.斑马鱼在中药毒性靶器官研究中的进展[J].中国新药杂志, 2016, 25(12):1343-1347.
[18]许冰洁,张立将,李春启,等.斑马鱼胚胎评价5种药物的发育毒性与模型验证[J].中国药理学通报, 2016, 32(1):74-79.
[19]汪艳平,戴德雄,谢媛媛,等.骨刺胶囊HPLC指纹图谱[J].中成药, 2016, 38(1):98-103.
[20]赵静,秦振娴,彭冰,等.基于UPLC-Q-TOF MS技术的三七中皂苷类成分质谱裂解规律研究[J].质谱学报,2017, 38(1):97-108.
[21]赵何峰,王永林,郑林,等.UPLC-PDA-ESI-MS分析杜仲中化学成分[J].中国实验方剂学杂志, 2014, 20(3):59-62.
[22] Yang Y, Yin X J, Guo H M, et al. Identification and comparative analysis of the major chemical constituents in the extracts of single Fuzi herb and Fuzi-Gancaoheb-pair by UFLC-IT-TOF/MS[J]. Chin J Nat Med, 2014, 12(7):542-553.