【作者】 熊静悦；

【导师】 赵军宁；

【作者基本信息】 成都中医药大学 ， 中药学， 2017， 博士

【摘要】 目的:利用基于发光细菌的Microtox测试技术,对国家药品不良反应监测中心数据库指引的高风险品种--参麦注射液的生物毒性实现定量化表征,初步探索影响其毒性测试结果的物质基础及临床意义,以期为参麦注射液的质量控制和不良反应风险预警提供新型、快速、灵敏的检测方法和技术平台。方法:应用Plackett-Burman法优化设计基于费氏弧菌CS234、明亮发光杆菌502以及青海弧菌Q67的测试体系中影响发光强度的6个因素,分别为:平衡时间、复苏稀释液体积、复苏时间、发光细菌菌液体积、待测样品体积以及反应时间,选择11因子两水平的实验设计。使用1.0 mol·L^-1的盐酸溶液或氢氧化钠溶液调节费氏弧菌CS234、明亮发光杆菌502以及青海弧菌Q67复苏稀释液的pH值,测试系列pH值复苏稀释液对基于上述三种发光细菌Microtox测试体系的影响。采用七水硫酸锌对基于费氏弧菌CS234的Microtox测试体系的测试方法学进行验证。分别考察该测试体系的特异性、标准曲线及定量范围、灵敏度、精密度（批内以及批间）、准确性、室温及4℃密封保存条件下七水硫酸锌储备液及质量控制样品中锌离子的稳定性。应用基于费氏弧菌CS234的Microtox测试体系对6个厂家共计15个批次的参麦注射液市售产品进行测试,得到相应的Microtox测试参数半数抑制浓度（%）,并且随行七水硫酸锌的标准曲线和质量控制样品,用以确保测试过程的正确性以及测试数据的可靠性。采用高效液相色谱法对上述参麦注射液中三种代表性的人参皂苷Re、人参皂苷Rg₁以及人参皂苷Rb₁进行含量测定,分析Microtox测试参数半数抑制浓度（%）与人参皂苷Re、人参皂苷Rg₁、人参皂苷Rb₁以及三种人参皂苷总含量的相关性。采用肥大细胞脱颗粒模型观察上述参麦注射液和人参皂苷Re对人肥大细胞系HMC-1细胞释放组胺以及β-氨基己糖苷酶的影响,倒置显微镜下观察参麦注射液、人参皂苷Re及Compond 48/80作用于HMC-1细胞1小时前后细胞形态的变化,ELISA酶联免疫试剂盒检测HMC-1细胞上清液中组胺以及β-氨基己糖苷酶的含量。结果:经Plackett-Burman方法优化,以发光衰减系数的均值偏差小于3%为判断标准,构建得到费氏弧菌CS234、明亮发光杆菌502以及青海弧菌Q67各自最适宜的发光体系为:⑴费氏弧菌CS234冻干粉于15℃恒温恒湿箱中平衡15min后转移至15℃预温槽,立即加入复苏稀释液1 ml,移液器轻柔吹打混匀10min,各取100μl菌液测试初始发光强度后,立即分别加入1 ml复苏稀释液（对照管）或待测样品,反应10 min后再次测试对照管以及样品管发光强度;⑵明亮发光杆菌502冻干粉于15℃恒温恒湿箱中平衡10 min后转移至15℃预温槽,立即加入复苏稀释液2 ml,移液器轻柔吹打混匀15 min,各取100μl菌液测试初始发光强度后,立即分别加入1 ml复苏稀释液（对照管）或待测样品,反应15 min后再次测试对照管以及样品管发光强度;⑶青海弧菌Q67冻干粉于15℃恒温恒湿箱中平衡10 min后转移至15℃预温槽,立即加入复苏稀释液2 ml,移液器轻柔吹打混匀10 min,各取100μl菌液测试初始发光强度后,立即分别加入2 ml复苏稀释液（对照管）或待测样品,反应10 min后再次测试对照管以及样品管发光强度。上述操作均在15℃±1℃条件下完成。调节上述三种发光细菌复苏稀释液的pH值后发现:⑴复苏稀释液pH值在4.5-8.0范围内,费氏弧菌CS234发光强度的波动小于±10%;⑵复苏稀释液pH值在3.6-3.8时,对明亮发光杆菌502发光强度的影响由抑制转为促进;⑶复苏稀释液pH值在5.0-6.0范围内时,对青海弧菌Q67发光强度的影响小于±15%。测试方法学特异性的考察结果显示:重复测试三次,复苏稀释液、渗透压调节液以及七水硫酸锌储备液的发光强度均在0.01 RLU-0.02 RLU之间,费氏弧菌CS234的发光强度在1152 RLU-1171 RLU之间;标准曲线、定量范围、精密度（含批内、批间）以及准确性的测试结果显示:重复测试三次,七水硫酸锌溶液在3.86mg·L^-1-77.27 mg·L^-1范围内标准曲线方程分别为y=20.48Ln（x）-13.34、y=20.48Ln（x）-13.83以及y=21.78Ln（x）-15.14,对应的相关系数和半数抑制浓度分别为0.992、0.990、0.998以及22.0 mg·L^-1、22.6 mg·L^-1、19.90 mg·L^-1;七水硫酸锌质量控制样品溶液90.0 mg·L^-1、50.0 mg·L^-1、15.0 mg·L^-1(终浓度分别为69.55 mg·L^-1、38.64 mg·L^-1以及11.59 mg·L^-1)以及定量下限样品溶液5.0 mg·L^-1(终浓度为3.86mg·L^-1)批内精密度的相对标准差小于5%、批间精密度的相对标准差小于6%,准确性在85.8%至103.2%范围内。对七水硫酸锌储备液及其质量控制样品溶液中锌离子的稳定性分别连续考察120小时及8小时,相对标准差均小于2%。对6个厂家15批次参麦注射液的Microtox测试结果显示:除SCSH制药股份有限公司的受试样品无法计算半数抑制浓度外（即最高浓度受试样品的抑制率未达到50%）,其余厂家样品的半数抑制浓度从46.7%至72.3%不等,所得浓度（%）-发光抑制率（%）动力学曲线相关性良好,相关性均在0.97以上。对5个厂家（除SCSH制药股份有限公司）半数抑制浓度进行单因素方差分析,结果显示厂家间半数抑制浓度存在显著性差异,这种差异具有统计学意义（P<0.05）。随行七水硫酸锌质控样品精密度的相对标准差小于4%,准确性在87.03%至97.76%范围内。对上述参麦注射液中人参皂苷Re、人参皂苷Rg₁和人参皂苷Rb₁含量测定的结果显示:各厂家参麦注射液中人参皂苷Rg₁和人参皂苷Rb₁的含量分别在0.11mg·ml^-1-0.18 mg·ml^-1以及0.14 mg·ml^-1-0.33 mg·ml^-1范围内,对上述两种人参皂苷含量分别进行非参数检验,结果显示厂家间含量无显著性差异;人参皂苷Re的含量和三种人参皂苷的总含量分别在0.07 mg·ml^-1-0.14 mg·ml^-1以及0.34 mg·ml^-1-0.60 mg·ml^-1范围内,非参数检验显示厂家间含量均存在显著性差异（P<0.05）。Microtox测试的半数抑制浓度（%）分别和人参皂苷Re、人参皂苷Rg₁、人参皂苷Rb₁含量及三者总含量进行Pearson相关性分析,结果显示,各厂家参麦注射液半数抑制浓度（%）和人参皂苷Re的含量之间存在显著的正相关关系（P=0.001）,相关系数为0.823。对肥大细胞脱颗粒模型的作用结果显示:人参皂苷Re含量为0.10 mg·ml^-1（终浓度）时,能够显著增加HMC-1细胞上清液中组胺的含量（P<0.01）;人参皂苷Re终浓度为0.06 mg·ml^-1时,对细胞外组胺及β-氨基己糖苷酶的含量均无明显影响。各参麦注射液中人参皂苷Re的终浓度在0.04 mg·ml^-1-0.08 mg·ml^-1范围内,因此未观察到促进HMC-1细胞释放组胺及β-氨基己糖苷酶的作用。结论:优化了基于费氏弧菌CS234、明亮发光杆菌502和青海弧菌Q67的Microtox测试体系的反应条件,发现基于费氏弧菌CS234的Microtox测试体系具有最广的pH值适应性,能够适应测试环境pH值4.5-8.0的变化,可满足绝大多数中药注射剂的pH值范围（p H 4.0-pH 9.0）。测试方法学验证结果显示:基于费氏弧菌CS234的Microtox测试体系具有良好的特异性、精密度、准确度、灵敏度以及稳定性,并且参比毒物七水硫酸锌的定量范围明确,线性关系良好。本测试体系可用半数抑制浓度（%）或参比毒性定量表征参麦注射液的生物毒性,进一步的研究发现,人参皂苷Re为显著影响半数抑制浓度（%）的物质之一,并且人参皂苷Re在一定剂量下(终浓度0.10 mg·ml^-1)对人肥大细胞系HMC-1细胞有显著的促进组胺释放的作用,提示参麦注射液中人参皂苷Re含量控制的必要性。综上所述,基于费氏弧菌CS234的Microtox测试体系及七水硫酸锌质量控制系统有望成为参麦注射液质量控制及不良反应风险预警新型、快速、灵敏的检测技术平台,以期最终为临床安全用药提供参考和保障。更多还原

【Abstract】 Objective:Microtox assay based on luminous bacteria was used to detect biotoxicity of Shenmai injection which was one of the high-risk varieties published by National Adverse Drug Reaction Monitoring Center.A tentative exploration was carried out to clear the correlative material basis and its clinic significance so as to provide a new,rapid and sensitive detection platform for quality control and predicting warnings,such as allergic reactions during clinical application of Shenmai injection.Methods:The 11 factors and 2 levels experimental design of Plackett-Burman trials were carried out to optimize the 6 agents which affected luminescent intensity of Vibrio fischeri CS234,Photosbacterium.Phosphoreum 502 and Vibrio qinghaiensis Q67,such as:warm-up time of luminous bacteria,volume of rehydration solution,rehydration time of luminous bacteria,volume of luminous bacteria suspension,volume of testing sample and reaction time.pH of Vibrio fischeri CS234,Photosbacterium.Phosphoreum 502 and Vibrio qinghaiensis Q67 rehydration solutions were adjusted by 1.0 mol·L^-11 HCl solution or1.0 mol·L^-11 NaOH solution.Effect of series pH rehydration solutions on corresponding microtox assay systems were detected respectively.ZnSO₄·7H₂O was chosen as a reference substance and quality control substance to validate the specificity,standard curve,quantitative range,sensitivity,precision,accuracy and stability of Zn²⁺in ZnSO₄·7H₂O stock solution and quality control samples of microtox assay system based on Vibrio fischeri CS234.Half maximal inhibitory concentrations(IC₅₀)of Shenmai injections purchased from 6 manufacturers with 15 batches were detected by microtox assay based on luminous bacterium Vibrio fischeri CS234 and along with ZnSO₄·7H₂O quality control samples.According to chromatographic conditions in National Drug Standards of SM injection（WS3-B-3428-98-2010Z）issued by State Food and Drug Administration（SFDA）,contents of ginsenoside Re,ginsenoside Rg₁ and ginsenoside Rb₁ in each Shenmai injection were assayed by high performance liquid chromatography（HPLC）.The relationships between ginsenoside Re,ginsenoside Rg_1,ginsenoside Rb₁,sum of them and corresponding IC₅₀0 values of those Shenmai injections were analyzed by Pearson’s correlation method,respectively.Degranulation of HMC-1 cells caused by ginsenoside Re and those Shenmai injections were also detected.Changes of cell morphology before and after administration for 1 hour were detected by inverted microscope.The contents of histamine andβ-hexosaminidase in the supernatant of cultured HMC-1 cells were detected by enzyme-linked immuno sorbent assay.Results:According to results of Plackett-Burman trials and taking the mean deviation of light intensity attenuation coefficient<3%as the criteria of judgment,the corresponding suitable reaction conditions were as followed.（1）At a temperature of15℃±1℃,Vibrio fischeri CS234 were placed at a thermostat for 15 min,then,1ml rehydration solution（3%NaCl solution）was pour quickly into each vial and blended them into suspensions.After a waiting time of 10 min,added 100μl bacteria suspension into the test tube to measure the luminescence intensity,immediately before the addition of 1.0 ml rehydration solution or test samples.The same time intervals（10s）were used for later intensity measurement.After a contact time of 10 min,the luminescence intensity of each vial was measured again.（2）Photosbacterium.Phosphoreum 502 vials were placed at a thermostat for 10min,then,2 ml rehydration solution（3%NaCl solution）was pour quickly into each vial and blended them into suspensions.After a waiting time of 15 min,added 100μl bacteria suspension into the test tube to measure the luminescence intensity,immediately before the addition of 1ml rehydration solution or test samples.The same time intervals（10s）were used for later intensity measurement.After a contact time of 15 min,the luminescence intensity of each vial was measured again.（3）At a temperature of 15℃±1℃,Vibrio qinghaiensis Q67 vials were placed at a thermostat for 10 min,then,2 ml rehydration solution（0.85%NaCl solution）was pour quickly into each vial and blended them into suspensions.After a waiting time of 10 min,added 100μl bacteria suspension into the test tube to measure the luminescence intensity,immediately before the addition of 2ml rehydration solution or test samples.The same time intervals（10s）were used for later intensity measurement.After a contact time of 10 min,the luminescence intensity of each vial was measured again.The influences of series pH rehydration solutions on the three microtox assay systems were different.（1）pH ranged from 4.5 to pH 8.0 inhibited lower than±10%luminescence intensity of Vibrio fischeri CS234.（2）Luminescence intensity of Photosbacterium.Phosphoreum 502 was reversed from inhibition to enhancement from pH 3.6 to pH 3.8.（3）pH ranged from 5.0 to pH 6.0 inhibited lower than±15%luminescence intensity of Vibrio qinghaiensis Q67.The results of methodology validation were as follow.（1）The luminescence intensity of 3%NaCl solution,20%NaCl solution（osmotic pressure regulator）and ZnSO₄·7H₂O 100 mg·L^-1 fluctuated between 0.01 RLU and 0.02 RLU,respectively,and which in Vibrio fischeri CS234 was from 1152 RLU to 1177 RLU,showed no interference to the assay system.（2）Repeated the sets for 3 times,a good linearity showed over the concentrations of ZnSO₄·7H₂O ranged from 5.0 mg·L^-1 to 100.0mg·L^-1(the final concentrations were 3.86 mg·L^-11 to 77.27 mg·L^-1),standard curve equations were y=20.48Ln（x）-13.34,y=20.48Ln（x）-13.83 and y=21.78Ln（x）-15.14,the corresponding correlation coefficient（R²）were 0.992,0.990 and 0.998.Meanwhile,IC₅₀ of ZnSO₄·7H₂O to Vibrio fischeri CS234 were 22.0 mg·L^-1,22.6 mg·L^-1 and 19.90mg·L^-1.（3）The precisions of quality controls samples(90.0 mg·L^-1,50.0 mg·L^-1,15.0mg·L^-11 and the final concentrations were 69.55 mg·L^-1,38.64 mg·L^-1,11.59 mg·L^-1)and the lower limit of quantification(LLOQ,5.0 mg·L^-1 and the final concentration was 3.86 mg·L^-1)were all lower than 6%while the accuracy ranged from 85.8%to103.2%.（4）Zn²⁺in ZnSO₄·7H₂O stock solution(100.0 mg·L^-1)and its quality control samples were stable at 120 h and 8 h,respectively.The precisions of them were all lower than 2%（RSD%）.IC₅₀ of Shenmai injections purchased from 5 manufacturers with 12 batches were ranged from 46.7%to 72.3%,except which purchased from SCSH Pharmaceutical Co.,LTD.The correlation of test sample concentrations（%）to inhibitory effects on luminescent bacteria（%）were all higher than 0.97.One-way variance analysis（ANOVA）showed that IC₅₀0 among different manufactories had a significantly difference（P<0.05）.The accompanying quality control samples of ZnSO₄·7H₂O showed a good precision which RSD%was lower than 4%while the accuracy ranged from 87.03%to 97.76%.Contents of ginsenoside Rg₁ and ginsenoside Rb₁ in 15 batches Shenmai injections ranged from 0.11 mg·ml^-1 to 0.18 mg·ml^-1 and 0.14 mg·ml^-1 to 0.33 mg·ml^-1,respectively.Contents of ginsenoside Re and sum of the three ginsenosides were 0.07mg·ml^-1 to 0.14 mg·ml^-1 and 0.34 mg·ml^-1 to 0.60 mg·ml^-1,respectively.Nonparametric tests showed that contents of ginsenoside Re and sum of the three had significantly differences among different manufactories（P<0.05）.There was a positive correlation between content of ginsenoside Re and IC₅₀（r=0.823,P<0.01）.In degranulation model of mast cell,ginsenoside Re increased the extracellular histamine cHMC-1 cells at a final concentration of 0.10 mg·ml^-1（P<0.01）,however,0.06 mg·ml^-1 was not obviously.Those Shenmai injections didn’t lead to increased degranulation of HMC-1 cells because the final concentration of ginsenoside Re in those samples ranged from 0.04 mg·ml^-1 to 0.08 mg·ml^-1.Conclusions:Reaction conditions of microtox assay systems based on Vibrio fischeri CS234,Photosbacterium.Phosphoreum 502 and Vibrio qinghaiensis Q67 were optimized,respectively.And the microtox assay systems based on Vibrio fischeri CS234 had an adaption of pH which changed from 4.5 to 8.0.Good specificity,precision,accuracy,sensitivity,stability,linearity and clear quantitative range were observed in methodology validation of microtox assay based on Vibrio fischeri CS234.This microtox assay system used IC₅₀ or reference toxicity of ZnSO₄·7H₂O to represent biotoxicities of Shenmai injection.Results of statistical analysis showed that IC₅₀0 was significantly affected by content of ginsenoside Re.Moreover,ginsenoside Re promoted histamine release from HMC-1 cells at a final dosage of 0.10 mg·ml^-1.All results above pointed out necessity of ginsenoside Re content control in Shenmai injection.The microtox assays based on Vibrio fischeri CS234 was suggested that it can be used as a new platform for quality control and adverse drug reaction pre-warning in Shenmai injection,in order to provide guarantees for clinical safety of medication.更多还原