熊静悦 李孝容 鄢良春 赵军宁

[摘要]基于費氏弧菌CS234的Microtox微毒测试体系优化与方法学考察，为该方法更加可靠地应用于中药注射剂综合急性毒性的测试打下基础。采用PlackettBurman法优化设计影响费氏弧菌发光的因素，建立以七水硫酸锌为标准毒物和质量控制样品的反应体系，得到最优发光条件为：费氏弧菌冻干粉平衡15 min后加入复苏液1 mL，混匀10 min，取100 μL菌液测试初始发光度，随后立即加入1 mL复苏液或待测样品，反应10 min后再次测试相应发光强度，上述操作均在（15±1）℃条件下完成；复苏稀释液、渗透压调节液及七水硫酸锌储备液不干扰费氏弧菌发光强度的测定，该方法特异性良好；质量控制样品、定量下限样品批内精密度<5%，批间精密度<10%，准确度在858%～1032%。七水硫酸锌溶液在386～7727 mg·L-1，标准曲线方程为y=2178lnx-1514，R2=0998。该方法下ZnSO4·7H2O对费氏弧菌的IC50为1990 mg·L-1；对七水硫酸锌储备液及其质量控制样品分别连续考察120，8 h，其RSD<2%，提示其在室温及4 ℃保存条件下稳定性良好；pH 45～80时，费氏弧菌发光的波动控制在±10%，该pH可满足绝大多数中药注射剂的测试需要。针对费氏弧菌CS234建立的微毒测试体系进行优化后，具有良好的特异性、稳定性、灵敏度、准确度及适应性，提示可应用于中药注射剂综合急性毒性的测试。

[关键词]中药注射剂；微毒测试；急性毒性；费氏弧菌CS234

[Abstract]Vibrio fischeri CS234 was used to establish and optimize microtox assay system， laying a foundation for the application of this method in comprehensive acute toxicity test of traditional Chinese medicine （TCM） injections Firstly， the PlackettBurman method was carried out to optimize the factors which would affect Vibrio fischeri CS234 luminescence Secondly， ZnSO4·7H2O was chosen as reference substance to establish its reaction system with quality control samples The optimal luminescence conditions were achieved as follows： ①At a temperature of （15±1） ℃， Vibrio fischeri CS234 lyophilized powders were balanced for 15 min， then， 1 mL resuscitation fluid was added and blended for 10 min 100 μL bacteria suspension was taken to measure the initial luminescence intensity， and then 1 mL resuscitation fluid or test sample was immediately added；after reaction for 10 min， corresponding luminescence intensity was measured again Resuscitation diluent， osmotic pressure regulator and ZnSO4·7H2O stock solution showed no interference to the determination of Vibrio fischeri CS234 luminescence intensity， so this method was of good specificity The withinand betweenbatch precisions of quality controls and the lower limit of quantification （LLOQ） samples were <5% and <10% respectively， while the accuracy ranged between 858% and 1032% The standard curve equation of ZnSO4·7H2O ranged from 386 mg·L-1 to 7722 mg·L-1 （final concentrations） was y=2178lnx-1514， R2=0998； meanwhile， IC50 of ZnSO4·7H2O to Vibrio fischeri CS234 was 1990 mg·L-1 ZnSO4·7H2O stock solution and its quality controls were continuously investigated for 120 h and 8 h respectively， and their RSD was lower than 2%， indicating stability at room temperature and 4 ℃ storage conditions Between pH 4580， luminescence intensity of Vibrio fischeri CS234 was controlled within ±10%， and such pH value range could meet the testing needs of the vast majority of traditional Chinese medicine injections The Vibrio fischeri strain CS234 assay system was specific， stable， sensitive， accurate and adaptable after optimization， so it was suitable for the comprehensive acute toxicity assessment of TCM injections

[Key words]traditional Chinese medicine injection； microtox assay； acute toxicity； Vibrio fischeri CS234

doi：10.4268/cjcmm20160909

微毒测试技术是一种利用发光细菌进行的生物活体检测技术。20世纪70至80年代初，国外科学家首次从海鱼体表分离和筛选出对人体无害、对环境敏感的发光细菌，用于抗生素、麻醉药物的筛选及水体生物毒性的检测等，现已成为一种简便、快速的生物急性毒性检测手段[1]。本课题组在国内外首次将微毒生物测试技术扩展到中药注射剂综合急性毒性的检测领域，试图解决现阶段仅对已知有效成分的测定远不能满足作为中药注射剂质量控制标准的问题[2]。本研究在前期工作基础上，对微毒测试体系方法学进行优化与验证，进一步提高该方法的应用范围与可靠性。

1材料

费氏弧菌Vibrio fischeri（Vf）CS234冻干粉试剂盒，编号D15G046，D15G037，D15G038，D15G039，D15G056，D15G033，北京滨松光子技术股份有限公司，-20 ℃避光冻存。……