| 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 Rg1,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 and?-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 100?l 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 100?l 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 100?l 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. |
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