Study On Sensitized Material Basis Of Shuanghuanglian For Injection Based On RBL-2H3 Cells

Objective Based on the experimental results of RBL-2H3 cells to establish allergenic detection method.To establish the fingerprints of SHLI and serum fingerprint by UPLC,studying their correlation in order to study the sensitized material basis for SHLI.Method First,the culture conditions of RBL-2H3 cells and optimum experimental time was studied.MTT method was used to determine the inhibitory rate of RBL-2H3 cells and calculation IC50 to determinate the dosage of SHLI and drug-containing serum to RBL-2H3 cells.Andβ-hexosaminidase release rate was measured to establish a method for the detection of allergy for SHLI and drug-containing serum.Then,investigating the chromatographic conditions of mobile phase,detection wavelength,operation time,column temperature and flow rate by UPLC,to establish an analytical method for the fingerprint in vitro of SHLI.By investigating the tests on the repetitiveness,accuracy and the stability to determine the reliability of analytical methods for the fingerprint in vitro of SHLI.And by investigating the processing method of drug-containing serum samples,the tests on the repetitiveness,accuracy and stability to determine the reliability of analytical methods for SHLI fingerprint analysis method of in vivo.Finally,β-hexosaminidase release rate and fingerprint of SHLI and drug-containing serum were determined.And using bivariate correlation analysis to study the chemical data of fingerprints and the data of RBL-2H3 cells,acquired the most closely related chemical composition for the sensitized material basis.Result Using 1.0×105m/L the cell concentration of vaccination,inoculation 24h after dosing.MTT method was used to determine the inhibitory rate of RBL-2H3 cells and calculation IC50 to determinate the dosage of SHLI and drug-containing serum to RBL-2H3 cells:SHLI group was 28.3mg/L,SHLI drug-containing serum group was 36.4mg/L.The results of P-hexosaminidase release rate:Positive control group was 0.6942±0.0328;SHLI group was 0.4753±0.0224;Positive control drug-containing serum group was 0.8374±0.0248,SHLI drug-containing serum group was 0.5348±0.0273,compared with the blank control group,Significant difference(p<0.05and p<0.01).The analysis method of SHLI UPLC fingerprint in vitro:DIKMA UPLC EndeavorsilC18 chromatographic column(50mm×2.1mm,1.8μm);The mobile phase consisted of acetonitrile(A)and 0.1%formic acid(B);The mobile phase was delivered at a flow-rate of 0.3 mL/min;UV detection wave length was set at 243nm;Column temperature was kept at 30℃;Sample quantity was 2μL.The optimal gradient elution mode was as follows:0-lmin,5%A;1-3.5min,5%-13%A;3.5-7.5min,13%-23%A;7.5-9min,13%-29%A;9.14min;9%-50%A.The variation of main peaks relative retention time and relative peak area RSD were less than 3%by measuring the precision,stability and reproducibility.Each batch of fingerprint similarity above 0.9,and 17 common peak was determined.And we confirmed that 5、6、11、15、16、17 respectively of chlorogenic acid,the lilacs glycosides,st John’s wort ester glucoside,A baicalin,st John’s wort glycosides,mignonette element.Serum treatment method for methanol-acetonitrile deposition method.Precision,repeatability,stability,the retention time and peak area of each common peak RSD(relative standard derivation,RSD)is less than 3%.Each batch of fingerprint similarity above 0.9,and 22 common peak was determined.And we confirmed that 5、6、14、19、20、21 respectively of chlorogenic acid,the lilacs glycosides,st John’s wort ester glucoside,A baicalin,st John’s wort glycosides,mignonette element.MTT method was used to determine the inhibitory rate of RBL-2H3 cells and calculation IC50 to determinate the dosage of dismantle square group of SHLI and drug-containing serum to RBL-2H3 cells:the group of honeysuckle was 1.87mg/L,the group of fructus forsythiae was 0.96mg/L,the group of radix scutellariae was 4,02mg/L,the group of honeysuckle and fructus forsythiae was 0.19mg/L,the group of honeysuckle and radix scutellariae was 7.93mg/L,the group of fructus forsythiae and radix scutellariae was 0.91 mg/L,the drug-containing serum group of honeysuckle was 2.32mg/L,the drug-containing serum group of fructus forsythiae was 1.47mg/L,the drug-containing serum group of radix scutellariae was 6.89mg/L,the drug-containing serum group of honeysuckle and fructus forsythiae was 0.67mg/L,the drug-containing serum group of honeysuckle and radix scutellariae was 11.75mg/L,the drug-containing serum group of fructus forsythiae and radix scutellariae was 2.08mg/L.The result ofβ-hexosaminidase release rate:the group of honeysuckle was 0.5036±0.0238,the group of fructus forsythiae was 0.1621±0.0113,the group of radix scutellariae was 0.2235±0.0097,the group of honeysuckle and fructus forsythiae was 0.5476±0.0364,the group of honeysuckle and radix scutellariae was 0.2865±0.0373,the group of fructus forsythiae and radix scutellariae was 0.1537±0.0241,the drug-containing serum group of honeysuckle was 0.5291 士0.0116,the drug-containing serum group of fructus forsythiae was 0.1894±0.0425,the drug-containing serum group of radix scutellariae was 0.248 8±0.0223,the drug-containing serum group of honeysuckle and fructus forsythiae was 0.5845士0.0153,the drug-containing serum group of honeysuckle and radix scutellariae was 0.3092±0.0442,the drug-containing serum group of fructus forsythiae and radix scutellariae was 0.1865±0.0691,compared with the blank control group,Significant difference(p<0.05andp<0.01).All dismantle square groups of SHLI and drug-containing serum fingerprint similarity above 0.9,has a good similarity.Analysis of the correlation between fingerprint and allergic reaction show that the 5、6、7、13、14 peak of SHLI fingerprint in vitro is close to the peak and sensitization(p<0.05),bivariate correlation analysis the Pearson correlation coefficient were 0.856、0.709、0.555、0.925、0.683.After comparison,these peaks are mainly from the honeysuckle,perhaps some parts are from the fructus forsythiae,The 5、6、7、8、17、18 of SHLI fingerprint in vivo is close to the peak and sensitization(p<0.05),bivariate correlation analysis the Pearson correlation coefficient were 0.673、0.653、0.781、0.724、0.735、0.652.After comparison,these peaks are mainly from the metabolic components of the honeysuckle perhaps and some parts are from the metabolic components of the fructus forsythiae.Conclusion The analysis method established of RBL-2H3 cells allergenic detection method and SHLI fingerprint has the advantages of simple and accurate,reproducibility and reliability,allergic reaction and fingerprint has a very good correlation,which laid the foundation for further study the basis of SHLI allergenic substances.And to provide a new method and thought for the detection of allergic method of TCMI.