Study On Quality Evalution Of Zushima Injection

The quality control of traditional Chinese medicine injection is one of the key problems in the modernization of TCM.Quality control is the basis and guarantee for the safety and effectiveness of TCM injections,and scientific and reasonable quality standard is an important part of the drug quality assurance system.Taking Zushima injection as the research object,this paper combines modern instrumental analysis with chemometrics to establish a comprehensive method for quality evaluation of traditional Chinese medicine injection from the chemical and biological perspectives.The main deliverables of this research are:1.Firstly,the HPLC fingerprint and chemical pattern recognition model of Zushima injection were established to provide reference for the evaluation of the quality consistency of Zushima injection.Agilent C18 chromatographic column（250 mm×4.6 mm,5 m）was used.Mobile phase A（0.1%formic acid water）-B（acetonitrile）,gradient elution 0～10 min95%～90%（A）;10～28 min,90%～87%（A）;28～40 min,87%～86%（A）;40～50 min,86%～70%（A）;50 to 55 min,70%～60%（A）;Flow rate 1.0 m L·min^-1;The detection wavelength was 0～30 min and 300 nm.30～35 min,254 nm;35～55 min,300 nm;Column temperature 30℃;The injection volume is 10μL.The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint（2012 version）was used to investigate the similarity of 15 batches of zusi ma injection,combining principal component analysis（PCA）,cluster analysis（CA）and orthogonal partial least squares discriminant analysis（orthogonal partial least squares discriminant analysis）.OPLS-DA)evaluated the overall quality of Zushima injection.The results showed that the similarity of 15 batches of samples in the HPLC fingerprint of Zushima injection ranged from 0.882 to 0.998.By PCA,CA and OPLS-DA analysis,15 batches of samples were clustered into 3 categories,S1 to S8from the same manufacturer and clustered into 2 categories.S9～S14 from the same manufacturer were grouped into 1 category,and four of the 12 common peaks were identified as Q-marker for Zushima injection,which were 5,8,7（Daphnetin）and 1peak,respectively.It is suggested that the manufacturer pay attention to these four markers in the production process.In this study,the samples of the high temperature group,the light group and the exposure group were examined by chemical fingerprints,and the four groups of Zushima injection were analyzed by clustering.It is recommended that the drug be sealed during transportation and storage,and be prepared before use.It can be seen that the method of combining chemical pattern recognition with fingerprint is simple and reproducible,which can quickly evaluate the stability and consistency of different batches of Zushima injection from different manufacturers,and is suitable for the evaluation of the overall quality of Zushima injection.2.A new in vitro anaphylaxis detection model was established to rapidly evaluate the preclinical safety of different batches of Zushima injection from different manufacturers.RBL-2H3 cell line of basophilic leukemia was selected as the cell model for in vitro detection of anaphylactic reactions,Compound 48/80 was selected as the positive sensitizing drug,and toluidine blue staining was used to observe the changes of morphology and skeleton of the cells after Compound 48/80 action,so as to verify the degranulation of RBL-2H3 cells Conditions.The appropriate inoculation density of RBL-2H3 cells and the cytotoxicity of Zushima injection were determined by CCK-8method.Real-time cell analysis（RTCA）system was used to detect the changes in cell index（CI）value caused by drug intervention,and the cell fingerprint corresponding to the drug was obtained.At the same time,clustering method（CA）was used to statistically analyze the cell fingerprint of 15 batches of Zushima injection.At the same time,the samples（S9～S15）without anaphylactic reactions were treated with high temperature,light and exposure,followed by cell fingerprint detection and cluster analysis.The results showed that the suitable inoculation density of RBL-2H3 cells was1.0x10⁵/m L by CCK-8 method.The appropriate concentration of Zushima injection was determined by CCK-8 method at 5:95（drug volume:medium volume）.Compound 48/80（20μg/m L）significantly altered both the cell morphology and cytoskeleton,leading to significant degranulation.Moreover,it showed a rapid decline within 30 min in the cell fingerprint,followed by a slow decline,which was in sharp contrast with the blank control,suggesting that RTCA system could rapidly and sensitively evaluate the degranulation effect of RBL-2H3 cells.The results of 15batches of Zushima injection combined with cluster analysis（CA）showed that Zushima injection from different manufacturers had different effects on RBL-2H3cells.S1～S8 and Compound 48/80 were grouped into one group,suggesting that the sample might have potential clinical allergic reactions.S9～S15 and the blank control group were grouped into one group,indicating that the sample had no anaphylactic reaction.It can be seen from the test results of samples in the high temperature group,the light group and the exposure group that the exposure group has a great influence on the safety of samples and produces a significant trend of anaphylaxis,which is consistent with the test results of chemical fingerprint.In this study,a rapid in vitro evaluation technique of anaphylaxis based on RTCA system and pattern recognition was established,which can be used for rapid in vitro evaluation of anaphylaxis in traditional Chinese medicine injections.