Quality Control Evaluation On Asini Corii Colla By Biothermal Fingerprint Spectrum

Asini Corii Colla(ACC)is a commonly used animal medicine.But in recently years,the negative reports about ACC and the related products frequently appeared,causing hot discussion at home and abroad.The quality of ACC is often the core of controversy.It is well known that consistency and stability of drug quality are important guarantees for the safety and effectiveness of clinical medication.For traditional Chinese medicine(TCM),especially animal drugs such as ACC,the fluctuation of the quality of ACC may be caused by the influence of raw material sources,process formulation,production process or other conditions.At present,the quality control of ACC is mainly based on the Chinese Pharmacopoeia to determination of L-hydroxyproline,glycine,alanine and L-proline,and to prove the presence or absence of characteristic peptide segments.However,the composition of ACC is complex,the efficacy is diverse,and the pharmacodynamic material basis has not been fully clarified now.The activity(or toxicity)of TCM is often not determined by the content of one or several components,and whether there are any components in ACC.The effect of interaction on the activity of ACC is unknown.It is not enough to effectively guarantee the quality consistency of ACC by controlling the content of four amino acids and lacking the evaluation of the integrity and specificity of the efficacy of ACC.Therefore,in order to ensure the clinical efficacy,this study intends to establish a quality evaluation method related to the biological activity of ACC,especially for the quality consistency of ACC.Then carry out relevant detection and analysis.To provide technical support for the quality control of ACC and reference for the quality control of animal drugs.Aim at the above research objectives,this study mainly carries out the following research contents:Part 1 The representative samples collection and the physiochemical analysis.Objective:Collect samples of ACC from different manufacturers and batches.The chemical analysis of ACC representative samples were carried out based on the quality control index detection in Chinese Pharmacopoeia,and were carried out physicochemical analysis from NIRS.Method:The L-hydroxyproline,glycine,alanine and L-proline were determined according to the method of amino acid detection from ACC in Chinese Pharmacopoeia,and the data were analyzed mainly by cluster analysis.The samples were scanned by NIRS,and the data were collected and analyzed mainly by principal component analysis.Result:40 batches of representative ACC products from 11 manufacturers were collected in this study.According to the chemistry detection results,35 were qualified and 5 were unqualified.The qualified rate was 87.5%.The similarity of the first 5 principal components from 40 batches were 60.1% after analyzing the results of NIRS,which indicated that there were great differences among the ACC samples.Conclusion:Based on the quality control index of Chinese Pharmacopoeia and the quality analysis of NIRS,the difference of ACC could be identified and there were unqualified products in the samples of ACC sold in the market.Part 2 Quality evaluation of ACC based on biothermal activity fingerprint spectrumObjective:To construct a quality evaluation model of ACC based on biothermal fingerprint spectrum and detect the difference activity of different ACC.Method:Escherichia coli(E.coli)was used as intestinal model bacteria to evaluate the regulatory effect of ACC from different manufacturers on intestinal bacteria,and the characteristic spectrum of biothermal activity was established.The characteristics could distinguish the quality differences among different manufacturers by principal component analysis.Result:The best detection method was determined.The experimental results showed that there was a stable dose-effect relationship between the concentration of ACC and the growth and metabolism of E.coli in the range of 0.625-20.0 mg/ml.After methodological investigation,the selected detection dose(5 mg/ml)could indicate the difference of ACC from different sources.Conclusion:Guided by the clinical efficacy of ACC in regulating intestinal flora,this study established a fingerprint method for evaluating the biothermal activity of ACC,which can be used to evaluate the consistency of ACC quality.Part 3 Biological evaluation for the quality control of Asini Corii Colla based on macrophage phagocytosis modelObjective:To establish a method for evaluating the biological activity of ACC based on the macrophage phagocytosis model.Method:The murine RAW 264.7 macrophage cell line and GFP-Escherichia coli were used to construct the model.The cell concentration and incubation time,as well as the appropriate concentration of ACC,were determined by CCK-8 method.The macrophage phagocytosis rate and single cell phagocytosis index affected by ACC were also analyzed by investigating the multiplicity of infection(MOI)and the dosage of ACC with high-content screening(HCS)technology.Result:To investigate the effect of ACC on macrophage cells,the cells were cultured with ACC solution for 24 hours,and then 200-fold GFP-Escherichia coli(MOI = 200)were added and stimulated for 2 hours before HCS analyzing.The results showed that ACC had double effects on the proliferation and phagocytosis of macrophages,but the dose-effect curves of the two effects were slightly different.Compared with the control group,the proliferation of macrophages could be promoted by ACC at an increased concentration from 0.125 to 2.0 mg/ml,and the strongest proliferation effect was shown with 0.5 mg/ml.Furthermore,there is no obvious promotion on the phagocytic ability in single cell for the ACC concentration of 0.25-1.0 mg/ml,but it had a dose-response effect on the holistic phagocytic rate of macrophages and had the strongest ability to promote the phagocytosis rate of macrophages at 0.25 mg/ml.Conclusion:Guided by the clinical efficacy of ACC in regulating immunity,this study established an in vitro biological method for evaluating the quality of ACC with high-content screening technology,which has the potential to detect the quality and consistence of different ACC.Part 4 Evaluation of ACC quality consistency based on effective Near-Infrared spectroscopyObjective:The biothermal activity fingerprint spectrum and NIRS were carried out the correlation to explore and establish the rapid detection methods for the related biological activity of ACC.Method:NIRS was used to scan the samples.Data were collected,and analyzed by principal component analysis.Then,combined with the NIRS detection method,the spectral correlation analysis between the two indices obtained from the biothermal activity characteristic spectrum and the NIRS was carried out,and the characteristic bands with high correlation with the activity were selected as the quality characteristic spectrum.Result:After analyzing the results of 18 batches of NIRS,combined with the biothermal activity fingerprint spectrum,the characteristic bands with high activity correlation were selected,which were 3695-4011 cm-1,5245-5268 cm-1,6711-6950 cm-1,and these could be used as the e NIRS to evaluating the consistency of ACC.Conclusion:The near-infrared band obtained by correlation with biothermal activity fingerprint spectrum combined with NIRS could be used as an e NIRS for on-line control in the production of ACC.Part 5 Exploration of quality evaluation method for ACC based on metabolic expression profile of macrophagesObjective:To explore an evaluation method of ACC quality based on macrophage metabolic characteristic spectrum.Method:The activity of ACC was detected and analyzed by HCS after multiple batches of cell viability.Then,cell samples were collected for metabolic detection.Metabonomics data were pretreated by Profinder and filtered and normalized by Gene Spring.The normalized data were imported into SIMCA for multivariate statistical analysis,including PCA and OPLS-DA.The values in OPLS-DA analysis were selected as VIP > 1 and | P(corr)|(> 0.5).Metabolites were used as candidate indicators;SPSS 22.0 was used to do the T-test between each drug group and non-drug group,screen the metabolites with significant differences(p<0.05,fold change>1.5),and METLIN database was used to identify the metabolites.The identified metabolites were used as biomarkers for the characteristics of ACC products.Result:The different manufacturer of ACC on macrophage function were analyzed by metabonomics,and the related were screened to construct the metabolic expression profile of macrophages.The results showed that 10 manufacturer of ACC had better aggregation degree,and the cell metabolic characteristics among different manufacturer of samples were specific,indicated that the fingerprint of cell metabolism could be used to distinguish different manufacturers of ACC.Conclusion:The quality evaluation method of ACC based on the characteristic spectrum of macrophage metabolism could be used to distinguish different manufacturers,and it also need further exploration and research.