Peptide Mapping Combined With Mass Spectrometry To Screen Specific Peptides And Identification Studies Of The Famous Chinese Medicine Ejiao

Asini Corii Colla(E'jiao)is prepared from the skin of Equus asinus by decoction and concentration and thus becomes the glue.It is load in the beginning of ?Sheng Nong's herbal classic?.Tao hongjing said:"From DongE,it is called E'jiao."E'jiao behaves flat, tastes sweet, act on lung, liver, kidney, nourishing Yin and enrich the blood,it is used for blood deficiency chlorosis, dizziness palpitations, muscle impotent helpless and upset sleepless,wind moving restlessly,cough of phlegm-heat,fatigue syndromes haemoptysis, vomiting blood or bloody urine, metrorrhagia and metrostaxis, abortion.At present, the quality evalution of Asini Corii Colla mainly depends on the appearance, color, texture, smell and amino acids to evaluate the authenticity.Now because of the great demand of Asini Corii Colla, other animal skins processed products emerge in endlessly, adulteration is serious. Because of its main ingredients are similar in collagen amino acid composition, existing methods are difficult to identify effectively.Chinese pharmacopoeia 2015 uses HPLC-MS to establish an identification of trypsin digested Asini Corii Colla,it can effectively solve the problem of adulteration of Asini Corii Colla, preliminary but still exsists standardization problems, so stable and effective identification and quality control method is very necessary. So the main work is as follows:1.Review on the Quality Evaluation of Asini Corii CollaQuality Evaluation of Asini Corii Colla mainly based on physical and chemical properties.Using kinematic viscosity method, thin layer chromatography, HPLC fingerprint method, spectral method, electrophoresis method to identify and evaluate its quality.Chinese pharmacopoeia 2015 using mass spectrometry,as the latest methods,to identify the Asini Corii Colla.2.Pretreatment conditions of collagen enzyme solution of Asini Corii CollaBy querying the NCBI databases, we found the amino acid sequence of collagen of donkeys, cattle, sheep.By using DNAMAN 6.0 to BLAST the collagen alpha-1 type I chain,we found more than 95% similarity.Then use ExPASy PeptideMass software system for Trypsin enzymolysis,enzymolysis results show that there are 7 specific peptides in Asini Corii Colla, mass-to-charge ratio are 3647.7804,2140.0417,2073.0107,1792.9340, 1752.9027,1100.4966,1752.9027.we can consider them as the specific peptides to identify Asini Corii Colla after verification.Dissolved by heating method and extraction system is methylene chloride-hexane and ultrafiltration purification processing samples of Asini Corii Colla.To verify the method of Chinese pharmacopoeia 2015, meanwhile,optimize the enzyme solution.The optimized enzymolysis conditions is:temperature 38 ?, pH range of 7.5?8.0, enzymolysis time more than 12 h, ratio of object and enzyme is 4:1-1:1 (250 u/mg Trypsin), thus collagen enzymolysis method is established.3.Select the specific peptides of Asini Corii Colla and establish the standard of identificationUsing mass spectrometry to find specific peptides of Asini Corii Colla, the mass-to-charge ratio is 536.7653,765.9070,834.9236,714.3693, and calculate the convolution, it's confirmed that they are all double charge.According to the results of the previous bioinformatics simulation enzymlysis,1 ions is close to it and the other three molecular weight can be added to the standard of identification until they were validated.Establishment of the specific peptides evaluation method:Separate with C18 chromatographic column (2.1 mm* 100 mm,2.7microns);acetonitrile is mobile phase A, 0.1% formic acid solution is mobile phase B,Elution condition is 0-5 min,5% acetonitrile;5-8 min,5%?10% acetonitrile;8-32 min,10%?20% acetonitrile;32-38 min, 20%?95% acetonitrile.The flow rate of 0.30 mL/min;Column temperature 30 ?. Sample quantity 1.0?L.Anion (ESI+) model is adopted to improve the detection. Use mass-to-charge ratio (m/z) 536.7653 (double charge),765.9070 (double charge),834.9236 (double charge) to test for the ions of samples, chromatographic retention time should be at the same time to present consistent and kurtosis larger chromatographic peaks.4. Verification of quality evaluation of the specific peptidesExtract ions m/z 536.7653,765.9070,834.9236 can effectively identify the collected 11 Asini Corii Colla samples, and has the same identification ability which Chinese pharmacopoeia 2015 mentioned in the extract ion of m/z539.8, according to the m/z 539.8 as target identification No.1,2,5 are not fake. The preliminary authentication scheme can effectively identify the collected 11 Asini Corii Colla samples.11 groups of samples, only 3 companies' production meet the criterion, the other 8 companies' production have different degree of adulteration.