Preparation Technology And Quality Study Of Deer Antler Extract

Purpose:1.Improve the extraction efficiency of sika deer antler extract and efficiently use antler medicinal materials.2.Provide a new direction for the rational development and scientific utilization of deer antler.3.The research on antler extract provides experimental basis for antler quality research.Material and method:1.In this study,sika deer antler was used as the research object,and the optimal extraction process parameters of velvet antler polysaccharide in this research group were used to prepare polysaccharide extract.The enzymatic hydrolysis temperature,p H,amount of enzyme,time and other factors of the polypeptide enzymatic hydrolysis system were investigated.The polypeptide content was used as the evaluation index to optimize the velvet antler polypeptide enzymatic hydrolysis process.The membrane separation technology was used to select a 5k Da ultrafiltration membrane to perform the enzymatic hydrolysis.After separation and purification,the freeze-dried small-molecule peptides obtained after freeze-drying are peptide extracts,which provide a material basis for later experiments.2.Starting from the peptide extract,using Tricine-SDS-PAGE analysis technology to determine the concentration of different separation gels by using tris（hydroxymethyl）methylglycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis（Tricine-SDS-PAGE）Optimum electrophoresis conditions,detect the molecular weight distribution of the separated and purified polypeptide extracts,and use high-performance gel filtration chromatography（HPGFC）to determine the chromatographic conditions of HPGFC by investigating the chromatographic column,detection wavelength,and mobile phase.The relationship between the logarithm of the relative molecular mass of the four standards of albumin,cytochrome C,insulin and bacitracin and the retention time of the chromatographic peak,draw a standard curve,calculate the molecular weight,and determine the molecular weight distribution of the velvet antler polypeptide extract;finally use The Folin phenol method is used to determine the content of polypeptides in polypeptide extracts,hoping to provide experimental basis for the quality research of sika deer antler polypeptide extracts.3.Starting from the polysaccharide extract,the purified polysaccharide is obtained after protein removal by Sevage method and H₂O₂decolorization,which is pre-column derivatized with 1-phenyl-3-methyl-5-pyrazolone（PMP）reagent and subjected to HPLC Analyze and determine the monosaccharide composition and content of the polysaccharide extract,and then use the phenol-sulfuric acid method to determine the polysaccharide content in the polysaccharide extract,hoping to provide an experimental basis for the quality research of the sika deer antler polysaccharide extract and provide a basis for the quality evaluation of traditional Chinese medicine.Results:1.By investigating the temperature,p H,amount of enzyme added,time and other factors of the polypeptide enzymatic hydrolysis system,it is determined to establish a three-factor three-level response surface experimental design based on enzymatic temperature,p H,and enzyme amount,and the peptide content is used as the evaluation index Optimize the enzymatic hydrolysis process of velvet antler polypeptide.The best enzymatic hydrolysis process of velvet antler polypeptide is:under the conditions of 50℃,p H8.0,add 3.5%alkaline protease to react for 2h.2.The yellow polysaccharide powder obtained by the water extraction and alcohol precipitation method is the polysaccharide extract.The drug residue is extracted by the best enzymatic hydrolysis process,and then ultrafiltration and freeze-dried to obtain the brown polypeptide freeze-dried powder,which is the polypeptide extract.Polysaccharide extraction The yield of the product was 8.020%,and the yield of the polypeptide extract was 26.64%.It has been verified that the average yield of polysaccharide extracts is 8.264%,and the average yield of polypeptide extracts is26.16%.3.Determine the conditions of Tricine-SDS-PAGE by examining the concentration of the separation gel,and select the molecular weight range of the peptide standard 2～245k Da protein marker determines the molecular weight distribution of the polypeptide extract.The results show that the molecular weight distribution of the polypeptide extract after ultrafiltration is below 15k Da,and the ultrafiltration has two less bands of about 25k Da and 63k Da than the polypeptide extract without ultrafiltration,Which shows that ultrafiltration can effectively trap macromolecules.4.The chromatographic conditions of HPGFC are determined by the inspection of the chromatographic column,detection wavelength and mobile phase:(Column:COSMOSIL 5Dio L-120-II（7.5mm×300mm,5μm）Mobile phase:phosphate buffer（p H 6.5）,Wavelength:220nm,Flow rate 1.0ml/min,Column temperature:30℃,Injection volume:10μl.Draw a standard curve based on the relationship between the logarithm of the relative molecular weight of each substance and the retention time of the chromatographic peak in a mixed reference product prepared with four standard products of ovalbumin,cytochrome C,insulin and bacitracin,using the standard curve:log（MW）=-0.245t+6.8135（r=0.9721）to calculate the molecular weight.The results measured that there are 60k Da and 40k Da macromolecules without ultrafiltration.The molecular weight of the polypeptides after ultrafiltration is widely distributed in the range of 1.6k Da-7k Da,accounting for 81.814%of the entire peak area,indicating that ultrafiltration can effectively filter polypeptide macromolecules.5.Using bovine serum albumin as the protein standard solution,the content of peptides in the peptide extract was determined by the Folin method,and the linear regression equation was calculated based on the mass concentration of the standard solution（C）and the corresponding absorbance（A）,and the standard curve obtained was A=2.3663C+0.0529（r=0.9994）,the RSD values of the precision,stability,and repeatability tests are all less than 2.0%,the sample recovery rate is between96.16～101.2%,and the RSD value is less than 2.0%.The average content of the polypeptide in the polypeptide extract measured by this method is 470.0mg/g.6.Pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone（PMP）was used for HPLC analysis.The monosaccharide composition in the polysaccharide extract was mannose and glucosamine hydrochloride before and after retention time.Glucuronic acid,galacturonic acid,galactosamine hydrochloride,glucose,and galactose.The seven monosaccharides have been completely separated.The content of these seven monosaccharides is mannose>glucuronic acid>glucuronic acid>glucosamine hydrochloride>amino hydrochloride.Galactose>glucose>galacturonic acid>galactose.Mannose,glucosamine hydrochloride,and glucuronic acid are the main monosaccharides of velvet antler polysaccharides;the contents of galacturonic acid,galactosamine hydrochloride,glucose,and galactose are relatively close.7.Use glucose as the standard solution to determine the content of polysaccharide in the polysaccharide extract by phenol-sulfuric acid method,draw the standard curve with glucose content（mg）as the abscissa and absorbance（A）as the ordinate to obtain the regression equation:y=8.563x+0.0389（r=0.9997）.The RSD values of the precision,stability,and repeatability tests are all less than 3.0%,the sample recovery rate is between 95.61%and 102.5%,and the RSD value is less than 3.0%.The average content of polysaccharides in the polysaccharide extract measured by this method is 9.956 mg/g.Conclusion:1 The best enzymatic hydrolysis process of velvet antler polypeptide is:under the conditions of 50℃,p H8.0,add 3.5%alkaline protease to react for 2h.The yield of polysaccharide extract was 8.020%,and the yield of polypeptide extract was 26.64%.It has been verified that the average yield of polysaccharide extracts is 8.264%,and the average yield of polypeptide extracts is 26.16%.2 Tricine-SDS-PAGE method has better separation rate,accuracy and intuitiveness for small molecule peptides;HPGFC method is simpler and faster,both of which can prove that ultrafiltration can effectively trap large molecules and can be used for peptides.Study on the distribution of molecular weight.The average content of the polypeptide in the polypeptide extract measured by the Folin method was 470.0 mg/g.3 The monosaccharide composition of polysaccharides from high to low are mannose>glucuronic acid>glucosamine hydrochloride>galactosamine hydrochloride>glucose>galacturonic acid>galactose.The pre-column derivatization high performance liquid chromatography method is accurate and reliable,High stability.The average content of polysaccharide in polysaccharide extract measured by phenol-sulfuric acid method is 9.956mg/g,which can be used for the content determination and quality control of velvet antler polysaccharide.