| Purpose: In this study,the growth factor-active peptides of commercial medicinal materials of Cervus elaphus were selected for study.Respectively,by water extraction and ethanol precipitation of modern extraction technology,membrane separation and purification technology of ultrafiltration membrane and nanofiltration membrane,Tricine-SDS-PAGE electrophoresis analysis and identification technology and amino acid pre-column derivatization assay technique were used to determine the biological properties of velvet antler polypeptides.In order to provide experimental basis and reference direction for the further study of nature of velvet antler polypeptides.Materials and methods:1.In this experiment,water was used as the solvent to combine ultrasonic extraction with ethanol precipitation.The L9(34)orthogonal design experiment was carried out by using Coomassie brilliant blue method to determine the content of valvet antler polypeptides as the evaluation index.The effects of four factors,such as size?solid-liquid ratio?extraction times and extraction time had influence on the water extraction process and study the impact of three factors of the relative concentration of ethanol precipitation prior to the liquid,the concentration of ethanol and alcohol precipitation time on the ethanol precipitation process,in order to optimize the extraction process of velvet antler polypeptides,provide the material basis for the later experiment.At the same time was determined by the Kay nitrogen analyzer for content of protein polypeptide on antler powder and water residue after extraction.The difference was the extracted polypeptide content,which further verified the determination method of Coomassie brilliant blue.2.Tricine-SDS-PAGE gel electrophoresis was used to study the molecular weight distribution of alcohol-precipitated peptides extracted from water,which provided experimental evidence for polypeptides membrane separation.The polypeptides were separated and purified by membrane separation technique.The deer peptide was divided into seven sections using 100 KD,50 KD,10 KD,5 KD,3 KD ultrafiltration membrane and nanofiltration membrane of 0.2KD,respectively.The effect of mass fraction and the transmembrane pressure difference on the flux and rejection of ultrafiltration membrane and the influence of volume concentration ratio on the desalination rate of nanofiltration membrane were studied.The single factor experiment was used to optimize the membrane separation process parameters of velvet antler active polypeptides and determine the optimal membrane separation and purification technology.3.With the method of pre-column derivatization,automatic amino acid analyzer was used to determine of the contents and compositions of hydrolysis and free amino acids in different molecular weight velvet antler peptide segments.The amino acid content of the polypeptide was determined by the difference of the hydrolyzed amino acid and the free amino acid,and the content of different peptides was analyzed to provide reference for the determination of the polypeptide content of velvet antler.Results:1.The optimum technological conditions for extraction:size is 80~100 mesh,solid-liquid ratio is 1:12,extraction 3 times and extraction 20 minutes each time;The optimum technological conditions for alcohol precipitation: the relative concentration of the liquid is 0.5g·m L-1(crude drug),concentration of ethanol is 65%,each time for 4 hours.Determination of extracted peptide content by the Kjeldahl method was close to the content determined by Coomassie brilliant blue method.2.Tricine-SDS-PAGE gel electrophoresis analysis showed that the majority of polypeptides were distributed between 5 KD and 75 KD,a small number distributed between 100 KD and150 KD,and 9 bands with higher resolution.There were two relatively high content of the bands at 60 KD and 13 KD.Ultrafiltration membrane separation and purification conditions:Mass fraction of velvet antler peptide were 2%;The optimal operating pressures of the ultrafiltration membranes with molecular weight 100 KD,50KD,10 KD,5KD and 3KD were0.06 MPa,0.08 MPa,0.15 MPa,0.15 MPa and 0.15 MPa,respectively;The contents of peptides were 38.5%,65.2%,23%,17.7%,20.6% and 8.3%,respectively.The average desalination ratio of nanofiltration membranes with volume concentration ratios of 1,2,3,4,5 were 74.91%,76.58%,78.83%,86.00%,87.94%;The optimum volume concentration ratio was 4.3.17 kinds of amino acids were detected in the velvet antler peptides,and all the amino acids had good linear relationship,r were greater than 0.9990;The RSD of precision,stability,repeatability were less than 3%;The recoveries were between 96.6% and 104.7% with RSD values less than 2.4%.The amino acid content and composition of peptides with different molecular weight were different.The amino acid content of 50~100KD molecular weight peptides was the highest.The pre-column derivatization method is reasonable and accurate,and can be used to measure the content of different amino acids,which provides a technical basis for the quality evaluation of traditional Chinese medicine and identification of traditional Chinese medicine.Conclusion: Water extraction and ethanol precipitation process of velvet antler polypeptide is stable,convenient,reasonable and feasible.The Tricine-SDS-PAGE electrophoresis was used to study the molecular weight range of the ethanol-precipitated peptides,which provided a theoretical basis for membrane separation.The separation and purification of velvet antler polypeptides by using ultrafiltration membrane and the desalination of velvet antler polypeptide by nanofiltration membrane have the advantages of simple operation,low cost and protection of the original activity of heat-sensitive substances.Cell viability assay was used to evaluate the activity of different peptides and to provide theoretical basis for the characterization of different peptides.The pre-column derivatization method has good reproducibility and high accuracy,and can be used for the determination of hydrolyzed amino acids and free amino acids in commercial antler peptides. |
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