Study On The Effect Of Buckskin Peptide On Bone Marrow Cells And Immune Cells

Objective: To expand the use of deerskin peptides in the development of immunomodulatory peptides,anti-deficiency peptides and health food products,the data on deerskin oligopeptides were used to optimise the screening of deerskin immunoactive components and to identify their active components.Methods: Using deer skin as the object of study,the activity screening of different polar fractions of deer skin was carried out using the systematic solvent method.Petroleum ether,methylene chloride,ethyl acetate,saturated n-butanol,a certain proportion of ethanol and pure water were applied to extract the deer skin,and the hydroxyl radical scavenging rate,HS-5 cell proliferation rate,RAW264.7 proliferation rate,and the effect of nitric oxide secretion by macrophage RAW264.7 cells as the screening index for immune activity,so as to determine the polar site with the best activity.The best polar solvent was selected to extract the collagen solution of deer skin using flash extraction method,and separated into total extraction,>10KDa solution,<1KDa solution and other molecular weight deer skin protein peptides using membrane separation technique;the effect of hydroxyl radical scavenging rate,HS-5 cell proliferation rate,macrophage proliferation rate and NO secretion by macrophages were used as the parameters for determining the immune activity for screening;the hydrolysis degree and hydroxyproline content were used to The specific conditions for the enzymatic digestion of deerskin were optimised by response surface methodology using hydrolysis degree and hydroxyproline content as indicators.In order to further identify the effective sites of immunomodulation and blood tonicity of deer skin,the active components of deer skin were separated by glucose gel chromatography Sephadex G-25,and their possible chemical structures were deduced by LC-ESI-MS/MS,and their immunological and blood tonic activities were evaluated by MTT method to further identify the main active components in deer fetal skin.Results: The results showed that the aqueous layer was slightly more active than the other fractions,so the aqueous layer was selected to continue the exploration of the next level of fractions.After comparing the immunological activity and blood tonic activity of each part,the experimental data showed that the combined activity of each fraction on the immune cells was,in descending order,less than 1KDa,<1KDa,1-3KDa,3-10KDa and >10KDa,and the immunity enhancing effect of the samples with enzymatic digestion of less than 1KDa and less than 1KDa were the best site for immunomodulatory activity was identified by the screening of deer skin oligopeptides.The most suitable parameters for the enzymatic extraction were determined by the response surface method: hydrolysis temperature of 60℃,hydrolysis time of 6h,p H=9,enzyme to substrate ratio of 5%,hydroxyproline content predicted response value of 6.48% and hydrolysis degree predicted response value of 46.09%,where the enzyme addition had the greatest effect on the hydrolysis effect,followed by p H value and finally the temperature of hydrolysis.The amino acid sequences of eight of the oligopeptides were determined by LC-ESI-MS/MS.The amino acid sequence of oligopeptide DT1 was determined to be NH₂-Asp-Asn-Val-COOH,the amino acid sequence of oligopeptide DT2 was determined to be NH₂-Met-Asp-Ser-COOH and the amino acid sequence of oligopeptide DT3 was determined to be The amino acid sequences of the enzymatically soluble oligopeptides were determined as follows: the amino acid sequence of oligopeptide DM1 was determined to be NH₂-Arg-Arg-COOH,the amino acid sequence of DM2 was determined to be NH₂-Ser-Try-COOH or NH₂-Asn-Pro-Met-COOH or NH₂-Arg-Met-Ile/ Leu-COOH,the amino acid sequence of DM3 was determined to be NH₂-Arg-Ile/Leu-COOH.Conclusion: Deer skin oligopeptides have significant immunomodulatory and pharmacological activities in the treatment of blood deficiency,and the active site <1KD components were screened,and eight deer skin oligopeptides were separated and identified.By studying the effects of RAW264.7 cells and bone marrow hematopoietic cells SH-5 on immune regulation and the hematopoietic function of bone marrow hematopoietic cells SH-5,it provides a reference for the development of deer skin immunomodulatory peptides and anti-blood deficiency peptides.It provides a theoretical basis in the field of new drug development.