| Objective: The purpose of this paper was to study the extraction technology of protein and peptide components from sika deer penis and screen the optimal component with the activity of tonifying kidney and bone,using sika deer penis as the raw material.Secondly,based on enzyme engineering technology and peptide bond thermal oscillation theory,the correlation between the dynamic changes of the activity and components of Penis et Testis Cervi before and after enzymolysis,and before and after processing was analyzed to explore the pharmacodynamic material basis.Finally,the optimal active components were further separated and purified to analyze the active components of Penis et Testis Cervi in tonifying kidney and strengthening bone,which would provide a theoretical basis for the development and new application of Penis et Testis Cervi in the fields of medicine,health care products and other products in the future.Methods: First,response surface methodology was used to optimize the extraction,enzymolysis and processing technology of Penis et Testis Cervi protein peptides.Then,the total extracts of the three technologies were divided into four components(total extract,> 10 k Da component,1-10 k Da component and < 1 k Da component)according to different molecular weights by ultrafiltration.The effects of different ultrafiltration components on the activities of TM3 cells and MC3T3-E1 cells were investigated,and the best active components were screened out.The effects of the best components on the secretion of Testosterone(T)and dihydrotestosterone(DHT)in TM3 cells and on the activity of Alkaline phosphatase(ALP)and the secretion of Osteocalcin(OCN)in MC3T3-E1 cells were determined.Subsequently,the chemical fingerprints of different batches of Penis et Testis Cervi were established by amino acid analysis and high performance liquid chromatography(HPLC)analysis technology.Combined with the kidney-strengthening and bonestrengthening activities of each group of samples,the dynamic changes of activity-component correlation of Penis et Testis Cervi before and after enzymatic hydrolysis and before and after processing were analyzed,and the iconic differences before and after enzymatic hydrolysis and before processing were obtained.Finally,Sephadex LH-20 was used to further separate and purify the best active components,and the best active fractions were screened at the cell level.Liquid chromatography y-diode array detector-electrospray ionization mass spectrum/mass spectrum(LC-DAD-ESI-MS2)was used to analyze the active components of Penis et Testis Cervi.Results: The ultrasonic method was adopted for the extraction of protein from Penis et Testis Cervi.The optimal extraction conditions were optimized by response surface methodology as follows: ultrasonic time 64 min,solid-to-liquid ratio 1: 14(g/m L),and ultrasonic power 300 W.Under these conditions,the protein content was(40.12±0.18)%;Alkaline protease was selected for extraction in the enzymatic hydrolysis process.The optimal enzymatic hydrolysis process was optimized as follows: temperature 51℃,p H8.7,time 4.1 h,enzyme dosage 1300 U/g,and the degree of hydrolysis was(40.36±0.22)%;The high temperature and high pressure extraction were adopted in the processing technology.The optimal processing technology after optimization was as follows: extraction temperature116℃,solid-to-liquid ratio 1: 18(g/m L),extraction time 57 min,and particle size 20 mesh.Under these conditions,the peptide content was(48.54±0.32)%.The screening results of the optimal active components showed that each component of the three deer whips with different processes had a certain proliferation effect on TM3 cells and MC3T3-E1 cells,and the components of natural oligopeptide,enzymolysis oligopeptide and processed oligopeptide(<1k Da)of deer whips had the strongest proliferation effect on TM3 cells and MC3T3-E1 cells.In addition,the three cervi penis oligopeptides were able to promote the secretion of T and DHT by TM3 cells,increase the ALP activity of MC3T3-E1 cells and increase the secretion of OCN.The comparative results of oligopeptide content and activity before and after enzymatic hydrolysis of Penis et Testis Cervi showed that the content and yield of the hydrolyzed oligopeptide were higher than those of the natural oligopeptide,but the activity of the natural oligopeptide was superior to that of the hydrolyzed oligopeptide.The correlation analysis of the dynamic changes of the activity-components of Penis et Testis Cervi oligopeptide before and after enzymatic hydrolysis showed that the compounds that had the greatest impact on the drug efficacy were Gly,Arg,Gly-Trp.Comparison of the content and activity of oligopeptides from Penis et Testis Cervi before and after processing showed that the content,yield and activity of the processed oligopeptides were superior to those of the natural oligopeptides.The correlation analysis of dynamic changes of the activitycomponents of the oligopeptides from Penis et Testis Cervi before and after processing showed that the compounds that had the greatest impact on the drug efficacy were Gly,LysAsn or Gln-Asn.After separation and purification,the optimal active fractions of natural oligopeptide,enzymolysis oligopeptide and processed oligopeptide of Penis et Testis Cervi were analyzed,and 10 compounds were obtained: natural oligopeptide(Gln/Lys-Asn,ProThr,Gly-Trp,Leu/Ile-Lys/Gln-Pro);Enzymatic hydrolysis of oligopeptides(Val-Phe,Val-Ser,Gly-TRP);Processed oligopeptides(Ala-His,Lys/Gln-Asn,Pro-Asn-Ala-Lys/Gln).Conclusion: In this study,we screened that the main material basis for the activity of tonifying kidney and strengthening bone of Penis et Testis Cervi raw products,enzymatic products and processed products was the oligopeptide(<1 k Da)component.The activity of Penis et Testis Cervi was slightly decreased after enzymolysis,but the oligopeptide content and yield were significantly increased.The enzymolysis oligopeptide could be considered as a substitute for natural oligopeptide.The activity of processed Penis et Testis Cervi was enhanced,and the oligopeptide content was also increased,which was presumably due to the generation of more small molecular peptides by peptide bond cleavage and the increase of active oligopeptide components after processed Penis et Testis Cervi.The effects of enzymolysis and processing on the activities of tonifying kidney and bone of Penis et Testis Cervi were preliminarily explored.The oligopeptide components of Penis et Testis Cervi raw product,enzymolysis product and processed product were separated and purified to obtain 17 groups of fractions,the optimal fraction for tonifying kidney and strengthening bone activity was screened out,and 10 compounds were resolved: Gln/Lys-Asn,Pro-Thr,Gly-Trp,Leu/IleLys/Gln-Pro(raw fraction);Val-Phe,Val-Ser,Gly-Trp(enzymatic fraction);Ala-His,Lys/Gln-Asn,and Pro-Asn-Ala-Lys/Gln(fractions of processed products),and thus confirmed the pharmacodynamic material basis of Penis et Testis Cervi,and provided a scientific theoretical basis for the development and application of products of Penis et Testis Cervi in tonifying kidney and strengthening bone. |
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