| Snakehead（Channa argus）is one of the most important freshwater fish species cultivated in ponds,paddy fields and reservoirs in China.The annual aquaculture production of snakehead in China reached 548500 tons in 2021.Snakehead with few bone spurs is tender and smooth in meat,delicious in taste,and its edible rate is as high as 63%.It is rich in protein,vitamins,polyunsaturated fatty acids,minerals（Ca,P,Fe）and other nutrients.Snakehead extract has been proved to have the ability to promote skin wound healing,and a variety of fish collagen peptides have also been shown to promote skin wound healing.Therefore,in this study,the collagen peptide with high antioxidant activity was prepared from snakehead skin by acid hydrolysis and enzymolysis.The relationship between snakehead skin collagen peptide（SSCP）and wound healing was analyzed by determining the structure of SSCP and the changes of antioxidant activity before and after simulated digestion in vitro.The animal model further proved that different doses of SSCP can significantly promote skin wound healing,which provides important information for SSCP to become a valuable nutritional food to promote wound healing.The main conclusions are as follows:（1）Protein gel from snakehead skin was prepared by acid hydrolysis method,and then flavourzyme was selected to prepare SSCP.The optimal extraction conditions of SSCP were determined by single factor and response surface experiments with the DPPH·scavenging rate of SSCP as screening index.The results show that the activity of flavourzyme was 3.15×104 U/g,and the DPPH·scavenging rate and hydrolysis degree of SSCP prepared by flavourzyme were the highest.The optimum extraction conditions of SSCP were as follows:enzyme dosage 7900 U/g,pH 7,enzymolysis temperature 45℃and enzymolysis time 4.7 h.The DPPH·scavenging rate of SSCP prepared by this process is 27.37%.（2）By measuring amino acid composition and content,molecular weight distribution,characteristic peptide sequence and antioxidant activity of SSCP,it was preliminatically concluded that SSCP has the potential to promote wound healing from the aspects of structure and antioxidant capacity.The results show that SSCP is rich in Gly,Hyp,Arg and hydrophobic amino acids which can promote wound healing.142-911 Da component was the main component of SSCP,accounting for 62.41%of the total peptide content.Thirty-two characteristic peptides of SSCP were identified by LC-MS/MS,all of which were composed of 4-10 amino acids,and their molecular weights ranged from 434.24-921.48 Da.SSCP has strong antioxidant activity,and its DPPH·scavenging rate,·OH scavenging rate,Fe2+chelating ability and reducing ability are 52.38%,61.52%,34.28%and 0.252,respectively.After simulated digestion in vitro,they were significantly improved,which were 54.61%,65.90%,99.37%and0.261,respectively.（3）The effects of different doses of SSCP on wound closure,inflammatory response,angiogenesis,collagen deposition,and cell proliferation and migration were studied in the skin wound of ICR mice with full thickness excision as an in vivo model,further demonstrating the significant promoting effect of SSCP on skin wound healing.The results showed that all three SSCP dose groups could promote skin wound healing in mice,and the H-SSCP group(1.5 g·kg-1·bw-1)had the best wound healing.Oral administration of SSCP can shorten the inflammatory response time by reducing the expression of inflammatory cytokines IL-1βand TNF-α.Increase the content of Hyp in the body,promote collagen synthesis and collagen deposition in wound tissue.In the early and middle stage of wound healing,the expressions of CD31 and PCNA and the levels of FGF-2 and TGF-β1 were promoted,and the migration and proliferation of endothelial cells,keratinocytes and fibroblasts were promoted,so as to shorten the formation time of granulation tissue and promote the formation of new blood vessels and epithelialization process.In the later stage of wound healing,the formation of keloids was inhibited by down-regulating the level of TGF-β1. |
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