Effect Of Totoaba Macdonaldi-meat Collagen Peptides On Wound Healing In Mice

Totoaba macdonaldi-meat was used as raw material.After hydrolyzed by combinative enzyme,Totoaba macdonaldi-meat collagen peptides(TMCP)was obtained.In this study,TMCP's effect on wound healing and its mechanism were explored by using cell experiments and skin wound model in mice by oral administration.The main methods and results are as follow:(1)Preparation of TMCP:Using combinative enzyme(Alcalase alkaline protease and Flavourzyme compound flavor protease)and selecting the hydrolysis degree as index to evaluate.Based on the results of the single factor experiments,the four factors and three levels response surface method was developed to get the optimum conditions:the enzymolysis temperature is 55 ?,the time is 4 h,the pH is 7.0,the dosage of enzyme is 1.0%,and the test degree of hydrolysis is 63.90%under this condition.Using Waters gel permeation chromatography,the molecular weight(Mw)of the TMCP is 837 Da,and TMCP is composed of 5?8 amino acids.22 kinds of amino acids are detected in TMCP,the content of hydroxyproline,?-aminobutyric acid,taurine,ornithine were 1.24%,1.03%,0.74%,0.37%respectively,so it has high nutritive and medical values.(2)In vitro:Research on the effect of TMCP on NIH/3T3 cells' proliferation,migration and collagen synthesis.The CCK8 assay showed that the positive effect of TMCP on cell proliferation and content of hydroxyproline were significantly higher than that of the control group(p<0.01)and were equal to that of bFGF group when the TMCP concentration is 25?100 ?g/mL.The 50 ?g/mL group showed the best accelerative effect on two indexes,and 50 ?g/mL was selected for the cell migration test.During 36 hours monitoring,the cell mobility of TMCP group was significantly increased since 12 h(p<0.01),the same to bFGF group.(3)In vivo:Research on the effect of TMCP on wound healing in mice.Forty healthy male ICR mice were underwent surgical full-thickness incision and were divided into 4 groups,which were normal control group(deionized water)and 0.66,1.33,2.0 g/kg·bw TMCP groups.The healing rate of the wound were observed every 2 days.Necropsy was performed on postoperative day 7 and 14.The wound tissues were excised for HE staining,Masson staining,hydroxyproline measurement and immunohistochemical analysis.At day 7 and 14,TMCP treatment groups were better than the control group on wound healing rate,collagen content,VEGF and bFGF expression,and both middle and high dose groups showed significant differences(p<0.01).At day 14,the TMCP low dose group has a very significant effect(p<0.01)on the above indexes,compared with the control group.Overall,in vivo and in vitro experiments,TMCP can promote fibroblasts proliferation,collagen producing and tissue remodeling of skin wound in mice.TMCP have potential to promote wound healing and can be used in high-quality oral healthy products,providing theoretical basis for high-efficient utilization.