| The polypeptide chain is hovered to form a protein with a certain spatial structure,and polypeptide is composed of 20 kinds of amino acids in different proportions,because it has good anti-oxidation,anti-aging,cell regeneration and many unique physiological activities in medicine,Which are widely used in medicine,functional foods and cosmetics.Fish skin is an ideal raw material for collagen peptide extraction.70% of the dry matter of tilapia skin is collagen.In this paper,tilapia skin collagen polypeptide(TSCP)was prepared by mixed enzymolysis of papain and neutral protease used tilapia skin as raw material.The microfiltration system and low pressure loop ultrafiltration system were used to hydrolyze enzymatic hydrolysate.Using reversed-phase high-performance liquid chromatography(RP-HPLC)purification and tandem mass spectrometry(MS/MS)analysis,the polypeptide sequence was finally identified by Max quant software search.And then determine its amino acid composition and content,toxic substances,heavy metals and microbial content,and scavenging rates for DPPH,OH,and O2-.At the same time,human fibroblasts(HDF)in vitro scratch fusion assay was used to evaluate the activity of TSCP.UVB and UVA were used as the radiation source to establish the skin photoaging model of BALB/c nude mice.The back of the mice was coated with TSCP to treat the skin photoaging.A macroscopic score is given by the apparent change in the back of a nude mouse,detection of changes in skin moisture content and HYP content preliminary examination of TSCP anti-aging skin photodynamic efficacy.In order to further study the mechanism of skin photoaging induced by TSCP,the enzyme activity of SOD,GSH-Px,CAT and MDA content in mouse skin were measured by biochemical method.The content of MMP-1/3 was analyzed by immunohistochemical staining.And combined with HE staining,Masson staining,Victorin blue staining were used to observe the effects of morphology,content and distribution of collagen fibers and elastic fibers.The main findings are as follows:(1)Ratio of solid to liquid 1:3,temperature 50°C,0.3% neutral protease and 0.05% papain mixed enzymatic hydrolyzed tilapia skin,the enzymatic hydrolyzate was centrifuged by 200?m ceramic membrane microfiltration,1k Da molecular membrane filtration system filtration,And the product prepared by post-concentration spray drying is mainly a collagen polypeptide,which is light yellow and has an earthy smell.The content of chromium,cadmium,and lead in TSCP was lower than the national standard,and the chromium content was 0.31 mg/kg,among which cadmium and lead were not detected.The toxic substances were inorganic arsenic,methylmercury,N-dimethyl nitrite,and polychlorinated.Biphenyl was not detected,and the total number of bacteria was 40 cfu/g,which was far below the national microbiological limit of 1000 cfu/g.No mold,yeast,coliform bacteria,Salmonella,and Staphylococcus aureus were detected.TSCP molecular weight is mainly <1000Da polypeptide,its mass fraction is 92.96%,identified 175 peptide chains,contains 17 kinds of amino acids,8 amino acid peptide chain is 151 segments,ion or molecular fragment mass-to-charge ratio(m/z)is 720~3500.The TSCP was isolated and purified by RP-PHLC.Two components,TSCP-3 and TSCP-4,were collected and identified by ESI-MS/MS as tetrapeptides Asn-His-Arg-Tyr(454.2 Da)and Gly-Asn-Arg-Gly(470.3 Da).(2)Activity of TSCP in vitro showed that it has good antioxidant activity and was able to inhibit active free radicals,including DPPH,· OH,and · O2-,with VC as a positive control.The half-inhibitory rate(IC50)values of TSCP and VC for DPPH were 3.875 mg/m L,and 2.289 mg/m L,respectively.And the IC50 values for superoxide radicals were 0.849 mg/m L,and 0.242 mg/m L,,respectively.The IC50 value for scavenging hydroxyl radicals of TSCP was 0.443 mg/m L.The results of in vitro scratch test of skin fibroblasts(HDF)showed that the TSCP concentration was between 50?g/m L-150?g/m L,which significantly promoted the HDF at 12 h,24h and 36 h respectively,to compare with the normal group.The effect of trace fusion(P<0.05 vs normal group)was dependent on the concentration.When the concentration was 200 ?g/m L,the HDF fusion was not significant(P<0.05 vs normal group);50 ?g/m L,100 ?g/m L,and 150 ?g/m L significantly promoted the HDF fusion rate(P<0.05 vs normal group)at 24 hours of culture,and increased by 26.67% and 35.00%,respectively,compared to the blank group at the same culture time.And 13.66%,36 h culture,the role of 50-150?g/m L HDF fusion rate has reached 100%.(3)In vivo initial pharmacodynamic studies showed that TSCP significantly improved the appearance of photoaged skin in mice,and proper TSCP(200,400,800 mg/ml)increased skin elasticity and hydration of collagen(P<0.05 vs model group),The apparent score was improved.In the high-dose group mice wrinkles were reduced,no damage and inflammation,rosy and glossy,the apparent recovery was close to the normal group(P<0.05 vs model group),followed by the low-dose group.In addition,the skin of mice in the VE group was significantly improved,Which indicating that it had a certain repair effect.The reason may be that VE has a strong free radical scavenging activity,but can not provide the basic unit structure of synthetic collagen-amino acids.Hydroxyproline(HYP)is a precursor amino acid that synthesizes skin collagen.It promotes protein regeneration and delays skin aging and repairs damage to the skin's metabolism.In the TSCP treatment group(especially the high-dose group),the HYP content in the dorsal skin of the mice was significantly increased(P<0.05 vs model group),the epidermis was abnormally thickened,and the skin moisture content gradually returned to normal levels,and the collagen content was also increased.The increase in dose indicates that TSCP has a significant effect on photoaging of the skin.The mechanism may be that it inhibits the oxidative damage of proteins,provides the amino acids required for the synthesis of proteins by skin cells,repairs damaged or aged collagen networks,and maintains the elasticity and physiological function of the skin.(4)Biochemical and pathological structures indicate that TSCP can regulate collagen synthesis in mouse skin,promote the secretion of new collagen(type I collagen,elastin)to build a collagen elastic fiber network and inhibit the skin's peroxidation,And maintain the normal physiological characteristics of the inner structure of mouse skin.The results of antioxidant studies suggest that TSCP can significantly increase the activity of SOD,GSH-Px,and CAT(P<0.05 vs model group),decrease the content of MDA(P<0.05 vs model group),and inhibit UV-induced abnormal expression of MMP-1 and MMP-3(P<0.05 vs model group).Collagen fibers are an important part of maintaining skin elasticity and normal physiological functions.Normal collagen fibers are arranged in order and structurally intact.In the model group,the skin collagen of mice was severely broken,and most of them were distorted,arranged disorderly and irregularly,showing typical photoaging pathological features.Histopathological staining showed that TSCP could significantly improve the UV-induced degeneration of the intrinsic structure of mouse skin(especially the reduction of collagen fiber content,the distortion of elastic fibers),repair of the contact surface between the stratum corneum and the dermis layer,and lightening of the dermis.Layers of inflammatory cell infiltration enhance the homeostasis environment of extracellular matrix ECM.In summary,the results show that topical application of TSCP through anti-oxidation,synergistic cell regulator MMP-1/3 directly affect the synthesis and secretion of matrix metalloproteinases,inhibit collagen degradation,promote the construction of collagen-elastic fiber network,and then confront UV irradiation accumulates chronic skin photoaging. |
PDF Full Text Request