Preparation Of Spirulina Protein-Derived Bioactive Peptides And Their Antiphotoaging Mechanisms On Skin

In this study,the peptide components with in vitro antioxidant and anti-photoaging activity were obtained from the dried powder of Spirulina by using low-temperature high-pressure continuous flow cell breakage extraction,enzymatic hydrolysis and gel filtration chromatography.Subsequently,six peptide sequences were determined with use of MALDI-TOF-MS/MS and PEAKS software.And then,the peptide sequences were synthesised by a solid phase procedure using Fmoc-protected amino acid synthesis methods.With the aim of screening the anti-photoaging peptides on the ultraviolet radiation B?UVB?-aged Human skin fibroblast?HSF?and Human immortalized epidermic cell?Hacat?,the MTT assay,flow cytometry analysis of apoptosis and Sirius red-picric acid staining were used to determine cell viability,apoptosis rate and collagen production,respectively.Furthermore,the mice model with UVB-aged dorsal skin was carried out to evaluate the anti-photoaging effect of peptides.Meanwhile,an isobaric tag for relative and absolute quantitation?iTRAQ?proteomic was applied to reveal the working mechanisms relating to anti-photoaging.For scavenging ABTS radical,IC₅₀ values of Trypsin <3 kDa,Pepsin <3 kDa and Papain <3 kDa were 76.16±3.33 ?g/mL,756.36±11.42 ?g/mL and 555.99±11.96 ?g/mL,respectively.Using the value 100 as the cell proliferation and collagen production of UVB-aged HSF,the cell proliferation rate treated by the positive control Matrixyl,Trypsin <3 kDa,Pepsin <3 kDa and Papain <3 kDa were 100.80 ± 7.40,129.11 ± 1.85,121.97 ± 11.25 and 120.86 ± 11.32,respectively;collagen production were 156.68 ± 10.87,122.87 ± 8.16,102.19 ± 2.36 and 151.04 ± 8.16,respectively.In summary,Trypsin <3 kDa,Pepsin <3 kDa and Papain <3 kDa possessed preferred antioxidant effect and in vitro anti-photoaging effect by either promoting proliferation or improving collagen production.Peptide components T₃,P₃ and A₆,screened out from Trypsin <3 kDa,Pepsin <3 kDa and Papain <3 kDa,were proved to possess potential in vitro antioxidant and anti-photoaging effects.Moreover,six peptide sequences were identified as follows:???GMCCSR;???FFEFF;???EYFDALA;???VTAPAASVAL;???ANAAFRPR;???WVAGLGYFTKNGGPK.Results showed that peptides ?,? and ? possessed strong antioxidant activity,among them,peptide ? presented the best hemolysis protection effect to AAPH-induced erythrocytes.Meanwhile,peptide ? could either promote the proliferation or improve collagen production of UVB-aged HSF.Taking the poor skin permeability of peptides ?,? and ? into consideration,the acetylated and amidated modification,and palmitoylated modification were applied to improve the situation.Compared the unmodified peptides ? and ?,acetylated and amidated ones and palmitoylated ones showed a gradually increase of ?-helix and random coil,and a gradually decrease of ?-sheet.And such trends in palmitoylated peptides ? and ? were significantly different from the unmodified ones.In terms of biological activity,anti-tumor activity of palmitoylated peptides ? and ? were significantly the unmodified ones and their inhibitory effects on proliferation of the melanoma cell A375 were stronger than the Matrixyl.Compared with peptide ?,peptide ? possessed lower toxicity to Hacat under the same sequnence modification.Acetylation and amidated peptide ? could significantly promote the proliferation of UVB-aged Hacat,and reduce the proportion of apoptotic peak,and antagonize the formation of S-phase arrest caused by UVB,and increase the proportion of cells in G₁ phase,thus to protect UVB-aged Hacat.Results from in vivo anti-photoaging experiment showed that acetylated and amidated peptide ??P₁?achieved its anti-photoaging effect by reducing the content of MDA in skin tissues and the expression of MMP-1 and MMP-3 in cytoplasmic matrix;increasing the collagen production and activities of antioxidant enzymes SOD,GSH-Px and CAT.Palmitoylated peptide ??P₂?exerted its anti-photoaging effect by increasing the collagen production in tissues.Results from iTRAQ differential proteomics gave a comprehensive analysis on GO functions and KEGG metabolic pathways relating to skin photoaging in mice.P₁ possessed strong antioxidant activity,thus to adjust mitochondrial electron transport function.P₂ possessed strong anti-tumor activity,thus to adjust p53 apoptotic signaling pathways.Based on the photoaging model,the signaling pathway involved the most differential proteins treated by P₁ and P₂ were Parkinson's disease and Alzheimer's disease,and the Two-component system was the only pathway to possess significant correlation with both of P₁ and P₂.