Preparation,in Vitro Antioxidant Damage And In Vivo Anti-aging Effects Of Deerskin Collagen Peptide And Its Mechanism

Purpose:Collagen peptides have potential anti-aging effects and have been shown to promote health,delay aging and improve quality of life.We obtained deer skin collagen peptides（DCPs）by screening the optimal process of enzymatic digestion of deer skin collagen（DC）and investigated their anti-aging effects to provide a strong basis for the use of DCPs as anti-aging systemic supplements.Methods:After enzymatic digestion of DCs by several common proteases,the yield of each DCPs was measured,the physicochemical properties were studied（effect of p H and Na Cl on solubility of DCPs,study of oil absorption,emulsification and emulsion stability of DCPs）,in vitro antioxidant activity（DPPH,ABTS,hydroxyl radical scavenging assay）,and the molecular weight of different DCPs was determined to select the best enzymatic digestion process.DCPs were prepared by using papaya complex trypsin,characterization of DCPs,including appearance examination of DCPs,determination of molecular weight range of DCPs by SDS-PAGE,and analysis of amino acid composition of DCPs.The cytotoxicity of H₂O₂ and DCPs on Hep G2 cells was detected by the CCK8method,and the effect of H₂O₂ on the morphology of Hep G2 cells was observed by crystalline violet staining.The protective effect of DCPs on oxidative damage in Hep G2cells was detected by CCK8 method,the effect of DCPs on intracellular ROS production was detected by ROS fluorescent probe method,the effect of DCPs on apoptosis in Hep G2 cells was detected by flow cytometry,and the activities of antioxidant enzymes（SOD,GSH-Px,CAT）and malondialdehyde（MDA）content in cells were detected by ELISA.The blood biochemical indexes of mice were detected by automatic biochemical analyzer,and the activities of SOD,GSH-Px,CAT and MDA contents in serum,liver and kidney of mice were detected by ELISA method,and the histopathological examination of liver and kidney of mice was performed by HE staining method.The expression levels of Nrf2,SOD1,CAT and HO-1 genes in liver tissues were detected by RT-PCR.p Akt,Akt,HO-1 and Nrf2 protein expression levels in mouse liver tissues were detected by Western blot assay.Prescription screening of the collagen peptide oral solution was performed to determine the optimal volume ratios of the oral solution,including the optimal additions of DCPs solution,flavor blends,de-bitters and stabilizers.Results:The peptide yield of the DCPs digested with papaya complex trypsin was the highest at 42.67±2.97%,and the DCPs with the best physicochemical properties,the strongest in vitro antioxidant activity and the lowest molecular weight were selected for the follow-up study.KDA;DCPs contained 18 amino acids.DCPs at 5.0-250μg/m L had no significant toxic effect on Hep G2 cells;the survival rate decreased with increasing dose after treatment with 1.0-1.5 mmol/L H₂O₂,and was56.77±5.98%when the H₂O₂ concentration was 1.3 mmol/L.Morphological analysis of cells by crystalline violet staining confirmed The toxic effects of H₂O₂ were confirmed by morphological analysis of cells by crystalline violet staining;DCPs（25.0-100.0μg/m L）significantly increased cell viability,reduced intracellular ROS accumulation,attenuated apoptosis and upregulated intracellular SOD,GSH-Px and CAT activities,and decreased MDA content.DCPs significantly improved the blood biochemical indexes of D-galactose-induced aging mice,upregulated the activities of SOD,GSH-Px and CAT in serum,liver and kidney,reduced the content of MDA and exerted protective effects on liver and kidney of mice.Realtime-PCR showed that administration of DCPs up-regulated the expression of Nrf2 and its downstream target genes SOD1,CAT,and HO-1 m RNA in mouse liver tissues.western blot showed that administration of DCPs increased the ratio of p Akt/Akt and the protein of Nrf2 transcription factor and its downstream target protein HO-1 in mouse liver tissues.expression increased,indicating that DCPs could activate Nrf2 through Akt signaling and attenuate liver injury.By optimizing the parameters through orthogonal experiments,the best formulation of the oral liquid was obtained as follows:DCPs solution addition of 20%,citric acid addition of 0.8%,sucrose addition of 8%,honey addition of 5%,xanthan gum,CMC-Na,EDTA-2Na addition of 0.1%and potassium sorbate addition of 0.1%.The resulting oral liquid is uniform in color and tasty in taste.Conclusion:Successfully extracted DCPs with good physicochemical properties,strong antioxidant activity,low molecular weight and abundant amino acids;good protection against oxidative damage in Hep G2 cells and aging in mice;anti-aging effect through activation of Akt/Nrf2/HO-1 pathway;successful preparation of collagen peptide oral solution.