Study On Preparation Of Pumpkin Seed Peptides And Their Anti-aging Effect

With people's increasing concern about skin aging,"anti-aging"is no longer a professional term.Now it has entered the era of"anti-aging and anti-oxidation"for the people.More and more anti-aging ingredients have been added to cosmetics.Products with anti-aging and anti-oxidation functions occupy a large number of markets in various countries,and also shine in the Chinese cosmetics market.Polypeptides have advantages in biocompatibility,easy absorption,safety,water solubility,and small molecular weight.They also promote cell proliferation,remove excess free radicals in cells,and repair oxidatively damaged cells.Pumpkin seed meal,as a by-product of squash seed oil,is currently only used as a soy sauce production auxiliary and a feed with low economic value,but its protein content is very high,containing 8 amino acids necessary for human body,is a high-quality protein resource.The current research on pumpkin seed protein mainly focuses on the antioxidant properties of the enzymatic hydrolysis products and the evaluation methods are chemical methods.It is not possible to evaluate the biological activity of pumpkin seed peptides and their effects on human cells.In this paper,pumpkin seed meal was prepared by using cheap and readily available pumpkin seed meal as the raw material,and its anti-aging properties were mainly investigated at the cellular level.The main contents are as follows:?1?Using pumpkin seed meal as raw material to prepare pumpkin seed protein and enzymatic hydrolysis method to prepare pumpkin seed polypeptide.Pumpkin seed protein was prepared by alkaline extraction and acid precipitation method,with a protein content of 86.27%;essential amino acids in the human body accounted for 27.948%.The pumpkin seed polypeptide obtained by alkaline-trypsin hydrolysis has a degree of hydrolysis of 27.23%,a peptide yield of 39.20%,a polypeptide content with a relative molecular mass of less than 1000 Da,and a content of 92.72%.And 10.57%are the best among the five enzymatic hydrolysis products,and their performance is improved compared with the traditional alkaline protease hydrolysate.HSF cells and HaCat cells were used to evaluate the bioavailability of pumpkin seed peptide and its effect on in vitro cells.The results show that the peptide can not only improve cell viability,up to 111.38%,110.91%,but also repair oxidatively damaged cells and cell survival rate.The highest reached 86.54%and 91.54%respectively.?2?The effect of pumpkin seed peptides with different molecular weights on anti-aging properties was investigated at the cellular level.The pumpkin seed peptides P-1,P-2 and P-3with different molecular weight distribution were successfully prepared by controlling the enzymolysis time.Pumpkin seed peptide P-3 with the smallest relative molecular mass has the best performance in promoting HSF cell viability,reaching 112.22%;it also has a significant repair effect on oxidative stress cells induced by H₂O₂,which improves cell survival and shows typical fibroblasts.Morphology;cell lifespan reached 1.36 times the highest in the model group.According to the senescence-related?-galactosidase kit,it was found that after 24 hours of incubation with P-3,the senescent cells were significantly reduced.P-3 can effectively inhibit H2O2-induced premature senescence of HSF cells.From the perspective of the increase of SOD enzyme activity and the increase of GSH content,the reason of P-3 anti-cell aging was explained.?3?Evaluate the proliferation ability of pumpkin seed peptides on human skin cells from different angles,and evaluate its anti-aging performance from the perspective of cell secretion.The effects of P-3 on human skin cell growth were evaluated by MTT method,cell morphology observation,cell cycle,and growth curve drawing.It was found that the highest proliferation rates of P-3 on HSF cells and HaCat cells were 113.1%and 114.87%,respectively;the peptide can reduce the proportion of cells in the stationary phase and increase the proportion of cells in the DNA synthesis phase;the cell density increased significantly,the number of cell populations increased significantly,and division The number of phase cells increased and the voids in the pores decreased.Finally,the cell growth curve was drawn to find that the number of HSF cells and HaCat cells was 3.6 times and 2.5 times that of the initial test.Due to the addition of P-3,the collagen content and elastin content in HSF cells increased by 18.07%and39.09%respectively compared with the control group;the content of hyaluronic acid in HaCat cells increased by 40.69%compared with the control group.Therefore,P-3 can promote the synthesis of collagen and elastin in HSF cells and the expression of hyaluronic acid in HaCat cells.?4?Isolation,purification and identification of active ingredients.P-3 was separated and purified by ultrafiltration,dextran gel G-10,DEAE-52 ion-exchange chromatography,and PR-HPLC,respectively.Promoting human skin cell proliferation as a screening index,the final component PU-3-b-2-i promotes the proliferation rate of HSF cells and HaCat cells up to 139.06%and131.31%,respectively.Using MALDI-TOF-MS to identify the structure of PU-3-b-2-i components and sequence the amino acid of the polypeptide,the amino acid sequence It is ASPNEPEDNPDGF?Ala-Ser-Pro-Asn-Glu-Pro-Gly-Asp-Asn-Pro-Asp-Gly-Phe?,and the measured molecular weight is 1315.39.