Study On Preparation Of Peach Seed Antioxidant Peptides And Their Antioxidant Effect

Oxidation occurs in human body during normal metabolic progresses,which lead to the generation of a large amount of reactive oxygen species?ROS?.Excess of ROS can cause many chronic diseases such as skin aging,adiposity and diabetes mellitus.As a highly reactive natural antioxidant,antioxidant peptides become one of the focuses of research owing to their properties of high safety,small molecular weight and easily absorbed and utilized by bodies.In this paper,protein extracted from peach seed was used to prepare peach seed antioxidant peptides?PSAP?by enzyme hydrolysis method.In-vitro antioxidant capacities were used as a target to the separation,purification and structural identification of PSAP.And the antioxidant capacities and mechanisms in cells were further studied.The main content was as follows:?1?Protein from peach seed was extractd by ultrasonic-assisted alkali extraction acid precipitation method,and the extraction rate of protein was used as a target.The extraction conditions were optimized by single-factor and orthogonal experiments.The optimal conditions were as follows:solid-liquid ratio of 1:30 g/mL,pH=10,temperature of 50?and time of 2 h.In this condition,the extraction rate of protein was 79.51%,and the purity of protein was 73.56%.?2?Peach seed antioxidant peptides?PSAP?were prepared by enzyme hydrolysis method.Degree of hydrolysis and DPPH radical scavenging rate were used as targets.Alkaline was then selected as the most suitable protease,and the conditions were optimized by single-factor experiment and response surface analysis.The optimal conditions were as follows:substrate concentration of 4.6%,ratio of enzyme to substrate of 2.8%,and time of87.5 min.In this condition,the DPPH radical sacvening rate of 5 mg/mL of PSAP was92.01%.?3?PSAP were successively separated and purified by ultrafiltration,sephadex column chromatography and RP-HPLC,and in-vitro antioxidant capacities including DPPH radical,superoxide anion radical,hydroxyl radical scavenging rate and reducing power were used as targets.PSAP-3,PSAP-3-a and PSAP-3-a-i were the most active fractions of each step,and PSAP-3 is the most active fraction among them.The IC₅₀0 of DPPH·and·OH were 1.44 and1.04 mg/mL,respectively.The O₂^-·scavenging rate and absorbance value of reducing power of 4 mg/mL PSAP-3 were 24.71%and 0.481,respectively.Then PSAP-3-a-i was identified by MALDI-TOF-MS.Result showed that there were four peptides with the amino acid sequence of Gly-Pro-Val-Ala-Glu-Val-Ala?GPVAEVA?,His-Asn-Gly-Val-Leu-Gln?HNGVLQ?,Thr-Pro-Val-Ala-Ala-Arg?TPVAAR?and His-Asn-Ala-Val-Leu-Ala-Asp-Leu-Phe?HNAVLADL F?.?4?The cytotoxicity experiment showed that 0.05-1.60 mg/mL of PSAP and PSAP-3had no cytotoxicity effect on HSF cells,instead,had proliferation effect.And 0.05-0.80mg/mL of PSAP and PSAP-3 both had protective effect against oxidative damage in HSF cells induced by H₂O₂.In addition,CAA assay showed that PSAP-3 can scavenge ROS in cells with the half scavenging rate of 53.94?g/mL.And also,PSAP-3 can work by improving the enzyme activities of SOD and CAT and the content of GSH.200?g/mL of PSAP-3 can improve the SOD activity,CAT activity and GSH content to 771.86 U/mg pro,25.39 U/mg pro and 3.87 mg/L,respectively.