Protective Effects Of British Red Kidney Bean Antioxidant Peptide On H₂O₂ Induced Oxidative Stress Damage In PC12 Cells

In recent years,chronic diseases related to oxidative damage have seriously affected human health.When the body is stimulated by the outside world,the increase of reactive oxygen species（ROS）in the body will lead to the imbalance of the redox system and the accumulation of excessive ROS.Excessive ROS will damage cells,tissues and systems,and then cause a series of diseases.The proper use of antioxidants can inhibit or slow down the occurrence of oxidation.However,because synthetic antioxidants have greater toxicity,natural antioxidants have attracted more and more attention due to their less side effects and strong antioxidant capacity,among which antioxidants peptides are new types of natural antioxidants.Antioxidant peptides have become a hot research topic at home and abroad because of their strong antioxidant activity and high biological safety.In this paper,British red kidney beans were used as raw materials.First,the British red kidney bean antioxidant peptides are prepared by alkaline protease enzymatic hydrolysis,and then the antioxidant peptides were separated by ultrafiltration method and the vitro antioxidant activity of the different peptides were studied.Sephadex G-15 chromatography was used to separate and purify the ultrafiltration components with higher antioxidant activity.Finally,they were applied to PC12 cells.The cell model was used to investigate the protective effects and the mechanism of anti-oxidation effects of British red kidney bean antioxidant peptides on H₂O₂-induced oxidative stress damage in PC12 cells and the mechanism of anti-oxidation effects were explored.In order to provide a theoretical basis for the application of British red kidney bean antioxidant peptides in the fields of food health and medicine,the main research contents and results are as follows:（1）First,British red kidney bean protein were extracted by alkaline extraction and acid precipitation,and then the protein were hydrolyzed by alkaline protease to prepare British red kidney bean antioxidant peptide.The antioxidant peptides were classified by ultrafiltration.The classification components were:Less than 1 k D,1 k D-3 k D,3 k D-5 k D,and greater than 5 k D.Secondly,the vitro antioxidant activity of each ultrafiltration component was analyzed.The results of the study showed that the British red kidney bean antioxidant peptide with less than 1k D had the strongest antioxidant capacity,with hydroxyl radical scavenging rate of 49.26%,DPPH free radical scavenging rate of 27.46%,and reducing power of 0.501.Finally,Sephadex G-15 chromatography was used to separate and purify the British red kidney bean antioxidant peptides less than 1 k D.Two components were separated and purified,as BRKBAPC-1 and BRKBAPC-2 with peak areas of 932.65 and 1440.33,respectively.（2）Based on the preparation,purification and vitro antioxidant activity determination of the preliminary materials.First,a hydrogen peroxide（H₂O₂）cell oxidative damage model was established.After PC12 cells were treated with 125,250,500,1 000,2 000,4 000μmol/L H₂O₂for 4 h,the cell survival rate decreased to 77.81%,54.33%,13.83%,5.13%,1.94%,and 1.77%,respectively.In subsequent experiments,250μmol/L H₂O₂ was selected as the injury model concentration.Second,the cell survival rate was used to determine the low,medium,and high doses concentration gradients of the antioxidant peptides as 50,100,200μg/m L,and the group was found compared with BRKBAPC-1,BRKBAPC-2 has a stronger protective effect and growth-promoting effect.Therefore,the BRKBAPC-2 component was selected to further study the protective effect of the antioxidant peptide component on H₂O₂-induced oxidative stress damage in PC12 cells.（3）Taking BRKBAPC-2 components as the research object to investigate the protective effect and mechanism of H₂O₂-induced oxidative stress damage in PC12 cells.The results showed that the antioxidant peptide component BRKBAPC-2 can alleviate the cell growth inhibitory effect of H₂O₂ on PC12,maintain cell morphology,reduce intracellular reactive oxygen species（ROS）levels,reduce intracellular malondialdehyde（MDA）content and extracellular Lactate dehydrogenase（LDH）activity,increase the enzyme activity of intracellular superoxide dismutase（SOD）,catalase（CAT）and intracellular reduced glutathione（GSH）content,inhibit mitochondrial membrane potential（MMP）reduces,improve cell cycle arrest,and prevent the activation of key apoptosis enzymes Caspase-3 and Caspase-9.In conclusion,the oxidative stress damage induced by 250μmol/L H₂O₂ in PC12 cells was ameliorated by pretreatment of BRKBAPC-2 fraction for 24 h.Therefore it can be seen that the antioxidant peptide component BRKBAPC-2 has a certain protective effect on oxidative damage of cells.