Structural Characterization Of Tartary Buckwheat Antioxidant Peptide And The Effect On Caco-2 Cells Under Oxidative Stress

Oxidative stress is the inducement of many diseases.Peptides with good antioxidant activity and high safety have become a research hotspot.Tartary buckwheat,as a kind of food and medicine mixed grain,has high nutritional value,rich protein content and reasonable amino acid composition.In this paper,six peptides with antioxidant activity were obtained by hydrolysis of Tartary Buckwheat globulin with alkaline protease.The protective effect of tartary buckwheat polypeptide on Caco-2 cells under oxidative stress was studied.The mechanism of its action under oxidative stress was studied by metabonomics.The main experimental results are as follows:?1?separation and purification of crude extracts from Tartary Buckwheat globulin by dextran gel G-75.After hydrolysis by alkaline protease,six peptides with molecular weights below 3000 Da were identified and identified.The sequences were: CR-8?CTGFVAVR?,LR-8?LRGENDQR?,GK-10?GFIVQAQDLK?SR-12?SFFLAGQSQQGR?,SR-17?SEAGVTEIWDHNTPEFR?and YR-26?YVIQPGGLLLPSYSNAPYITFVEQGR?.Through Py MOL simulation of the three-dimensional structure of peptides,we can see that there are irregular curls and ?-folds in all six peptides,and there are some differences in the structure of C-terminal and N-terminal.There were significant differences in DPPH and ABTS free radical scavenging ability among the six peptides,among which cr-8 had the strongest scavenging activity of free radicals,and four buckwheat peptides,cr-8,lr-8,gk-10 and sr-12,were selected for cell experiments.?2?H₂O₂ was used as oxidative stress inducer of Caco-2 cells to construct oxidative damage model.The protective effects of tartary buckwheat polypeptide on oxidative stress were studied by studying cell activity,apoptosis and necrosis,cell cycle,total antioxidant capacity T-AOC,malondialdehyde content?MAD?,superoxide dismutase?SOD?and catalase?CAT?.It was found that tartary buckwheat polypeptide had no effect on the proliferation of Caco-2 cells at the concentration of 500 ?g/ml.H₂O₂ can induce apoptosis of Caco-2 cells,decrease enzyme activity in cells,and increase MDA content,resulting in cell membrane damage.Tartary buckwheat polypeptide intervention can reduce the apoptosis rate of Caco-2 cells,improve mitochondrial membrane potential and enzyme activity in cells,significantly increase the total antioxidant capacity of cells,reduce lipid peroxidation,and protect the normal physiological activities of cells.Among them,tartary buckwheat polypeptide CR-8 has the best effect on the recovery of oxidative damaged cells.?3?Through the fitting of software model and the search of metabolite database,the metabolic pathway of tartary buckwheat polypeptide cr-8 intervention on cell marker differential metabolites and its effects under oxidative stress was obtained.The intervention of Tartary Buckwheat active polypeptide on oxidative stress of Caco-2 cells was analyzed from the perspective of metabonomics.By multivariate analysis of PCA,PLS-DA and opls-da metabolites,we can see that cr-8 can improve the oxidative stress state of cells after tartary buckwheat peptide intervention.Ten different metabolites were screened by S-plot,including L-acetylcarnitine,L-tryptophan,acetylspermidine,lysophosphatidic acid?20:4?,lysophosphatidic acid?20:3?,lysophosphatidic acid?16:0?,pantothenic acid,glycerophosphate ethanolamine,L-histidine and L-alanine.Metaboanalyst analysis showed that cr-8 may be involved in 10 cell metabolism pathways,of which the highest contribution is glycerophospholipid metabolism.Tartary buckwheat polypeptide CR-8 can interfere with fatty acid metabolism,amino acid metabolism and other related pathways to improve cell oxidative stress.