Isolation,Purification And Bioactivity Study Of OAT Antioxidant Peptides

Numerous studies have shown that excess reactive oxygen species could induce cancer,cardiovascular diseases,diabetes,neurological diseases and Alzheimer’s disease,etc.When the body’s antioxidant defense capacity is reduced or the antioxidant defense system is damaged,the body will not be able to remove the excess reactive oxygen species and needs to be assisted by exogenous substances.Excessive or incorrect addition of synthetic antioxidants can be toxic and carcinogenic to the body.The screening and evaluation of natural resources with strong antioxidant activity has become a new trend in bioresearch,medical research and food science research.On the other hand,natural antioxidants have the advantages of wide source of raw materials,high efficiency and safety in antioxidant properties.Therefore,in this study,oat antioxidant peptides were prepared by enzymatic digestion,purified by ultrafiltration and gel column chromatography,identified and analyzed by mass spectrometry,and screened for activity using the Peptide Ranker database.Then,the protective effect of oat antioxidant peptides on oxidative damage in HepG2 cells was investigated by establishing an H₂O₂-induced oxidative stress model.Finally,the molecular mechanism of oat antioxidant peptides in preventing oxidative damage was preliminarily analyzed by transcriptomics.The results of the study were as follows:（1）Four peptide fractions were obtained from the purification of oat peptides by ultrafiltration,namely OP-Ⅰ（MW>5 k Da）,OP-Ⅱ（3 k Da<MW<5 k Da）,OP-Ⅲ（1 k Da<MW<3 k Da）and OP-Ⅳ（MW<1 k Da）,of which OP-Ⅳhad the strongest antioxidant activity,and its IC₅₀values for scavenging rate of DPPH·and ABTS⁺were 2.25 and 0.098mg/m L,and the reducing power was 0.52 at a concentration of 5 mg/m L;OP-IV was separated by dextran gel G-15,and three fractions OP-IVA,OP-IVB and OP-IVC were obtained,of which OP-IVC had the strongest antioxidant activity,and the IC₅₀values of scavenging rate DPPH·and ABTS⁺were 0.175 and 0.030 mg/m L,and the reducing power was 0.64 at a concentration of 5 mg/m L;the amino acid sequence identification of OP-ⅣC was performed using Q-Exactive plus,and the two peptides with the highest scores of Peptide Ranker were OP-1 and OP-2,whose amino acid sequences were His-Trp-Pro-Leu-Pro-Pro-Phe（892.46 Da）and Gly-Trp-Leu-Asp-Arg-Phe-Pro-Met-Phe（1167.55 Da）.（2）A model of oxidative stress injury was established by stimulating HepG2 cells with H₂O₂at a concentration of 0.8 m M,and a study of the protective ability of OP-1 and OP-2against oxidative damage was conducted.The results showed that both OP-1 and OP-2 in the concentration range of 0.06-1.0 mg/m L had no toxic effects on cells after pretreatment for 24 h.The protective effects of OP-1 and OP-2 at a concentration of 1.0 mg/m L were the strongest,but there was no significant difference between OP-1 and OP-2 groups（p>0.05）.The results of measuring the antioxidant indexes（ROS,MDA,CAT,SOD and GSH-PX）in HepG2 cells showed that OP-1 and OP-2 at a concentration of 1.0 mg/m L could significantly reduce the content of ROS and MDA in HepG2 cells（p<0.05）,and significantly increase the activity of CAT,SOD and GSH-PX（p<0.05）.Preliminary experimental results indicate that OP-1 and OP-2 are two novel antioxidant peptides that can effectively scavenge reactive oxygen species,enhance the activity of related antioxidant enzymes and protect cells from damage caused by cellular oxidative stress.（3）RNA-seq analysis showed that the GO functions of differentially expressed genes in OP-1,OP-2 and the model group were mainly annotated to immune processes,growth metabolism and molecular functions,indicating that OP-1 and OP-2 may exert antioxidant effects to mitigate cellular oxidative damage by regulating biological processes,molecular function regulation and other processes.KEGG analysis showed that the pathways involved in the antioxidant activity of OP-1 are mainly the Hippo signaling pathway and necrotic apoptosis,and upregulated expression of differentially expressed genes Id2 and SNAI2 could inhibit ROS-induced apoptosis and reduce oxidative damage;downregulated expression of TLR4 and CAMK2D was beneficial to reduce necrotic apoptosis and mitigate the damage caused by oxidative stress.In addition,OP-2 may exert antioxidant effects through estrogen signaling pathway and IL-17 signaling pathway.Upregulation of CALML6 and CXCL2expression promoted cell proliferation and reduced cellular oxidative stress levels.Upregulation of UGT1A7 expression indicated that the presence of OP-2 may reduce the effect of ROS on HepG2 cells.FOS expression was downregulated,indicating that OP-2 reduced ROS levels,suggesting an ameliorative effect on cellular oxidative damage.