Research On The Quantitative Structure-Activity Relationship And Antioxidant Molecular Mechanism Of Antioxidant Tripeptide

Unbalanced metabolism of free radicals can induce oxidative stress damage in the body,leading to the occurrence of various diseases.The role of antioxidant peptides in scavenging free radicals has been widely recognized.As a new type of safe antioxidant,antioxidant peptides have shown broad application prospects in functional foods,medicine and other fields.However,the sequence characteristics,active site and antioxidant mechanism of the highly active antioxidant peptide has not been fully clarified,so it is difficult to guide the design and screening of the highly active antioxidant peptide,which hinders its large-scale application.This paper studies the quantitative structure-activity relationship?QSAR?of antioxidant tripeptides,which is used to predict its activity,and then guides the design of highly active antioxidant tripeptides.At the same time,various in vitro activity evaluation systems were used to screen highly active antioxidant tripeptides and cell models are used to elucidate the mechanism of action of antioxidant tripeptides which can lay a theoretical foundation for the development of highly active antioxidant tripeptides.The main findings of this paper are as follows:?1?In view of the unclear characteristics of high-activity antioxidant tripeptide sequences,2D-QSAR models were established to predict the activity of antioxidant tripeptides and to guide the design of highly active antioxidant tripeptides.91antioxidant tripeptides was screened for the antioxidant tripeptides database.The antioxidant tripeptides were characterized by 7 physicochemical properties of amino acids which were screened from 134 physicochemical properties of amino acids using stepwise regression.The method combined the stepwise regression and multiple linear regression?SWR-MLR?,partial least squares?PLS?,support vector machine?SWR-SVM?and and random forest?SWR-RF?algorithm were used to construct QSAR model for 91 tripeptides.The models of SWR-MLR,PLS,SWR-SVM and SWR-RF have good fitting ability and stability(²_?64?9)>0.8,²>0.7,²₀)>0.8).Among these models,the fitting ability of PLS model is superior(²_?64?9)=0.902)and the stability of SWR-MLR is the best(²=0.798,²_-17)?9?>0.792).The normalized regression coefficients?NRC?of STR-MLR can discover the do minant position which displayed C-terminal a mino acids are Pro,Tyr,Trp;the N-terminal a mino acids are Cys,Tyr,Trp;the 2 a mino acids are Ile,Val,Lys,and Leu are favorable amino acids for each point.19 antioxidant tripeptides were synthesized to verify the predictive ability of each model.The results shows the total antioxidant capacity?ABTS assays?of the synthetic tripeptide was found to be higher than the predicted value.The contribution trend of the do minant position amino acid of the tripeptide total antioxidant activity?ABTS assays?is consistent with the trend predicted by the normalized regression coefficients?NRC?of STR-MLR.?2?In order to further study the sequence characteristics of highly active antioxidant tripeptides,27 antioxidant tripeptides and 18 dipeptides which with C-ter minus or N-ter minal were removed according to the do minant a mino acids were synthesis.High activity antioxidant tripeptides were screened by linoleic acid oxidation inhibition method,DPPH assays,ABTS assays,ORAC assays and FRAP assays.The results showed that YXY?X=Ile,Val,Lys?is a high activity antioxidant tripeptide characteristic sequence in the linoleic acid oxidation inhibition,ABTS,ORAC evaluation system.In the evaluation of linoleic acid oxidation inhibition,YVY has the highest activity,and its linoleic acid oxidation inhibition rate is equivalent to?-tocopherol.YIY has the highest activity in ORAC and ABTS evaluation system,ORAC value is 4.53±0.12?M TE/?M which is equivalent to GSH?4.85±0.13?M TE/?M?and ABTS value is 6.56±0.33?M TE/?M which is higher than GSH.?1.86±0.24?M TE/?M?.YXC?X=Ile,Val,Lys?is a high activity tripeptide characteristic sequence in DPPH system,and YKC has the highest activity(IC₅₀=76.235?M).PXY?X=Ile,Val,Lys?is a high-activity peptide characteristic sequence in the FRAP evaluation system,and PVY(3.75±0.33 Fe²⁺?M/?M)shows the strongest antioxidant activity.YIY,YVY,YKC and PVY were screened as the highest-activity antioxidant tripeptides among the 27 antioxidant tripeptides in vitro activity evaluation.?3?The antioxidant activities of YIY,YVY,YKC and PVY were further evaluated in a rabbit red blood cell oxidative hemolysis model.The results showed that YIY,YVY,YKC and PVY could effectively inhibit AAPH-induced rabbit red blood cell hemolysis.2 mg/mL of YIY,YVY,YKC and PVY reduced the oxidative hemolysis rate of rabbit red blood cells by 61.19%,51.14%,31.96%and 45.81%,respectively.The four antioxidant tripeptides inhibit the lipid oxidation of rabbit red blood cells,reduce intracellular ROS production,increase the activity of SOD and GSH-Px enzymes in rabbit red blood cells,increase the ratio of GSH/GSSG in rabbit red blood cells,and reduce rabbits.Red blood cell oxidative damage,protective effect showed a concentration-dependent trend.According to hemolysis inhibition,YIY and YVY with strong antioxidant activity in rabbit model of erythrocyte oxidative damage were selected for follow-up study.?4?In-depth study of the molecular mechanism of antioxidant activity and antioxidant activity of YIY and YVY.The protective effects of YIY and YVY on Caco-2cells oxidative damage and their molecular mechanisms were investigated by cell morphology observation,kit detection,flow cytometry,Real-time PCR and Western blotting.It was proved that the concentration of 0.5³ mg/mL YIY and YVY had no obvious toxicity to Caco-2 cells.1 mg/mL YIY and YVY can significantly inhibit the decrease of Caco-2 cell activity in oxidative damage,reduce the formation of MDA and the leakage of LDH in cells,and the concentration-dependent trend of protection.2mg/mL YIY and YVY significantly inhibited the activity of CAT,GSH-Px,SOD and GR in Caco-2 oxidative damage cells,increased the ratio of GSH/GSSG in cells and restored the redox balance of cells.It was also confirmed that 3 mg/mL YIY and YVY pre-incubation can up-regulate the expression of downstream antioxidant-related genes Ho-1,Nqo 1 and Gstm1 regulated by Keal-Nrf2-ARE signaling pathway in Caco-2 cells,and promote expression of HO-1?NQO1 and GSTM1.In this paper,the anti-oxidation tripeptide activity prediction model was established by combining QSAR theory in bioinformatics,and four high-activity antioxidant tripeptides of YIY,YVY,YKC and PVY were designed and screened based on SWR-MLR model,which revealed at the cellular level.YIY and YVY can exert anti-oxidation by regulating the expression of downstream antioxidant-related genes and proteins regulated by Keap1-Nrf2-ARE signaling pathway.It is indicated that the anti-oxidation tripeptide design and screening method based on QSAR model is feasible and efficient,and plays an important role in discovering new antioxidant tripeptides.