Study On The Structure-Activity Relationship And Molecular Mechanism Of Watermelon Seed Antioxidant Peptides

Watermelon is one of the main fruits produced in China,with an annual output of about 63 million tons.Watermelon seed,the main by-product of watermelon processing,rich in protein and antioxidant amino acids,is a high-quality resource for preparing antioxidant peptides.It is known that antioxidant peptides can balance free radicals in the body and prevent related chronic diseases caused by oxidative stress,but the research on watermelon seed antioxidant peptides is still focused on the preparation and activity evaluation of peptide mixtures.Due to the lack of information on the sequence spectrum and structure-activity relationship of watermelon seed peptides,the mechanism of their action still remains unclear.Based on this,watermelon seed protein(WSP)was used to prepare watermelon seed protein hydrolysates(WSPHs)by slit divergent ultrasonic assisted enzymatic hydrolysis in this study.The structure,structure-activity relationship,specific action sites and modes of antioxidant peptides were studied through chromatography and spectroscopy techniques,solid-phase synthesis techniques,quantum chemistry and molecular docking techniques;The digestion,absorption and transmembrane transport of antioxidant peptides were elucidated by cell model in vitro.The role and mechanism of antioxidant peptides in preventing oxidative stress damage were studied at the molecular level by molecular biology techniques.This study may lay a new theoretical foundation and basis for the development of watermelon seed antioxidant peptides and provide theoretical guidance for the intensive processing and value-added research of watermelon seed.The main research contents and results are as follows:(1)Effect of Ultrasonic Pretreatment on the Structure of Watermelon Seed Protein and Its Enzymatic HydrolysisTaking hydrolysis degree of WSP and reducing power of WSPHs as the inspection indicators,a slit divergent dual-frequency 20/28 k Hz was screened out as the best ultrasonic pretreatment mode,and its technical parameters were optimized as follows:material concentration 20 g/L,ultrasonic time 60 min,and power density 100 W/L.Under these conditions,the hydrolysis degree of WSP and the reducing power of WSPHs were 16.72%and 0.52,respectively,which were 75.26%and 550%higher than that of traditional enzymatic hydrolysis.Using stepwise multiple regression analysis,a quantitative relationship between WSP structure and WSPHs reducing power was established.During the ultrasonic pretreatment process,the protein-SH content and?-turn angle can predict the reducing power of WSPHs,which can be used to judge the endpoint of ultrasonic treatment.(2)Study on Antioxidant Activity of Watermelon Seed Protein HydrolysatesThe WSPHs were separated by ultrafiltration membranes of 5 kDa and 1 kDa to obtain WSPHs-I(M_W<1 kDa),WSPHs-II(1 kDa<M_W<5 kDa)and WSPHs-III(M_W>5kDa)with different molecular weights,the external antioxidant activity of WSPHs and its ultrafiltration components through in vivo and in vitro experiments was evaluated.The results showed that the antioxidant capacity of WSPHs-I was the strongest,and its IC₅₀ values for DPPH·and ABTS+·clearance rates were 1.15 mg/m L and 2.21 mg/m L,respectively;The ability of chelating Fe²⁺and reducing power of WSPHs-I(1 mg/m L)were 20.04 and 0.66 respectively;Compared with model group,WSPHs-I can significantly prevent cell oxidative damage induced by H₂O₂(p<0.05),increase cell survival rate and reduce malondialdehyde(MDA)content(p<0.05);In addition,WSPHs-I can significantly increase the activity of Superoxide dismutase(SOD),Catalase(CAT),Glutathione peroxidase(GSH-Px)and glutathione(GSH)in the liver tissue of rats with oxidative damage induced by D-galactose(p<0.05),reduce the content of MDA in liver tissue(p<0.05).(3)Isolation,Purification and Structural Identification of Watermelon Seed Antioxidant Peptide Components(WSPHs-I)WSPHs-I was separated and purified by chromatography combined with biological activity tracking to screen for high-activity antioxidant peptide fragments,identify the sequence of the peptide by using LC-MS/MS,and predict the physical and chemical properties of the peptide by computer simulation.Three peptide components(WSPHs-IA,WSPHs-IB and WSPHs-IC)were separated from WSPHs-I by Sephadex G-15.Among them,WSPHs-IB has the strongest antioxidant activity.The IC₅₀ values of WSPHs-IB on DPPH·and ABTS⁺·clearance rates were 0.76 mg/m L and 1.80 mg/m L,respectively,and the ORAC value was 68.72?M TE/mg peptide;WSPHs-IB was separated by semi-preparative liquid phase to obtain 20 peptide components.WSPHs-IB-15 was screened to have the strongest antioxidant activity.After LC-MS/MS separation and identification,it was found that WSPHs-IB-15 contained five peptides(P1-P5),the relative molecular masses were 800.38,774.42,858.43,802.39,and975.45 Da,respectively;and their amino acid sequences were RDPEER,KELEEK,DAAGRLQE,LDDDGRL and GFAGDAPRA.(4)Study on the Structure-Activity Relationship of Watermelon Seed Antioxidant PeptidesUse quantum chemistry to perform molecular simulations on the chemical structure of P1-P5,calculate the frontier molecular orbital distribution and energy,atomic net charge distribution and bond length of P1-P5,and infer the active sites of P1-P5;Use molecular docking to study the optimal docking configuration and interaction of P1-P5with DPPH·and ABTS⁺·,respectively.The results showed that the order of P1-P5energy parameters was P2>P3>P5>P4>P1,and the predicted order of its antioxidant activity was P1>P4>P5>P3>P2.It was verified by experiments that P1 had the strongest antioxidant activity;The order of the frontier orbital energy level difference of P1-P5was P2>P3>P5>P4>P1,the active site of P1 is located at C₆H₁₄ on Arg,the active site of P2 is located at C₂₉H₃₆ on Glu,the active site of P3 is located at C₄₇H₅₅ on Gly,the active site of P4 is located at C₇₅H₈₅ on Arg,and the active site of P5 is located at C₆₃H₆₉on Asp.These sites act as hydrogen donors to provide hydrogen atoms to achieve the purpose of scavenging free radicals.P1-P4 can bind to DPPH·through hydrogen bond and hydrophobic interaction,P5 mainly binds to DPPH·through hydrophobic interaction;P1-P5 binds to ABTS⁺·through hydrogen bond and hydrophobic interaction force.(5)Study on the Digeation,Absorption and Transport Mechanism of Antioxidant Peptide P1The simulated two-stage digestion method was used to study the digestive stability of P1 in the gastrointestinal tract,and the Caco-2 monolayer cell model was used to evaluate the absorption and transport of P1 at different time.The main pathway of P1transmembrane transport was studied by adding promoters or inhibitors.The composition and structure of metabolites after P1 trans-cell transport were analyzed by LC-MS/MS.The results showed that the apparent permeability coefficient of different concentrations of P1 was 4.53×10^-6-4.72×10^-6,and the transport rate was 3.65%-3.81%,which had a high transport rate and showed a concentration and time-dose effect;P1transported across the membrane mainly through the cell alternative pathway;Part of P1 was degraded by brush border membrane enzymes on the top side of the Caco-2 cell membrane and new peptide components RDP and EER were produced.(6)Preventive Effect of Antioxidant Peptide P1 on Oxidative Stress Damage and its Molecular MechanismBased on the Hep G2 cell oxidative damage model induced by H₂O₂,the effect of P1 on preventing cell oxidative damage was evaluated.Transcriptomics analysis combined with RT-q PCR and Western Blot verification was used to study the antioxidant pathway of P1 and its molecular mechanism.The results show that,compared with the model group,P1 can significantly increase the activity of intracellular antioxidant enzymes and the ratio of GSH/GSSH(p<0.05),reduce cell membrane damage,intracellular calcium ion concentration and MDA content;After P1pretreatment,a total of 175 genes were differentially expressed in Hep G2 cells,of which 148 genes were up-regulated,27 genes were significantly down-regulated,and42 genes were co-expressed in the model group and the P1 group.Through the KEGG pathway and GO function enrichment analysis,P1 can exert its antioxidant effect by regulating molecular functions,biological processes,signal transduction,organelles and other functions,and mainly regulates the p38 MAPK signal pathway to inhibit the downstream apoptosis pathway and activate the endogenous antioxidant signalling pathway Nrf2-Keap1 to prevent cell oxidative damage.