An oligopeptide, its preparation method and application

Abstract

This invention discloses an oligopeptide, its preparation method, and its applications. The optimal oligopeptide, with the amino acid sequence LYVPHWN, was obtained through LC-MS / MS analysis, computer-aided screening, and network pharmacology. This oligopeptide exhibits high affinity for key targets such as MMP9, TNF, BCL2, and STAT3, and demonstrates good biocompatibility and low toxicity. At concentrations ≤1600 μg / mL, it shows no significant toxicity to Caco-2 cells. Furthermore, it significantly increases GSH content, decreases MDA content, and enhances SOD activity in Caco-2 cells in an oxidative stress model, effectively regulating the intracellular antioxidant system and alleviating oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an oligopeptide, its preparation method, and its application. Background Technology

[0002] Reactive oxygen species (ROS) are byproducts of normal cellular metabolism, playing a crucial role in regulating cellular signal transduction and immune responses. However, under stress, excessive ROS accumulation can exceed the regulatory capacity of the intracellular antioxidant system, leading to oxidative stress, DNA damage, lipid peroxidation, and protein dysfunction. This, in turn, disrupts cellular homeostasis, induces aging, and contributes to various chronic diseases. Therefore, maintaining ROS balance in vivo is essential for protecting organismal health and delaying oxidative damage. Antioxidants, as an effective means of regulating ROS levels, have been widely used in pharmaceuticals, food, and cosmetics. Although synthetic antioxidants (such as TBHQ, BHT, and BHA) are widely used in industry, they possess potential toxicity and side effects, such as allergic reactions, gastrointestinal discomfort, and hepatotoxicity. In contrast, antioxidant peptides derived from natural proteins, due to their good biocompatibility, higher absorption rate, low molecular weight, and lower toxicity, have become a current research hotspot in antioxidants.

[0003] Food-derived antioxidant peptides, as an important source of natural antioxidants, possess advantages such as high antioxidant activity, high safety, easy absorption, and good tolerability. They exert their antioxidant protective effects primarily through multiple mechanisms, including scavenging free radicals, providing protons to block chain reactions, chelating metal ions, regulating the activity of antioxidant enzymes (such as SOD, CAT, and GSH-Px), reducing aminotransferase (AST and ALT) levels, and regulating glutathione metabolism and related gene expression. Currently, various antioxidant peptides have been isolated from animals, plants, and microorganisms, with animal-derived peptides being the most widely used. However, plant processing often generates large quantities of inexpensive byproducts, such as walnut meal and rice bran. These byproducts are rich in protein and have been considered an important source of plant-derived antioxidant peptides. For example, several peptides with antioxidant activity have been found in walnut meal, including QGRPWG, PSRADY, AYNIPVNIAR, IFW, IIPF, IVAF, IIFY, ILAFF, IFIP, PGHFE, YWSPNDEQFR, RNHPQF, YRYL, and NPDDEFRPQ. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a thin-shelled pecan oligopeptide with antioxidant potential.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the thin-shelled pecan oligopeptide with antioxidant potential.

[0006] The final technical problem to be solved by this invention is to provide the application of oligopeptides or the products described herein in the preparation of antioxidant products.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides an oligopeptide, wherein the amino acid sequence of the oligopeptide is LYVPHWN.

[0008] The present invention also provides a method for preparing the aforementioned oligopeptide, the method comprising enzymatically hydrolyzing thin-shelled pecan meal using a complex protease, followed by dilution and ultrafiltration.

[0009] The enzymatic hydrolysis conditions are as follows: hydrolysis time of 1-5 hours, concentration of thin-shelled pecan cake meal of 1-20% (m / v), and addition amount of compound protease of 2000-10000 u / g.

[0010] Preferably, the optimal conditions for the enzymatic hydrolysis of the complex protease in the method are a hydrolysis time of 4.68 h, a substrate concentration of 11.35% (m / v), and an enzyme addition of 7000 u / g.

[0011] The oligopeptide is a polypeptide with good oxidative stress relief properties, screened by combining computer-aided screening, network pharmacology and molecular docking methods, and its amino acid sequence is LYVPHWN.

[0012] The present invention also provides a product containing the oligopeptide, wherein the product is a food, health product or pharmaceutical.

[0013] The food mentioned herein is a general food, a functional food, or a food for special medical purposes.

[0014] The oligopeptides are used alone or as additives in general foods, health products, functional foods or foods for special medical purposes.

[0015] The present invention also provides the application of the oligopeptides or the products described herein in the preparation of antioxidant products.

[0016] The amount of oligopeptide added was 6.351 mg / mL.

[0017] The oligopeptide is used in the preparation of products that increase GSH levels and / or decrease intracellular MDA content and / or increase SOD activity levels.

[0018] The oligopeptides are used in the preparation of products that inhibit lipid peroxidation and / or reduce and protect against damage to cell membrane structures.

[0019] The present invention also includes functional peptide sequence analysis: the amino acid sequence of functional peptides was analyzed by LC-MS / MS.

[0020] The invention also includes virtual screening, and the methods include computer-aided screening, network pharmacology, and molecular docking.

[0021] The network pharmacology analysis involved in this invention includes peptide-target prediction using the Swiss TargetPrediction database (http: / / www.swisstargetprediction.ch / ), and searching for target information related to oxidative stress and antioxidation using the GeneCards database (https: / / www.genecards.org / ). A network of oxidative stress targets and peptide-predicted targets is constructed using CytoScape 3.10.4 software (http: / / www.cytoscape.org / ).

[0022] The peptide activity prediction and bioinformatics analysis involved in this invention include using the Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) database to predict the potential biological activity of identified peptides, using the functional annotation and pathway enrichment tools of the David database (https: / / david.ncifcrf.gov) to screen key targets for bioinformatics analysis, and using a bioinformatics database (http: / / www.bioinformatics.com.cn / ) for data analysis and visualization.

[0023] The molecular docking method of this invention includes obtaining the 3D X-ray crystal structure of the core target protein using the protein PDB database (https: / / www.rcsb.org / ); preprocessing the receptor protein using the AutoDockTools toolkit; predicting the two-dimensional (2D) structure of the peptide using the ChemBio3D Ultra 20.0 software package and converting it into a three-dimensional structure using ChemBio3D 20.0; predicting the protein binding site using DeepPocket; performing docking using AutoDockVinal.2.7; and visualizing the results using PyMol and Discovery Studio 2025.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The present invention optimizes the enzymatic hydrolysis process for preparing antioxidant peptides from thin-shelled pecans. The optimal conditions for preparing the oligopeptides from thin-shelled pecans in the present invention are: enzymatic hydrolysis of thin-shelled pecan cake meal using a complex protease, with the following hydrolysis conditions: hydrolysis time 4.68 h, enzyme addition amount 7000 U / g, and substrate concentration 11.35% (m / v). Under these conditions, the peptide yield is 96.25±1.54 mg / g, and the DPPH free radical scavenging rate is 66.1%; (2) The present invention obtains the optimal oligopeptide by LC-MS / MS analysis, computer-aided screening, and network pharmacology, and its amino acid sequence is LYVPHWN. The oligopeptide has high affinity for core targets such as MMP9, TNF, BCL2, and STAT3, and has good biocompatibility and low toxicity. It has no significant toxicity to Caco-2 cells at a concentration of ≤1600 μg / mL. It can significantly increase the GSH content, reduce the MDA content, and increase the SOD activity of Caco-2 cells in the oxidative stress model, effectively regulate the intracellular antioxidant system and relieve oxidative stress, proving that the antioxidant peptide can be well used for the treatment of oxidative stress; (4) The oligopeptide derived from thin-shelled pecan described in this invention has outstanding antioxidant potential and can be used to prepare functional preparations to achieve the effects of regulating the intracellular antioxidant system and relieving oxidative stress. Attached Figure Description

[0025] Figure 1 To investigate the effects of different proteases on the DPPH free radical scavenging rate and polypeptide content of the products after enzymatic hydrolysis of thin-shelled pecan cake;

[0026] Figure 2 The effects of enzymatic hydrolysate of thin-shelled pecan meal on DPPH radical scavenging rate and peptide content; (A) Effect of hydrolysis time on DPPH radical scavenging rate and peptide content; (B) Effect of enzyme dosage on DPPH radical scavenging rate and peptide content; (C) Effect of substrate concentration on DPPH radical scavenging rate and peptide content.

[0027] Figure 3 The results of response surface methodology optimization are as follows: (A) the effect of the interaction of hydrolysis time, (B) substrate concentration, and (C) enzyme dosage on the yield of pecan polypeptide; (D) the effect of the interaction of hydrolysis time, (E) substrate concentration, and (F) enzyme dosage on the DPPH radical scavenging rate of pecan polypeptide.

[0028] Figure 4 The effect of different polypeptide molecular weight distributions on DPPH free radical scavenging rate;

[0029] Figure 5Construction of an antioxidant peptide-target network; (A) Venn diagram of peptides and disease targets; (B) PPI network diagram of intersection targets; (C) Peptide-disease target network for screening key targets;

[0030] Figure 6 Figures for GO enrichment analysis and KEGG signaling pathway enrichment analysis; (A) GO enrichment analysis; the length and color of the bands represent the number of genes involved in their respective biological processes; (B) Bubble diagram of KEGG pathway analysis. Changes in bubble color represent changes in P-value, and bubble size represents the number of genes;

[0031] Figure 7 Molecular models of the binding of peptide LYVPHWN to predicted protein crystals; (A) 3D model and 2D interaction diagram of LYVPHWN docking with MMP9 protein crystal structure; (B) 3D model and 2D interaction diagram of LYVPHWN docking with TNF protein crystal structure; (C) 3D model and 2D interaction diagram of LYVPHWN docking with BCL2 protein crystal structure; (D) 3D model and 2D interaction diagram of LYVPHWN docking with STAT3 protein crystal structure;

[0032] Figure 8 High-performance liquid chromatography (HPLC) chromatogram and mass spectrum of LYVPHWN synthesis; (A) HPLC chromatogram of LYVPHWN; (B) Mass spectrum of LYVPHWN;

[0033] Figure 9 The in vitro DPPH free radical scavenging rate of LYVPHWN;

[0034] Figure 10 The effect of LYVPHWN on Caco-2 cell proliferation;

[0035] Figure 11 The effects of LYVPHWN on intracellular antioxidant markers (A) GSH, (B) MDA, and (C) SOD. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The terminology used in this invention, unless otherwise stated, generally has the meaning commonly understood by those skilled in the art.

[0038] The experimental measurement methods used in the embodiments of the present invention are as follows:

[0039] 1. Free radical scavenging ability of 1,1-Diphenyl-2-picrylhydrazyl (DPPH)

[0040] Dissolve 0.0079 g of DPPH powder (Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30629) thoroughly in anhydrous ethanol, mix well, and bring the volume to 100 mL to prepare DDPH free radical working solution. Store in a brown volumetric flask at room temperature. Take 1 mL of the extracted sample, mix thoroughly with 1 mL of DPPH free radical working solution, and let stand in the dark for 30 min. Use 1 mL of anhydrous ethanol instead of DPPH free radical working solution as a control group, and use 1 mL of anhydrous ethanol instead of the sample solution as a blank group. Measure the absorbance at a wavelength of 517 nm. Each group of samples has 3 replicates. The scavenging rate is calculated using the following formula:

[0041]

[0042] Note: A i A represents the absorbance of the experimental group. j A0 represents the absorbance value of the blank group, and A1 represents the absorbance value of the control group.

[0043] 2. Determination of polypeptide content

[0044] 2.1 Construction of Standard Curve for Polypeptide Content Determination

[0045] The biuret method was used for determination. An appropriate amount of glutathione (Shanghai Maclean Biochemical Technology Co., Ltd., G6268) was prepared into standard solutions of 0 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL using distilled water. 1 mL of each standard solution was taken, and 4 mL of biuret reagent (Biuret reagent A: 0.1 g / mL sodium hydroxide aqueous solution; Biuret reagent B: 0.01 g / mL copper sulfate aqueous solution) (Shanghai Maclean Biochemical Technology Co., Ltd., Biuret reagent A: S861527, Biuret reagent B: C861528) was added. The mixture was thoroughly mixed using a vortex mixer, allowed to stand for 10 min, and centrifuged at 8000×g for 10 min. The absorbance of the supernatant was measured at 540 nm.

[0046] 2.2 Determination of peptide content in samples

[0047] Mix the sample solution with an equal volume of 10% (w / v) trichloroacetic acid (Shanghai Aladdin Biochemical Technology Co., Ltd., T104257), let stand for 30 min, and centrifuge at 6000 ×g for 15 min. Take 1.5 mL of the supernatant, add 1 mL of biuret reagent (0.75 mL NaOH, 0.25 mL CuSO4), mix thoroughly, and let stand for 30 min. Centrifuge at 8000 ×g for 10 min, and measure the absorbance of the supernatant at 540 nm.

[0048] 3. Network pharmacology analysis

[0049] 3.1 Peptide Disease Target Prediction

[0050] The oligopeptide LYVPHWN was input into the SwissADME database (http: / / www.swissadme.ch / index.php) to obtain the SMILES number, and then the SMILES number was imported into the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch / ) for target prediction.

[0051] 3.2 Collection of colorectal cancer-related targets

[0052] By entering the keywords "Oxidative Stress" and "Antioxidant" into the GeneCards database (https: / / www.genecards.org / ), relevant disease targets were collected. Duplicate targets were removed to obtain the final disease targets.

[0053] 3.3 Protein-Protein Interaction Network (PPI)

[0054] The intersection of disease targets and peptide-targets was obtained using Veen database analysis (https: / / bioinfogp.cnb.csic.es / tools / venny / index.html). These intersection targets were then imported into the STRING database (https: / / cn.string-db.org / ) to construct a protein-protein interaction (PPI) network. Finally, CytoScape 3.10.4 software was used to plot the component-target interaction network.

[0055] 3.4 Enrichment Analysis of GO and KEGG

[0056] Core targets were identified using functional annotation and pathway enrichment tools from the David database (https: / / david.ncifcrf.gov), followed by G0 and KEGG pathway enrichment analysis. A p<0.05 threshold was used to screen for potential signaling pathways and anti-colon cancer mechanisms of walnut peptides. Data analysis and visualization were performed using a bioinformatics database (http: / / www.bioinformatics.com.cn).

[0057] 4. Molecular docking

[0058] The two-dimensional (2D) structure of peptides was predicted using the ChemBio3D Ultra 20.0 software package, and then converted into a three-dimensional structure using ChemBio3D20.0. After importing the target peptide structure into the AutoDockTools platform, the system performed the following automated processing: first, intelligent allocation of charge distribution and atom types; then, defining all rotatable chemical bonds within the molecule as flexible states; and finally, outputting a PDBQT format ligand file that meets the requirements for molecular docking. The structures of MMP9 (PDBID: 20VX), TNF (PDB ID: 2AZ5), BCL2 (PDB ID: 4MAN), and STAT3 (PDB ID: 6HJS) were downloaded from the PDB database. The receptor proteins were preprocessed using the AutoDockTools software package, removing water of crystallization molecules and small molecule ligands, adding hydrogen atoms, performing charge allocation and atom type definition, and finally saving the data as a PDBQT format receptor model for molecular docking. The DeepPocket software was used to predict the active pockets of the receptor proteins. Autodock Vina 1.2.7 was used to simulate the interaction between the peptide and the receptor protein, and the output posture was evaluated using the Gird scoring function. The lowest score was selected as the predicted interaction mode. The binding site and interaction were visualized using PyMol and Discovery Studio 2025 software.

[0059] 5. Caco-2 cell culture

[0060] 5.1 Cell resuscitation

[0061] Remove the frozen cells from the -80℃ freezer and place them in a 37℃ water bath. Shake the cryovials to thaw the cryopreservation solution. Prepare cell culture medium, preheat to 37℃, with a culture system of MEM medium: fetal bovine serum: penicillin-streptomycin solution (double antibiotic) = 100:20:1 (v / v / v). Centrifuge at 4℃, 888 ×g for 5 min, remove the cryopreservation solution, resuspend the cells in 1 mL of cell culture medium, transfer to a T25 culture flask, and add 3 mL of culture medium. Incubate at 37℃ in a 5% CO2 incubator, changing the cell culture medium every 48 h.

[0062] 5.2 Cell digestion and passage

[0063] When cell confluence reaches 80-90%, remove the culture medium from the T25 culture flask, add trypsin for rinsing, add 1.5 mL of 0.25% trypsin-EDTA solution, let stand for about 10 min, and when cell detachment is observed, add 3 mL of cell culture medium to stop digestion, gently pipette, and collect the cells. Centrifuge at 888 ×g for 5 min at 4℃, remove the supernatant, and resuspend in 2 mL of cell culture medium. Passage at a 1:2 (v / v) ratio, transfer the cells to a new T25 culture flask, add 4 mL of cell culture medium, and incubate in an incubator, changing the culture medium every 48 h.

[0064] 6. MTT Experiment

[0065] The final concentration is 1×10 4 100 μL / well of Caco-2 cells / mL were added to each well of a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 h. Then, 10 μL of the aforementioned peptide solutions of different concentrations were added, and the cells were cultured for another 2 h. 50 μL of 1×MTT solution (Nanjing Jiancheng Bioengineering Institute, G020-1-1) was added to each well, and the cells were incubated for another 4 h. After removing the cell culture medium, 150 µL of DMSO was added to each well and the cells were shaken at 37°C in the dark for 10 min to dissolve the formazan. The absorbance was measured at 570 nm. Wells without samples served as the control group. Cell viability was calculated using the following formula. To induce oxidative stress in Caco-2 cells, after culturing for 24 h, culture medium containing different concentrations (20, 50, 100, 200, 400, 600 μM) of H2O2 was added to each well, and the cells were cultured for another 2 h. Cell viability was also measured using the MTT assay.

[0066]

[0067] 7. Different groups

[0068] Caco-2 cells cultured in complete culture medium were then subjected to a 1×10⁻⁶ induction reaction. 5 Caco-2 cells were seeded at a density of 10 cells / mL in 6-well plates (2 mL per well) and cultured for 24 h. To establish an oxidative stress model, Caco-2 cells were treated with 200 μM H2O2 for 2 h; after treatment, the medium was replaced with fresh medium, and the cells were cultured for another 24 h to obtain the model group. In the peptide intervention group, Caco-2 cells were seeded at a density of 1 × 10⁻⁶ cells / mL. 5Caco-2 cells were seeded at a density of 10 cells / mL in 6-well plates (2 mL per well) and cultured for 24 h. To establish an oxidative stress model, Caco-2 cells were treated with 200 μM H2O2 for 2 h, and then cultured in fresh medium containing 400 μg / mL peptide to obtain the peptide intervention group. The control group was treated with PBS instead of H2O2 and peptide. Cell viability was measured using the MTT assay. After culture, cells were collected and lysed, and intracellular GSH (Nanjing Jiancheng Bioengineering Institute, A006-2-1), MDA (Beijing Solarbio Science & Technology Co., Ltd., BC0025), and SOD (Nanjing Jiancheng Bioengineering Institute, A001-1-1) activities were measured using a kit.

[0069] Example 1: Preparation of thin-shelled pecan oligopeptides

[0070] (1) Effects of different proteases on DPPH free radical scavenging rate and peptide content

[0071] Using thin-shelled pecan meal (provided by Jiangsu Academy of Agricultural Sciences) as the substrate, enzymatic hydrolysis was carried out at an optimal pH and temperature for different proteases (all purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog numbers shown in Table 1) at a substrate concentration of 15% and an enzyme addition of 8000 u / g. The reaction lasted for 3 h, and the specific reaction parameters are shown in Table 1. After the enzymatic hydrolysis process, the enzymatic reaction was terminated by heating in a 95℃ water bath for 10 min. The reaction solution was centrifuged at 8000×g for 20 min at 4℃ to obtain the supernatant, which was then clarified and filtered through a 0.22 μm microporous membrane. The resulting filtrate was freeze-dried and stored at −20℃. Figure 1 It can be seen that the DPPH radical scavenging rate and polypeptide content in the enzymatic hydrolysis products differed significantly after enzymatic hydrolysis by different proteases. Among them, the sample hydrolyzed by the compound protease had the highest DPPH radical scavenging rate and polypeptide content, which were (65.9±1.308)% and (90.77±2.392) mg / g, respectively, which were significantly higher than those of other proteases.

[0072] Table 1 Optimal Enzymatic Hydrolysis Conditions for Proteases

[0073]

[0074] (2) Effects of different enzymatic hydrolysis times on DPPH free radical scavenging rate and peptide content

[0075] Following the method in (1), a complex protease hydrolysis fermentation broth was selected. The amount of complex protease added, temperature (50℃), pH (7.5), and substrate concentration (4%) were fixed. The effects of different hydrolysis times (1, 2, 3, 4, and 5 h) on DPPH free radical scavenging rate and peptide content were investigated. Figure 2 As shown in A, with the extension of enzymatic hydrolysis time, the DPPH free radical scavenging rate and peptide content show a trend of increasing and then decreasing, reaching their highest values ​​after 4 hours of enzymatic hydrolysis. Therefore, the optimal enzymatic hydrolysis time for preparing antioxidant peptides from thin-shelled pecans using the complex protease is 4 hours.

[0076] (3) Effects of different enzyme addition amounts on DPPH free radical scavenging rate and peptide content

[0077] Following the method described in (1), with a fixed temperature of 50℃, pH 7.5, time of 4 h, and substrate concentration of 4%, the effects of different enzyme addition amounts (2000 u / g, 4000 u / g, 6000 u / g, 8000 u / g, 10000 u / g) on ​​DPPH free radical scavenging rate and peptide content were investigated. Figure 2 As shown in section B, with the increase of enzyme quantity, the probability of its contact with the substrate increases, and the DPPH free radical scavenging rate and peptide content increase accordingly. When the enzyme addition amount is greater than 8000 u / g, the DPPH free radical scavenging rate and peptide content decrease with the increase of enzyme addition amount. Therefore, the optimal enzyme addition amount for preparing thin-shelled pecan antioxidant peptides by compound protease is 8000 u / g.

[0078] (4) Effects of different substrate concentrations on DPPH radical scavenging rate and peptide content

[0079] Following the method described in (1), with fixed enzymatic hydrolysis time, enzyme dosage, temperature 50℃, and pH 7.5, the effects of different substrate concentrations (1%, 5%, 10%, 15%, 20%) on DPPH free radical scavenging rate and peptide content were investigated. Figure 2 As shown in C, with increasing substrate concentration, the DPPH free radical scavenging rate and peptide content rapidly increase within the range of 1%-15%. However, when the substrate concentration reaches 20%, the scavenging rate and peptide content begin to decrease. Therefore, the optimal substrate concentration for preparing thin-shelled pecan antioxidant peptides using complex proteases is 15%.

[0080] (5) Response surface optimization

[0081] The enzymatic hydrolysis protease was fixed as a complex protease, the hydrolysis pH was 7.5, and the temperature was 50℃. The experiment used hydrolysis time, enzyme dosage, and substrate concentration as independent variables, with Y1 representing peptide yield and Y2 representing DPPH radical scavenging rate. The response surface methodology optimization experimental design and results are shown in Table 2. DesignExpert 13 software was used for experimental design and result analysis in exploring the optimization of peptide content and DPPH radical scavenging rate during the enzymatic hydrolysis of thin-shelled pecan meal. Through in-depth statistical regression analysis of the experimental data, quadratic regression models describing the relationship between peptide content (Y1), DPPH radical scavenging rate (Y2), and different process parameters were successfully constructed. The quadratic regression equations are as follows:

[0082] Y1=95.95-0.1154A+5.92B-3.10C+2.27AB-0.8164AC+7.54BC-7.30A2-10.68B2-11.45C2

[0083] Y2=66.52+0.1875A+2.70B-1.74C+1.50AB-0.1750AC+3.10BC-3.57A2-4.85B2-5.12C2

[0084] Table 3 shows that the quadratic polynomial fitting model for peptide yield has an F=8.27 and a p=0.0055, indicating that the regression model is significant. The lack-of-fit test p=0.5852>0.05 indicates that the lack-of-fit term is not significant, suggesting high model reliability and strong experimental method dependability. The correlation coefficient R of the peptide content regression model is also shown. 2 =0.9140, greater than 0.9. As shown in Table 4, the quadratic polynomial fitting model of DPPH free radical scavenging rate has F=6.59 and p=0.0106, indicating that this regression model is significant; the lack-of-fit test p=0.6695>0.05, the lack-of-fit term is not significant, indicating that the model has high credibility and this experimental method is highly reliable.

[0085] Table 2 Response Surface Optimization Experimental Design and Results

[0086]

[0087] Table 3. Significance test and analysis of variance for the peptide content regression model.

[0088]

[0089] Table 4. Significance test and analysis of variance of the regression model for DPPH free radical scavenging rate

[0090]

[0091] Note: * indicates a significant difference (P<0.05); ** indicates an extremely significant difference (P<0.01).

[0092] like Figure 3 The optimal process conditions for producing functional peptides from thin-shelled pecan meal via enzymatic hydrolysis, as determined by Design-Expert 13 software analysis and optimization, are: hydrolysis time 4.68 h, enzyme dosage 7000 U / g, and substrate concentration 11.35% (m / v). Under these conditions, the predicted peptide yield is 96.801 ± 0.934 mg / g, and the DPPH radical scavenging rate is 66.944%. To verify the feasibility of the model prediction, a validation experiment was conducted using the determined optimal process parameters. Three repeated experiments yielded an average peptide yield of 96.25 ± 1.54 mg / g and a DPPH radical scavenging rate of 66.1%, with an error within 3%. The experimental and theoretical values ​​show good agreement.

[0093] Example 2: Isolation and Identification of Oligopeptides from Thin-Shelled Pecans

[0094] (1) Ultrafiltration separation

[0095] To facilitate experimental procedures, the optimized conditions were rounded down. Thin-shelled pecan meal with a substrate concentration of 11% was hydrolyzed with 7000 u / g of complex protease for 4.5 h to obtain a hydrolysate. An appropriate amount of the hydrolysate was then sequentially separated using ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 5 kDa, and 3 kDa to obtain four polypeptide fractions with different relative molecular mass ranges (greater than 10 kDa, 10-5 kDa, 5-3 kDa, and less than 3 kDa).

[0096] (2) Effect of different polypeptide molecular weights on DPPH free radical scavenging rate

[0097] The DPPH free radical scavenging capacity of the four obtained polypeptide fractions (greater than 10 kDa, 10⁻⁵ kDa, 5⁻³ kDa, and less than 3 kDa) was determined. Figure 4 It was found that, compared with other fractions with molecular weights exceeding 3000 Da, fractions with molecular weights below 3000 Da exhibited significantly higher DPPH radical scavenging rates (p<0.05). Therefore, fractions with molecular weights <3 kDa were selected for LC-MS / MS mass spectrometry analysis.

[0098] Example 3: Network pharmacological analysis of antioxidant peptides from thin-shelled pecans

[0099] (1) PPI network analysis

[0100] LC-MS / MS mass spectrometry analysis, combined with PeptideRanker scoring (partial results are shown in Table 5), selected the peptide LYVPHWN with the highest score. Further investigation into its antioxidant activity and mechanism was conducted, including network pharmacology analysis.

[0101] The Homo sapiensi target of LYVPHWN was predicted using SwissTargetPrediction (https: / / swisstargetprediction.ch / ) and PharmMapper (https: / / www.lilab-ecust.cn / pharmmapper / index.html), respectively. After removing duplicates, 369 targets were obtained. Simultaneously, using the median method, 1509 antioxidant-related targets were screened in the GeneCards database using "antioxidant" and "oxidative stress" as keywords, after removing duplicate targets. Interaction analysis between oxidative targets and peptide targets identified 133 overlapping targets. Figure 5 (A) Further analysis of the interactions of 133 intersecting target points was conducted using the STRING database, and a PPI network was constructed (…). Figure 5 B in the dataset contains 1905 edges, with an enrichment P-value < 1.0e-16. Furthermore, Cytoscape analysis was used to analyze 133 intersecting target points. Based on the criteria of a betweenness value greater than 124.98, a closeness value greater than 0.0040, and a degree value greater than 28.86, 29 core target points were identified. Figure 5 (C in the text). Among them, the degree values ​​of MMP9, TNF, BCL2, and STAT3 are greater than 28, which means that these target proteins play a relatively more important role in the network.

[0102] Table 5. PeptideRanker scores for partial peptide sequences

[0103]

[0104] (2) Bioinformatics analysis

[0105] To systematically elucidate the pharmacological mechanisms of peptide therapy for oxidative stress, G0 biological functions and KEGG pathway enrichment analyses were performed on core targets using the DAVID database and a microbiology platform. A total of 672 G0 items with P < 0.05 were obtained, including 505 biological processes (BP), 54 cellular components (CC), and 113 molecular functions (MF). Figure 6The "A" column lists the top 10 most significantly enriched G0 items for each category. In terms of biological processes, the targets primarily involve protein degradation, proteasome-mediated ubiquitin-dependent protein degradation, positive regulation of cell apoptosis, positive regulation of cell proliferation, and apoptosis. From a cellular component perspective, the targets are mainly related to the nucleus, cytoplasm, and plasma membrane. Analysis of molecular functions indicates that these targets are primarily involved in protein binding, identical protein binding, and endonuclease activity. Further analysis of the enriched KEGG pathway was conducted to identify potential signaling pathways in which these targets may participate. Figure 6 The KEGG pathway bubble diagram in B shows 20 enriched pathways based on core targets.

[0106] Example 4 Molecular docking verification

[0107] To verify the interaction between the peptide and the first four core targets (MMP9, TNF, BCL2, and STAT3), molecular docking was performed. A binding energy < -5 indicates stable binding between the ligand and receptor, while a binding energy < -7 indicates even more stable binding. Lower binding energies indicate higher affinity between the receptor and ligand, and a greater probability of interaction. As shown in Table 6, the peptide LYVPHWN exhibits very low binding energies with receptors MMP9, TNF, BCL2, and STAT3, with affinity all less than -7 kcal / mol, indicating that LYVPHWN has good binding affinity and stable interaction with these receptors.

[0108] To further elucidate the precise binding sites and interactions between the peptide and receptor protein crystals, docking analysis and visualization were performed using PyMOL and Discovery Studio 2025. Figure 7 ).

[0109] Table 6. Docking analysis between LYVPHWN and key targets

[0110]

[0111] Example 5: Synthesis of Peptides and In Vitro Antioxidant Validation

[0112] (1) Synthesizing polypeptides

[0113] To verify the antioxidant activity of the thin-shelled pecan polypeptide LYVPHWN, it was synthesized in a solid phase by Shanghai Aminolink Biotechnology Co., Ltd. The liquid chromatography and mass spectra are shown below. Figure 8 As shown, the polypeptide has a molecular weight of 928.04 and a purity of 94.60%.

[0114] (2) In vitro antioxidant activity experiment of LYVPHWN

[0115] The DPPH free radical scavenging rate of oligopeptide LYVPHWN (0, 2, 4, 6, 8, 10 mg / mL) at different concentrations was determined using the aforementioned method for determining free radical scavenging capacity.

[0116] The results are as follows Figure 9 As shown, the DPPH radical scavenging rate of LYVPHWN increases with increasing peptide concentration. The half-inhibitory concentration (IC50) was calculated using Graphpad software. 50 The concentration was 6.351 mg / mL.

[0117] (3) LYVPHWN toxicity test

[0118] Following the aforementioned MTT assay method, Caco-2 cells were treated with different concentrations (50, 100, 200, 400, 800, 1600 μg / mL) of the oligopeptide LYVPHWN for 24 hours, and their survival rate was measured. Figure 10 As shown, in the low concentration range (50-200 μg / mL), cell viability was close to 100%, indicating that the oligopeptide LYVPHWN has good biocompatibility and did not exhibit significant cytotoxicity. The cell viability at 1600 μg / mL was greater than 50%, indicating that the IC50 of LYVPHWN is high. 50 A value greater than 1600 μg / mL indicates a low-toxicity or non-toxic polypeptide.

[0119] (4) LYVPHWN's regulation of the antioxidant defense system in Caco-2 cells

[0120] Referring to the aforementioned groupings, including the peptide intervention group, normal control group, and model group, the results are as follows: Figure 11 As shown.

[0121] Figure 11 As shown in Figure A, compared with the normal control group (62.61±2.44 μmol / gprot), the intracellular GSH content in the model group was significantly reduced to 13.62±1.73 μmol / gprot, indicating that H2O2 treatment led to a significant depletion of intracellular reducing reserves. After peptide intervention, LYVPHWN significantly increased the intracellular GSH level, restoring it to 31.61±4.37 μmol / gprot, suggesting that LYVPHWN can effectively alleviate oxidative stress-induced GSH depletion.

[0122] Figure 11As shown in Figure B, the MDA level in the Control group remained at a low level, while it significantly increased to 13.76 ± 0.72 nmol / mgprot in the Model group, indicating that oxidative stimulation triggered a significant lipid peroxidation response. Compared with the Model group, after LYVPHWN treatment, the intracellular MDA content significantly decreased to 9.42 ± 1.20 nmol / mgprot (p < 0.05), indicating that the peptide has a significant protective effect in inhibiting lipid peroxidation and reducing cell membrane structural damage.

[0123] Figure 11 As shown in C, under the condition of 200 μg / mL, the SOD activity of the LYVPHWN group increased to 104.02±11.22 U / mgprot, indicating that the peptide can enhance the functional level of the antioxidant enzyme system in damaged Caco-2 cells.

Claims

1. An oligopeptide, characterized in that, The amino acid sequence of the oligopeptide is LYVPHWN.

2. The method for preparing the oligopeptide according to claim 1, characterized in that, The preparation method includes enzymatic hydrolysis of thin-shelled pecan cake meal using a complex protease, followed by dilution and ultrafiltration.

3. The method for preparing oligopeptides according to claim 2, characterized in that, The enzymatic hydrolysis conditions are as follows: hydrolysis time 1-5 h, concentration of thin-shelled pecan cake meal 1-20% (m / v), and addition amount of compound protease 2000-10000 u / g.

4. A product containing the oligopeptide of claim 1, characterized in that, The products mentioned are food, health products, or medicines.

5. The product according to claim 4, characterized in that, The food products mentioned are general food products, functional food products, or food products for special medical purposes.

6. The product according to any one of claims 4 to 5, characterized in that, The oligopeptides are used alone or as additives in general foods, health products, functional foods or foods for special medical purposes.

7. The use of the oligopeptide of claim 1 or the product of any one of claims 4 to 5 in the preparation of antioxidant products.

8. The application according to claim 5, characterized in that, The amount of oligopeptide added was 6.351 mg / mL.

9. The application according to claim 7, characterized in that, The oligopeptides are used in the preparation of products that increase GSH levels and / or decrease intracellular MDA content and / or increase SOD activity levels.

10. The application according to claim 7, characterized in that, The oligopeptides are used in the preparation of products that inhibit lipid peroxidation and / or reduce and protect against damage to cell membrane structures.

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