Antioxidant polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and particularly relates to an antioxidant polypeptide as well as a preparation method and application thereof. The antioxidant polypeptide provided by the invention contains at least one of LMGPR, GFDGD, FGFDGDF and GHNGLDGL. The antioxidant polypeptide has better oxidation resistance, can relieve oxidative damage and inflammation, plays a role in protecting oxidative stress cells, is non-toxic to cells, and can be used as an active component of an antioxidant product. The four peptide fragments also have a synergistic effect in the anti-oxidation aspect, and an excellent anti-oxidation effect can be generated by compounding use of the four peptide fragments.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and in particular relates to an antioxidant polypeptide and a preparation method and application thereof. Background Art

[0002] Excessive free radicals produced during oxidation pose a major threat to human health. Excessive free radicals can cause oxidative stress reactions, which in turn damage the body's health. The use of antioxidant active substances can reduce the level of free radicals in the body, reduce oxidative damage, and help delay aging and prevent related diseases. Some synthetic antioxidants such as butylated hydroxytoluene, tert-butylhydroquinone and butylated hydroxyisoalcohol can reduce the level of free radicals in food, but they are potentially toxic to human health, which limits their application in food. The development of safe and harmless antioxidants has attracted much attention.

[0003] Bioactive peptides have gradually become important ingredients in food and biomedicine due to their high safety and different biological activities. Among them, antioxidant peptides have the function of scavenging free radicals and have broad development prospects in many fields such as food, medicine, and cosmetics. Therefore, obtaining antioxidant peptides with antioxidant activity through natural screening or artificial synthesis can provide more choices for the active ingredients of antioxidant products, which has broad significance for the development and application of peptide products and antioxidant products. Summary of the invention

[0004] In view of the above technical problems, the present invention provides an antioxidant polypeptide and a preparation method and application thereof. The antioxidant polypeptide provided by the present invention has high antioxidant activity, has a protective effect on oxidative stress cells, is safe and non-toxic, and can be used to prepare antioxidant products.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: The first aspect of the present invention provides an antioxidant polypeptide, which contains at least one of LMGPR, GFDGD, FGFDGDF and GHNGLDGL; wherein the amino acid sequence of the LMGPR is: Leu-Met-Gly-Pro-Arg (i.e., leucine-methionine-glycine-proline-arginine, the sequence is shown in SEQ ID No.1); the amino acid sequence of the GFDGD is: Gly-Phe-Asp-Gly-Asp (i.e., glycine-phenylalanine-aspartic acid-glycine-aspartic acid, the sequence is shown in SEQ ID No.2); the amino acid sequence of the FGFDGDF is: Phe-Gly-Phe-Asp-Gly-Asp-Phe (i.e., phenylalanine-glycine-phenylalanine-aspartic acid-glycine-aspartic acid-phenylalanine, the sequence is shown in SEQ ID The amino acid sequence of GHNGLDGL is: Gly-His-Asn-Gly-Leu-Asp-Gly-Leu (i.e., glycine-histidine-asparagine-glycine-leucine-aspartic acid-glycine-leucine, the sequence is shown in SEQ ID No.4).

[0006] The results of molecular docking studies showed that the above four peptides can block the interaction of Keap-Nrf2 and promote the accumulation of Nrf2, which may cause the expression of antioxidant enzyme genes. The scavenging rate analysis of DPPH free radicals, ABTS free radicals, and hydroxyl free radicals showed that the above four peptides had good antioxidant properties. The results of cytotoxicity experiments showed that the above four peptides had no toxic effect on HepG2 cells after being treated with HepG2 cells in the concentration range of 0.1-2.5 mg / mL. In addition, the cell oxidative damage experiment proved that the above four peptides could reduce oxidative damage and inflammation, and had a certain protective effect on oxidative stress cells.

[0007] The molecular weights of the above four peptides are all less than 1 kDa, making them easier to be absorbed and utilized by the body to exert their effects.

[0008] Preferably, the total content of LMGPR, GFDGD, FGFDGDF and GHNGLDGL in the antioxidant polypeptide is not less than 90%.

[0009] Preferably, the antioxidant polypeptide contains at least one of LMGPR, GFDGD and FGFDGDF. FGFDGDF has better DPPH free radical scavenging activity than LMGPR, FGFDGDF has better hydroxyl free radical activity than GFDGD, and FGFDGDF has better ABTS free radical scavenging activity than LMGPR.

[0010] Preferably, the antioxidant polypeptide contains two, three or four of LMGPR, GFDGD, FGFDGDF and GHNGLDGL.

[0011] After the above four peptides were compounded in pairs, the DPPH free radical scavenging activity and ABTS free radical scavenging activity were significantly better than those of a single peptide at the same concentration.

[0012] When any three of the above four peptides are mixed, the DPPH free radical scavenging activity and ABTS free radical scavenging activity are also significantly better than that of a single peptide at the same concentration, and the hydroxyl free radical activity is better than that of a combination of two peptides at the same concentration.

[0013] After the above four peptides are compounded, that is, the antioxidant polypeptide contains LMGPR, GFDGD, FGFDGDF and GHNGLDGL, and the DPPH free radical scavenging activity, ABTS free radical scavenging activity and hydroxyl free radical activity are significantly better than those of a single peptide at the same concentration, and better than the free radical scavenging effect of a compound of two or three peptides at the same concentration.

[0014] More preferably, the peptide segments are compounded in equal mass ratios.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned antioxidant polypeptide, which specifically comprises the following steps: S1. According to the amino acid sequence of the peptide segment, a crude peptide is synthesized using an automatic peptide synthesizer; S2. The crude polypeptide obtained in S1 was dissolved in a 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse-phase high performance liquid chromatography to obtain a purified polypeptide.

[0016] Preferably, when preparing the LMGPR, the chromatographic conditions of the preparative reverse-phase high performance liquid chromatography described in S2 include: mobile phase A is 0.09%~0.11% formic acid aqueous solution, and mobile phase B is 0.09%~0.11% formic acid acetonitrile solution; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, mobile phase B is linearly eluted from 8%→33%, and the eluate is collected at 13~14min.

[0017] Further preferably, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution.

[0018] Preferably, when preparing the GFDGD, the chromatographic conditions of the preparative reverse-phase high performance liquid chromatography in S2 include: mobile phase A is 0.09%~0.11% formic acid aqueous solution, and mobile phase B is 0.09%~0.11% formic acid acetonitrile solution; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, the mobile phase B is linearly eluted from 10%→35%, and the eluent is collected at 12~13.5 min.

[0019] Further preferably, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution.

[0020] Preferably, when preparing the FGFDGDF, the chromatographic conditions of the preparative reverse-phase high performance liquid chromatography described in S2 include: mobile phase A is a 0.09%~0.11% formic acid aqueous solution, mobile phase B is an acetonitrile aqueous solution containing 0.09%~0.11% formic acid, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 83%~85%; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, the mobile phase B is linearly eluted from 24%→49%, and the eluate is collected at 13~14min.

[0021] Further preferably, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is an acetonitrile aqueous solution containing 0.1% formic acid, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%.

[0022] Preferably, the mobile phase A for preparing the GHNGLDGL is 0.09% to 0.11% formic acid aqueous solution, the mobile phase B is 0.09% to 0.11% formic acid in acetonitrile aqueous solution, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 83% to 85%; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, the mobile phase B is linearly eluted from 17% to 42%, and the eluate is collected at 10.5~11.5min.

[0023] Further preferably, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is an acetonitrile aqueous solution containing 0.1% formic acid, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%.

[0024] The third aspect of the present invention provides the use of the above antioxidant polypeptide in the preparation of antioxidant products, the active ingredients of which include the above antioxidant polypeptide.

[0025] Preferably, the antioxidant products include food additives, antioxidant drugs and antioxidant cosmetics.

[0026] The beneficial effects of the present invention are: 1. The present invention predicts the binding sites and docking energies of the above four peptides with the receptor protein Keap1 through molecular docking simulation technology. The lower the docking energy, the better the binding. The results show that the docking energies of LMGPR, GFDGD, FGFDGDF, GHNGLDGL with Keap1 are -68.8519, -115.582, -141.445, and -136.723 kcal / mol, respectively, proving that the above peptides have good antioxidant effects.

[0027] 2. The present invention evaluated the in vitro antioxidant effect of the above four peptides by scavenging DPPH free radicals, ABTS free radicals and hydroxyl free radicals, and the results showed that the four peptides in the antioxidant polypeptide had significant antioxidant effects. At the same time, the above four peptides had a significant protective effect on oxidative damage of HepG2 cells.

[0028] 3. The antioxidant polypeptide provided by the present invention provides a new option for developing safe new antioxidant polypeptide products, and has broad application prospects in medicines, cosmetics, health foods and food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the LC-MS / MS mass spectrum and purity detection diagram of the antioxidant polypeptide LMGPR synthesized by solid phase method in Example 1 of the present invention; Figure 2 It is the LC-MS / MS mass spectrum and purity detection diagram of the antioxidant polypeptide GFDGD synthesized by solid phase method in Example 2 of the present invention; Figure 3 It is the LC-MS / MS mass spectrum and purity detection diagram of the antioxidant polypeptide FGFDGDF synthesized by solid phase method in Example 3 of the present invention; Figure 4 It is the LC-MS / MS mass spectrum and purity detection diagram of the antioxidant polypeptide GHNGLDGL synthesized by solid phase method in Example 4 of the present invention; Figure 5 The molecular docking results of LMGPR and Keap1 protein in Test Example 2 of the present invention; Figure 6 The molecular docking results of GFDGD and Keap1 protein in Test Example 2 of the present invention; Figure 7 The molecular docking results of FGFDGDF and Keap1 protein in Test Example 2 of the present invention; Figure 8 The molecular docking results of GHNGLDGL and Keap1 protein in Test Example 2 of the present invention; Fig. 9The antioxidant activity of the antioxidant polypeptides of Examples 1 to 4 in Test Example 3 of the present invention; Fig.10 The antioxidant activity of the antioxidant polypeptides of Examples 5 to 10 in Test Example 4 of the present invention; Fig.11 The antioxidant activity of the antioxidant polypeptides of Examples 11 to 15 in Test Example 4 of the present invention; Fig.12 The effect of different concentrations of H2O2 on the viability of HepG2 cells in Test Example 5 of the present invention; Fig.13 The effects of LMGPR, GFDGD, FGFDGDF, and GHNGLDGL on the viability of HepG2 cells in Test Example 5 of the present invention; Fig.14 The effects of LMGPR, GFDGD, FGFDGDF, and GHNGLDGL on the survival rate of HepG2 cells induced by H2O2. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0031] Excessive free radicals produced during oxidation can cause oxidative stress, which in turn damages the body's health. Currently, antioxidants are mainly chemically synthesized products with potential toxicity.

[0032] The embodiment of the present invention provides an antioxidant polypeptide, which contains at least one of LMGPR, GFDGD, FGFDGDF and GHNGLDGL. The above four peptide segments have good antioxidant properties, can reduce oxidative damage and inflammation, have a certain protective effect on oxidative stress cells, and are non-toxic to cells.

[0033] The present invention also provides a method for preparing an antioxidant polypeptide, which specifically comprises the following steps: S1. According to the amino acid sequence of the peptide segment, a crude peptide is synthesized using an automatic peptide synthesizer; S2. The crude polypeptide obtained in S1 was dissolved in a 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse-phase high performance liquid chromatography to obtain a purified polypeptide.

[0034] The embodiment of the present invention also provides the use of the antioxidant polypeptide in the preparation of antioxidant products.

[0035] The scheme of the present invention is described below through specific embodiments.

[0036] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercial products obtained from commercial channels. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0037] Example 1 This embodiment provides an antioxidant polypeptide LMGPR and a preparation method thereof.

[0038] (1) Preparation of crude polypeptide: According to the amino acid sequence of the peptide: leucine-methionine-glycine-proline-arginine, the crude peptide was synthesized by conventional solid phase synthesis method; (2) Separation and purification of target peptides: The crude polypeptide obtained in step (1) was dissolved in 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse phase high performance liquid chromatography. The chromatographic conditions were as follows: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The liquid chromatography column was RP-C18 (4.6 mm×250 mm, Column Technology luc.). The liquid phase gradient elution was set as follows: 0→25 min, liquid B was linearly eluted from 8%→33%. The eluate was collected at 13~14 min.

[0039] The peptide sequence was identified by LC-MS / MS technology. The liquid chromatography conditions were consistent with the above separation and purification conditions. Then, a Q Exactive HF-X mass spectrometer (Thermo Fisher) was used for 60 min of positive ion mass spectrometry analysis. The mass-to-charge ratio of the peptide and its fragments was calculated by collecting 10 fragment spectra after each full scan. The raw file of mass spectrometry was retrieved from the corresponding database using the software MaxQuant 1.5.5.1. After calculation and analysis, the peptide sequence in the purified polypeptide was LMGPR, with a molecular mass of 572.72 and a purity of 98.47%. Its mass spectrum and liquid phase separation diagram are shown in the figure. Figure 1 shown.

[0040] Example 2 This embodiment provides an antioxidant polypeptide GFDGD and a preparation method thereof.

[0041] (1) Preparation of crude polypeptide: According to the amino acid sequence of the peptide: glycine-phenylalanine-aspartic acid-glycine-aspartic acid, a crude polypeptide was synthesized by conventional solid phase synthesis; (2) Separation and purification of target peptides: The crude polypeptide obtained in step (1) was dissolved in 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse phase high performance liquid chromatography. The chromatographic conditions were as follows: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The liquid chromatography column was RP-C18 (4.6 mm×250 mm, Column Technology luc.). The liquid phase gradient elution was set as follows: 0→25 min, liquid B was linearly eluted from 10%→35%, and the eluate was collected at 12~13.5 min.

[0042] The peptide sequence was identified by LC-MS / MS technology, wherein the liquid chromatography conditions were consistent with the above separation and purification conditions, and the mass spectrometry conditions were the same as those in Example 1. The peptide sequence in the purified polypeptide was obtained by calculation and analysis to be GFDGD, with a molecular mass of 509.46 and a purity of 92.12%. The mass spectrum and liquid phase separation diagram are shown in FIG. Figure 2 shown.

[0043] Example 3 This embodiment provides an antioxidant polypeptide FGFDGDF and a preparation method thereof.

[0044] (1) Preparation of crude polypeptide: According to the amino acid sequence of the peptide: phenylalanine-glycine-phenylalanine-aspartic acid-glycine-aspartic acid-phenylalanine, a crude polypeptide was synthesized by conventional solid phase synthesis; (2) Separation and purification of target peptides: The crude polypeptide obtained in step (1) was dissolved in 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse phase high performance liquid chromatography. The chromatographic conditions were as follows: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile accounted for 84%). The liquid chromatography column was RP-C18 (4.6 mm× 250 mm, Column Technology luc.). The liquid phase gradient elution was set as follows: 0→25 min, liquid B was linearly eluted from 24%→49%, and the eluate was collected at 13~14 min.

[0045] The peptide sequence was identified by LC-MS / MS technology, wherein the liquid chromatography conditions were consistent with the above separation and purification conditions, and the mass spectrometry conditions were the same as those in Example 1. The peptide sequence in the purified polypeptide was obtained by calculation and analysis to be FGFDGDF, with a molecular mass of 803.81 and a purity of 95.01%. The mass spectrum and liquid phase separation diagram are shown in FIG. Figure 3 shown.

[0046] Example 4 This embodiment provides an antioxidant polypeptide GHNGLDGL and a preparation method thereof.

[0047] (1) Preparation of crude polypeptide: According to the amino acid sequence of the peptide: glycine-histidine-asparagine-glycine-leucine-aspartic acid-glycine-leucine, a crude polypeptide was synthesized by conventional solid phase synthesis; (2) Separation and purification of target peptides: The crude polypeptide obtained in step (1) was dissolved in 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse phase high performance liquid chromatography. The chromatographic conditions were as follows: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile accounted for 84%). The liquid chromatography column was RP-C18 (4.6 mm×250 mm, Column Technology luc.). The liquid phase gradient elution was set as follows: 0→25 min, liquid B was linearly eluted from 17%→42%, and the eluate was collected at 10.5~11.5 min.

[0048] The peptide sequence was identified by LC-MS / MS technology, wherein the liquid chromatography conditions were consistent with the above separation and purification conditions, and the mass spectrometry conditions were the same as those in Example 1. The peptide sequence in the purified polypeptide was obtained by calculation and analysis to be GHNGLDGL, with a molecular mass of 781.81 and a purity of 96.37%. The mass spectrum and liquid phase separation diagram are shown in FIG. Figure 4 shown.

[0049] Example 5 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1 and the GFDGD of Example 2 in an equal mass ratio.

[0050] Example 6 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1 and the FGFDGDF of Example 3 in an equal mass ratio.

[0051] Example 7 This example provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1 and the GHNGLDGL of Example 4 in an equal mass ratio.

[0052] Example 8 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the GFDGD of Example 2 and the FGFDGDF of Example 3 in an equal mass ratio.

[0053] Example 9 This example provides an antioxidant polypeptide, which is prepared by compounding the GFDGD of Example 2 and the GHNGLDGL of Example 4 in an equal mass ratio.

[0054] Example 10 This example provides an antioxidant polypeptide, which is prepared by compounding the FGFDGDF of Example 3 and the GHNGLDGL of Example 4 in an equal mass ratio.

[0055] Embodiment 11 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1, the GFDGD of Example 2, and the FGFDGDF of Example 3 in an equal mass ratio.

[0056] Example 12 This example provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1, the GFDGD of Example 2, and the GHNGLDGL of Example 4 in an equal mass ratio.

[0057] Embodiment 13 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1, the FGFDGDF of Example 3 and the GHNGLDGL of Example 4 in an equal mass ratio.

[0058] Embodiment 14 This example provides an antioxidant polypeptide, which is prepared by compounding the GFDGD of Example 2, the FGFDGDF of Example 3, and the GHNGLDGL of Example 4 in an equal mass ratio.

[0059] Embodiment 15 This embodiment provides an antioxidant polypeptide, which is prepared by compounding the LMGPR of Example 1, the GFDGD of Example 2, the FGFDGDF of Example 3 and the GHNGLDGL of Example 4 in equal mass ratios.

[0060] Test Example 1 In this test example, the toxicity of the peptides in Examples 1 to 4 was predicted by the ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / index.html) online program, and the water solubility of the peptides in Examples 1 to 4 was determined by Innovagen (http: / / www.innovagen.com / proteomics-tools). The results showed that each peptide segment had no toxicity or allergic properties and had good water solubility.

[0061] Test Example 2 In this test example, molecular docking simulation technology is used to predict the binding sites and docking energies of the antioxidant polypeptides of Examples 1 to 4 with the receptor protein Keap1.

[0062] The 2D structural formula of the peptides of Examples 1 to 4 was drawn using ChemDraw 21.0.0 software, and then converted into 3D structures using Chem3D. MM2 in the Calculations function was selected to minimize the energy. The crystal structure of the complex of the Keap1 protein Kelch region and the Nrf2 fragment 16 peptide was obtained from the RCSB protein database (PDB ID: 2FLU), and other ligands such as water, solvent small molecules, and Nrf2 16-mer peptide in the protein structure were removed, and the protein was hydrogenated. The obtained peptide was used as a ligand to dock with the receptor protein Keap1 using the molecular docking mode technology. The receptor protein was set to rigid and the ligand small molecule was set to flexible. The center coordinates of the active pocket were x: 5, y: 9 and z: 1, and the radius was 15 Å. The molecular docking was evaluated based on the docking energy. The larger the absolute value of the docking binding energy obtained by the docking result, the stronger the interaction between the receptor and the ligand, that is, the stronger the potential antioxidant effect. The docking results were finally visualized and analyzed using PyMol 2.5.

[0063] The results showed that the docking energies of LMGPR, GFDGD, FGFDGDF, GHNGLDGL with Keap1 were -68.8519, -115.582, -141.445, and -136.723 kcal / mol, respectively. The molecular docking results of the antioxidant peptide LMGPR and Keap1 receptor are shown in Figure 5 The molecular docking results of GFDGD and Keap1 receptor are shown in Figure 6 The molecular docking results of FGFDGDF and Keap1 receptor are shown in Figure 7 The molecular docking results of GHNGLDGL and Keap1 receptor are shown in Figure 8 .

[0064] The binding modes and binding sites of the four antioxidant peptides and the receptor protein Keap1 show that the interaction between antioxidant peptides and the receptor protein Keap1 includes intermolecular hydrogen bonds, electrostatic and hydrophobic interactions. The position structure of the interaction between LMGPR and Keap1 protein is shown in Figure 2. Figure 5As shown, it forms salt bridges, electrostatic, traditional hydrogen bonds, carbon-hydrogen bonds and π-alkyl interactions with TYR-572, ARG-483, ARG-415 and SER-508 residues in the active site of Keap1. Keap1 interacts with the six active pockets of Nrf2, and the binding pocket can be divided into six sub-pockets, namely P1-P6. The LMGPR ligand binds to ARG-415, ARG-483 and SER-508 in the active pocket P1, and to TYR-572 in the active pocket P4. The position structure of the interaction between GFDGD and Keap1 protein is shown in Figure 6 As shown, it forms salt bridges, traditional hydrogen bonds, carbon-hydrogen bonds, electrostatic, π-anion, π-alkyl and π-π stacking interactions with ASN-414, ASN-382, ARG-380, TYR-334, ARG-415, TYR-525 and ALA-556 residues in the active site of Keap1. The GFDGD ligand binds to ARG-415 in the active pocket P1, ARG-380, ASN-382 and ASN-414 in the active pocket P2, ALA-556 in the active pocket P3, TYR-525 in the active pocket P4 and TYR-334 in P5. The positional structure of the interaction between FGFDGDF and Keap1 protein is shown in Figure 7 As shown, it forms traditional hydrogen bonds, carbon-hydrogen bonds, salt bridges, electrostatic and π-π stacking interactions with SER-508, ASN-382, ARG-380, ARG-483, ARG-415, ARG-380, GLY-509, SER-602 and TYR-334 in the active site of Keap1. The FGFDGDF ligand binds to ARG-415, ARG-483 and SER-508 in the active pocket P1 of Keap1, to ARG-380 and ASN-382 in the active pocket P2, to GLY-509 and SER-602 in P3, and to TYR-334 in P5. The position structure of GHNGLDGL interacting with Keap1 protein is shown in Figure 8As shown, it forms traditional hydrogen bonds, carbon-hydrogen bonds, salt bridges, electrostatic, alkyl, π-cation, π-anion and π-alkyl interactions with ARG-380, ASN-382, TYR-525, SER-555, ARG-483, PHE-478, ARG-415, HIS-436 and ILE-461 residues in the active site of Keap1. GHNGLDGL ligand binds to ARG-415, PHE-478, ILE-461 and ARG-483 in active pocket P1, ARG-380 and ASN-382 in active pocket P2, SER-555 in active pocket P3, TYR-525 in active pocket P4 and HIS-436 in active pocket P6.

[0065] The results showed that the interactions between the four peptides and the receptor protein Keap1 included intermolecular hydrogen bonds, electrostatic and hydrophobic interactions, and all formed new and stable complexes with Keap1, and all interacted with the six active pockets of Keap1 and the ligand Nrf2. The four peptides all contacted the residues of Keap1-Nrf2 interaction, and may competitively bind to Keap1 and release Nrf2, and exert antioxidant activity by activating the Keap1-Nrf2 / ARE pathway. From the above data, it can be seen that the above four peptides in <1 kDa can block the interaction of Keap-Nrf2 and promote the accumulation of Nrf2, which may cause the expression of antioxidant enzyme genes.

[0066] Test Example 3 This test example measured the free radical scavenging ability of the antioxidant polypeptides of Examples 1 to 4.

[0067] The antioxidant polypeptides obtained in Examples 1 to 4 were respectively dissolved in deionized water to prepare 5 mg / mL peptide solutions, and their DPPH free radical scavenging rates, ABTS free radical scavenging rates and hydroxyl free radical scavenging rates were respectively measured.

[0068] Take 100 μL of 5 mg / mL peptide solution and mix it with 100 μL of 0.1 mmol / L DPPH anhydrous ethanol solution, react at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate of the sample using the formula: Where: X—DPPH free radical scavenging rate, %; A X — indicates the absorbance value when the sample reacts with 0.1 mmol / L DPPH anhydrous ethanol solution; A0— indicates the absorbance value when the sample reacts with anhydrous ethanol solution; A1— indicates the absorbance value when deionized water reacts with 0.1 mmol / L DPPH anhydrous ethanol solution; A2— indicates the absorbance value when deionized water reacts with anhydrous ethanol solution.

[0069] Mix equal volumes of 7.4 mmol / L ABTS solution and 2.6 mmol / L K2S2O8 solution and keep in dark for 16 h, dilute with anhydrous ethanol to make the absorbance at 734 nm within the range of 0.70 ± 0.02 to obtain ABTS working solution. Mix 40 μL of 5 mg / mL peptide solution with 160 μL ABTS working solution, add to 96 microplates, react at room temperature in dark for 10 min, and measure the absorbance at 734 nm. Calculate the ABTS free radical scavenging rate of the sample using the formula: Where: X—ABTS free radical scavenging rate, %; A0—represents the absorbance value when deionized water reacts with ABTS working solution; A1—represents the absorbance value when the sample reacts with ABTS working solution.

[0070] Prepare 6.0 mmol / L salicylic acid solution and FeSO4 solution, take 0.5 mL of each and add them to a test tube, then add 0.5 mL of 5 mg / mL peptide solution and 0.5 mL of 6.0 mmol / L H2O2 solution, mix well, and react in a 37℃ water bath for 0.5 h. The absorbance is measured at 510 nm. The hydroxyl radical scavenging rate of the sample is calculated using the formula: Where: X—hydroxyl radical scavenging rate, %; A0—represents the absorbance value of FeSO4+H2O2+sample+salicylic acid; A1—represents the absorbance value of FeSO4+water+sample+salicylic acid; A—represents the absorbance value of FeSO4+H2O2+water+salicylic acid.

[0071] The results of DPPH, ABTS and hydroxyl radical scavenging activities of synthetic peptides are shown in Fig. 9 As shown in the results, FGFDGDF and LMGPR showed good DPPH free radical scavenging activity compared with the other three peptides, with significant differences, and the scavenging rates were 25.80% ± 1.82% and 23.56% ± 2.68%, respectively. FGFDGDF and LMGPR showed good ABTS free radical scavenging activity compared with the other two peptides, with significant differences, and the scavenging rates were 19.15% ± 1.59% and 14.28% ± 1.05%, respectively. FGFDGDF and GFDGD showed good hydroxyl free radical activity compared with the other two peptides, with significant differences, and the scavenging rates were 44.44% ± 1.94% and 41.61% ± 1.73%, respectively. In summary, FGFDGDF has the best free radical scavenging activity and can be used as an excellent antioxidant.

[0072] Test Example 4 This test example measured the free radical scavenging ability of the antioxidant polypeptides of Examples 5 to 15.

[0073] The detection method is the same as that of Test Example 3, and the results are shown in Tables 1 and Fig.10 , Fig.11 shown.

[0074] Table 1 Free radical scavenging ability of antioxidant peptides of Examples 5 to 15

[0075] As shown in Table 1, at the same concentration, the DPPH free radical scavenging activity and ABTS free radical scavenging activity of the above-mentioned peptides after being compounded in pairs are significantly better than those of a single peptide; after any three peptides are compounded, the DPPH free radical scavenging activity and ABTS free radical scavenging activity are also significantly better than those of a single peptide, and the hydroxyl free radical activity is better than that of a compound of two peptides; after the four peptides are compounded, the DPPH free radical scavenging activity, ABTS free radical scavenging activity and hydroxyl free radical activity are significantly better than those of a single peptide, and better than the free radical scavenging effect of the compound of two or three peptides. The above results show that the above-mentioned four peptides can produce better antioxidant effects when used in combination, and have a synergistic effect in antioxidant effects.

[0076] Test Example 5 This test example investigates the protective effect of the antioxidant polypeptides of Examples 1 to 4 on oxidative damage of HepG2 cells, and the specific steps include: (1) Establishment of H2O2-induced oxidative stress model in HepG2 cells HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin) solution and stored in a cell culture incubator at 37°C and 5% CO2. When the HepG2 cells adhered to the wall and the coverage rate reached more than 85%, they were digested and passaged using 0.25% trypsin solution.

[0077] HepG2 cells were cultured at 5×10 4 The cells were cultured in a 96-well plate at a density of 100 μL / mL for 24 hours. After the culture medium was removed, the pre-cultured cells were divided into a control group (C) and a model group (M). 100 μL of complete culture medium was added to the C group, and 100 μL of complete culture medium containing different concentrations (800, 1000, 1200, 1400, 1600 μM H2O2) was added to the M group. The culture medium was discarded after incubation for 2 hours. Finally, 90 μL of culture medium and 10 μL of CCK-8 reagent were added and cultured for 2 hours. The absorbance of the cells at 450 nm was measured by a microplate reader. The cell viability was measured by the CCK-8 method to determine the optimal H2O2 concentration for inducing the oxidative stress model.

[0078]

[0079] A0: absorbance value of the well with culture medium, CCK-8 solution, and no cells; As: absorbance values ​​of wells with cells, culture medium, CCK-8 solution, and drug solution; Ac: absorbance value of the wells containing cells, culture medium, CCK-8 solution, and no drug solution.

[0080] H2O2 is a hepatotoxic chemical with a long half-life. It can be converted into hydroxyl free radicals and oxygen free radicals and is an important cause of oxidative stress damage. Figure 8 As shown in the figure, the cell survival rate decreased with the increase of H2O2 concentration. Under the treatment of 800, 1000, 1200, 1400 and 1600 μM H2O2, the cell survival rates were 91.77% ± 4.53%, 80.28% ± 4.08%, 58.37% ± 3.81%, 51.29% ± 3.06% and 37.01% ±3.20%, respectively, which were significantly lower than those in the control group ( P <0.05), and when the concentration was 1400 µM, the cell survival rate was close to 50%. Fig.12 Therefore, in the subsequent experiments, 1400 μM H2O2 was selected to induce HepG2 cells to construct an oxidative damage model.

[0081] (2) Effects of antioxidant peptides on the survival of normal and oxidatively stressed cells HepG2 cells were divided into group C, LMGPR group, GFDGD group, FGFDGDF group, and GHNGLDGL group. Group C was added with 100 μL of complete culture medium, and the other groups were added with 100 μL of complete culture medium containing the antioxidant polypeptides LMGPR, GFDGD, FGFDGDF, and GHNGLDGL of Examples 1 to 4, at concentrations of 0.1, 0.2, 0.5, 1.0, and 2.5 mg / mL, respectively. The cells were incubated for 24 h, the culture medium was discarded, and the cell viability was evaluated using the CCK-8 method.

[0082] In another group of experiments, HepG2 cells were divided into C group, M group, LMGPR group, GFDGD group, FGFDGDF group, and GHNGLDGL group. Groups C and M were added with 100 μL of complete culture medium, and the other groups were added with culture medium containing the antioxidant polypeptides LMGPR, GFDGD, FGFDGDF, and GHNGLDGL of Examples 1 to 4, with concentrations of 0.1, 0.5, and 1 mg / mL. After the cells were incubated for 24 hours, the culture medium was removed. Group C continued to add 100 μL of complete culture medium, while group M and other groups were treated with 100 μL of culture medium containing 1400 μM H2O2 for 2 hours, and then the cell viability was determined using the CCK-8 method to study its preventive effect on oxidative stress.

[0083] After incubation of HepG2 cells with antioxidant peptides, the results were as follows Fig.13 As shown in Figure 2, within the concentration range of 0.1-2.5 mg / mL, the cell survival rate was higher than 90%, indicating that the antioxidant polypeptides of Examples 1-4 had no toxic effect on the normal proliferation of HepG2 cells. Fig.14 As shown, within the concentration range of 0.1-1.0 mg / mL, with the increase of the antioxidant polypeptide concentration, the survival rate of HepG2 cells also gradually increased. Compared with the M group, the survival rate of HepG2 cells had a significant difference, indicating that the antioxidant polypeptides of Examples 1-4 have a good damage protection effect on HepG2 cells.

[0084] (3) Effects of antioxidant peptides on antioxidant enzymes, malondialdehyde (MDA) and IL-8 levels in HepG2 cells induced by H2O2. The cells were cultured according to the method in (2), and the levels of SOD, CAT, MDA and IL-8 were determined strictly according to the instructions of the kit.

[0085] The results are shown in Table 2.

[0086] Table 2 Effects of antioxidant peptides on SOD, CAT, MDA and IL-8 levels

[0087] As shown in Table 2, after H2O2 induction, the activities of antioxidant enzymes SOD and CAT in HepG2 cells in group M were 6.17 ± 0.41 U / mgprot and 26.01 ± 1.78 U / mgprot, respectively, which were significantly lower than those in group C (14.35 ± 0.06 U / mgprot and 60.80 ± 1.10 U / mgprot). However, after pretreatment with antioxidant peptides (LMGPR, GFDGD, FGFDGDF, GHNGLDGL), the activities of SOD and CAT in cells were significantly higher than those in group M ( P <0.05). This indicates that LMGPR, GFDGD, FGFDGDF, and GHNGLDGL can regulate the activity of antioxidant enzymes and protect cells damaged by oxidation.

[0088] MDA is one of the key products of peroxidation of cell membrane lipids under oxidative stress conditions. Studies have shown that the higher the MDA content, the more serious the cell damage. As shown in Table 2, compared with the C group, the MDA content in HepG2 cells in the M group increased significantly ( P <0.05), indicating that lipid peroxidation occurred in HepG2 cells. In addition, after pretreatment with antioxidant peptides (LMGPR, GFDGD, FGFDGDF, GHNGLDGL), the content of MDA was significantly reduced (P <0.05). IL-8 is a pro-inflammatory cytokine. H2O2-induced oxidative stress can trigger cellular inflammatory responses. The results are shown in Table 1. Compared with group C, the IL-8 content in group M was significantly increased ( P <0.05), while after pretreatment with antioxidant peptides (LMGPR, GFDGD, FGFDGDF, GHNGLDGL), the intracellular IL-8 content was significantly reduced ( P <0.05), showing potential anti-inflammatory and antioxidant effects.

[0089] Based on the above results, it can be seen that the antioxidant peptides (LMGPR, GFDGD, FGFDGDF, GHNGLDGL) upregulated the activity of intracellular antioxidant enzymes to cope with hydrogen peroxide-induced oxidative stress, and the levels of MDA and IL-8 decreased. These indicate that the above antioxidant peptides can reduce oxidative damage and inflammation, and further have a certain protective effect on cells.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An antioxidant polypeptide, characterized in that: The antioxidant polypeptide contains at least one of LMGPR, GFDGD, FGFDGDF and GHNGLDGL; wherein the amino acid sequence of the LMGPR is: Leu-Met-Gly-Pro-Arg; the amino acid sequence of the GFDGD is: Gly-Phe-Asp-Gly-Asp; the amino acid sequence of the FGFDGDF is: Phe-Gly-Phe-Asp-Gly-Asp-Phe; the amino acid sequence of the GHNGLDGL is: Gly-His-Asn-Gly-Leu-Asp-Gly-Leu.

2. The antioxidant polypeptide according to claim 1, characterized in that The total content of LMGPR, GFDGD, FGFDGDF and GHNGLDGL in the antioxidant polypeptide is not less than 90%.

3. The antioxidant polypeptide according to claim 1 or 2, characterized in that The antioxidant polypeptide contains at least one of LMGPR, GFDGD and FGFDGDF.

4. The antioxidant polypeptide according to claim 1 or 2, characterized in that The antioxidant polypeptide contains two, three or four of LMGPR, GFDGD, FGFDGDF and GHNGLDGL.

5. The antioxidant polypeptide according to claim 4, characterized in that Each peptide was mixed in equal mass ratio.

6. The method for preparing the antioxidant polypeptide according to any one of claims 1 to 5, comprising the following steps: S1. According to the amino acid sequence of the peptide, a crude polypeptide is synthesized by solid phase synthesis; S2. The crude polypeptide obtained in S1 was dissolved in a 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated and purified by preparative reverse-phase high performance liquid chromatography to obtain a purified polypeptide.

7. The preparation method according to claim 6, characterized in that: When preparing the LMGPR, the chromatographic conditions of the preparative reversed-phase high performance liquid chromatography described in S2 include: mobile phase A is 0.09%~0.11% formic acid aqueous solution, mobile phase B is 0.09%~0.11% formic acid acetonitrile solution; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, mobile phase B is linearly eluted from 8%→33%, and the eluate is collected at 13~14 min; or When preparing the GFDGD, the chromatographic conditions of the preparative reversed-phase high performance liquid chromatography described in S2 include: mobile phase A is 0.09%~0.11% formic acid aqueous solution, mobile phase B is 0.09%~0.11% formic acid acetonitrile solution; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, mobile phase B is linearly eluted from 10%→35%, and the eluent is collected at 12~13.5 min; or When preparing the FGFDGDF, the chromatographic conditions of the preparative reversed-phase high performance liquid chromatography described in S2 include: mobile phase A is 0.09% to 0.11% formic acid aqueous solution, mobile phase B is acetonitrile aqueous solution containing 0.09% to 0.11% formic acid, and the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 83% to 85%; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, the mobile phase B is linearly eluted from 24% to 49%, and the eluent is collected at 13 to 14 min; or The mobile phase A for preparing the GHNGLDGL is 0.09% to 0.11% formic acid aqueous solution, and the mobile phase B is 0.09% to 0.11% formic acid in acetonitrile aqueous solution, wherein the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 83% to 85%; the liquid chromatography column is an octadecyl bonded silica gel chromatography column with a specification of 4.6 mm×250 mm; the elution gradient is: 0→25 min, the mobile phase B is linearly eluted from 17% to 42%, and the eluate is collected at 10.5 to 11.5 min.

8. The preparation method according to claim 7, characterized in that: The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is an acetonitrile aqueous solution containing 0.1% formic acid, wherein the volume percentage concentration of acetonitrile in the acetonitrile aqueous solution is 84%.

9. Use of the antioxidant polypeptide according to any one of claims 1 to 5 in the preparation of antioxidant products, characterized in that: The active ingredients thereof include the antioxidant polypeptide.

10. The use according to claim 9, characterized in that: The antioxidant products include food additives, antioxidant drugs and antioxidant cosmetics.

Images