Antioxidant peptide based on mussel byssus, preparation method, preparation and application thereof

Abstract

The invention relates to the technical field of bioactive peptides, in particular to an antioxidant peptide based on mussel byssus, a preparation method, a preparation and application of the antioxidant peptide based on mussel byssus, the antioxidant peptide is designed and synthesized by referring to the structural characteristics of mussel byssus proteins mfp-3, mfp-5 and mfp-6, the antioxidant peptide comprises four sequences of MFP3-AP1, MFP5-AP2, MFP6-AP3 and MFP-Hybrid, tyrosine in the peptide is modified into DOPA through enzymatic modification, and the antioxidant peptide is prepared through enzymatic modification. The stability and activity of the antioxidant peptide are enhanced through disulfide bond formation, metal ion coordination, PEGylation and other modifications, and the antioxidant peptide has high DPPH free radical scavenging activity, ABTS free radical scavenging activity, hydroxyl free radical scavenging activity and iron ion reducing capacity, has a protective effect on the cellular level, can be prepared into a liquid preparation, a freeze-dried preparation or a micro-capsule preparation, and can be used for preparing the antioxidant peptide. The compound is expected to be applied to the fields of antioxidant health care products, cosmetics and medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and particularly relates to an antioxidant peptide based on mussel byssus, a preparation method, a preparation and its application. Through bionic design, the present invention has developed a series of polypeptides with high antioxidant activity, which can be applied to the preparation of antioxidant health products, cosmetics or drugs for anti-aging, anti-fatigue or preventing ultraviolet damage and other aspects. Background Art

[0002] In recent years, with the acceleration of the population aging process and the improvement of health awareness, the research and development of antioxidants have received increasing attention. During the metabolic process of the human body, a large number of reactive oxygen species free radicals are generated, including superoxide anion radicals, hydroxyl radicals, etc. These free radicals are highly reactive and can attack biological macromolecules such as unsaturated fatty acids and proteins in biological membranes, resulting in damage to cell structure and function, and are closely related to the occurrence and development of various diseases such as aging, cardiovascular diseases, and cancers.

[0003] Currently, the antioxidants on the market mainly include synthetic antioxidants and natural antioxidants. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) etc. Although they have good antioxidant effects, there may be certain potential safety hazards in long-term use. Natural antioxidants mainly come from plant extracts, such as polyphenols, flavonoids, etc. Limited by resource scarcity and extraction costs, large-scale application faces challenges. Therefore, it is of great significance to develop new antioxidants with high efficiency and safety.

[0004] Marine biological resources are rich in bioactive substances and have become an important source for the research and development of new antioxidants. US Patent US20210077540A1 discloses a bioactive non-lipid extract extracted from the New Zealand green-lipped mussel (Perna canaliculus). This extract contains fragments with a molecular weight less than 10 kDa or less than 1 kDa and has various biological activities, including antioxidant activity, antihypertensive activity, and antimicrobial activity, etc. The extract contains various bioactive substances, including free amino acids, peptides, carbohydrate compounds, and phenolic compounds, etc. Among them, the molecules with a molecular weight less than 1 kDa exhibit significant antioxidant characteristics, but this patent mainly focuses on the extract of mussel flesh and does not involve the utilization of byssus.

[0005] In recent years, due to their high efficiency, safety, and easy absorption by the human body, bioactive peptides have gradually become a research hotspot in antioxidants. European Patent EP1787995A1 discloses a recombinant mussel foot protein and its preparation method. The patent points out that mussel foot silk has extraordinary mechanical properties, being able to behave like soft rubber at one end and like hard nylon at the other end, and these properties are seamlessly connected and gradually changed during the transition process. This patent mainly focuses on the structural function and recombinant expression of foot proteins, but does not involve their antioxidant activity and the development of related peptides.

[0006] Mussels are a common type of marine shellfish. Their foot silk is a protein fiber bundle secreted by mussels, which is used to firmly attach mussels to the surface of substrates such as rocks. Mussels live in an oxidative environment in the intertidal zone, and their foot silk has extremely strong antioxidant ability, being able to maintain structural integrity and functional stability in the highly oxidative seawater environment for a long time. Research shows that mussel foot silk contains various unique foot proteins (mussel foot proteins, mfps), among which mfp-3, mfp-5, and mfp-6 are rich in 3,4-dihydroxyphenylalanine (DOPA) and cysteine residues, and these amino acid residues play important roles in the antioxidant process.

[0007] Mussel foot silk is a material rich in collagen. However, due to its complex structural characteristics, it is challenging to extract the collagen component from it using food-grade technology. Collagen was extracted from the foot silk of the blue mussel (Mytilus edulis) with the assistance of endopeptidase proline, and the yield was 138.82 mg / g dry weight. The collagen extract was hydrolyzed with various food-grade enzyme preparations, and it was found that these hydrolysates had angiotensin-converting enzyme (ACE) inhibitory activity, dipeptidyl peptidase IV (DPP-IV) inhibitory activity, and antioxidant activity. However, this study only focused on the collagen hydrolysates of mussel foot silk, especially their inhibitory activities against ACE and DPP-IV, rather than specifically studying and developing their antioxidant active ingredients.

[0008] In addition, bioactive peptides with antioxidant activity have also been found in the protein hydrolysates of the New Zealand green-lipped mussel. By hydrolyzing mussel proteins with pepsin or alkaline protease, peptide segments with antioxidant activity and angiotensin-converting enzyme inhibitory activity were obtained. Research shows that the peptide (GPH) obtained after 30 minutes of pepsin hydrolysis exhibits the highest antioxidant and ACE inhibitory activities, and the protein component with a molecular weight less than 5 kDa has the strongest antioxidant and ACE inhibitory activities. However, these studies mainly focused on the mussel flesh proteins rather than the foot proteins.

[0009] Although the antioxidant properties of mussel byssus play a crucial role in its underwater attachment function, extracting antioxidant peptides directly from mussel byssus faces many challenges. First, the collection and processing of mussel byssus are difficult, and the extraction efficiency is low. Second, the byssus protein structure is complex, and the peptide segments obtained by direct hydrolysis have a complex composition and large differences in activity. Third, DOPA is prone to oxidation and is easily inactivated during the extraction process. Currently, there are no relevant reports on the design and development of highly efficient antioxidant peptides based on mussel byssus proteins.

[0010] In summary, how to design and develop a mussel byssus biomimetic antioxidant peptide with a clear structure, stable activity, and easy preparation, and overcome the problem of easy oxidation of DOPA, is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0011] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide an antioxidant peptide based on mussel byssus, its preparation method, preparation, and application. This peptide is designed based on the structural characteristics of mussel byssus proteins. By enzymatic modification, tyrosine is converted into DOPA, and through functionalization modifications such as disulfide bond formation, metal ion coordination, and PEGylation, the problems of insufficient activity and poor stability of antioxidant peptides in the prior art are solved.

[0012] To achieve the above purpose, the present invention provides the following technical solutions: An antioxidant peptide based on mussel byssus, the peptide is one or a combination of the following sequences: MFP3-AP1: GYCYGTYKC MFP5-AP2: KYTGYYKGK MFP6-AP3: CKYCGYYCG MFP-Hybrid: CGKYTCGYCK Among them, part or all of the tyrosine residues of the peptide are modified to 3,4-dihydroxyphenylalanine (DOPA).

[0013] Preferably, the peptide forms a stable three-dimensional structure through disulfide bond formation, and / or the N-terminus is coupled with polyethylene glycol (2-5 kDa), and / or forms a coordination complex with metal ions Fe 3+ or Zn 2+ to form a coordination complex.

[0014] The present invention also provides a preparation method for the above antioxidant peptide, including the following steps: (1) According to the structural characteristics of mussel byssus proteins mfp-3, mfp-5, and mfp-6, design and synthesize short peptides containing tyrosine and cysteine residues; (2) Use tyrosinase to convert tyrosine residues in the peptide into DOPA; (3) Promote the formation of intramolecular disulfide bonds in peptides through a redox system; (4) Enhance the stability and activity of peptides by metal ion coordination and / or PEGylation modification.

[0015] Preferably, in step (2), the tyrosinase is mushroom tyrosinase, the reaction system is 50 mM phosphate buffer (pH 6.8), the enzyme concentration is 100 - 200 U / mL, the reaction is carried out at 25 °C for 4 - 8 hours, and 0.5 mM ascorbic acid is added every 2 hours to prevent over-oxidation of DOPA.

[0016] Preferably, in step (3), the redox system is 0.5 mM oxidized glutathione (GSSG) and 0.1 U / mL protein disulfide isomerase (PDI), the reaction is carried out in 0.1 M Tris-HCl buffer (pH 8.0), and the reaction is carried out at 25 °C for 8 - 12 hours.

[0017] Preferably, in step (4), for metal ion coordination, FeCl 3 or ZnCl2 is used, the peptide:metal molar ratio is 3:1 or 2:1, and the reaction is carried out at room temperature for 2 - 4 hours in 10 mM HEPES buffer (pH 7.4); for PEGylation, mPEG-NHS (2 - 5 kDa) is used, the peptide:PEG molar ratio is 1:1.5, and the reaction is carried out at 4 °C for 12 hours in 0.1 M borate buffer (pH 8.5).

[0018] The present invention also provides a preparation containing the above antioxidant peptide, and the preparation is a liquid preparation, a freeze-dried preparation or a microcapsule preparation.

[0019] Preferably, the liquid preparation contains the following components: antioxidant peptide (1 - 5 mg / mL), ascorbic acid (0.1%), disodium EDTA (0.01%), mannitol (5%), polysorbate 80 (0.05%), phosphate buffer (20 mM, pH 7.2 - 7.4); Or, the freeze-dried preparation contains the following components: antioxidant peptide (10 - 50 mg / vial), trehalose (3%), mannitol (2%), ascorbic acid (0.1%), phosphate buffer (10 mM, pH 7.0 - 7.2).

[0020] The present invention also provides the application of the above antioxidant peptide in the preparation of antioxidant health products or cosmetics, and the application in the preparation of drugs for anti-aging, anti-fatigue or preventing ultraviolet damage.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. Based on the structural characteristics of mussel foot protein, a series of short peptides containing tyrosine and cysteine residues were designed. Through enzymatic modification, tyrosine was converted into DOPA, realizing the efficient preparation of antioxidant peptides and avoiding the difficulties of direct extraction from mussel foot silk.

[0022] 2. Through functional modifications such as disulfide bond formation, metal ion coordination, and PEGylation, the structural stability and antioxidant activity of the peptides were significantly improved. Among them, the MFP-Hybrid-DOPA-Fe 3+ peptide exhibited the best antioxidant activity, with an IC50 value of 72 μM for DPPH radical scavenging ability, significantly superior to various existing natural antioxidant peptides.

[0023] 3. The antioxidant peptides described in the present invention showed excellent antioxidant protection at the cellular level, effectively reducing H 2 O 2 -induced oxidative damage in HepG2 cells and increasing cell viability (85.3%), approaching the protective effect of the positive control vitamin C (82.7%).

[0024] 4. The antioxidant peptides developed in the present invention had good stability. After pH stability, thermal stability, protease resistance, and long-term stability tests, they all showed excellent stability, laying a foundation for their application in the fields of health products, cosmetics, and drugs. Specific Embodiments

[0025] The present invention will be further described in detail below through specific embodiments, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical methods and materials used in the present invention are the methods and materials well-known to those skilled in the art.

[0026] The core concept of the present invention is to design a series of short peptides containing tyrosine and cysteine residues based on the structural characteristics of mussel foot protein, convert tyrosine into DOPA through enzymatic modification, and improve the structural stability and antioxidant activity of the peptides through functional modifications such as disulfide bond formation, metal ion coordination, and PEGylation.

[0027] I. Design and Synthesis of Peptides The antioxidant peptides designed in the present invention mainly refer to the structural characteristics of mussel foot proteins mfp-3, mfp-5, and mfp-6. Analyses show that specific regions in these proteins are rich in DOPA and cysteine residues, and these regions play important roles in the antioxidant process. Therefore, based on the amino acid composition and arrangement characteristics of these regions, four candidate peptides were designed: MFP3-AP1: Antioxidant peptide 1 based on mfp-3, with the sequence GYCYGTYKC and a molecular weight of 1065.2 Da; MFP5-AP2: Antioxidant peptide 2 based on mfp-5, with the sequence KYTGYYKGK and a molecular weight of 1059.2 Da; MFP6-AP3: Antioxidant peptide 3 based on mfp-6, with the sequence CKYCGYYCG and a molecular weight of 1067.3 Da; MFP-Hybrid: Hybrid antioxidant peptide, with the sequence CGKYTCGYCK and a molecular weight of 1105.3 Da; The design of these peptides follows the following principles: (1) Each peptide contains 2 - 3 tyrosine residues for subsequent DOPA modification; (2) Contains 1 - 3 cysteine residues for forming stable disulfide bonds; (3) Add 1 - 2 lysine residues to improve water solubility; (4) Maintain an amino acid length of 8 - 12 to balance activity and synthesis difficulty; (5) The MFP-Hybrid peptide combines the structural features of mfp-3, mfp-5, and mfp-6, aiming to obtain better antioxidant activity.

[0028] Example 1: Synthesis and Characterization of Peptides 1.1 Peptide Synthesis The above four peptides were synthesized using the Fmoc solid-phase synthesis method. The specific steps are as follows: (1) Resin preparation: Use Rink Amide MBHA resin (substitution degree 0.5 mmol / g), weigh 0.2 mmol of resin and place it in a reactor, and swell it with DMF for 30 minutes.

[0029] (2) Fmoc deprotection: Add 20% piperidine / DMF solution, react at room temperature for 10 minutes, repeat once, and then wash the resin with DMF 5 times.

[0030] (3) Amino acid coupling: According to the order from the C-terminus to the N-terminus of the peptide, sequentially add DMF solutions of 4 equivalents of Fmoc-amino acid-OH, 4 equivalents of HBTU, 4 equivalents of HOBt, and 8 equivalents of DIEA, and react at room temperature for 60 minutes. For cysteine, extend the reaction time to 120 minutes. Monitor the coupling efficiency with Kaiser test or TNBS test after each coupling. If the efficiency is lower than 99.5%, perform re-coupling.

[0031] (4) Peptide chain elongation: Repeat steps (2) and (3) to complete the synthesis of the entire peptide chain.

[0032] (5) Terminal deprotection and cleavage: Remove the N-terminal Fmoc protecting group with 20% piperidine / DMF solution, and then treat with a mixed solution of TFA / TIS / H2O / EDT (92.5:2.5:2.5:2.5) at room temperature for 2 hours to cleave the peptide chain and remove the side-chain protecting groups.

[0033] (6) Peptide recovery: Filter the reaction mixture, precipitate the filtrate with cold ether, collect the precipitate by centrifugation, and dry it under vacuum to obtain the crude peptide.

[0034] 1.2 Peptide purification Purify the crude peptide by semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions are as follows: Chromatographic column: C18 column (250×21.2 mm, 10 μm) Mobile phase A: 0.1% aqueous TFA solution Mobile phase B: 0.1% TFA acetonitrile solution Gradient: 10-40% B, 30 minutes Flow rate: 10 mL / min Detection wavelength: 220 nm and 280 nm Collect the target peak and freeze-dry to obtain a peptide with a purity greater than 98%.

[0035] 1.3 Peptide characterization Characterize the purified peptide by analytical HPLC and mass spectrometry (MALDI-TOF MS or ESI-MS). The analysis results are as follows: MFP3-AP1: HPLC purity 99.1%, molecular weight measured by MS is 1065.3 Da (theoretical value is 1065.2 Da) MFP5-AP2: HPLC purity 98.7%, molecular weight measured by MS is 1059.4 Da (theoretical value is 1059.2 Da) MFP6-AP3: HPLC purity 98.5%, molecular weight measured by MS is 1067.5 Da (theoretical value is 1067.3 Da) MFP-Hybrid: HPLC purity 99.3%, molecular weight measured by MS is 1105.2 Da (theoretical value is 1105.3 Da) The above results indicate that the four peptides were successfully synthesized, and their purity and molecular weight meet the expectations.

[0036] II. Enzymatic DOPA modification of peptides In the present invention, tyrosinase is used to convert tyrosine residues in peptides into DOPA. Tyrosinase (EC 1.14.18.1) is a copper enzyme that can catalyze the hydroxylation of tyrosine to DOPA and is widely present in animals, plants, and microorganisms. In the present invention, tyrosinase derived from mushrooms is selected for enzymatic modification.

[0037] Example 2: Enzymatic DOPA Modification of Peptides 2.1 Enzymatic Reaction Taking the MFP-Hybrid peptide as an example, the process of enzymatic DOPA modification is described in detail: (1) Prepare the reaction solution: Dissolve the MFP-Hybrid peptide in 50 mM phosphate buffer (pH 6.8) to make the peptide concentration 5 mg / mL, and add 0.01% Tween-80 to increase the solubility of the peptide.

[0038] (2) Add the enzyme: Add mushroom tyrosinase to the reaction solution to make the final enzyme concentration 200 U / mL.

[0039] (3) Reaction: Stir the reaction at 25 ± 1 °C with a stirring speed of 250 rpm. Sample every 2 hours to monitor the reaction progress, and add 0.5 mM ascorbic acid to prevent excessive oxidation of DOPA.

[0040] (4) Terminate the reaction: After 6 hours of reaction, when the tyrosine conversion rate reaches over 92%, add 5 mM ascorbic acid and 1 mM EDTA to terminate the reaction, and adjust the pH to 4.5 to inhibit the enzyme activity.

[0041] (5) Removal of the enzyme: Use a 10 kDa ultrafiltration membrane to centrifuge (4000 g, 15 minutes) to remove the enzyme, and collect the filtrate.

[0042] 2.2 Purification of the Modified Peptide Purify the DOPA-modified peptide by ion exchange chromatography: (1) Packing preparation: Use SP Sepharose Fast Flow cation exchange packing and equilibrate it with 20 mM acetic acid buffer (pH 4.5).

[0043] (2) Loading: Adjust the sample to pH 4.5 and load it onto the chromatographic column.

[0044] (3) Elution: Elute the unbound substances with 20 mM acetic acid buffer (pH 4.5), and then elute the target peptide with the same buffer containing an NaCl gradient (0 - 500 mM).

[0045] (4) Fraction collection: Collect the fractions containing DOPA peptides based on the UV280 nm absorption and the Arnow reaction (a specific color reaction for DOPA).

[0046] (5) Desalting and concentration: Desalt and concentrate the collected fractions using a 3 kDa ultrafiltration membrane, and then lyophilize to obtain the DOPA-modified peptide powder.

[0047] 2.3 Determination of DOPA modification rate Determine the DOPA content using the Arnow reaction and calculate the DOPA modification rate: (1) Standard curve: Use pure DOPA as the standard to prepare a standard curve with concentrations ranging from 0.05 - 1.0 mg / mL.

[0048] (2) Sample preparation: Dissolve the DOPA-modified peptide in water to a concentration of 1 mg / mL.

[0049] (3) Arnow reaction: Take 0.5 mL of the sample or standard, and successively add 0.5 mL of 0.5 M HCl, 0.5 mL of sodium nitrite-molybdic acid reagent (a mixture of 5% sodium nitrite and 5% sodium molybdate), and 0.5 mL of 1 M NaOH. After mixing, measure the absorbance at a wavelength of 500 nm.

[0050] (4) Modification rate calculation: Calculate the DOPA content based on the standard curve. Modification rate = actual DOPA content / theoretical fully modified DOPA content × 100%.

[0051] The DOPA modification results of the four peptides are as follows: MFP3-AP1-DOPA: Modification rate is 90.5%, HPLC purity is 97.2%, and the molecular weight measured by MS is 1113.2 Da (theoretical value is 1113.2 Da) MFP5-AP2-DOPA: Modification rate is 89.8%, HPLC purity is 96.8%, and the molecular weight measured by MS is 1107.1 Da (theoretical value is 1107.2 Da) MFP6-AP3-DOPA: Modification rate is 91.2%, HPLC purity is 97.5%, and the molecular weight measured by MS is 1115.2 Da (theoretical value is 1115.3 Da) MFP-Hybrid-DOPA: Modification rate is 92.5%, HPLC purity is 98.1%, and the molecular weight measured by MS is 1137.1 Da (theoretical value is 1137.3 Da) The above results indicate that all four peptides have been successfully modified with DOPA, with modification rates above 90%, good purity, and molecular weights in line with expectations.

[0052] III. Functional Enhancement Modification of Peptides To further improve the stability and activity of peptides, the present invention performs three functional enhancement modifications on DOPA-modified peptides: disulfide bond formation, N-terminal PEGylation, and metal ion coordination.

[0053] Example 3: Disulfide Bond Formation of Peptides 3.1 Disulfide Bond Formation Reaction Taking the MFP-Hybrid-DOPA peptide as an example, the process of disulfide bond formation is described in detail as follows: (1) Prepare the reaction solution: Dissolve the MFP-Hybrid-DOPA peptide in 0.1 M Tris-HCl buffer (pH 8.0) to make the peptide concentration 2 mg / mL.

[0054] (2) Add the redox system: Add 0.5 mM oxidized glutathione (GSSG) and 0.1 U / mL protein disulfide isomerase (PDI) to the reaction solution.

[0055] (3) React: Gently stir the reaction at 25 °C for 10 hours.

[0056] (4) Terminate the reaction: Add 10 mM DTT to terminate the reaction and adjust the pH to 3.0.

[0057] 3.2 Purification of Disulfide-Bonded Peptides Purify the disulfide-bonded peptides by reverse-phase HPLC: (1) Chromatographic conditions: C18 column, gradient elution with 15 - 45% acetonitrile for 30 minutes, flow rate 5 mL / min.

[0058] (2) Fraction collection: Collect the target peak and freeze-dry to obtain the purified disulfide-bonded peptides.

[0059] 3.3 Determination of Disulfide Bond Formation Rate Determine the free sulfhydryl content using the Ellman reagent method and calculate the disulfide bond formation rate: (1) Sample preparation: Dissolve the peptide in 0.1 M phosphate buffer (pH 8.0) containing 1 mM EDTA to make the concentration 0.5 mg / mL.

[0060] (2) Ellman reaction: Take 0.5 mL of the sample, add 50 μL of 10 mM DTNB solution, react at room temperature for 15 minutes, and measure the absorbance at a wavelength of 412 nm.

[0061] (3) Formation rate calculation: Disulfide bond formation rate = (1 - free sulfhydryl content after disulfide bond formation / free sulfhydryl content before disulfide bond formation) × 100%.

[0062] The disulfide bond formation results of the four peptides are as follows: MFP3-AP1-DOPA: The disulfide bond formation rate is 95.3%, and the HPLC purity is 96.8% MFP6-AP3-DOPA: The disulfide bond formation rate is 94.5%, and the HPLC purity is 97.2% MFP-Hybrid-DOPA: The disulfide bond formation rate is 96.8%, and the HPLC purity is 97.8% Since MFP5-AP2-DOPA does not contain cysteine residues, disulfide bond formation modification is not carried out. The above results show that the three peptides containing cysteine residues have all successfully formed disulfide bonds, and the formation rates are all above 94%.

[0063] Example 4: N-terminal PEGylation modification of peptides 4.1 PEGylation reaction Taking the MFP-Hybrid-DOPA peptide as an example, the process of N-terminal PEGylation is described in detail: (1) Prepare the reaction solution: Dissolve the MFP-Hybrid-DOPA peptide in 0.1 M borate buffer (pH 8.5) to make the peptide concentration 5 mg / mL.

[0064] (2) Add the PEG reagent: Add mPEG-NHS (3.5 kDa) to the reaction solution to make the peptide:PEG molar ratio 1:1.5.

[0065] (3) React: Stir the reaction gently at 4 °C for 12 hours.

[0066] (4) Terminate the reaction: Add an excess of glycine (final concentration 50 mM) to terminate the reaction.

[0067] 4.2 Purification of the PEGylated peptide Purify the PEGylated peptide by ion exchange chromatography: (1) Prepare the packing: Use a Resource S cation exchange chromatography column and equilibrate it with 20 mM sodium acetate buffer (pH 5.0).

[0068] (2) Load the sample: Adjust the reaction mixture to pH 5.0 and load it onto the chromatography column.

[0069] (3) Elute: Elute with 20 mM sodium acetate buffer (pH 5.0) containing an NaCl gradient (0 - 500 mM) for 30 minutes.

[0070] (4) Fraction collection: Collect the fractions containing PEGylated peptides based on the absorption at UV280 nm.

[0071] (5) Desalting and concentration: Desalt and concentrate the collected fractions using a 5 kDa ultrafiltration membrane, and then lyophilize to obtain PEGylated peptide powder.

[0072] 4.3 Determination of PEGylation modification rate Analyze the PEGylation modification rate by SDS-PAGE and MALDI-TOF MS: (1) SDS-PAGE analysis: Use a 12% polyacrylamide gel, stain with Coomassie Brilliant Blue R-250, and determine the relative intensity of each band by a gel scanner.

[0073] (2) Mass spectrometry analysis: Determine the molecular weight of the PEGylated peptide by MALDI-TOF MS.

[0074] (3) Calculation of modification rate: PEGylation modification rate = peak area of PEGylated peptide / (peak area of PEGylated peptide + peak area of unmodified peptide) × 100%.

[0075] The PEGylation results of the four peptides are as follows: MFP3-AP1-DOPA-PEG: The modification rate is 91.2%, and the molecular weight measured by MALDI-TOF MS is about 4.6 kDa MFP5-AP2-DOPA-PEG: The modification rate is 89.5%, and the molecular weight measured by MALDI-TOF MS is about 4.6 kDa MFP6-AP3-DOPA-PEG: The modification rate is 90.8%, and the molecular weight measured by MALDI-TOF MS is about 4.6 kDa MFP-Hybrid-DOPA-PEG: The modification rate is 93.2%, and the molecular weight measured by MALDI-TOF MS is about 4.6 kDa The above results indicate that all four peptides have been successfully PEGylated, and the modification rates are all above 89%.

[0076] Example 5: Metal ion coordination modification of peptides 5.1 Metal ion coordination reaction Taking the MFP-Hybrid-DOPA peptide as an example, the process of metal ion coordination is described in detail: (1) Prepare the reaction solution: Dissolve the MFP-Hybrid-DOPA peptide in 10 mM HEPES buffer (pH 7.4) to make the peptide concentration 3 mg / mL.

[0077] (2) Metal ion coordination: Slowly add FeCl 3 solution to the reaction solution to make the peptide:Fe 3+ molar ratio 3:1, stir and react at room temperature for 3 hours.

[0078] (3) In addition, using the same method, use ZnCl 2 for metal ion coordination to make the peptide:Zn 2+ molar ratio 2:1.

[0079] 5.2 Purification of metal-coordinated peptides Purify the metal-coordinated peptides by gel filtration chromatography: (1) Chromatographic conditions: Sephadex G-25, elute with 10 mM HEPES buffer (pH 7.4), flow rate 1 mL / min.

[0080] (2) Fraction collection: Collect the fractions containing metal-coordinated peptides based on the absorption at UV280 nm.

[0081] (3) Concentration: Freeze-dry the collected fractions to obtain metal-coordinated peptide powder.

[0082] 5.3 Verification of metal coordination Verify metal coordination by UV-vis spectroscopy and ICP-MS: (1) UV-vis spectroscopy: Dissolve the peptide in 10 mM HEPES buffer (pH 7.4) at a concentration of 0.5 mg / mL, and scan the absorption spectrum in the wavelength range of 200 - 800 nm. DOPA-Fe 3+ coordination shows a characteristic absorption peak at 570 nm.

[0083] (2) ICP-MS analysis: Determine the metal content in the metal-coordinated peptide and calculate the peptide:metal molar ratio.

[0084] The results of the coordination of four peptides with Fe 3+ are as follows: MFP3-AP1-DOPA-Fe 3+ : peptide:Fe 3+ molar ratio 3.2:1, UV-vis spectroscopy shows a characteristic absorption peak at 570 nm MFP5-AP2-DOPA-Fe 3+ : peptide:Fe 3+ molar ratio 3.1:1, UV-vis spectroscopy shows a characteristic absorption peak at 570 nm MFP6-AP3-DOPA-Fe 3+ : peptide:Fe 3+The molar ratio is 3.3:1, and the UV-vis spectrum shows a characteristic absorption peak at 570 nm. MFP-Hybrid-DOPA-Fe 3+ : Peptide:Fe 3+ The molar ratio is 3.0:1, and the UV-vis spectrum shows a characteristic absorption peak at 570 nm. Four peptides and Zn 2+ The results of coordination are as follows: MFP3-AP1-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.1:1 MFP5-AP2-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.0:1 MFP6-AP3-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.2:1 MFP-Hybrid-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 1.9:1 The above results indicate that all four peptides have successfully undergone metal ion coordination modification, and the coordination ratio meets the expectations.

[0085] IV. Determination of Antioxidant Activity The present invention uses a variety of methods to determine the antioxidant activity of peptides, including DPPH radical scavenging activity, ABTS radical scavenging activity, hydroxyl radical scavenging activity, ferric ion reducing ability, and metal ion chelating ability, etc.

[0086] Example 6: Determination of Antioxidant Activity 6.1 DPPH Radical Scavenging Activity DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical with a characteristic absorption peak at 517 nm. Antioxidants can react with DPPH, resulting in a decrease in absorption, which can be used to evaluate the antioxidant ability.

[0087] The determination method is as follows: (1) Prepare the peptide solution: Dissolve the peptide in a mixed solution of ethanol and water (1:1, v / v) to prepare a concentration gradient of 10 - 500 μM.

[0088] (2) DPPH reaction: Take 100 μL of peptide solutions with different concentrations, mix them with an equal volume of 0.1 mM DPPH ethanol solution, and react in the dark for 30 minutes.

[0089] (3) Absorbance measurement: The absorbance was measured using a microplate reader at a wavelength of 517 nm.

[0090] (4) Calculation of scavenging rate: DPPH radical scavenging rate (%) = (Acontrol - Asample) / Acontrol × 100%, where Acontrol is the absorbance of the DPPH solution without peptide, and Asample is the absorbance of the DPPH solution with peptide.

[0091] (5) Calculation of IC50: A curve of scavenging rate vs. peptide concentration was plotted, and the peptide concentration required to achieve 50% scavenging rate (IC50) was calculated.

[0092] The results of DPPH radical scavenging activities of each peptide are as follows (expressed as IC50, unit: μM): Unmodified peptides: MFP3 - AP1: >500 MFP5 - AP2: >500 MFP6 - AP3: >500 MFP - Hybrid: >500 DOPA - modified peptides: MFP3 - AP1 - DOPA: 156 MFP5 - AP2 - DOPA: 188 MFP6 - AP3 - DOPA: 125 MFP - Hybrid - DOPA: 92 Disulfide - formed peptides: MFP3 - AP1 - DOPA - SS: 118 MFP6 - AP3 - DOPA - SS: 102 MFP - Hybrid - DOPA - SS: 85 PEGylation - modified peptides: MFP3 - AP1 - DOPA - PEG: 142 MFP5 - AP2 - DOPA - PEG: 172 MFP6 - AP3 - DOPA - PEG: 115 MFP - Hybrid - DOPA - PEG: 88 Metal - ion - coordinated peptides: MFP3 - AP1 - DOPA - Fe 3+ : 95 MFP5 - AP2 - DOPA - Fe 3+ : 105 MFP6 - AP3 - DOPA - Fe 3+ : 83 MFP-Hybrid-DOPA-Fe 3+ : 72 MFP3-AP1-DOPA-Zn 2+ : 128 MFP5-AP2-DOPA-Zn 2+ : 150 MFP6-AP3-DOPA-Zn 2+ : 106 MFP-Hybrid-DOPA-Zn 2+ : 79 Reference substance: Vitamin C: 25 Glutathione: 75 BHT: 18 The above results show that: (1) The unmodified peptide has almost no DPPH radical scavenging activity; (2) After DOPA modification, the peptide exhibits obvious antioxidant activity; (3) Disulfide bond formation, PEGylation, and metal ion coordination further enhance the antioxidant activity of the peptide; (4) Coordination with Fe 3+ is more effective in enhancing the antioxidant activity of the peptide than coordination with Zn 2+ ; (5) Among all the peptides tested, MFP-Hybrid-DOPA-Fe 3+ exhibits the strongest DPPH radical scavenging activity with an IC50 of 72 μM. Although it is lower than that of the synthetic antioxidant BHT (18 μM) and the natural antioxidant vitamin C (25 μM), it is superior to glutathione (75 μM) and various antioxidant peptides reported previously.

[0093] 6.2 ABTS radical scavenging activity ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) can form a blue-green ABTS radical cation under the action of an oxidant, which has a characteristic absorption peak at 734 nm. Antioxidants can scavenge the ABTS radical, resulting in a decrease in absorption.

[0094] The measurement method is as follows: (1) Preparation of ABTS radical: Mix 7 mM ABTS and 2.45 mM potassium persulfate in equal volumes and place in the dark for 16 hours to generate the ABTS radical. Dilute it with PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm.

[0095] (2) Reaction: Take 20 μL of peptide solutions with different concentrations, mix them with 180 μL of ABTS radical solution, and react at room temperature for 6 minutes.

[0096] (3) Absorbance measurement: The absorbance was measured using a microplate reader at a wavelength of 734 nm.

[0097] (4) Calculation of scavenging rate: ABTS radical scavenging rate (%) = (Acontrol - Asample) / Acontrol × 100%.

[0098] (5) Calculation of TEAC value: Using Trolox as the standard, the scavenging rate of the peptide was compared with the Trolox standard curve to calculate the Trolox equivalent antioxidant capacity (TEAC), with the unit of mmol Trolox / g peptide.

[0099] The results of the ABTS radical scavenging activities of each peptide are as follows (expressed as TEAC value, unit: mmol Trolox / g peptide): DOPA-modified peptides: MFP3-AP1-DOPA: 0.98 MFP5-AP2-DOPA: 0.85 MFP6-AP3-DOPA: 1.23 MFP-Hybrid-DOPA: 1.42 Metal ion coordination peptides: MFP3-AP1-DOPA-Fe 3+ : 1.35 MFP5-AP2-DOPA-Fe 3+ : 1.18 MFP6-AP3-DOPA-Fe 3+ : 1.56 MFP-Hybrid-DOPA-Fe 3+ : 1.65 Control substances: Vitamin C: 2.20 Glutathione: 1.45 BHT: 2.50 The above results indicate that MFP-Hybrid-DOPA-Fe 3+ showed the strongest ABTS radical scavenging activity, with a TEAC value of 1.65 mmol Trolox / g peptide, approaching the activity level of glutathione (1.45 mmol Trolox / g peptide).

[0100] 6.3 Hydroxyl radical scavenging activity Hydroxyl radical is one of the most reactive oxygen free radicals and has a strong oxidative damage effect on cells and tissues. Hydroxyl radicals can be generated through the Fenton reaction. Using 3-CCA (coumarin-3-carboxylic acid) as a probe, the scavenging ability of antioxidants against hydroxyl radicals can be measured.

[0101] The measurement method is as follows: (1) Reaction system: Mix 1 mL of peptide solution (10 - 250 μM), 1 mL of 3-CCA (0.5 mM), 1 mL of FeSO4 (0.5 mM), and 1 mL of H 2 O 2 (0.5 mM) and react at 37 °C for 1 hour.

[0102] (2) Fluorescence measurement: Measure the fluorescence intensity at an excitation wavelength of 395 nm and an emission wavelength of 440 nm.

[0103] (3) Calculation of scavenging rate: Hydroxyl radical scavenging rate (%) = (Fsample - Fblank) / (Fcontrol - Fblank) × 100%, where Fsample is the fluorescence intensity of the system containing the peptide, Fblank is the fluorescence intensity of the system without 3-CCA, and Fcontrol is the fluorescence intensity of the system without the peptide.

[0104] (4) Calculation of IC50: Plot the relationship curve between the scavenging rate and the peptide concentration, and calculate the peptide concentration (IC50) required to achieve a 50% scavenging rate.

[0105] The results of the hydroxyl radical scavenging activity of each peptide are as follows (expressed as IC50, unit: μM): DOPA-modified peptides: MFP3-AP1-DOPA: 158 MFP5-AP2-DOPA: 175 MFP6-AP3-DOPA: 132 MFP-Hybrid-DOPA: 118 Metal ion coordination peptides: MFP-Hybrid-DOPA-Fe 3+ : 95 Control substances: Vitamin C: 65 Glutathione: 85 The above results show that MFP-Hybrid-DOPA-Fe 3+ exhibits strong scavenging activity against hydroxyl radicals, with an IC50 of 95 μM, which is better than glutathione (85 μM) but weaker than vitamin C (65 μM).

[0106] 6.4 Ferric Ion Reducing Ability The ferric ion reducing ability is an important index for evaluating the electron donor ability of antioxidant substances. It can be evaluated by measuring the ability of a substance to reduce Fe 3+ to Fe 2+ to evaluate its antioxidant activity.

[0107] The measurement method is as follows: (1) Reaction system: Mix 1 mL of peptide solution (50 - 500 μg / mL), 2.5 mL of phosphate buffer (0.2 M, pH6.6) and 2.5 mL of [K 3 Fe(CN) 6 (1%) and incubate in a water bath at 50 °C for 20 minutes.

[0108] (2) Terminate the reaction: Add 2.5 mL of TCA (10%) and centrifuge to collect the supernatant.

[0109] (3) Color reaction: Take 2.5 mL of the supernatant and mix it with 2.5 mL of distilled water and 0.5 mL of FeCl 3 (0.1%).

[0110] (4) Absorbance measurement: Measure the absorbance at a wavelength of 700 nm. The higher the absorbance, the stronger the reducing ability.

[0111] (5) Calculation of reducing ability: Based on the standard curve of FeSO 4 , calculate the ferric ion reducing ability, with the unit of mmol Fe 2+ / g peptide.

[0112] The ferric ion reducing ability results of each peptide are as follows (unit: mmol Fe 2+ / g peptide): DOPA-modified peptides: MFP3-AP1-DOPA: 0.56 MFP5-AP2-DOPA: 0.48 MFP6-AP3-DOPA: 0.65 MFP-Hybrid-DOPA: 0.72 Metal ion coordination peptides: MFP-Hybrid-DOPA-Fe 3+ : 0.85 Control substances: Vitamin C: 1.50 Glutathione: 0.80 The above results show that MFP-Hybrid-DOPA-Fe 3+Exhibited strong ferric ion reducing ability, which was 0.85 mmol Fe 2+ / g peptide, close to glutathione (0.80 mmol Fe 2+ / g peptide), but weaker than vitamin C (1.50 mmol Fe 2+ / g peptide).

[0113] 6.5 Metal ion chelating ability Metal ions (such as Fe 2+） can catalyze lipid peroxidation, and antioxidants can inhibit lipid peroxidation by chelating metal ions. The Ferrozine method can be used to determine the chelating ability of substances to Fe 2+ .

[0114] The determination method is as follows: (1) Reaction system: Mix 1 mL of peptide solution (50 - 500 μg / mL), 0.1 mL of FeCl 2 (2 mM) and 3.7 mL of distilled water, react at room temperature for 10 minutes, and then add 0.2 mL of Ferrozine (5 mM).

[0115] (2) Absorbance measurement: After reacting for 10 minutes, measure the absorbance at a wavelength of 562 nm.

[0116] (3) Chelation rate calculation: The Fe 2+ chelation rate (%) = (A control - A sample) / A control × 100%, where A control is the absorbance of the system without peptide, and A sample is the absorbance of the system with peptide.

[0117] The results of the metal ion chelating ability of each peptide are as follows (expressed as the Fe 2+ chelation rate, unit: %): DOPA - modified peptide (500 μg / mL): MFP3 - AP1 - DOPA: 48.5% MFP5 - AP2 - DOPA: 42.3% MFP6 - AP3 - DOPA: 55.8% MFP - Hybrid - DOPA: 60.2% Metal ion coordination peptide (500 μg / mL): MFP - Hybrid - DOPA - Fe 3+ : 65.0% Control substance (500 μg / mL): EDTA: 98.5% Glutathione: 58.7% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ exhibits strong Fe 2+ chelating ability, with a chelation rate of 65.0%, which is better than glutathione (58.7%) but weaker than EDTA (98.5%).

[0118] V. Verification of antioxidant activity at the cellular level To verify the antioxidant activity of the peptide at the cellular level, the present invention uses a H 2 O 2 -induced oxidative damage model of HepG2 cells for research.

[0119] Example 7: Protection experiment against H 2 O 2 -induced oxidative damage in HepG2 cells 7.1 Cell culture The HepG2 human hepatoma cell line was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO 2 conditions.

[0120] 7.2 Determination of cell viability (1) Cell treatment: HepG2 cells were seeded in 96-well plates at a density of 1×104 cells / well. After 24 hours of culture, they were grouped and treated as follows: Control group: Normal culture Model group: Treated with 200 μM H 2 O 2 for 4 hours Low-dose peptide group: Pretreated with 10 μM peptide for 2 hours, then added H 2 O 2 Medium-dose peptide group: Pretreated with 50 μM peptide for 2 hours, then added H 2 O 2 High-dose peptide group: Pretreated with 100 μM peptide for 2 hours, then added H 2 O 2 Positive control group: Pretreated with 100 μM vitamin C for 2 hours, then added H 2 O 2 (2) Determination of cell viability by MTT method: After the treatment, 20 μL of MTT (5 mg / mL) was added to each well, and the cells were further cultured for 4 hours. The culture medium was aspirated, 150 μL of DMSO was added, and the mixture was shaken for 10 minutes. The absorbance was measured at a wavelength of 570 nm.

[0121] (3) Survival rate calculation: Cell survival rate (%) = absorbance of treatment group / absorbance of control group × 100%.

[0122] The results of cell survival rates in each group are as follows: Control group: 100% Model group: 48.2% MFP-Hybrid-DOPA-Fe 3+ Low-dose group (10 μM): 62.5% MFP-Hybrid-DOPA-Fe 3+ Medium-dose group (50 μM): 75.8% MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 85.3% Vitamin C group (100 μM): 82.7% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can protect HepG2 cells from H 2 O 2 induced oxidative damage in a dose-dependent manner, and the protective effect at 100 μM (85.3%) is better than that of vitamin C at the same concentration (82.7%).

[0123] 7.3 Determination of intracellular ROS level (1) Cell treatment: The same as 7.2.

[0124] (2) ROS detection: The DCFH-DA probe method was used. After the treatment, 10 μM DCFH-DA was added, incubated at 37 °C for 30 minutes, and washed 3 times with PBS.

[0125] (3) Fluorescence detection: The intracellular ROS level (green fluorescence) was observed under a fluorescence microscope, and the fluorescence intensity was quantitatively detected by flow cytometry.

[0126] (4) Calculation of relative fluorescence intensity: Taking the fluorescence intensity of the control group as 1, the relative fluorescence intensities of each group were calculated.

[0127] The results of relative fluorescence intensities in each group are as follows: Control group: 1.00 Model group: 3.76 MFP-Hybrid-DOPA-Fe 3+ Low-dose group (10 μM): 2.82 MFP-Hybrid-DOPA-Fe 3+ Medium-dose group (50 μM): 1.94 MFP-Hybrid-DOPA-Fe3+ High-dose group (100 μM): 1.45 Vitamin C group (100 μM): 1.52 The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can dose-dependently reduce the level of ROS in HepG2 cells induced by H 2 O 2 The effect at 100 μM (relative fluorescence intensity 1.45) is slightly better than that of vitamin C at the same concentration (relative fluorescence intensity 1.52).

[0128] 7.4 Detection of antioxidant enzyme activity (1) Cell treatment: The same as 7.2, but use a 6-well plate, and seed 2×105 cells in each well.

[0129] (2) Cell lysis: After the treatment, collect the cells, wash them twice with PBS, add lysis buffer, and lyse them on ice for 30 minutes. Centrifuge to collect the supernatant.

[0130] (3) Activity detection: Use a commercial kit to measure the activities of SOD, CAT, and GPx.

[0131] (4) Activity calculation: Take the activity of the control group as 100%, and calculate the relative activities of each group.

[0132] The relative activities of antioxidant enzymes in each group are as follows: Relative activity of SOD: Control group: 100% Model group: 65.3% MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 94.0% Vitamin C group (100 μM): 91.5% Relative activity of CAT: Control group: 100% Model group: 58.7% MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 91.1% Vitamin C group (100 μM): 88.6% Relative activity of GPx: Control group: 100% Model group: 62.4% MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 88.3% Vitamin C group (100 μM): 85.6% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can significantly increase the activity of antioxidant enzymes (SOD, CAT, and GPx) in HepG2 cells after H 2 O 2 treatment, and the effect is slightly better than that of vitamin C.

[0133] VI. Stability evaluation of peptides To evaluate the stability of the peptides, the present invention conducted pH stability, thermal stability, protease resistance, and long-term stability tests on the peptides.

[0134] Example 8: Stability test of peptides 8.1 pH stability (1) Sample preparation: Dissolve the MFP-Hybrid-DOPA-Fe 3+ peptide in buffers with different pH values (pH 2.0, 4.0, 6.0, 7.4, 9.0) to a concentration of 1 mg / mL.

[0135] (2) Incubation: Incubate at 25 °C for 24 hours.

[0136] (3) Activity determination: Use the DPPH method to determine the antioxidant activity of the peptide under each pH condition.

[0137] (4) Stability calculation: Take the activity at pH 7.4 as 100% and calculate the activity retention rate under other pH conditions.

[0138] The pH stability results are as follows: pH 2.0: 82.3% pH 4.0: 91.5% pH 6.0: 98.7% pH 7.4: 100% (reference) pH 9.0: 85.6% The above results indicate that the MFP-Hybrid-DOPA-Fe 3+ peptide has good stability in the pH range of 4.0 - 7.4, and the activity retention rate is above 90%; under the conditions of pH 2.0 and 9.0, the activity decreases, but still remains above 80%.

[0139] 8.2 Thermal stability (1) Sample preparation: Dissolve the MFP-Hybrid-DOPA-Fe 3+ peptide in PBS (pH 7.4) to a concentration of 1 mg / mL.

[0140] (2) Heat treatment: Place the samples in a water bath at different temperatures (4°C, 25°C, 37°C, 60°C, 80°C) for 30 minutes.

[0141] (3) Activity determination: The antioxidant activity of the peptides after treatment at each temperature was determined by the DPPH method.

[0142] (4) Stability calculation: Taking the activity at 4°C as 100%, calculate the activity retention rate under other temperature conditions.

[0143] The results of thermal stability are as follows: 4°C: 100% (reference) 25°C: 99.2% 37°C: 95.8% 60°C: 83.4% 80°C: 67.9% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ peptide has good thermal stability in the range of 25 - 37°C, and the activity retention rate is above 95%; at 60°C and 80°C, the activity decreases, but it still remains above 80% at 60°C.

[0144] 8.3 Protease resistance (1) Sample preparation: Dissolve MFP-Hybrid-DOPA-Fe 3+ peptide in a suitable buffer to a concentration of 2 mg / mL.

[0145] Pepsin digestion: 0.1 M HCl-KCl buffer (pH 2.0) Trypsin digestion: 0.1 M Tris-HCl buffer (pH 8.0) (2) Enzymolysis: Pepsin digestion: Add pepsin (enzyme: substrate = 1:50, w / w), and incubate at 37°C for 0, 0.5, 1, 2, 4 hours.

[0146] Trypsin digestion: Add trypsin (enzyme: substrate = 1:50, w / w), and incubate at 37°C for 0, 0.5, 1, 2, 4 hours.

[0147] (3) Activity determination: The antioxidant activity at each time point was determined by the DPPH method.

[0148] (4) Stability calculation: Taking the activity at 0 hour as 100%, calculate the activity retention rate at other time points.

[0149] The results of protease resistance are as follows: Pepsin digestion: 0 h: 100% 0.5 h: 95.2% 1 h: 88.6% 2 h: 75.3% 4 h: 58.4% Trypsin digestion: 0 h: 100% 0.5 h: 92.1% 1 h: 82.5% 2 h: 68.7% 4 h: 53.2% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ peptide has certain resistance to protease digestion. After 2 h of pepsin digestion, the activity retention rate is 75.3%; after 2 h of trypsin digestion, the activity retention rate is 68.7%.

[0150] 8.4 Long-term stability (1) Sample preparation: Liquid preparation: Dissolve MFP-Hybrid-DOPA-Fe 3+ peptide in PBS (pH 7.4) to a concentration of 2 mg / mL, filter through a 0.22 μm filter membrane, and fill aseptically.

[0151] Lyophilized preparation: Lyophilize the liquid preparation and seal it.

[0152] (2) Storage conditions: Storage at 4°C: Liquid preparation, 0, 1, 3, 6 months.

[0153] Storage at 25°C: Lyophilized preparation, 0, 3, 6, 12 months.

[0154] (3) Activity determination: The antioxidant activity at each time point was determined by the DPPH method.

[0155] (4) Stability calculation: Taking the activity at 0 month as 100%, calculate the activity retention rate at other time points.

[0156] The long-term stability results are as follows: Liquid preparation (4°C): 0 month: 100% 1 month: 98.5% 3 months: 95.2% 6 months: 88.7% Lyophilized preparation (25°C): 0 month: 100% 3 months: 98.7% 6 months: 97.2% December: 93.5% The above results indicate that MFP-Hybrid-DOPA-Fe 3+ peptide preparation has good long-term stability. After storing the liquid preparation at 4 °C for 6 months, the activity retention rate is 88.7%; after storing the lyophilized preparation at 25 °C for 12 months, the activity retention rate is 93.5%.

[0157] VII. Preparation Development For convenient storage and use, the present invention has developed two dosage forms: liquid preparation and lyophilized preparation.

[0158] Example 9: Preparation of Peptide Preparation 9.1 Liquid Preparation (1) Formulation composition: Active ingredient: MFP-Hybrid-DOPA-Fe 3+ peptide, 2 mg / mL Stabilizer: Ascorbic acid (0.1%), Disodium EDTA (0.01%) Isotonicity regulator: Mannitol (5%) Surfactant: Polysorbate 80 (0.05%) Buffer system: 20 mM phosphate (pH 7.2 - 7.4) Water for injection: Appropriate amount (2) Preparation process: Preparation of buffer solution: Weigh appropriate amounts of disodium hydrogen phosphate and sodium dihydrogen phosphate and dissolve them in 80% of the final volume of water for injection.

[0159] Dissolution of excipients: Add disodium EDTA, mannitol, and ascorbic acid in sequence and stir until completely dissolved.

[0160] Dissolution of peptide: Slowly add peptide powder and stir at low speed until completely dissolved.

[0161] Add polysorbate 80 and stir evenly.

[0162] pH adjustment: Adjust to pH 7.3 ± 0.1 with 1 M NaOH or 1 M HCl.

[0163] Volume fixation: Add water for injection to the final volume.

[0164] Filtration and sterilization: Filter through a 0.22 μm PVDF membrane.

[0165] Filling: 5 mL brown glass bottle, replace the headspace with nitrogen, and seal with a crimped cap.

[0166] 9.2 Lyophilized Preparation (1) Formulation composition: Active ingredient: MFP-Hybrid-DOPA-Fe 3+ Peptide, 20 mg / vial Lyoprotectant: Trehalose (3%), Mannitol (2%) Buffer system: 10 mM Phosphate (pH 7.0 - 7.2) Stabilizer: Ascorbic acid (0.1%) Water for injection: q.s. (2) Preparation process: Formulation solution preparation: Prepare a formulation solution containing 20 mg / mL of peptide according to the method for liquid preparations.

[0167] Filling: Precisely quantify into 10 mL brown ampoules, filling volume 1 mL / vial.

[0168] Pre-freezing: -40°C, 4 hours

[0169] Primary drying: First stage: -35°C, pressure 100 Pa, 6 hours Second stage: -20°C, pressure 80 Pa, 6 hours Third stage: -5°C, pressure 60 Pa, 6 hours Fourth stage: 10°C, pressure 40 Pa, 6 hours Fifth stage: 25°C, pressure 20 Pa, 6 hours Secondary drying: 25°C, pressure <10 Pa, 8 hours.

[0170] Stoppering: Automatically stopper in the freeze dryer, displace with nitrogen, and crimp the cap.

[0171] 9.3 Quality control of the preparation (1) Liquid preparation: Appearance: Colorless or light yellow clear liquid, without visible foreign matters pH: 7.2 - 7.4 Content: 1.9 - 2.1 mg / mL Purity (HPLC): ≥95% Sterility test: Passed Endotoxin: <5 EU / mg peptide (2) Freeze-dried preparation: Appearance: White or off-white loose blocky solid Redissolution property: Completely dissolve within 30 seconds of shaking after adding 1 mL of water for injection at room temperature pH after redissolution: 7.0 - 7.2 Content: 19 - 21 mg / vial Purity (HPLC): ≥95% Sterility test: Passed Endotoxin: <5 EU / mg peptide Water content: ≤3% VIII. Optimization of Peptide Modification Methods In order to obtain the best peptide modification effect, the present invention has carried out optimization research on modification methods such as PEGylation, disulfide bond formation, and metal coordination.

[0172] Example 10: PEGylation Optimization 10.1 PEG Molecular Weight Optimization (1) Experimental design: PEGylate the MFP-Hybrid-DOPA peptide with mPEG-NHS of different molecular weights (2, 3.5, 5 kDa).

[0173] (2) Reaction conditions: 0.1 M borate buffer (pH 8.5), peptide:PEG molar ratio of 1:1.5, react at 4°C for 12 hours.

[0174] (3) Determination of modification rate and activity: Determine the modification rate and DPPH free radical scavenging activity after PEG modification of different molecular weights.

[0175] The results are as follows: 2 kDa mPEG-NHS: Modification rate 93.8%, activity retention rate 96.2% 3.5 kDa mPEG-NHS: Modification rate 95.7%, activity retention rate 92.3% 5 kDa mPEG-NHS: Modification rate 94.2%, activity retention rate 87.5% The above results show that 3.5 kDa mPEG-NHS is the optimal choice, with the highest modification rate and the activity retention rate still above 90%.

[0176] 10.2 Peptide:PEG Molar Ratio Optimization (1) Experimental design: Carry out PEGylation modification with different peptide:PEG molar ratios (1:1.2, 1:1.5, 1:2.0).

[0177] (2) Reaction conditions: 3.5 kDa mPEG-NHS, 0.1 M borate buffer (pH 8.5), react at 4°C for 12 hours.

[0178] (3) Determination of modification rate and activity: Determine the modification rate and DPPH free radical scavenging activity under different molar ratio conditions.

[0179] The results are as follows: Peptide: PEG = 1:1.2, modification rate 89.5%, activity retention rate 93.8% Peptide: PEG = 1:1.5, modification rate 95.7%, activity retention rate 92.3% Peptide: PEG = 1:2.0, modification rate 96.5%, activity retention rate 91.2% The above results show that the peptide:PEG molar ratio of 1:1.5 is a more suitable condition, with a modification rate as high as 95.7% and an activity retention rate of 92.3%.

[0180] Example 11: Optimization of disulfide bond formation method 11.1 Optimization of oxidation system (1) Experimental design: Explore the effect of different oxidation systems on disulfide bond formation: Air oxidation: 0.1 M Tris-HCl (pH 8.5), 48 hours GSSG oxidation: 0.5 mM GSSG, 0.1 M Tris-HCl (pH 8.0), 10 hours Iodine oxidation: 10 mM I2 / KI, pH 7.5, 2 hours GSSG + PDI: 0.5 mM GSSG + 0.1 U / mL PDI, 0.1 M Tris-HCl (pH 8.0), 10 hours (2) Determination of disulfide bond formation rate and activity: Determine the disulfide bond formation rate and DPPH radical scavenging activity under different oxidation systems.

[0181] The results are as follows: Air oxidation: Disulfide bond formation rate 62.3%, activity retention rate 85.2% GSSG oxidation: Disulfide bond formation rate 95.8%, activity retention rate 92.5% Iodine oxidation: Disulfide bond formation rate 94.2%, activity retention rate 78.3% GSSG + PDI: Disulfide bond formation rate 96.8%, activity retention rate 94.8% The above results show that the GSSG + PDI system is the optimal choice, with the highest disulfide bond formation rate and the highest activity retention rate.

[0182] Example 12: Optimization of metal coordination 12.1 Selection of metal ions (1) Experimental design: Compare the effect of different metal ions (Fe 3+ , Zn 2+ , Cu 2+） on the antioxidant activity of the peptide.

[0183] (2) Reaction conditions: MFP-Hybrid-DOPA peptide, peptide: metal molar ratio of 3:1, 10 mM HEPES (pH 7.4), reaction at room temperature for 3 hours.

[0184] (3) Activity assay: Measure the DPPH radical scavenging activity after coordination with different metal ions.

[0185] The results are as follows (expressed as IC50, unit: μM): Uncoordinated peptide: 92 Fe 3+ Coordinated peptide: 72 Zn 2+ Coordinated peptide: 79 Cu 2+ Coordinated peptide: 85 The above results show that Fe 3+ coordination can maximize the antioxidant activity of the peptide, and the IC50 is reduced to 72 μM.

[0186] 12.2 Peptide: metal molar ratio optimization (1) Experimental design: Compare the effects of different peptide:Fe 3+ molar ratios (5:1, 3:1, 2:1, 1:1) on the antioxidant activity of the peptide.

[0187] (2) Reaction conditions: MFP-Hybrid-DOPA peptide, 10 mM HEPES (pH 7.4), reaction at room temperature for 3 hours.

[0188] (3) Activity assay: Measure the DPPH radical scavenging activity under different molar ratio conditions.

[0189] The results are as follows (expressed as IC50, unit: μM): Peptide:Fe 3+ =5:1: 83 Peptide:Fe 3+ =3:1: 72 Peptide:Fe 3+ =2:1: 75 Peptide:Fe 3+ =1:1: 78 The above results show that when the peptide:Fe 3+ molar ratio is 3:1, the antioxidant activity is the best, and the IC50 is 72 μM.

[0190] IX. Peptide sequence optimization comparison To verify the superiority of the MFP-Hybrid peptide, the present invention systematically compared the antioxidant activities of four designed peptides.

[0191] Example 13: Study on the peptide sequence-activity relationship 13.1 Comparison of the antioxidant activities of four peptides Comparison of the DPPH and ABTS radical scavenging activities of four DOPA-modified peptides and their Fe 3+ coordinated peptides: IC50 (μM) by DPPH method: MFP3-AP1-DOPA: 156 MFP5-AP2-DOPA: 188 MFP6-AP3-DOPA: 125 MFP-Hybrid-DOPA: 92 MFP3-AP1-DOPA-Fe 3+ : 95 MFP5-AP2-DOPA-Fe 3+ : 105 MFP6-AP3-DOPA-Fe 3+ : 83 MFP-Hybrid-DOPA-Fe 3+ : 72 TEAC value (mmol Trolox / g peptide) by ABTS method: MFP3-AP1-DOPA: 0.98 MFP5-AP2-DOPA: 0.85 MFP6-AP3-DOPA: 1.23 MFP-Hybrid-DOPA: 1.42 MFP3-AP1-DOPA-Fe 3+ : 1.35 MFP5-AP2-DOPA-Fe 3+ : 1.18 MFP6-AP3-DOPA-Fe 3+ : 1.56 MFP-Hybrid-DOPA-Fe 3+ : 1.65 The above results show that among the four peptides, the MFP-Hybrid peptide exhibits the strongest antioxidant activity, and Fe 3+ coordination further enhances its activity.

[0192] 13.2 Structure-activity relationship analysis (1) Influence of DOPA content: The number of DOPA residues in the peptide is positively correlated with the antioxidant activity. MFP-Hybrid-DOPA contains 3 DOPA residues and has the highest activity; MFP5-AP2-DOPA also contains 3 DOPA residues, but its activity is relatively low, indicating that the number of DOPA alone cannot fully explain the activity difference.

[0193] (2) Influence of cysteine number: The peptides containing cysteine (MFP3-AP1, MFP6-AP3, and MFP-Hybrid) generally have higher activities than the peptides without cysteine (MFP5-AP2). Among them, MFP-Hybrid contains 3 cysteines and can form a more stable disulfide bond structure, with the highest activity.

[0194] (3) Influence of peptide chain length: The lengths of the four peptides are all 9 - 10 amino acids, and within this range, the length has no significant influence.

[0195] (4) Influence of amino acid arrangement: The DOPA and cysteine in the MFP-Hybrid peptide are distributed at intervals, forming a spatial conformation that is more conducive to antioxidant activity.

[0196] (5) Rationality of overall design: The MFP-Hybrid peptide successfully integrates the advantageous domains of mfp-3 (rich in DOPA), mfp-5 (rich in DOPA), and mfp-6 (rich in cysteine), achieving the maximization of antioxidant activity.

Claims

1. An antioxidant peptide based on mussel byssus, characterized in that: The peptide is one of the following sequences or a combination thereof: MFP3-AP1: GYCYGTYKC MFP5-AP2: KYTGYYKGK MFP6-AP3: CKYCGYYCG MFP-Hybrid: CGKYTCGYCK Wherein, the tyrosine residues of the peptide are partially or completely modified into 3,4-dihydroxyphenylalanine DOPA.

2. The antioxidant peptide according to claim 1, characterized in that: The peptide forms a stable three-dimensional structure through a disulfide bond, and / or is coupled to polyethylene glycol at the N-terminus, and / or forms a coordination complex with metal ions Fe³⁺ or Zn²⁺.

3. A method for preparing an antioxidant peptide based on mussel byssus according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Based on the structural characteristics of mussel byssus proteins mfp-3, mfp-5, and mfp-6, short peptides containing tyrosine and cysteine ​​residues were designed and synthesized; (2) using tyrosinase to convert the tyrosine residues in the peptide into DOPA; (3) promoting intrapeptide disulfide bond formation through a redox system; (4) Use metal ion coordination and / or PEGylation modification to enhance the stability and activity of peptides.

4. The preparation method according to claim 3, characterized in that: In step (2), the tyrosinase is mushroom tyrosinase, the reaction system is 50 mM phosphate buffer at pH 6.8, the enzyme concentration is 100-200 U / mL, the reaction is carried out at 25°C for 4-8 hours, and 0.5 mM ascorbic acid is added every 2 hours to prevent excessive oxidation of DOPA.

5. The preparation method according to claim 3, characterized in that: In step (3), the redox system is 0.5 mM oxidized glutathione GSSG and 0.1 U / mL protein disulfide isomerase PDI, and the reaction is carried out in 0.1 M Tris-HCl buffer at pH 8.0 at 25°C for 8-12 hours.

6. The preparation method according to claim 3, characterized in that: In step (4), FeCl3 or ZnCl2 was used for metal ion coordination, with a peptide:metal molar ratio of 3:1 or 2:1, and the reaction was carried out in 10 mM HEPES buffer at pH 7.4 at room temperature for 2-4 hours; mPEG-NHS, 2-5 kDa, with a peptide:PEG molar ratio of 1:1.5, was used for PEGylation in 0.1 M borate buffer at pH 8.5 at 4°C for 12 hours.

7. A preparation containing the antioxidant peptide according to any one of claims 1 to 2, characterized in that: The preparation is a liquid preparation, a freeze-dried preparation or a microcapsule preparation.

8. The preparation according to claim 7, characterized in that: The liquid preparation comprises the following components: antioxidant peptide 1-5 mg / mL, ascorbic acid 0.1%, disodium EDTA 0.01%, mannitol 5%, polysorbate 80 0.05%, phosphate buffer 20 mM, pH 7.2-7.4; Alternatively, the lyophilized preparation comprises the following components: antioxidant peptide 10-50 mg / bottle, trehalose 3%, mannitol 2%, ascorbic acid 0.1%, phosphate buffer 10 mM, pH 7.0-7.

2.

9. Use of the antioxidant peptide according to any one of claims 1 to 2 in the preparation of antioxidant health products or cosmetics.

10. Use of the antioxidant peptide according to any one of claims 1 to 2 in the preparation of anti-aging, anti-fatigue or anti-ultraviolet damage drugs.