An antioxidant peptide based on mussel byssus, preparation method, preparation and application thereof

Abstract

The present invention relates to the technical field of bioactive peptides, and particularly relates to an antioxidant peptide based on mussel foot thread, a preparation method, a preparation and its application. The antioxidant peptide is designed and synthesized with reference to the structural characteristics of mussel foot thread proteins mfp-3, mfp-5 and mfp-6, and contains four sequences, namely MFP3-AP1, MFP5-AP2, MFP6-AP3 and MFP-Hybrid. Tyrosine in the peptide is modified to DOPA by enzymatic means, and its stability and activity are enhanced through modifications such as disulfide bond formation, metal ion coordination and PEGylation. The antioxidant peptide has strong DPPH radical scavenging activity, ABTS radical scavenging activity, hydroxyl radical scavenging activity and ferric ion reducing ability, has a protective effect at the cellular level, and can be prepared into liquid preparations, freeze-dried preparations or microcapsule preparations, and is expected to be applied in the fields of antioxidant health products, cosmetics and drugs.

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 prevention of ultraviolet damage and other aspects. Background Art

[0002] In recent years, with the acceleration of the aging process of the population 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 free radicals are generated, including superoxide anion free radicals, hydroxyl free 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 when used for a long time. 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 contain rich 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 show significant antioxidant characteristics, but this patent mainly focuses on the mussel flesh extract 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 threads have 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 change 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 threads are protein fiber bundles secreted by mussels to firmly attach the mussels to the surface of substrates such as rocks. Mussels live in an oxidative environment in the intertidal zone, and their foot threads have extremely strong antioxidant capabilities, being able to maintain structural integrity and functional stability in the highly oxidative seawater environment for a long time. Research shows that mussel foot threads contain a variety of 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 threads are a material rich in collagen. However, due to their complex structural characteristics, it is challenging to extract the collagen component from them using food-grade techniques. Using endopeptidase proline to assist in extracting collagen from the foot threads of the blue mussel (Mytilus edulis), the yield is 138.82 mg / g dry weight, and the collagen extract is hydrolyzed with a variety of food-grade enzyme preparations. It is found that these hydrolysates have angiotensin-converting enzyme (ACE) inhibitory activity, dipeptidyl peptidase IV (DPP-IV) inhibitory activity, and antioxidant activity. However, this study only focuses on the collagen hydrolysates of mussel foot threads, especially their inhibitory activities against ACE and DPP-IV, rather than specifically researching 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 using pepsin or alkaline protease to hydrolyze mussel proteins, peptide segments with antioxidant activity and angiotensin-converting enzyme inhibitory activity are obtained. Research shows that the peptide obtained after 30 minutes of pepsin hydrolysis (GPH) exhibits the highest antioxidant and ACE inhibitory activities, and the protein fraction with a molecular weight less than 5 kDa has the strongest antioxidant and ACE inhibitory activities. However, these studies mainly focus on the mussel meat 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 has a complex structure, 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. The peptide is designed based on the structural characteristics of mussel byssus protein, tyrosine is converted to DOPA by enzymatic modification, and functional modifications such as disulfide bond formation, metal ion coordination, and PEGylation are carried out to solve the problems of insufficient activity and poor stability of antioxidant peptides in the prior art.

[0012] To achieve the above purpose, the present invention provides the following technical solutions:

[0013] An antioxidant peptide based on mussel byssus, the peptide is one of the following sequences or a combination thereof:

[0014] MFP3-AP1: GYCYGTYKC

[0015] MFP5-AP2: KYTGYYKGK

[0016] MFP6-AP3: CKYCGYYCG

[0017] MFP-Hybrid: CGKYTCGYCK

[0018] Among them, part or all of the tyrosine residues of the peptide are modified to 3,4-dihydroxyphenylalanine (DOPA).

[0019] 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.

[0020] The present invention also provides a preparation method for the above antioxidant peptide, including the following steps:

[0021] (1) Design and synthesize short peptides containing tyrosine and cysteine residues according to the structural characteristics of mussel foot protein mfp-3, mfp-5, and mfp-6;

[0022] (2) Use tyrosinase to convert tyrosine residues in the peptide into DOPA;

[0023] (3) Promote the formation of disulfide bonds within the peptide through an oxidation-reduction system;

[0024] (4) Enhance the stability and activity of the peptide by metal ion coordination and / or PEGylation modification.

[0025] 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.

[0026] Preferably, in step (3), the oxidation-reduction 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.

[0027] Preferably, in step (4), for metal ion coordination, FeCl3 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).

[0028] 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.

[0029] 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);

[0030] Alternatively, 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).

[0031] The present invention also provides the application of the above antioxidant peptides in the preparation of antioxidant health products or cosmetics, as well as in the preparation of anti-aging, anti-fatigue or anti-ultraviolet damage drugs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. Based on the structural characteristics of mussel foot thread protein, the present invention designs a series of short peptides containing tyrosine and cysteine residues, and converts tyrosine into DOPA through enzymatic modification, realizing the efficient preparation of antioxidant peptides and avoiding the difficulty of directly extracting from mussel foot threads.

[0034] 2. Through functional modifications such as disulfide bond formation, metal ion coordination and PEGylation, the present invention significantly improves the structural stability and antioxidant activity of the peptides. Among them, the MFP-Hybrid-DOPA-Fe 3+ peptide exhibits the best antioxidant activity, and the IC50 value of the DPPH free radical scavenging ability is 72 μM, which is significantly better than many existing natural antioxidant peptides.

[0035] 3. The antioxidant peptides described in the present invention show excellent antioxidant protection at the cellular level, can effectively reduce the oxidative damage of HepG2 cells induced by H2O2, and improve the cell survival rate (85.3%), approaching the protection effect of the positive control vitamin C (82.7%).

[0036] 4. The antioxidant peptides developed by the present invention have good stability. After pH stability, thermal stability, protease resistance and long-term stability tests, they all show excellent stability, laying a foundation for their application in the fields of health products, cosmetics and drugs. Detailed implementation manners

[0037] The following further elaborates on the present invention through specific examples in detail, 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.

[0038] The core concept of the present invention lies in, based on the structural characteristics of mussel foot thread protein, designing a series of short peptides containing tyrosine and cysteine residues, converting tyrosine into DOPA through enzymatic modification, and through functional modifications such as disulfide bond formation, metal ion coordination and PEGylation, improving the structural stability and antioxidant activity of the peptides.

[0039] I. Design and synthesis of peptides

[0040] The antioxidant peptides designed in the present invention mainly refer to the structural characteristics of mussel foot thread proteins mfp-3, mfp-5, and mfp-6. Analysis shows 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, the present invention designed four candidate peptides:

[0041] MFP3-AP1: Antioxidant peptide 1 based on mfp-3, with the sequence GYCYGTYKC and a molecular weight of 1065.2 Da;

[0042] MFP5-AP2: Antioxidant peptide 2 based on mfp-5, with the sequence KYTGYYKGK and a molecular weight of 1059.2 Da;

[0043] MFP6-AP3: Antioxidant peptide 3 based on mfp-6, with the sequence CKYCGYYCG and a molecular weight of 1067.3 Da;

[0044] MFP-Hybrid: Hybrid antioxidant peptide, with the sequence CGKYTCGYCK and a molecular weight of 1105.3 Da;

[0045] The design of these peptides follows the following principles:

[0046] (1) Each peptide contains 2 - 3 tyrosine residues for subsequent DOPA modification;

[0047] (2) Contains 1 - 3 cysteine residues for forming stable disulfide bonds;

[0048] (3) Add 1 - 2 lysine residues to improve water solubility;

[0049] (4) Maintain an amino acid length of 8 - 12 to balance activity and synthesis difficulty;

[0050] (5) The MFP-Hybrid peptide combines the structural characteristics of mfp-3, mfp-5, and mfp-6, aiming to obtain better antioxidant activity.

[0051] Example 1: Synthesis and Characterization of Peptides

[0052] 1.1 Synthesis of Peptides

[0053] The above four peptides were synthesized by the Fmoc solid-phase synthesis method. The specific steps are as follows:

[0054] (1) Resin preparation: Use Rink Amide MBHA resin (substitution degree of 0.5 mmol / g), weigh 0.2 mmol of resin and place it in a reactor, and swell it with DMF for 30 minutes.

[0055] (2) Fmoc deprotection: Add 20% piperidine / DMF solution and react at room temperature for 10 minutes. Repeat once, then wash the resin with DMF 5 times.

[0056] (3) Amino acid coupling: According to the order from the C-terminus to the N-terminus of the peptide, successively 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.

[0057] (4) Peptide chain elongation: Repeat steps (2) and (3) to complete the synthesis of the entire peptide chain.

[0058] (5) Terminal deprotection and cleavage: Remove the N-terminal Fmoc protecting group with 20% piperidine / DMF solution, 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.

[0059] (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.

[0060] 1.2 Purification of the peptide

[0061] Purify the crude peptide by semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions are as follows:

[0062] Chromatographic column: C18 column (250×21.2 mm, 10 μm)

[0063] Mobile phase A: 0.1% aqueous TFA solution

[0064] Mobile phase B: 0.1% TFA acetonitrile solution

[0065] Gradient: 10 - 40% B, 30 minutes

[0066] Flow rate: 10 mL / min

[0067] Detection wavelength: 220 nm and 280 nm

[0068] Collect the target peak and freeze-dry to obtain a peptide with a purity greater than 98%.

[0069] 1.3 Characterization of the peptide

[0070] Characterize the purified peptide by analytical HPLC and mass spectrometry (MALDI-TOF MS or ESI-MS). The analysis results are as follows:

[0071] MFP3-AP1: HPLC purity is 99.1%, and the molecular weight measured by MS is 1065.3 Da (theoretical value is 1065.2 Da).

[0072] MFP5-AP2: HPLC purity is 98.7%, and the molecular weight measured by MS is 1059.4 Da (theoretical value is 1059.2 Da).

[0073] MFP6-AP3: HPLC purity is 98.5%, and the molecular weight measured by MS is 1067.5 Da (theoretical value is 1067.3 Da).

[0074] MFP-Hybrid: HPLC purity is 99.3%, and the molecular weight measured by MS is 1105.2 Da (theoretical value is 1105.3 Da).

[0075] The above results indicate that all four peptides were successfully synthesized, and their purity and molecular weight meet the expectations.

[0076] II. Enzymatic DOPA Modification of Peptides

[0077] 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.

[0078] Example 2: Enzymatic DOPA Modification of Peptides

[0079] 2.1 Enzymatic Reaction

[0080] Taking the MFP-Hybrid peptide as an example, the process of enzymatic DOPA modification is described in detail:

[0081] (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.

[0082] (2) Add the enzyme: Add mushroom tyrosinase to the reaction solution to make the final enzyme concentration 200 U / mL.

[0083] (3) Reaction: Stir the reaction at 25 ± 1 °C with a stirring speed of 250 rpm. Take samples every 2 hours to monitor the reaction progress, and add 0.5 mM ascorbic acid to prevent over-oxidation of DOPA.

[0084] (4) Termination of reaction: After 6 hours of reaction, when the conversion rate of tyrosine reached over 92%, 5 mM ascorbic acid and 1 mM EDTA were added to terminate the reaction, and the pH was adjusted to 4.5 to inhibit the enzyme activity.

[0085] (5) Removal of enzyme: The enzyme was removed by centrifugation (4000 g, 15 minutes) using a 10 kDa ultrafiltration membrane, and the filtrate was collected.

[0086] 2.2 Purification of modified peptide

[0087] Ion exchange chromatography was used to purify the DOPA-modified peptide:

[0088] (1) Preparation of packing material: SP Sepharose Fast Flow cation exchange packing material was used and equilibrated with 20 mM acetate buffer (pH 4.5).

[0089] (2) Loading: The sample was adjusted to pH 4.5 and loaded onto the chromatography column.

[0090] (3) Elution: Unbound substances were eluted with 20 mM acetate buffer (pH 4.5), and then the target peptide was eluted with the same buffer containing an NaCl gradient (0 - 500 mM).

[0091] (4) Fraction collection: Fractions containing the DOPA peptide were collected based on the absorbance at UV280 nm and the Arnow reaction (DOPA-specific color reaction).

[0092] (5) Desalting and concentration: The collected fractions were desalted and concentrated using a 3 kDa ultrafiltration membrane, and then freeze-dried to obtain the DOPA-modified peptide powder.

[0093] 2.3 Determination of DOPA modification rate

[0094] The Arnow reaction was used to determine the DOPA content and calculate the DOPA modification rate:

[0095] (1) Standard curve: Using pure DOPA as the standard, a standard curve of 0.05 - 1.0 mg / mL was made.

[0096] (2) Sample preparation: The DOPA-modified peptide was dissolved in water to a concentration of 1 mg / mL.

[0097] (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.

[0098] (4) Modification rate calculation: Calculate the DOPA content based on the standard curve. Modification rate = actual DOPA content / theoretical fully modified DOPA content × 100%.

[0099] The DOPA modification results of the four peptides are as follows:

[0100] MFP3 - AP1 - DOPA: The modification rate is 90.5%, the HPLC purity is 97.2%, and the molecular weight measured by MS is 1113.2 Da (the theoretical value is 1113.2 Da)

[0101] MFP5 - AP2 - DOPA: The modification rate is 89.8%, the HPLC purity is 96.8%, and the molecular weight measured by MS is 1107.1 Da (the theoretical value is 1107.2 Da)

[0102] MFP6 - AP3 - DOPA: The modification rate is 91.2%, the HPLC purity is 97.5%, and the molecular weight measured by MS is 1115.2 Da (the theoretical value is 1115.3 Da)

[0103] MFP - Hybrid - DOPA: The modification rate is 92.5%, the HPLC purity is 98.1%, and the molecular weight measured by MS is 1137.1 Da (the theoretical value is 1137.3 Da)

[0104] The above results show that all four peptides have been successfully modified with DOPA, the modification rates are all above 90%, the purity is good, and the molecular weights are in line with expectations.

[0105] III. Functional enhancement modification of peptides

[0106] To further improve the stability and activity of the peptides, the present invention performs three functional enhancement modifications on the DOPA - modified peptides: disulfide bond formation, N - terminal PEGylation, and metal ion coordination.

[0107] Example 3: Disulfide bond formation of peptides

[0108] 3.1 Disulfide bond formation reaction

[0109] Taking the MFP - Hybrid - DOPA peptide as an example, the process of disulfide bond formation is described in detail:

[0110] (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.

[0111] (2) Add redox system: Add 0.5 mM oxidized glutathione (GSSG) and 0.1 U / mL protein disulfide isomerase (PDI) to the reaction solution.

[0112] (3) Reaction: Stir the reaction gently at 25 °C for 10 hours.

[0113] (4) Terminate the reaction: Add 10 mM DTT to terminate the reaction and adjust the pH to 3.0.

[0114] 3.2 Purification of disulfide - containing peptides

[0115] Purify the peptides with disulfide bonds formed by reversed - phase HPLC:

[0116] (1) Chromatographic conditions: C18 column, gradient elution with 15 - 45% acetonitrile for 30 minutes, flow rate 5 mL / min.

[0117] (2) Fraction collection: Collect the target peak and freeze - dry to obtain the purified disulfide - containing peptide.

[0118] 3.3 Determination of disulfide - forming rate

[0119] Determine the content of free sulfhydryl groups by Ellman reagent method and calculate the disulfide - forming rate:

[0120] (1) Sample preparation: Dissolve the peptide in 0.1 M phosphate buffer (pH 8.0) containing 1 mM EDTA to a concentration of 0.5 mg / mL.

[0121] (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.

[0122] (3) Formation rate calculation: Disulfide - forming rate=(1 - content of free sulfhydryl groups after disulfide bond formation / content of free sulfhydryl groups before disulfide bond formation)×100%.

[0123] The disulfide - forming results of four peptides are as follows:

[0124] MFP3 - AP1 - DOPA: Disulfide - forming rate is 95.3%, HPLC purity is 96.8%

[0125] MFP6 - AP3 - DOPA: Disulfide - forming rate is 94.5%, HPLC purity is 97.2%

[0126] MFP - Hybrid - DOPA: Disulfide - forming rate is 96.8%, HPLC purity is 97.8%

[0127] Since MFP5-AP2-DOPA does not contain cysteine residues, no disulfide bond formation modification occurs. The above results indicate that all three peptides containing cysteine residues have successfully formed disulfide bonds, and the formation rate is above 94%.

[0128] Example 4: N-terminal PEGylation Modification of Peptides

[0129] 4.1 PEGylation Reaction

[0130] Taking the MFP-Hybrid-DOPA peptide as an example, the process of N-terminal PEGylation is described in detail as follows:

[0131] (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.

[0132] (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.

[0133] (3) React: Gently stir the reaction at 4 °C for 12 hours.

[0134] (4) Terminate the reaction: Add an excess of glycine (final concentration 50 mM) to terminate the reaction.

[0135] 4.2 Purification of PEGylated Peptides

[0136] Purify the PEGylated peptides by ion exchange chromatography:

[0137] (1) Prepare the packing: Use a Resource S cation exchange chromatography column and equilibrate it with 20 mM sodium acetate buffer (pH 5.0).

[0138] (2) Load the sample: Adjust the reaction mixture to pH 5.0 and load it onto the chromatography column.

[0139] (3) Elute: Elute with 20 mM sodium acetate buffer (pH 5.0) containing an NaCl gradient (0 - 500 mM) for 30 minutes.

[0140] (4) Collect the fractions: Collect the fractions containing the PEGylated peptides based on the UV280 nm absorption.

[0141] (5) Desalt and concentrate: Use a 5 kDa ultrafiltration membrane to desalt and concentrate the collected fractions, and then lyophilize to obtain the PEGylated peptide powder.

[0142] 4.3 Determination of PEGylation Modification Rate

[0143] The PEGylation modification rate was analyzed by SDS - PAGE and MALDI - TOF MS:

[0144] (1) SDS - PAGE analysis: A 12% polyacrylamide gel was used, stained with Coomassie Brilliant Blue R - 250, and the relative intensities of each band were measured by a gel scanner.

[0145] (2) Mass spectrometry analysis: The molecular weight of the PEGylated peptide was determined by MALDI - TOF MS.

[0146] (3) Calculation of the modification rate: PEGylation modification rate = (peak area of PEGylated peptide) / (peak area of PEGylated peptide + peak area of unmodified peptide) × 100%.

[0147] The PEGylation results of the four peptides are as follows:

[0148] MFP3 - AP1 - DOPA - PEG: The modification rate was 91.2%, and the molecular weight measured by MALDI - TOF MS was approximately 4.6 kDa

[0149] MFP5 - AP2 - DOPA - PEG: The modification rate was 89.5%, and the molecular weight measured by MALDI - TOF MS was approximately 4.6 kDa

[0150] MFP6 - AP3 - DOPA - PEG: The modification rate was 90.8%, and the molecular weight measured by MALDI - TOF MS was approximately 4.6 kDa

[0151] MFP - Hybrid - DOPA - PEG: The modification rate was 93.2%, and the molecular weight measured by MALDI - TOF MS was approximately 4.6 kDa

[0152] The above results indicate that all four peptides were successfully PEGylated, and the modification rates were all above 89%.

[0153] Example 5: Metal ion coordination modification of peptides

[0154] 5.1 Metal ion coordination reaction

[0155] Taking the MFP - Hybrid - DOPA peptide as an example, the process of metal ion coordination was described in detail:

[0156] (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.

[0157] (2) Metal ion coordination: Slowly add FeCl3 solution to the reaction solution to make the peptide:Fe 3+ The molar ratio is 3:1, and stir the reaction at room temperature for 3 hours.

[0158] (3) In addition, using the same method, perform metal ion coordination with ZnCl2 to make the peptide:Zn 2+ The molar ratio is 2:1.

[0159] 5.2 Purification of metal-coordinated peptides

[0160] Purify the metal-coordinated peptides by gel filtration chromatography:

[0161] (1) Chromatographic conditions: Sephadex G-25, elute with 10 mM HEPES buffer (pH 7.4), flow rate 1 mL / min.

[0162] (2) Fraction collection: Collect the fractions containing metal-coordinated peptides based on the absorption at UV280 nm.

[0163] (3) Concentration: Freeze-dry the collected fractions to obtain the metal-coordinated peptide powder.

[0164] 5.3 Verification of metal coordination

[0165] Verify metal coordination by UV-vis spectroscopy and ICP-MS:

[0166] (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.

[0167] (2) ICP-MS analysis: Determine the metal content in the metal-coordinated peptide and calculate the peptide:metal molar ratio.

[0168] The results of the coordination of four peptides with Fe 3+ are as follows:

[0169] MFP3-AP1-DOPA-Fe 3+ : peptide:Fe 3+ The molar ratio is 3.2:1, and the UV-vis spectrum shows a characteristic absorption peak at 570 nm

[0170] MFP5-AP2-DOPA-Fe 3+ : peptide:Fe 3+ The molar ratio is 3.1:1, and the UV-vis spectrum shows a characteristic absorption peak at 570 nm

[0171] 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

[0172] 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

[0173] The results of the coordination of four peptides with Zn 2+ are as follows:

[0174] MFP3-AP1-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.1:1

[0175] MFP5-AP2-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.0:1

[0176] MFP6-AP3-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 2.2:1

[0177] MFP-Hybrid-DOPA-Zn 2+ : Peptide:Zn 2+ The molar ratio is 1.9:1

[0178] The above results indicate that all four peptides have been successfully modified by metal ion coordination, and the coordination ratio meets the expectations.

[0179] IV. Determination of antioxidant activity

[0180] In the present invention, multiple methods are used to determine the antioxidant activity of the peptides, including DPPH free radical scavenging activity, ABTS free radical scavenging activity, hydroxyl free radical scavenging activity, ferric ion reducing ability, and metal ion chelating ability, etc.

[0181] Example 6: Determination of antioxidant activity

[0182] 6.1 DPPH free radical scavenging activity

[0183] 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 capacity.

[0184] The determination method is as follows:

[0185] (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.

[0186] (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.

[0187] (3) Absorbance measurement: Measure the absorbance using an enzyme - linked immunosorbent assay (ELISA) reader at a wavelength of 517 nm.

[0188] (4) Calculation of scavenging rate: The 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 containing peptide.

[0189] (5) Calculation of IC50: Plot a curve of the scavenging rate against the peptide concentration and calculate the peptide concentration (IC50) required to achieve a 50% scavenging rate.

[0190] The results of the DPPH radical scavenging activities of each peptide are as follows (expressed as IC50, unit: μM):

[0191] Unmodified peptide:

[0192] MFP3 - AP1: >500

[0193] MFP5 - AP2: >500

[0194] MFP6 - AP3: >500

[0195] MFP - Hybrid: >500

[0196] DOPA - modified peptide:

[0197] MFP3 - AP1 - DOPA: 156

[0198] MFP5 - AP2 - DOPA: 188

[0199] MFP6 - AP3 - DOPA: 125

[0200] MFP - Hybrid - DOPA: 92

[0201] Disulfide - forming peptide:

[0202] MFP3 - AP1 - DOPA - SS: 118

[0203] MFP6 - AP3 - DOPA - SS: 102

[0204] MFP-Hybrid-DOPA-SS: 85

[0205] PEGylated peptide:

[0206] MFP3-AP1-DOPA-PEG: 142

[0207] MFP5-AP2-DOPA-PEG: 172

[0208] MFP6-AP3-DOPA-PEG: 115

[0209] MFP-Hybrid-DOPA-PEG: 88

[0210] Metal ion coordination peptide:

[0211] MFP3-AP1-DOPA-Fe 3+ : 95

[0212] MFP5-AP2-DOPA-Fe 3+ : 105

[0213] MFP6-AP3-DOPA-Fe 3+ : 83

[0214] MFP-Hybrid-DOPA-Fe 3+ : 72

[0215] MFP3-AP1-DOPA-Zn 2+ : 128

[0216] MFP5-AP2-DOPA-Zn 2+ : 150

[0217] MFP6-AP3-DOPA-Zn 2+ : 106

[0218] MFP-Hybrid-DOPA-Zn 2+ : 79

[0219] Control substance:

[0220] Vitamin C: 25

[0221] Glutathione: 75

[0222] BHT: 18

[0223] The above results indicate 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) Fe 3+ coordination can enhance the antioxidant activity of the peptide more than Zn 2+ coordination; (5) Among all the tested peptides, MFP-Hybrid-DOPA-Fe 3+ exhibits the strongest DPPH radical scavenging activity with an IC50 of 72 μM. Although it is lower than the synthetic antioxidant BHT (18 μM) and the natural antioxidant vitamin C (25 μM), it is superior to glutathione (75 μM) and various reported antioxidant peptides.

[0224] 6.2 ABTS radical scavenging activity

[0225] 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. Antioxidant substances can scavenge ABTS radicals, resulting in a decrease in absorption.

[0226] The determination method is as follows:

[0227] (1) Preparation of ABTS radicals: Mix 7 mM ABTS and 2.45 mM potassium persulfate in equal volumes and place in the dark for 16 hours to generate ABTS radicals. Dilute with PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm.

[0228] (2) Reaction: Take 20 μL of peptide solutions with different concentrations and mix them with 180 μL of ABTS radical solution, and react at room temperature for 6 minutes.

[0229] (3) Absorbance measurement: Measure the absorbance using a microplate reader at a wavelength of 734 nm.

[0230] (4) Calculation of scavenging rate: ABTS radical scavenging rate (%) = (Acontrol - Asample) / Acontrol × 100%.

[0231] (5) Calculation of TEAC value: Using Trolox as a standard, compare the scavenging rate of the peptide with the Trolox standard curve to calculate the Trolox equivalent antioxidant capacity (TEAC), with the unit of mmol Trolox / g peptide.

[0232] The results of the ABTS radical scavenging activity of each peptide are as follows (expressed as TEAC value, with the unit of mmol Trolox / g peptide):

[0233] DOPA-modified peptide:

[0234] MFP3-AP1-DOPA: 0.98

[0235] MFP5-AP2-DOPA: 0.85

[0236] MFP6-AP3-DOPA: 1.23

[0237] MFP-Hybrid-DOPA: 1.42

[0238] Metal ion coordination peptide:

[0239] MFP3-AP1-DOPA-Fe 3+ : 1.35

[0240] MFP5-AP2-DOPA-Fe 3+ : 1.18

[0241] MFP6-AP3-DOPA-Fe 3+ : 1.56

[0242] MFP-Hybrid-DOPA-Fe 3+ : 1.65

[0243] Control substance:

[0244] Vitamin C: 2.20

[0245] Glutathione: 1.45

[0246] BHT: 2.50

[0247] The above results show that MFP-Hybrid-DOPA-Fe 3+ exhibits 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).

[0248] 6.3 Hydroxyl radical scavenging activity

[0249] Hydroxyl radical is one of the most reactive reactive oxygen species 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 was measured.

[0250] The measurement method is as follows:

[0251] (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 H2O2 (0.5 mM), and react at 37 °C for 1 hour.

[0252] (2) Fluorescence measurement: Measure the fluorescence intensity at an excitation wavelength of 395 nm and an emission wavelength of 440 nm.

[0253] (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.

[0254] (4) IC50 calculation: 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.

[0255] The results of the hydroxyl radical scavenging activities of each peptide are as follows (expressed as IC50, unit: μM):

[0256] DOPA-modified peptides:

[0257] MFP3-AP1-DOPA: 158

[0258] MFP5-AP2-DOPA: 175

[0259] MFP6-AP3-DOPA: 132

[0260] MFP-Hybrid-DOPA: 118

[0261] Metal ion coordination peptides:

[0262] MFP-Hybrid-DOPA-Fe 3+ : 95

[0263] Control substances:

[0264] Vitamin C: 65

[0265] Glutathione: 85

[0266] The above results indicate that MFP-Hybrid-DOPA-Fe 3+ shows strong scavenging activity against hydroxyl radicals, with an IC50 of 95 μM, which is superior to glutathione (85 μM) but weaker than vitamin C (65 μM).

[0267] 6.4 Ferric ion reducing ability

[0268] The ferric ion reducing ability is an important indicator 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 assess its antioxidant activity.

[0269] The measurement method is as follows:

[0270] (1) Reaction system: Mix 1 mL of peptide solution (50 - 500 μg / mL), 2.5 mL of phosphate buffer (0.2 M, pH 6.6), and 2.5 mL of [K3Fe(CN)6] (1%), and incubate in a water bath at 50 °C for 20 minutes.

[0271] (2) Terminate the reaction: Add 2.5 mL of TCA (10%), and centrifuge to collect the supernatant.

[0272] (3) Color reaction: Mix 2.5 mL of the supernatant with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%).

[0273] (4) Absorbance measurement: Measure the absorbance at a wavelength of 700 nm. The higher the absorbance, the stronger the reducing ability.

[0274] (5) Calculation of reducing ability: Based on the FeSO4 standard curve, calculate the ferric ion reducing ability, with the unit of mmol Fe 2+ / g peptide.

[0275] The ferric ion reducing ability results of each peptide are as follows (unit: mmol Fe 2+ / g peptide):

[0276] DOPA-modified peptides:

[0277] MFP3-AP1-DOPA: 0.56

[0278] MFP5-AP2-DOPA: 0.48

[0279] MFP6-AP3-DOPA: 0.65

[0280] MFP-Hybrid-DOPA: 0.72

[0281] Metal ion coordination peptides:

[0282] MFP-Hybrid-DOPA-Fe 3+ : 0.85

[0283] Control substances:

[0284] Vitamin C: 1.50

[0285] Glutathione: 0.80

[0286] The above results indicate that MFP-Hybrid-DOPA-Fe 3+ exhibits strong ferric ion reducing ability, which is 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).

[0287] 6.5 Metal ion chelating ability

[0288] 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+ .

[0289] The determination method is as follows:

[0290] (1) Reaction system: Mix 1 mL of peptide solution (50 - 500 μg / mL), 0.1 mL of FeCl2 (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).

[0291] (2) Absorbance measurement: After reacting for 10 minutes, measure the absorbance at a wavelength of 562 nm.

[0292] (3) Chelation rate calculation: The chelation rate of Fe 2+ (%) = (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.

[0293] The results of the metal ion chelating ability of each peptide are as follows (expressed as the chelation rate of Fe 2+ , unit: %):

[0294] DOPA-modified peptide (500 μg / mL):

[0295] MFP3-AP1-DOPA: 48.5%

[0296] MFP5-AP2-DOPA: 42.3%

[0297] MFP6-AP3-DOPA: 55.8%

[0298] MFP-Hybrid-DOPA: 60.2%

[0299] Metal ion coordination peptide (500 μg / mL):

[0300] MFP-Hybrid-DOPA-Fe 3+ : 65.0%

[0301] Control substance (500 μg / mL):

[0302] EDTA: 98.5%

[0303] Glutathione: 58.7%

[0304] The above results show 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%).

[0305] V. Verification of antioxidant activity at the cellular level

[0306] To verify the antioxidant activity of the peptide at the cellular level, the present invention uses an H2O2-induced oxidative damage model of HepG2 cells for research.

[0307] Example 7: Protection experiment on H2O2-induced oxidative damage of HepG2 cells

[0308] 7.1 Cell culture

[0309] 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% CO2.

[0310] 7.2 Determination of cell viability

[0311] (1) Cell treatment: HepG2 cells were seeded in 96-well plates at a density of 1×104 cells / well and cultured for 24 hours, then grouped for treatment:

[0312] Control group: Normal culture

[0313] Model group: Treated with 200 μM H2O2 for 4 hours

[0314] Low-dose peptide group: Pretreated with 10 μM peptide for 2 hours, then added H2O2

[0315] Medium-dose peptide group: Pretreated with 50 μM peptide for 2 hours, then added H2O2

[0316] High-dose peptide group: Pretreated with 100 μM peptide for 2 hours, then added H2O2

[0317] Positive control group: Pretreat with 100 μM vitamin C for 2 hours, and then add H2O2

[0318] (2) Determination of cell viability by MTT method: After the treatment, add 20 μL of MTT (5 mg / mL) to each well, continue to culture for 4 hours, aspirate the medium, add 150 μL of DMSO, shake for 10 minutes, and measure the absorbance at a wavelength of 570 nm.

[0319] (3) Calculation of viability: Cell viability (%) = absorbance of treatment group / absorbance of control group × 100%.

[0320] The results of cell viability in each group are as follows:

[0321] Control group: 100%

[0322] Model group: 48.2%

[0323] MFP-Hybrid-DOPA-Fe 3+ Low-dose group (10 μM): 62.5%

[0324] MFP-Hybrid-DOPA-Fe 3+ Medium-dose group (50 μM): 75.8%

[0325] MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 85.3%

[0326] Vitamin C group (100 μM): 82.7%

[0327] The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can protect HepG2 cells from H2O2-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%).

[0328] 7.3 Determination of intracellular ROS level

[0329] (1) Cell treatment: The same as 7.2.

[0330] (2) ROS detection: Using the DCFH-DA probe method, after the treatment, add 10 μM DCFH-DA, incubate at 37 °C for 30 minutes, and wash 3 times with PBS.

[0331] (3) Fluorescence detection: Observe the intracellular ROS level (green fluorescence) under a fluorescence microscope and quantitatively detect the fluorescence intensity by flow cytometry.

[0332] (4) Relative fluorescence intensity calculation: Taking the fluorescence intensity of the control group as 1, calculate the relative fluorescence intensity of each group.

[0333] The relative fluorescence intensity results of each group are as follows:

[0334] Control group: 1.00

[0335] Model group: 3.76

[0336] MFP-Hybrid-DOPA-Fe 3+ Low-dose group (10 μM): 2.82

[0337] MFP-Hybrid-DOPA-Fe 3+ Medium-dose group (50 μM): 1.94

[0338] MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 1.45

[0339] Vitamin C group (100 μM): 1.52

[0340] The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can dose-dependently reduce the intracellular ROS level in HepG2 cells induced by H2O2, and 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).

[0341] 7.4 Detection of antioxidant enzyme activity

[0342] (1) Cell treatment: The same as 7.2, but use a 6-well plate, and seed 2×105 cells in each well.

[0343] (2) Cell lysis: After the treatment, collect the cells, wash them 2 times with PBS, add lysis buffer, and lyse them on ice for 30 minutes, then centrifuge to collect the supernatant.

[0344] (3) Activity detection: Use a commercial kit to measure the activities of SOD, CAT and GPx.

[0345] (4) Activity calculation: Taking the activity of the control group as 100%, calculate the relative activity of each group.

[0346] The relative activity results of antioxidant enzymes in each group are as follows:

[0347] Relative activity of SOD:

[0348] Control group: 100%

[0349] Model group: 65.3%

[0350] MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 94.0%

[0351] Vitamin C group (100 μM): 91.5%

[0352] Relative activity of CAT:

[0353] Control group: 100%

[0354] Model group: 58.7%

[0355] MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 91.1%

[0356] Vitamin C group (100 μM): 88.6%

[0357] Relative activity of GPx:

[0358] Control group: 100%

[0359] Model group: 62.4%

[0360] MFP-Hybrid-DOPA-Fe 3+ High-dose group (100 μM): 88.3%

[0361] Vitamin C group (100 μM): 85.6%

[0362] The above results indicate that MFP-Hybrid-DOPA-Fe 3+ can significantly increase the activities of antioxidant enzymes (SOD, CAT, and GPx) in HepG2 cells after H2O2 treatment, and the effect is slightly better than that of vitamin C.

[0363] VI. Evaluation of Peptide Stability

[0364] To evaluate the stability of the peptide, the present invention conducted pH stability, thermal stability, protease resistance, and long-term stability tests on the peptide.

[0365] Example 8: Stability Test of Peptide

[0366] 8.1 pH Stability

[0367] (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.

[0368] (2) Incubation: Incubate at 25 °C for 24 hours.

[0369] (3) Activity assay: The antioxidant activity of the peptide under various pH conditions was determined by the DPPH method.

[0370] (4) Stability calculation: Taking the activity at pH 7.4 as 100%, the activity retention rate under other pH conditions was calculated.

[0371] The pH stability results are as follows:

[0372] pH 2.0: 82.3%

[0373] pH 4.0: 91.5%

[0374] pH 6.0: 98.7%

[0375] pH 7.4: 100% (reference)

[0376] pH 9.0: 85.6%

[0377] 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%.

[0378] 8.2 Thermal stability

[0379] (1) Sample preparation: Dissolve the MFP-Hybrid-DOPA-Fe 3+ peptide in PBS (pH 7.4) to a concentration of 1 mg / mL.

[0380] (2) Heat treatment: Place the sample in a water bath at different temperatures (4 °C, 25 °C, 37 °C, 60 °C, 80 °C) for 30 minutes.

[0381] (3) Activity assay: The antioxidant activity of the peptide after treatment at each temperature was determined by the DPPH method.

[0382] (4) Stability calculation: Taking the activity at 4 °C as 100%, the activity retention rate under other temperature conditions was calculated.

[0383] The thermal stability results are as follows:

[0384] 4 °C: 100% (reference)

[0385] 25 °C: 99.2%

[0386] 37 °C: 95.8%

[0387] 60 °C: 83.4%

[0388] 80 °C: 67.9%

[0389] 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.

[0390] 8.3 Protease Resistance

[0391] (1) Sample Preparation: Dissolve MFP-Hybrid-DOPA-Fe 3+ peptide in a suitable buffer to a concentration of 2 mg / mL.

[0392] Pepsin Digestion: 0.1 M HCl-KCl buffer (pH 2.0)

[0393] Trypsin Digestion: 0.1 M Tris-HCl buffer (pH 8.0)

[0394] (2) Enzymatic Hydrolysis:

[0395] Pepsin Digestion: Add pepsin (enzyme: substrate = 1:50, w / w), and incubate at 37 °C for 0, 0.5, 1, 2, 4 hours.

[0396] Trypsin Digestion: Add trypsin (enzyme: substrate = 1:50, w / w), and incubate at 37 °C for 0, 0.5, 1, 2, 4 hours.

[0397] (3) Activity Assay: Use the DPPH method to measure the antioxidant activity at each time point.

[0398] (4) Stability Calculation: Take the activity at 0 hour as 100%, and calculate the activity retention rate at other time points.

[0399] The results of protease resistance are as follows:

[0400] Pepsin Digestion:

[0401] 0 hour: 100%

[0402] 0.5 hour: 95.2%

[0403] 1 hour: 88.6%

[0404] 2 hours: 75.3%

[0405] 4 hours: 58.4%

[0406] Trypsin Digestion:

[0407] 0 hours: 100%

[0408] 0.5 hours: 92.1%

[0409] 1 hour: 82.5%

[0410] 2 hours: 68.7%

[0411] 4 hours: 53.2%

[0412] The above results show that MFP-Hybrid-DOPA-Fe 3+ peptide has a certain resistance to protease digestion. After 2 hours of pepsin digestion, the activity retention rate is 75.3%; after 2 hours of trypsin digestion, the activity retention rate is 68.7%.

[0413] 8.4 Long-term stability

[0414] (1) Sample preparation:

[0415] 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.

[0416] Lyophilized preparation: Lyophilize the liquid preparation and seal it.

[0417] (2) Storage conditions:

[0418] Storage at 4°C: Liquid preparation, 0, 1, 3, 6 months.

[0419] Storage at 25°C: Lyophilized preparation, 0, 3, 6, 12 months.

[0420] (3) Activity determination: The antioxidant activity at each time point was determined by the DPPH method.

[0421] (4) Stability calculation: Taking the activity at 0 month as 100%, calculate the activity retention rate at other time points.

[0422] The long-term stability results are as follows:

[0423] Liquid preparation (4°C):

[0424] 0 month: 100%

[0425] 1 month: 98.5%

[0426] 3 months: 95.2%

[0427] 6 months: 88.7%

[0428] Lyophilized preparation (25 °C):

[0429] 0 months: 100%

[0430] 3 months: 98.7%

[0431] 6 months: 97.2%

[0432] 12 months: 93.5%

[0433] 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%.

[0434] VII. Preparation Development

[0435] For convenient storage and use, the present invention has developed two dosage forms: liquid preparation and lyophilized preparation.

[0436] Example 9: Preparation of Peptide Preparation

[0437] 9.1 Liquid Preparation

[0438] (1) Formulation composition:

[0439] Active ingredient: MFP-Hybrid-DOPA-Fe 3+ peptide, 2 mg / mL

[0440] Stabilizer: Ascorbic acid (0.1%), Disodium EDTA (0.01%)

[0441] Isotonicity regulator: Mannitol (5%)

[0442] Surfactant: Polysorbate 80 (0.05%)

[0443] Buffer system: 20 mM phosphate (pH 7.2 - 7.4)

[0444] Water for injection: Appropriate amount

[0445] (2) Preparation process:

[0446] Buffer preparation: Weigh appropriate amounts of disodium hydrogen phosphate and sodium dihydrogen phosphate, and dissolve them in 80% of the final volume of water for injection.

[0447] Auxiliary material dissolution: Add disodium EDTA, mannitol, and ascorbic acid in sequence, and stir until completely dissolved.

[0448] Peptide dissolution: Slowly add the peptide powder and stir at low speed until completely dissolved.

[0449] Add polysorbate 80 and stir well to mix evenly.

[0450] pH adjustment: Adjust to pH 7.3 ± 0.1 with 1 M NaOH or 1 M HCl.

[0451] Volume fixation: Add water for injection to the final volume.

[0452] Filter sterilization: Filter through a 0.22 μm PVDF membrane.

[0453] Filling: 5 mL brown glass bottles, displace the headspace with nitrogen, and seal with a crimp cap.

[0454] 9.2 Lyophilized preparation

[0455] (1) Formulation composition:

[0456] Active ingredient: MFP-Hybrid-DOPA-Fe 3+ Peptide, 20 mg / vial

[0457] Lyoprotectant: Trehalose (3%), mannitol (2%)

[0458] Buffer system: 10 mM phosphate (pH 7.0 - 7.2)

[0459] Stabilizer: Ascorbic acid (0.1%)

[0460] Water for injection: Appropriate amount

[0461] (2) Preparation process:

[0462] Formulation solution preparation: Prepare a formulation solution containing 20 mg / mL of peptide according to the method of the liquid preparation.

[0463] Filling: Precisely quantify 10 mL brown ampoules, with a filling volume of 1 mL / vial.

[0464] Pre-freezing: -40°C, 4 hours.

[0465] Primary drying:

[0466] First stage: -35°C, pressure 100 Pa, 6 hours

[0467] Second stage: -20°C, pressure 80 Pa, 6 hours

[0468] Third stage: -5°C, pressure 60 Pa, 6 hours

[0469] Fourth stage: 10°C, pressure 40 Pa, 6 hours

[0470] Fifth stage: 25°C, pressure 20 Pa, 6 hours

[0471] Post-drying: 25 °C, pressure < 10 Pa, 8 hours.

[0472] Stoppering: Automatic stoppering inside the freeze dryer, nitrogen replacement, capping.

[0473] 9.3 Quality control of the preparation

[0474] (1) Liquid preparation:

[0475] Appearance: Colorless or light yellow clear liquid, no visible foreign matters

[0476] pH: 7.2 - 7.4

[0477] Content: 1.9 - 2.1 mg / mL

[0478] Purity (HPLC): ≥ 95%

[0479] Sterility test: Passed

[0480] Endotoxin: < 5 EU / mg peptide

[0481] (2) Freeze-dried preparation:

[0482] Appearance: White or off-white loose block solid

[0483] Redissolution property: Add 1 mL of water for injection at room temperature, and it can be completely dissolved within 30 seconds of shaking.

[0484] pH after redissolution: 7.0 - 7.2

[0485] Content: 19 - 21 mg / vial

[0486] Purity (HPLC): ≥ 95%

[0487] Sterility test: Passed

[0488] Endotoxin: < 5 EU / mg peptide

[0489] Water content: ≤ 3%

[0490] VIII. Optimization of peptide modification methods

[0491] 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.

[0492] Example 10: Optimization of PEGylation

[0493] 10.1 Optimization of PEG molecular weight

[0494] (1) Experimental design: PEGylation modification of MFP-Hybrid-DOPA peptide was carried out using mPEG-NHS with different molecular weights (2, 3.5, 5 kDa).

[0495] (2) Reaction conditions: 0.1 M borate buffer (pH 8.5), peptide:PEG molar ratio of 1:1.5, reaction at 4 °C for 12 hours.

[0496] (3) Determination of modification rate and activity: The modification rate and DPPH radical scavenging activity after PEG modification with different molecular weights were determined.

[0497] The results are as follows:

[0498] 2 kDa mPEG-NHS: Modification rate 93.8%, activity retention rate 96.2%

[0499] 3.5 kDa mPEG-NHS: Modification rate 95.7%, activity retention rate 92.3%

[0500] 5 kDa mPEG-NHS: Modification rate 94.2%, activity retention rate 87.5%

[0501] The above results indicate that 3.5 kDa mPEG-NHS is the optimal choice, with the highest modification rate and the activity retention rate still above 90%.

[0502] 10.2 Optimization of peptide:PEG molar ratio

[0503] (1) Experimental design: PEGylation modification was carried out using different peptide:PEG molar ratios (1:1.2, 1:1.5, 1:2.0).

[0504] (2) Reaction conditions: 3.5 kDa mPEG-NHS, 0.1 M borate buffer (pH 8.5), reaction at 4 °C for 12 hours.

[0505] (3) Determination of modification rate and activity: The modification rate and DPPH radical scavenging activity under different molar ratio conditions were determined.

[0506] The results are as follows:

[0507] Peptide:PEG = 1:1.2: Modification rate 89.5%, activity retention rate 93.8%

[0508] Peptide:PEG = 1:1.5: Modification rate 95.7%, activity retention rate 92.3%

[0509] Peptide:PEG = 1:2.0: Modification rate 96.5%, activity retention rate 91.2%

[0510] The above results indicate that a 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%.

[0511] Example 11: Optimization of the disulfide bond formation method

[0512] 11.1 Optimization of the oxidation system

[0513] (1) Experimental design: Explore the effect of different oxidation systems on the formation of disulfide bonds:

[0514] Air oxidation: 0.1 M Tris-HCl (pH 8.5), 48 hours

[0515] GSSG oxidation: 0.5 mM GSSG, 0.1 M Tris-HCl (pH 8.0), 10 hours

[0516] Iodine oxidation: 10 mM I2 / KI, pH 7.5, 2 hours

[0517] GSSG + PDI: 0.5 mM GSSG + 0.1 U / mL PDI, 0.1 M Tris-HCl (pH 8.0), 10 hours

[0518] (2) Determination of the disulfide bond formation rate and activity: Measure the disulfide bond formation rate and DPPH radical scavenging activity under different oxidation systems.

[0519] The results are as follows:

[0520] Air oxidation: Disulfide bond formation rate 62.3%, activity retention rate 85.2%

[0521] GSSG oxidation: Disulfide bond formation rate 95.8%, activity retention rate 92.5%

[0522] Iodine oxidation: Disulfide bond formation rate 94.2%, activity retention rate 78.3%

[0523] GSSG + PDI: Disulfide bond formation rate 96.8%, activity retention rate 94.8%

[0524] The above results indicate that the GSSG + PDI system is the optimal choice, with the highest disulfide bond formation rate and the highest activity retention rate.

[0525] Example 12: Optimization of metal coordination

[0526] 12.1 Selection of metal ions

[0527] (1) Experimental design: Compare different metal ions (Fe 3+ , Zn 2+ , Cu2+） Effect on the antioxidant activity of peptides.

[0528] (2) Reaction conditions: MFP-Hybrid-DOPA peptide, peptide: metal molar ratio of 3:1, 10 mM HEPES (pH 7.4), react at room temperature for 3 hours.

[0529] (3) Activity determination: Determine the DPPH radical scavenging activity after coordination with different metal ions.

[0530] The results are as follows (expressed as IC50, unit: μM):

[0531] Uncoordinated peptide: 92

[0532] Fe 3+ Coordinated peptide: 72

[0533] Zn 2+ Coordinated peptide: 79

[0534] Cu 2+ Coordinated peptide: 85

[0535] The above results show that Fe 3+ coordination can maximize the antioxidant activity of the peptide, and the IC50 is reduced to 72 μM.

[0536] 12.2 Optimization of peptide: metal molar ratio

[0537] (1) Experimental design: Compare the effect of different peptide: Fe 3+ molar ratios (5:1, 3:1, 2:1, 1:1) on the antioxidant activity of the peptide.

[0538] (2) Reaction conditions: MFP-Hybrid-DOPA peptide, 10 mM HEPES (pH 7.4), react at room temperature for 3 hours.

[0539] (3) Activity determination: Determine the DPPH radical scavenging activity under different molar ratio conditions.

[0540] The results are as follows (expressed as IC50, unit: μM):

[0541] Peptide: Fe 3+ =5:1:83

[0542] Peptide: Fe 3+ =3:1:72

[0543] Peptide: Fe 3+ =2:1:75

[0544] Peptide: Fe 3+ =1:1:78

[0545] The above results indicate that for the peptide:Fe 3+ When the molar ratio is 3:1, the antioxidant activity is optimal, and the IC50 is 72 μM.

[0546] IX. Comparison of Peptide Sequence Optimization

[0547] To verify the superiority of the MFP-Hybrid peptide, the present invention systematically compared the antioxidant activities of four designed peptides.

[0548] Example 13: Study on the Relationship between Peptide Sequence and Activity

[0549] 13.1 Comparison of the Antioxidant Activities of Four Peptides

[0550] Compare the DPPH and ABTS radical scavenging activities of four DOPA-modified peptides and their Fe 3+ coordinated peptides:

[0551] IC50 (μM) by DPPH method:

[0552] MFP3-AP1-DOPA: 156

[0553] MFP5-AP2-DOPA: 188

[0554] MFP6-AP3-DOPA: 125

[0555] MFP-Hybrid-DOPA: 92

[0556] MFP3-AP1-DOPA-Fe 3+ : 95

[0557] MFP5-AP2-DOPA-Fe 3+ : 105

[0558] MFP6-AP3-DOPA-Fe 3+ : 83

[0559] MFP-Hybrid-DOPA-Fe 3+ : 72

[0560] TEAC value (mmol Trolox / g peptide) by ABTS method:

[0561] MFP3-AP1-DOPA: 0.98

[0562] MFP5-AP2-DOPA: 0.85

[0563] MFP6-AP3-DOPA: 1.23

[0564] MFP-Hybrid-DOPA: 1.42

[0565] MFP3-AP1-DOPA-Fe 3+ : 1.35

[0566] MFP5-AP2-DOPA-Fe 3+ : 1.18

[0567] MFP6-AP3-DOPA-Fe 3+ : 1.56

[0568] MFP-Hybrid-DOPA-Fe 3+ : 1.65

[0569] The above results show that among the four peptides, MFP-Hybrid peptide exhibits the strongest antioxidant activity, and the coordination of Fe 3+ further enhances its activity.

[0570] 13.2 Structure-activity relationship analysis

[0571] (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 DOPA number alone cannot fully explain the activity difference.

[0572] (2) Influence of cysteine number: Peptides containing cysteine (MFP3-AP1, MFP6-AP3, and MFP-Hybrid) generally have higher activities than 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.

[0573] (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.

[0574] (4) Influence of amino acid arrangement: DOPA and cysteine in MFP-Hybrid peptide are distributed at intervals, forming a spatial conformation more favorable for antioxidant activity.

[0575] (5) Rationality of overall design: 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 foot thread, characterized in that: The antioxidant peptide has the following sequence: MFP-Hybrid: CGKYTCGYCK, wherein, part or all of the tyrosine residues of the antioxidant peptide are modified to 3,4-dihydroxyphenylalanine DOPA.

2. A modified product of an antioxidant peptide, characterized in that: It is obtained by the antioxidant peptide forming a stable three-dimensional structure through disulfide bonds, and / or the N-terminus being coupled with polyethylene glycol, and / or forming a coordination complex with metal ions Fe³⁺ or Zn²⁺. The antioxidant peptide has the following sequence: MFP-Hybrid: CGKYTCGYCK, wherein, part or all of the tyrosine residues of the antioxidant peptide are modified to 3,4-dihydroxyphenylalanine DOPA.

3. The preparation method of the modified product of the antioxidant peptide according to claim 2, characterized in that, It includes the following steps: (1) According to the structural characteristics of mussel foot thread proteins mfp-3, mfp-5, and mfp-6, design and synthesize a short peptide MFP-Hybrid: CGKYTCGYCK containing tyrosine and cysteine residues; (2) Use tyrosinase to convert the tyrosine residues in the peptide into DOPA; (3) Promote the formation of disulfide bonds within the peptide through an oxidation-reduction system; (4) Use metal ion coordination and / or PEGylation modification to enhance the stability and activity of the peptide.

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 over-oxidation of DOPA.

5. The preparation method according to claim 3, characterized in that: In step (3), the oxidation-reduction 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 at pH 8.0, and the reaction is carried out at 25 °C for 8-12 hours.

6. The preparation method according to claim 3, characterized in that: In step (4), for metal ion coordination, FeCl3 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 at pH 7.4; for PEGylation, mPEG-NHS with a molecular weight of 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 at pH 8.

5.

7. A preparation containing the antioxidant peptide described in claim 1 or a preparation containing a modified product of the antioxidant peptide described in claim 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 contains the following components: 1-5 mg / mL of the antioxidant peptide according to claim 1 or 2, 0.1% ascorbic acid, 0.01% disodium EDTA, 5% mannitol, 0.05% polysorbate 80, 20 mM phosphate buffer, pH 7.2-7.4; Or, the freeze-dried preparation contains the following components: The antioxidant peptide described in claim 1 or 2 is 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 described in claim 1 or a modified product of the antioxidant peptide described in claim 2 in the preparation of antioxidant health products or cosmetics.

10. Use of the antioxidant peptide described in claim 1 or a modified product of the antioxidant peptide described in claim 2 in the preparation of drugs for anti - aging and preventing ultraviolet damage.