Collagen peptide with antioxidant function and hesperetin composition thereof

Abstract

The invention discloses a collagen peptide and a dressing preparation formed by combining the collagen peptide and hesperetin. And the collagen peptide is obtained by carrying out enzymolysis on amino acid sequences as shown in SEQ ID NO.5-SEQ ID NO.8. The collagen peptide is prepared from the collagen peptide. The polypeptide and the preparation thereof have the functions of resisting oxidation and repairing skin injury.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide preparation, and particularly to a polypeptide and its composition with antioxidant and skin damage repair functions. Background Art

[0002] Collagen peptide, as a natural bioactive substance, has been widely used in the field of skin repair. It has excellent biocompatibility, degradability, and the ability to promote cell proliferation and migration, and can significantly accelerate the regeneration of epithelial tissues. However, the effect of using collagen peptide alone still has certain limitations, especially its weak antioxidant and anti-inflammatory effects, which are difficult to meet the needs of complex wound repair.

[0003] Hesperetin is a natural flavonoid compound with various biological activities such as significant antioxidant, anti-inflammatory, and promoting skin cell repair. Research shows that hesperetin can effectively improve the wound microenvironment and accelerate epithelial repair by mechanisms such as scavenging free radicals, inhibiting the release of inflammatory factors, and promoting collagen synthesis. However, hesperetin has the property of being poorly soluble in water, which greatly limits its bioavailability in practical applications. In the prior art, CN113181114B proposed a method of improving the solubility of hesperetin using basic amino acids (such as arginine), but this method promotes the dissolution of hesperetin in an oily solution. How to solve the problem of promoting the dissolution of hesperetin in an aqueous solvent is still an urgent problem to be solved.

[0004] To solve the above problems, the present invention provides a composition containing collagen peptide and hesperetin. The inventors successfully obtained collagen peptides with unique solubility properties from earthworm collagen through specific enzymatic hydrolysis conditions, and found that hesperetin can be efficiently dissolved in this collagen peptide solution to form a stable complex. This composition not only solves the problem of the poor solubility of hesperetin, but also significantly improves the epithelial repair effect through synergistic effects. Comparative experiments show that the composition of the present invention is superior to using only collagen peptide or commercially available collagen products in promoting wound healing, reducing inflammatory reactions, and improving tissue regeneration.

[0005] Based on the above findings, the present invention proposes a novel epithelial barrier dressing. This dressing utilizes the synergistic effect of collagen peptide and hesperetin, can effectively accelerate skin wound repair, improve the wound microenvironment, and has good biocompatibility and stability. The present invention not only provides a new solution for the application of hesperetin, but also provides important technical support for the development of skin repair materials, and has broad application prospects and market value. Summary of the Invention

[0006] The present invention aims to provide a novel epithelial barrier dressing, which utilizes the synergistic effect of collagen peptides and hesperetin, can effectively accelerate the repair of skin wounds, improve the wound microenvironment, and has good biocompatibility and stability. To achieve the above object, the present invention provides the following technical solutions.

[0007] First, the present invention provides a protein, which can be enzymatically cleaved to form a protein with antioxidant functional polypeptides. It is characterized in that the sequence of the protein is shown in any one of SEQ ID NO.5 to SEQ ID NO.8, and the enzyme consists of any one or a combination of trypsin, papain, and alkaline protease.

[0008] Optionally, the preparation method of the protein includes but is not limited to genetic engineering expression, natural extraction, etc.

[0009] Second, the present invention provides a gene encoding the above protein sequence.

[0010] Third, the present invention provides a polypeptide with antioxidant function, which is characterized in that the polypeptide is obtained by enzymatically cleaving the aforementioned protein with a protease, and the protease consists of any one of trypsin, papain, and alkaline protease.

[0011] Optionally, the protease is enzymatically cleaved according to the following combinations and conditions

[0012] Table 1 Enzymatic cleavage combination conditions

[0013]

[0014] Preferably, enzyme combination 4 is used for the enzymatic hydrolysis of SEQ ID NO.5 and / or SEQ ID NO.7.

[0015] Preferably, enzyme combination 6 is used for the enzymatic hydrolysis of SEQ ID NO.6 and / or SEQ ID NO.7.

[0016] Preferably, enzyme combination 5 is used for the enzymatic hydrolysis of SEQ ID NO.8, SEQ ID NO.5 and / or SEQ ID NO.7.

[0017] Third, the present invention provides a composition with skin injury repair function, which is characterized in that the composition contains the aforementioned polypeptide.

[0018] Preferably, the composition further contains hesperetin.

[0019] Preferably, the composition is an external preparation.

[0020] Optionally, the dosage form of the composition can be gel, cream / ointment, spray, patch / membrane, powder, foam, hydrogel, microgel, nano - preparation, etc.

[0021] Optionally, the composition further comprises other active ingredients and / or excipients.

[0022] Optionally, the other active ingredients include hyaluronic acid, growth factors, antibacterial components, vitamins.

[0023] Optionally, different dosage forms can be selected from, but are not limited to, the following excipients

[0024] Excipients for gel dosage form:

[0025] Gel matrix: Carbomer, hydroxypropyl methylcellulose (HPMC), sodium alginate, agar, etc.

[0026] Thickening agents: xanthan gum, polyvinyl alcohol (PVA), etc.

[0027] Humectants: glycerol, propylene glycol, hyaluronic acid, etc.

[0028] Excipients for cream / ointment dosage form

[0029] Emulsifiers: glyceryl stearate, polysorbate (Tween series), span (Span series), etc.

[0030] Oil matrices: petrolatum, lanolin, mineral oil, etc.

[0031] Stabilizers: disodium ethylenediaminetetraacetate (EDTA-2Na), antioxidants (such as vitamin E), etc.

[0032] Excipients for spray dosage form

[0033] Solvents: ethanol, water, propylene glycol, etc.

[0034] Solubilizers: polyethylene glycol (PEG), Tween (Tween series), etc.

[0035] Preservatives: phenoxyethanol, parabens, etc.

[0036] Excipients for patch / membrane dosage form

[0037] Film-forming materials: polyvinyl alcohol (PVA), chitosan, gelatin, etc.

[0038] Plasticizers: glycerol, sorbitol, etc.

[0039] Adhesives: polyacrylate, sodium carboxymethyl cellulose (CMC-Na), etc.

[0040] Excipients for powder dosage form

[0041] Fillers: lactose, microcrystalline cellulose, starch, etc.

[0042] Anticaking agents: silica, talc, etc.

[0043] Dispersants: Polyvinylpyrrolidone (PVP), Hydroxypropyl cellulose (HPC), etc.

[0044] Nanopreparation excipients

[0045] Carrier materials: Liposomes (such as phospholipids, cholesterol), Polymer nanoparticles (such as PLGA, chitosan), etc.

[0046] Surfactants: Polysorbate (Tween series), Polyethylene glycol (PEG), etc. Stabilizers: Mannitol, Trehalose, etc.

[0047] Hydrogel formulation excipients

[0048] Crosslinking agents: Glutaraldehyde, Calcium ions (for sodium alginate), etc.

[0049] Humectants: Glycerol, Hyaluronic acid, etc.

[0050] Thickeners: Xanthan gum, Carbomer, etc.

[0051] Preferably, the composition is a gel preparation, and the composition components are as follows:

[0052] Collagen peptide: 1% - 15% (w / w), preferably 1% - 10% (w / w).

[0053] Hesperetin: Saturated solubility.

[0054] Gel matrix (such as sodium alginate, gelatin or sodium carboxymethylcellulose): 10% - 20% (w / w).

[0055] Plasticizers (such as glycerol or propylene glycol): 5% - 10% (w / w).

[0056] Crosslinking agents (such as calcium chloride or glutaraldehyde): 1% - 3% (w / w).

[0057] Humectants (such as hyaluronic acid or aloe extract): 2% - 5% (w / w).

[0058] Antibacterial agents (such as phenoxyethanol or tea tree oil): 0.5% - 1% (w / w).

[0059] Furthermore, the present invention relates to the use of the aforementioned protein or polypeptide in the preparation of a drug for antioxidant and / or skin damage repair.

[0060] Furthermore, the present invention relates to a method for preparing a composition having a skin damage repair function, characterized by comprising the following steps:

[0061] Step 1, Preparation of a collagen peptide - hesperetin complex, wherein the collagen peptide is the aforementioned polypeptide;

[0062] Step 2, preparation of the gel matrix;

[0063] Step 3: Add other functional components;

[0064] Step 4: Molding of the gel dressing.

[0065] Optionally, the gel matrix is selected from, but not limited to, any one of sodium alginate, gelatin, or sodium carboxymethylcellulose.

[0066] Optionally, the other functional components are selected from, but not limited to, plasticizers, humectants, or antibacterial agents.

[0067] Preferably, the specific steps of the preparation method are as follows,

[0068] Step 1: Preparation of the collagen peptide - hesperetin complex

[0069] Solvent: Take deionized water.

[0070] Solute: Add collagen peptide (SEQ ID NO.7 + enzyme combination 4), stir until completely dissolved to form a 5% (w / w) collagen peptide solution.

[0071] Add hesperetin: Add a sufficient amount of hesperetin, stir and heat to 40 - 50 °C, and continuously stir for 2 - 4 hours to fully dissolve hesperetin and form a complex.

[0072] Filtration: Filter with a 0.45 μm filter membrane to remove undissolved hesperetin to obtain a clear collagen peptide - hesperetin complex solution.

[0073] Step 2: Preparation of the gel matrix

[0074] Add sodium alginate, heat to 50 - 60 °C, stir until completely dissolved to form a 10% (w / v) gel solution.

[0075] Step 3: Add functional components

[0076] Plasticizer: Add glycerol and stir evenly. The mass ratio of glycerol to the deionized water in Step 1 is 50:1.

[0077] Humectant: Add hyaluronic acid and stir until completely dissolved. The mass ratio of hyaluronic acid to the deionized water in Step 1 is 20:1.

[0078] Antibacterial agent: Add phenoxyethanol and stir evenly. The mass ratio of hyaluronic acid to the deionized water in Step 1 is 5:1.

[0079] Step 4: Molding of the gel dressing

[0080] Crosslinking: Pour the mixed solution into a mold and spread it evenly into a thin layer (thickness 1 - 2 mm).

[0081] Solidification: Spray with 2% (w / v) calcium chloride solution (or use glutaraldehyde vapor) for crosslinking and let stand for 10 minutes.

[0082] Drying: Dry at 40 - 50 °C until the gel dressing forms a transparent film.

[0083] Cutting: Cut into appropriate sizes (such as 5 cm × 5 cm) according to requirements.

[0084] Step 5: Post - treatment

[0085] Packaging: Seal the gel dressing in a sterile packaging bag and store it in a cool and dry place.

[0086] Sterilization: Can be sterilized by ultraviolet irradiation or γ - ray irradiation to ensure the sterility of the product.

[0087] Compared with the prior art, the present invention has the following advantages:

[0088] 1) The present invention obtains polypeptides and compositions with antioxidant and wound - repair - promoting effects.

[0089] 2) The collagen peptide - hesperetin microgel product with high solubility, stability and sustained - release effect meets various application requirements.

[0090] 3) The present invention proposes a novel epithelial barrier dressing, which utilizes the synergistic effect of collagen peptide and hesperetin, can effectively accelerate skin wound repair, improve the wound microenvironment, and has good biocompatibility and stability. Description of the Drawings

[0091] The following combines the drawings and specific embodiments to detail the method of the present invention and its beneficial effects.

[0092] Figure 1 Shown is the PAGE gel electrophoresis diagram (the size of the Marker is 14

[0093] 4 - 97.4 kDa), Figure A is the protein after primary purification, and Figure B is the protein after secondary purification.

[0094] Figure 2 Rat wound healing diagrams, Figure A is the gel of collagen peptide + hesperetin, Figure B is the collagen gel without hesperetin, and Figure C is the commercially available recombinant human type I / III collagen gel. Detailed Embodiments

[0095] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0096] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0097] Example 1 Transcriptome Sequencing and Identification of Collagen-Related Genes

[0098] After selecting Pheretima aspergillum and extracting RNA in segments, the RNA was mixed to construct a library for second-generation sequencing. After quality control of the sequencing data, a total of 10.4G clean Data was obtained. Further assembly and annotation were performed. According to the annotation results of the nr and swiss-prot databases, 94 potential collagen-related transcripts were obtained. According to the expression level and annotation results, 4 candidate transcripts were selected for subsequent research. The read coverage depths of the 4 sequences were 1181.9 (SEQ ID NO.1), 630.3 (SEQ ID NO.2), 420.5 (SEQ ID NO.3), and 80.3 (SEQ ID NO.4), respectively. The nucleotide sequences of the coding frames of the candidate genes are shown in SEQ ID NO.1 to SEQ ID NO.4:

[0099] SEQ ID NO.1:

[0100]

[0101] SEQ ID NO.2:

[0102] ATGGCCATGCCATTTATGTGGCGCGGTGTGTCGAGCCTTACAGACAGACTTCGCTCATTGGCGTACCTTCGTTACCTGCTCGTTGTTCCATTCCTCGTGGTTCTCGCAGTTGGAACCTACAGCTTCGGTCGATTCTCAGGAATATCGAAGCCTGCAAGCGTAAATGGCGAAGAATTGTTGCACAACTTGCACCTGAGAGA AAGGAGAGAAGCTGCTACTCTTGAATCTCCTTCTCTCCAAGCGGAGCAGAGAGCTTCACCCACAGGGGGTGCTGTTTACACGCGCTGGGGTAGAACTGTCTGCCCCAACGGAAGCGACTTGGTTTACGCCGGTATAACAGGTGGTTCGTTCTTCAGCAACCACGGCGGCGGCAGCAATCCGCTCTGCCTCCCGATGGACCCGCAGTGGAGCACGTACAACGAGAACGTCGAGAAGGGCTCGAAGCTGTACGGAGCCGAGTACGAGGCCCCCTCCGGGTTCGATCTCGGGAACGCCGGCGGCCGATCGCTCCACGACCACAACATCCCGTGCGCCGTCTGCCGCAGCCCGACCAGGGCCTCGGCGCTGATGATCCCCGCCCGGAACCTCTGCCACGCCGACGACTGGCGCGTCGAGTACGCCGGGTACCTGATGTCGTCGCACCCGGGTCACCAGGGCCGCTCGCAGTTCGTCTGCGTCGACGCCGGTCCCGAGTCGGACCCGGCCGGGTTCAGGGACGAGAACGGGGCGCTGTTCTACAACGTCGAGGGGGTCTGCGGATCGCTTCCGTGCCCCCCGTACGTACAGCGCCGAGAAATCGTTTGCGTCGTCTGCTCGAAATAG

[0103] SEQ ID NO.3:

[0104] ATGGCTTTTATGTGGCGCGGGTGTTCTACTCAGGCCGAGAAATTCTTTTCGCATCCGTACCTTCGGTACCTCCTCGTTGTTTCATGTCTAGTGGTTTTCGTCATCGGAACCTACAGCCTTGGACGTGTTTCGGGAAAGTCGTCGAAAGTTAATGGTGAAGAGTCAACGTTGCTGAGGGAGAGAAGAGCAGTCGACAATACTCCTCCTTCACCTCCTACGCCTGCTCCTCTTCCTACCCAGTCTACTCAGATTATTTCAGCCGCAGGGGGAGCAGTTTATACCCGCTGGGGTCGAACGGAATGCCCTACGGGTAGTGACGTGGTTTACGCCGGCGTAACTGGAGGTTCATTATTCTCCACCCACGGCGGCGGAAGCAATCCTCTTTGTCTGCCGACGGACCCCCTCTGGGGCAAGTACAACGAAAACGTGGAGAAGGGGGCTAAACTCTACGGGGCCGAGTACGAGTCTCCTTTGGGTTTCGACTTCTCGAACGTCGGCAACCGGTCGCCGCAAGACCACAACATCCCGTGCGTCGTCTGCCGAAGCCAGACGAGGAGTTCGGTGCTGATGATTCCCGCCAGGAACGTCTGCTACGACGACTGGAACGTCGAGTACACCGGGTACCTGATGACCGGACATCAGACTCACGCCGGCCGATCGCAGTTCGTCTGCGTCGACGGAAGTCCGGAGTCGAATCCGGCGGGATTCCGGGACGACAACGGAGTGCTGTTCTACAACGTCGAGGGGGTCTGCGGATCGCTCCCGTGTCCTCCGTACGTCCCACGTCGAGAGATCGTCTGTGCCGTCTGCACGAAATAA

[0105] SEQ ID NO.4:

[0106] ATGGCTTATATGTGGCGCGGGTTTTCTAATCTGGCCGAGAAGTTCTTTTCGCATCCGTACCTTCGATACATCCTCGTTGTTCCATTTCTTGTCGTTTTTGTCATCGGAACCTACAGCCTCGGACGAGTCTCCGGAAAGTCATCGCAAGTTAATGGTGAAGAGTCAACGGTGCTGAGGGAGAGAAGAGATGTCGTCAATCCTCCTTCACCTCCTACGCCTGCTCCTCTTCCTACCCAGTCTACTCAGATTGTTTCTTCCGCAGGGGGAGCAGTTTATACCCGCTGGGGTCGAACGGAATGCCCTACGGGTAGTGACGTGGTGTACGCCGGTGTAACTGGAGGTTCATTATATTCCACCCACGGCGGCGGAAGCAATCCTCTTTGTCTGCCGATGGACCCCCTCTGGGGCAAGTACGACGAAAACGTGGAGAAGGGGTCCAAACTCTACGGGGCCGAGTACGAGTCTCCTTCGGGTTTCGACTTCGCGAACGTCGGCAACCGGTCGCCGCAGGATCACAACATCCCGTGCGTCGTCTGCCGCAATCCGACGAGGAGTTCGGCGCTGATGATTCCCGCCAGGAATGTCTGCTACGACGACTGGAACCTCGAGTACACCGGGTACCTGATGACCAGTCATCCGACGCACGCCGGGCGATCGCAGTTCGTCTGCGTCGACGGAAGTCCGGAGTCGGATCCGGCGGGATTCCGGGACGAGAACGGAGCGTTGTTCTACAACGTCGAGGGCGTCTGCGGATCGCTCCCGTGTCCTCCGTACGTCCAGCGTCGAGAGATCGTCTGCACAGTCTGCACGAAATAA。

[0107] The amino acid sequences of the coding frames of the candidate genes are shown in SEQ ID NO.5 to SEQ ID NO.8:

[0108] SEQ ID NO.5:

[0109] MERIFNIGGAVSMASLLQIFAGGSVPCRSCCRFLQDRSSKRRSSQNGTKMTIVWSGFSTFVEQFQWRRYFKLLLAVPFLVVLAVGSYKIGLSRREMTMEPKMAIVWSGFSTFVDQFQWRLYFKLLLAVPFLFVLAV DSYKIGHLRGEADKMNDYHPSESRVRRDVSQTPNYSSQTIIRGGGAIYTRWGRTTCPSGSDAVYSGVAGGSLYDHTGGGSNYLCLPTDPLWGQYNEGAEKASKLFGTEYELPSGIDLTNMRGRPAQDNNVPCVVCRSQTKTSVVMIPARNLCYGDWKLEYAGYLMSAASLHRGRTEFVCVDGSPEADPAGFRDENGALFYNVEGVCGSLPCPPYVPRREITCAVCTK;

[0110] SEQ ID NO.6:

[0111] MAMPFMWRGVSSLTDRLRSLAYLRYLLVVPFLVVLAVGTYSFGRFSGISKPASVNGEELLHNLHLRERREAATLESPSLQAEQRASPTGGAVYTRWGRTVCPNGSDLVYAGITGGSFFSNHGGGSNPLCLPMDPQWSTYNENVEKGSKLYGAEYEAPSGFDLGNAGGRSLHDHNIPCAVCRSPTRASALMIPARNLCHADDWRVEYAGYLMSSHPGHQGRSQFVCVDAGPESDPAGFRDENGALFYNVEGVCGSLPCPPYVQRREIVCVVCSK;

[0112] SEQ ID NO.7:

[0113] MAFMWRGCSTQAEKFFSHPYLRYLLVVSCLVVFVIGTYSLGRVSGKSSKVNGEESTLLRERRAVDNTPPSPPTPAPLPTQSTQIISAAGGAVYTRWGRTECPTGSDVVYAGVTGGSLFSTHGGGSNPLCLPTDPLWGKYNENVEKGAKLYGAEYESPLGFDFSNVGNRSPQDHNIPCVVCRSQTRSSVLMIPARNVCYDDWNVEYTGYLMTGHQTHAGRSQFVCVDGSPESNPAGFRDDNGVLFYNVEGVCGSLPCPPYVPRREIVCAVCTK;

[0114] SEQ ID NO.8:

[0115] MAYMWRGFSNLAEKFFSHPYLRYILVVPFLVVFVIGTYSLGRVSGKSSQVNGEESTVLRERRDVVNPPSPPTPAPLPTQSTQIVSSAGGAVYTRWGRTECPTGSDVVYAGVTGGSLYSTHGGGSNPLCLPMDPLWGKYDENVEKGSKLYGAEYESPSGFDFANVGNRSPQDHNIPCVVCRNPTRSSALMIPARNVCYDDWNLEYTGYLMTSHPTHAGRSQFVCVDGSPESDPAGFRDENGALFYNVEGVCGSLPCPPYVQRREIVCTVCTK。

[0116] Example 2 Preparation of Collagen Peptide Chain

[0117] □ Step 1: Construction and Identification of Recombinant Expression Vector

[0118] Fuse the SUMO tag protein to the N-terminus of the candidate protein and the His tag to the C-terminus, and insert the restriction enzyme site BamHI and the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln↓-Gly (TEV Protease recognition site) between the tag and the target protein. Optimize the codons of the candidate protein for Escherichia coli (https: / / www.novopro.cn / tools / codon-optimization.html) and send it to the company for whole gene synthesis to obtain the candidate gene sequence. Clone the gene into the NdeI and Xho restriction enzyme sites of the pET21a plasmid.

[0119] The recombinant plasmid was transformed into BL21(DE3) competent cells and spread on an LB plate containing ampicillin. The cells were cultured overnight at 37°C, and transformants were selected by resistance screening. The correct recombinant transformant strain was determined by colony PCR amplification and sequencing.

[0120] Step 2: Induced expression and enzyme isolation and purification

[0121] Positive clones were selected and cultured in an LB liquid medium containing ampicillin. When OD600 = 0.6 - 0.8 (using sterile LB medium as a control), isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the target protein was induced to express overnight at 16°C. The fermentation broth was centrifuged to collect the cell precipitate, which was suspended by adding a citrate-disodium hydrogen phosphate buffer and then sonicated, and the supernatant was collected. According to the His-Tag on the fusion protein, a nickel affinity chromatography column was used to purify the target protein, and the target protein was eluted with imidazole solutions of different concentrations. Finally, 10% SDS-PAGE electrophoresis was used to detect the molecular weight and purity of the target protein. Before secondary purification, TEV protease digestion was performed, and desalting was carried out using G25 gel chromatography to replace the buffer containing imidazole with 20 mmol / L Na2HPO4 buffer with a pH value of 7.5. For large-scale protein production, fed-batch fermentation in a 5 L fermenter was carried out for the positive strain.

[0122] The results are as Figure 1 shown. The protease solution after primary purification had a single band in the electrophoresis pattern, and its size was consistent with the molecular weight of the theoretical protein plus the recombinant tag ( Figure 2 A), indicating that the protein was successfully expressed in the supernatant. Two obvious bands were observed at molecular weights of 15 - 22 kD and about 30 - 43 kD during secondary purification, corresponding to SUMO and the target protein respectively ( Figure 2 B). The purified protein can be used for subsequent experiments.

[0123] Example 3 Preparation and antioxidant activity of collagen peptides

[0124] Step 1 Preparation of collagen peptides

[0125] Trypsin, papain, and alkaline protease were used to enzymatically hydrolyze the aforementioned 4 proteins under the optimal pH and temperature conditions. The specific enzymatic hydrolysis conditions are as follows:

[0126] Table 1 Enzymatic digestion combination conditions

[0127]

[0128]

[0129] Step 2 Determination of DPPH free radical scavenging rate (reference standard number: GB / T39100 - 2020)

[0130] Accurately weigh DPPH and prepare a DPPH solution with a concentration of 0.2 mmol / L, and store it in the dark at 0 - 4 °C. Add 100 μL of the DPPH solution and 100 μL of samples with different concentration gradients (after dialysis and ultrafiltration of the samples, protease and uncut proteins are removed) into a micro cuvette, and react in the dark for 30 min. Measure the absorbance at a wavelength of 517 nm. The calculation formula for the DPPH radical scavenging rate is as follows:

[0131]

[0132] In the formula: A1: absorbance value of the sample plus the DPPH solution; A2: absorbance value of the sample plus absolute ethanol solution; A3: absorbance value of the DPPH solution plus 95% ethanol solution.

[0133] The results are shown in the following table. After enzymatic digestion, all showed improved radical scavenging ability, especially in the case of combined enzymatic digestion with better performance:

[0134] Table 2. Half-maximal inhibitory concentration (IC50, mmol / L)

[0135]

[0136] Note: a - e are significant markers for group comparison, and all are highly significant between the enzymatic digestion combinations and the control (not marked).

[0137] Example 4: Solubilization effect of collagen peptide solution on hesperetin

[0138] The water solubility of hesperetin is very poor (almost insoluble in water, only 23.78 μg / Ml, Lu Xiang, Jiang Chengjun. Research progress on cocrystals of flavonoids [J]. Herald of Medicine, 2019, 38(7): 921 - 926.), which has caused quite a large limitation to their use. Existing technologies report that alcohols, cyclodextrins, etc. have the effect of increasing the solubility of hesperetin. There are also reports that basic amino acids have a certain solubilization effect. The present invention attempts to utilize the solubilization effect of polypeptides from different enzymatic digestion combinations of different proteins on hesperetin (1 g of polypeptide is added to every 100 mL of water). The results are shown in the following table, and each combination significantly increases the solubility of hesperetin in water at room temperature.

[0139] Protein and Enzyme Digestion Combinations Dosage Hesperetin Saturated Solubility (mg / mL) SEQ ID NO.5 + Enzyme Combination 4 1g 7.45 SEQ ID NO.5 + Enzyme Combination 5 1g 13.98 SEQ ID NO.5 + Enzyme Combination 6 1g 4.67 SEQ ID NO.6 + Enzyme Combination 4 1g 15.23 SEQ ID NO.6 + Enzyme Combination 5 1g 11.89 SEQ ID NO.6 + Enzyme Combination 6 1g 3.56 SEQ ID NO.7 + Enzyme Combination 4 1g 15.34 SEQ ID NO.7 + Enzyme Combination 5 1g 9.76 SEQ ID NO.7 + Enzyme Combination 6 1g 17.21 SEQ ID NO.8 + Enzyme Combination 4 1g 2.89 SEQ ID NO.8 + Enzyme Combination 5 1g 12.67 SEQ ID NO.8 + Enzyme Combination 6 1g 6.45 Control (Pure Water without Polypeptide) 0g 0.02

[0140] Example 5: Preparation of a gel dressing containing collagen peptide and hesperetin

[0141] Product ratio

[0142] Collagen peptide: 1% - 15% (w / w).

[0143] Hesperetin: maximum dissolution amount.

[0144] Gel matrix (such as sodium alginate, gelatin or sodium carboxymethylcellulose): 10%-20% (w / w).

[0145] Plasticizer (such as glycerol or propylene glycol): 5%-10% (w / w).

[0146] Cross-linking agent (such as calcium chloride or glutaraldehyde): 1%-3% (w / w).

[0147] Humectant (such as hyaluronic acid or aloe extract): 2%-5% (w / w).

[0148] Antibacterial agent (such as phenoxyethanol or tea tree oil): 0.5%-1% (w / w).

[0149] Water: the balance.

[0150] Preparation method:

[0151] Step 1: Preparation of collagen peptide - hesperetin complex

[0152] Solvent: Take 95 mL of deionized water.

[0153] Solute: Add 5 g of collagen peptide, stir at 50 °C until completely dissolved to form a 5% (w / w) collagen peptide solution.

[0154] Add hesperetin: Add 1 g of hesperetin, stir and heat to 40 - 50 °C, continuously stir for 2 - 4 hours to fully dissolve hesperetin and form a complex.

[0155] Filtration: Filter with a 0.45 μm filter membrane to remove undissolved hesperetin, and obtain a clear collagen peptide - hesperetin complex solution.

[0156] Step 2: Preparation of gel matrix

[0157] Add 10 g of sodium alginate, heat to 50 - 60 °C, stir until completely dissolved to form a 10% (w / w) gel solution.

[0158] Step 3: Add functional components

[0159] Plasticizer: Add 5 g of glycerol and stir evenly.

[0160] Humectant: Add 2 g of hyaluronic acid and stir until completely dissolved.

[0161] Antibacterial agent: Add 0.5 g of phenoxyethanol and stir evenly.

[0162] Step 4: Molding of gel dressing

[0163] Crosslinking: Pour the mixed solution into a mold and spread it evenly into a thin layer (thickness 1 - 2 mm).

[0164] Curing: Spray a 2% (w / w) calcium chloride solution (or use glutaraldehyde vapor) for crosslinking and let it stand for 10 minutes.

[0165] Drying: Dry at 40 - 50 °C until the gel dressing forms a transparent film.

[0166] Cutting: Cut into appropriate sizes according to requirements (such as 5 cm × 5 cm).

[0167] Step 5: Post - treatment

[0168] Packaging: Seal the gel dressing in a sterile packaging bag and store it in a cool and dry place.

[0169] Sterilization: Can be sterilized by ultraviolet irradiation or γ - ray to ensure the product is sterile.

[0170] Example 6 Repair effect of the gel dressing on rat skin wounds

[0171] Select male healthy SD rats with a body weight of 200 - 250 g for the skin wound repair experiment to evaluate the effect of different gel products on rat wound repair. Intraperitoneally inject a 2% sodium pentobarbital solution at 50 mg / kg body weight, and perform the operation after the rats are completely anesthetized. Use a scalpel to prepare a full - thickness skin wound with a diameter of 1 cm on the back of the rats (depth to the subcutaneous tissue). The first group ( Figure 2 A) Evenly apply the gel prepared in Example 5 of the present invention (SEQ ID NO.7 + enzyme combination 4) on the wound, with a thickness of about 1 - 2 mm, and cover it with a sterile gauze. The second group ( Figure 2 B) Evenly apply the gel containing only the collagen peptide of the present invention (prepared according to Example 5, without hesperidin) on the wound, with a thickness of about 1 - 2 mm, and cover it with a sterile gauze. The third group ( Figure 2 C) Evenly apply a commercially available recombinant human type I / III collagen gel on the wound, with a thickness of about 1 - 2 mm, and cover it with a sterile gauze. Change the dressing every 2 days, and take photos for comparison before and on the tenth day.

[0172] The results are as Figure 2 shown. All samples have the effect of promoting healing, but the gel of collagen peptide + hesperidin of the present invention has the best effect and the wound heals the fastest.

[0173] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above-described embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polypeptide with antioxidant function, characterized in that: The polypeptide is obtained by enzymatically cleaving a protein with an amino acid sequence such as any one of SEQ ID NO.1 to SEQ ID NO.4 with a protease, and the protease is composed of any one of trypsin, papain, and alkaline protease or a combination thereof.

2. Use of the polypeptide according to claim 1 in improving the water solubility of hesperetin.

3. A composition having skin damage repair function, characterized in that: The composition comprises the polypeptide of claim 1.

4. The composition according to claim 3, characterized in that The composition also includes hesperetin.

5. The composition according to claim 4, characterized in that The composition further includes auxiliary materials.

6. Use of the polypeptide according to claim 1 in the preparation of drugs for anti-oxidation and / or skin damage repair.

7. A method for preparing a composition having skin damage repair function, characterized in that it comprises the following steps: Step 1: Preparation of a collagen peptide-hesperidin complex, wherein the collagen peptide is the polypeptide according to claim 1; Step 2: Preparation of gel matrix; Step 3: Add other functional ingredients; Step 4: Forming of the gel dressing.

8. The method according to claim 7, characterized in that The gel matrix is ​​any one of sodium alginate, gelatin or sodium carboxymethyl cellulose.

9. The method according to claim 7, characterized in that: The other functional ingredients are plasticizers, moisturizers or antibacterial agents.

10. A protein that can be enzymatically cleaved to form an antioxidant polypeptide, characterized in that: The sequence of the protein is shown in any one of SEQ ID NO.5 to SEQ ID NO.8, and the enzyme is composed of any one of trypsin, papain, alkaline protease or a combination thereof.

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