Composition containing recombinant human type iii collagen and antioxidant peptide and use thereof

Abstract

The present application relates to the technical field of medical dressings, in particular to a composition containing recombinant human type III collagen and antioxidant peptides and application thereof. The present application uses a new type of antioxidant peptide LR and recombinant human type III collagen COL3A1 in a certain proportion, and selects the optimal composition through in vitro antioxidant index detection and repair experiments on UVB damaged cells. The final results show that the composition provided by the present application can achieve antioxidant and skin repair effects. The composition provided by the present application provides a new idea for improving the antioxidant activity and skin repair effect of type III collagen.

Description

Technical Field

[0001] This invention relates to the field of medical dressing technology, and in particular to compositions containing recombinant human type III collagen and antioxidant peptides and their applications. Background Technology

[0002] In recent years, with the improvement of people's living standards and the pursuit of beauty, therapeutic agents for reducing pigmentation and anti-aging and wrinkle removal have attracted great interest in the clinical and cosmetic fields. It has been reported that commercial antioxidants and whitening agents, including hydroquinone, arbutin, kojic acid, vitamin C, and some herbal medicines, have been banned due to various adverse side effects (such as contact dermatitis, irritation, white skin, hypopigmentation, and ochre syndrome) caused by long-term use. Antioxidant peptides, as one of the emerging antioxidants, have received much attention in the food and health fields in recent years due to their naturalness, effectiveness, bioavailability, and compatibility. Existing studies have shown that antioxidant peptides not only have the function of scavenging free radicals in the body, but also have a significant protective effect against free radical-induced lipid peroxidation. Their mechanism of action also includes providing hydrogen to antioxidant enzymes and chelating metal ions.

[0003] Collagen is the most abundant protein in the human body, distributed throughout various organs and tissues, with the highest concentration in the dermis of the skin. The smooth, plump, and elastic skin of young people is primarily due to their sufficient collagen levels. Compared to animal collagen, recombinant collagen has higher activity, better hydrophilicity and water retention, can improve skin metabolism, has no viral residue, no immunogenicity, and no endotoxins, making it safe for long-term use. Type III collagen has a wide range of applications in daily life, forming medical dressings such as sponge dressings, membrane dressings, and gel dressings, primarily used for treating wound infections, promoting tissue regeneration, and wound closure. Producing recombinant human type III collagen through genetic engineering is a novel method in recent years. Currently, recombinant human type III collagen is mostly expressed using small fragments, as full-length expression requires hydroxylation by proline hydroxylase. However, commonly used bacterial and yeast expression systems do not contain proline hydroxylase, resulting in products with very low stability and easy degradation, significantly limiting the use of recombinant collagen.

[0004] Therefore, it is particularly important to provide a stable combination of human type III collagen and antioxidant peptides that has both antioxidant and skin repair effects. Summary of the Invention

[0005] The purpose of this invention is to provide compositions containing recombinant human type III collagen and antioxidant peptides and their applications, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a composition containing recombinant human type III collagen and an antioxidant peptide, the composition comprising recombinant human type III collagen COL3A1 and an antioxidant peptide LR; the amino acid sequence of the recombinant human type III collagen COL3A1 is shown in SEQ ID NO.2; the amino acid sequence of the antioxidant peptide LR is shown in SEQ ID NO.15.

[0008] Optionally, the mass ratio of the recombinant human type III collagen COL3A1 to the antioxidant peptide LR in the composition is 1:(13.9~19).

[0009] Optionally, the mass ratio of the recombinant human type III collagen COL3A1 to the antioxidant peptide LR in the composition is 1:19.

[0010] The present invention provides a method for preparing the above-described composition, comprising the step of mixing the recombinant human type III collagen COL3A1 and the antioxidant peptide LR.

[0011] This invention provides the use of the above-described composition in the preparation of products with antioxidant and skin-repairing effects.

[0012] This invention provides a product with antioxidant and skin repair effects, the product comprising the above-described composition.

[0013] Optionally, the product may also include auxiliary materials.

[0014] Optionally, the product includes medical dressings.

[0015] Optionally, the dosage form of the product includes one or more of the following: aqueous solution, oil, emulsion, cream, ointment, and gel.

[0016] This invention provides the application of antioxidant peptide LR in the preparation of products with antioxidant and skin repair effects, wherein the amino acid sequence of the antioxidant peptide LR is shown in SEQ ID NO.15.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides a stable composition with both antioxidant and skin-repairing effects by combining a novel antioxidant peptide LR with recombinant human type III collagen COL3A1 in a specific ratio. In a specific embodiment, a strain capable of stably producing recombinant human type III collagen is first provided, and the type III collagen produced by this strain is a highly active recombinant human type III collagen modified by proline hydroxylase. Then, a novel antioxidant peptide LR is provided, and its antioxidant and anti-tyrosinase activities are evaluated by studying its protective effect against oxidative damage to cells, measuring physiological and biochemical indicators, and inhibiting browning in apples. Finally, this novel antioxidant peptide LR is combined with recombinant human type III collagen COL3A1 in a specific ratio, and the optimal composition is selected through in vitro antioxidant index detection and UVB-damaged cell repair experiments. The final results show that the composition provided by this invention can achieve both antioxidant and skin-repairing effects. The composition provided by this invention offers a new approach to improving the antioxidant activity and skin-repairing effects of type III collagen. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 These are plasmid maps; where A is the PIC9K plasmid map and B is the PICZA plasmid map.

[0021] Figure 2 The image shows the PCR identification results; where M: DNA molecular weight standard (200~4500bp), Cp-positive control: positive control strain carrying recombinant plasmid, Cp2: Pichia pastoris transformant that simultaneously integrates two recombinant plasmids, pPIC9K-COL3A1 and pPICZA-A085R, c3-22: single plasmid control (pPIC9K-COL3A1), 9k: pPIC9K empty vector as blank control;

[0022] Figure 3 The images show the SDS-PAGE results; where 1 to 5 represent 5 replicates.

[0023] Figure 4 This is a infographic of Western Blot results; CP1 and CP2 represent different bacterial colonies.

[0024] Figure 5The image shows the protein purification results; where 1 is the 1 mL collection of CP1 colony, 2 is the 2 mL collection of CP1 colony, 3 is the 3 mL collection of CP1 colony, NC: blank control, 4 is the 1 mL collection of CP2 colony, 5 is the 2 mL collection of CP2 colony, and 6 is the 3 mL collection of CP2 colony.

[0025] Figure 6 The results indicated collagen cytotoxicity; among them, ** P < 0.01, **** P < 0.0001;

[0026] Figure 7 The effect of collagen on UVB damage repair; where * P<0.05, *** P<0.001;

[0027] Figure 8 The CD spectrum of the sample is shown in the wavelength range of 190~260nm.

[0028] Figure 9 This is a full-wavelength ultraviolet scan.

[0029] Figure 10 The DPPH free radical scavenging rate of 10 peptide sequences derived from the skin mucus of the forest frog;

[0030] Figure 11 The results were used to determine the in vitro antioxidant index; where A represents the total antioxidant activity of LR; B represents the scavenging rate of LR against DPPH free radicals; ns P>0.05, ** P<0.01, *** P<0.001, **** P<0.0001; the x-axis represents the concentration of LR or GSH (μmol / mL).

[0031] Figure 12 To analyze the interaction between the antioxidant peptide LR and DPPH (A) and ABTS (B) based on molecular docking;

[0032] Figure 13 The structure-antioxidant activity relationship of LR;

[0033] Figure 14 This is a biosafety experiment; where A represents the hemolytic activity of LR on mouse erythrocytes; B represents the cytotoxicity of LR on NIH-3T3 cells; different letters represent significant differences (P<0.05).

[0034] Figure 15 LR represents the DPPH clearance rate under different treatment conditions; where A~E represent the DPPH clearance rate under different treatment conditions (temperature, pH, NaCl concentration, time, and in vitro digestion), respectively; different uppercase letters represent significant differences among LR groups (P<0.05); different lowercase letters represent significant differences among GSH groups (P<0.05).

[0035] Figure 16 The study aimed to protect LR from H2O2-induced oxidative damage in NIH-3T3 cells. In this study, A represents the effect of different concentrations of H2O2 on the viability of NIH-3T3 cells; B represents the effect of different concentrations of LR on the viability of cells damaged by oxidative damage. Different letters indicate significant differences (P<0.05).

[0036] Figure 17 The inhibitory effect of LR on browning of apples; where A is the browning index of apple slices treated with different concentrations of LR and GSH; B is the inhibition rate of tyrosinase activity in apples treated with different concentrations of LR and GSH; **** P<0.0001;

[0037] Figure 18 Visual appearance of apple slices treated with different concentrations of LR and GSH during 24 hours of storage;

[0038] Figure 19 The hemolysis rate of mouse erythrocytes by different proportions of the compound; different letters represent significant differences (P<0.05).

[0039] Figure 20 The scavenging rates of DPPH free radicals (A), ABTS scavenging rates (B), and their repair effects on UVB-damaged HaCaT cells (C) are represented by different proportions of the compound groups; different letters represent significant differences (P<0.05).

[0040] Figure 21 The effects of each group on MDA level (A) and the activities of antioxidant enzymes CAT (B) and SOD (C) in UVB-induced HaCaT cell damage were shown; different letters represent significant differences (P<0.05). Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Example 1: Recombinant human type III collagen COL3A1

[0047] 1. Gene design

[0048] Based on the gene sequence of human type III collagen in the GEO database, a cDNA sequence of recombinant human type III collagen core functional region (GenBank: BC028178.1) with the N-terminus and C-terminus removed was selected for easy extraction and purification. Optimized codons for expression by the host strain *Pichia pastoris* were used. During the design process, an α-MF signal peptide sequence and six histidine coding sequences were added to the N-terminus and C-terminus, respectively, to facilitate subsequent protein purification. The optimized nucleotide sequence of the COL3A1 gene is shown in SEQ ID NO. 1. Compared with the original gene sequence, this sequence shows that the codons of the recombinant human type III collagen are preferred by *Pichia pastoris*, which is beneficial for the efficient expression of this gene by the host strain *Pichia pastoris*. The amino acid sequence of the COL3A1 protein encoded by this optimized gene sequence is shown in SEQ ID NO. 2.

[0049] SEQ ID NO.1:

[0050]

[0051] SEQ ID NO.2:

[0052]

[0053] The nucleotide sequence of the viral A085R protein gene (GenBank: NP_048433.1, also known as the P4H gene), which is similar to the α subunit sequence of human P4H, is shown in SEQ ID NO.3. It is the gene sequence of proline hydroxylase.

[0054] SEQ ID NO.3:

[0055] .

[0056] 2. Gene synthesis and construction of recombinant plasmids

[0057] The nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.3 were sent to Jilin Kumei Biotechnology Co., Ltd. for whole-gene synthesis, and expression vectors were constructed. The theoretical map of the constructed plasmid vectors is as follows: Figure 1 As shown, specifically: using the PIC9K plasmid as the base vector, the gene sequence shown in SEQ ID NO.1 (COL3A1 gene) was inserted at the restriction sites SnaBI and NotI to obtain the recombinant plasmid pPIC9K-COL3A1; using the PICZA plasmid as the base vector, the gene sequence shown in SEQ ID NO.3 was inserted at the restriction sites EcoRI and KpnI to obtain the recombinant plasmid pPICZA-A085R.

[0058] 3. Preparation of a Pichia pastoris expression system containing the proline hydroxylase gene

[0059] (1) Recombinant plasmids pPIC9K-COL3A1 and pPICZA-A085R were transformed into Pichia pastoris GS115 competent cells.

[0060] The recombinant plasmids pPIC9K-COL3A1 and pPICZA-A085R were linearized using the restriction endonuclease sacI and transformed into Pichia pastoris GS115 competent cells by electroporation.

[0061] The steps for preparing competent cells are as follows:

[0062] Using an inoculation loop, streak Pichia pastoris GS115 glycerol culture onto a non-resistant YPD agar plate. Incubate until a single colony appears. Pick a single colony and inoculate it into 5 mL of YPD liquid medium. Incubate overnight at 30°C and 220 rpm. Take 500 μL of the bacterial culture and inoculate it into 50 mL of YPD liquid medium for further culture, expanding to OD at 30°C and 220 rpm. 600 =0.6~2.0, transfer the bacterial culture to a 50mL sterile centrifuge tube, centrifuge at 2500×g for 5min at room temperature, discard the supernatant, add deionized water (pre-cooled) to resuspend the bacterial cells, centrifuge at 2500×g for 5min at room temperature, discard the supernatant, add 1~2mL of deionized water according to the amount of bacterial cells to resuspend the bacterial cells, which are now competent cells, and place them on ice for later use.

[0063] The steps for linearizing the vector are as follows:

[0064] Before transformation, the recombinant plasmids pPIC9K-COL3A1 and pPICZA-A085R were linearized using the restriction endonuclease Sac I. The linearization system is shown in Table 1. The linearization was carried out at 37℃ for 2 hours.

[0065] Table 1 Linearized System

[0066]

[0067] The steps for electroporation to transform Pichia pastoris GS115 competent cells are as follows:

[0068] Under aseptic conditions, add 20 μL of linearized plasmid (linearized plasmid pPIC9K-COL3A1 and / or linearized plasmid pPICZA-A085R; when adding linearized plasmid pPIC9K-COL3A1 and linearized plasmid pPICZA-A085R, the mass ratio of linearized plasmid pPIC9K-COL3A1 to linearized plasmid pPICZA-A085R is 1:1) and 80 μL of competent cells to a pre-cooled electroporation cuvette. Incubate on ice for 15 min, at 1500 V, 25 mF, 200 Ω, 6 ms / cycle, and electroporate once. After transformation, the plasmid is spread on YPD medium containing 100 μg / mL bleomycin resistance, inverted in a 30℃ incubator, and incubated for 2-3 days until white colonies appear.

[0069] (2) Screening of recombinant yeast

[0070] The genome of the transformed colonies was extracted using a DNA extraction kit from Tiangen Biotech Co., Ltd. Positive strains were screened by PCR amplification using universal yeast primers (as shown in SEQ ID NO.4~SEQ ID NO.6). The reaction conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 60 s, 72℃ for 3 min, 30 cycles; 72℃ for 10 min, and stored at 4℃. The pPIC9K empty vector was used as a control, and Pichia pastoris cells GS115 were used as a blank control. The positive recombinant that successfully recombined into the yeast cell genome was identified as strain GS115 / pPIC9K-COL3A1-pPICZA-A085R.

[0071] 5' Sequencing primers and sequence: 5'AOX1:5'-GACTGGTTCCAATTGACAAGC-3', SEQ ID NO.4;

[0072] alpha-Factor-F: 5'-TACTATTGCCAGCATTGCTGC-3', SEQ ID NO.5;

[0073] 3' Sequencing primers and sequence: 3'AOX1:5'-GCAAATGGCATTCTGACATCC-3', SEQ ID NO.6.

[0074] The results are as follows Figure 2The results showed that the recombinant plasmid had been successfully transformed into Pichia pastoris GS115. PCR amplification was performed on two clones (CPs) co-expressing the human COL3A1 and P4H genes using primers with nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.6, yielding two bands of 2250 bp and 741 bp, respectively. A single band of 3994 bp was obtained using primers with nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6, consistent with expectations. The α-factor signal peptide has been correctly fused with the COL3A1 gene, providing a molecular basis for the subsequent secretory expression of recombinant collagen. PCR identification results are as follows: Figure 2 As shown, the band size analysis showed that the actual value matched the theoretical value, proving that the recombinant human type III collagen expression system was successfully constructed in this embodiment. The recombinant bacteria that successfully transformed the recombinant plasmid is designated as GS115 / pPIC9K-COL3A1-pPICZA-A085R.

[0075] 4. Induced expression of recombinant human type III collagen

[0076] (1) Take the bacterial strain GS115 / pPIC9K-COL3A1-pPICZA-A085R stored at -80℃ for activation. Use a glass triangular spreading stick to dip the bacterial solution and spread it evenly on a YPD solid plate. Incubate at 30℃ for 2-3 days. Pick a single colony and place it in YPD liquid medium. Incubate at 30℃ for about 24 hours until OD reaches 0.5. 600 When the concentration reaches the range of 0.6 to 2.0, it is considered a first-grade seed culture. The seed culture concentration can be adjusted according to the size of the culture volume.

[0077] (2) Inoculate the seed culture at a rate of 10% (v / v) into glycerol medium (0.2% biotin, 1% yeast nitrogen source, 1% glycerol, 1% yeast extract, 2% peptone, 100mM potassium phosphate), with a volume of 50mL / 250mL. Incubate at 30℃ for 24h. Centrifuge the bacterial culture at 4℃, 5000×g for 20min to obtain a large amount of bacterial precipitate. Discard the supernatant and transfer the bacterial precipitate into 200mL of pre-prepared methanol induction medium (0.2% biotin, 1% methanol, 1% yeast nitrogen source, 1% yeast extract, 2% peptone, 100mM potassium phosphate) for further culture (aseptic operation is required during centrifugation and transfer in this step). After 24h of culture, add methanol to the methanol induction medium to make the methanol concentration in the methanol induction medium 1% (if the total volume of the methanol induction medium is 200mL, add 2mL of methanol). Add methanol every 24h to maintain the methanol concentration at 1%. After culturing for 120 hours, collect all the supernatant (the supernatant needs to be stored at 4℃), which is the bacterial supernatant protein sample.

[0078] 5. SDS-PAGE protein detection

[0079] Sample preparation: Collect the bacterial supernatant protein sample from step 4. Add loading buffer at a ratio of bacterial supernatant protein sample: 5 × loading buffer = 4:1 (v:v), mix thoroughly, boil in a water bath for 5 minutes, and allow to cool naturally for later use. Load the sample onto an 8% separating gel and electrophoresis at 80V for 30 minutes to concentrate the sample to a horizontal line. Then, electrophoresis at 120V for 40-60 minutes until the bromophenol blue band reaches the bottom of the gel.

[0080] Coomassie Brilliant Blue G250 staining: (1) Fixative preparation: 50 mL anhydrous ethanol, 10 mL glacial acetic acid, 40 mL distilled water; (2) Staining solution preparation: 0.25 g Coomassie Brilliant Blue 250 added to 45 mL methanol, 10 mL glacial acetic acid, and 45 mL distilled water. (3) Destaining solution preparation: 25 mL ethanol, 8 mL glacial acetic acid, and distilled water to a final volume of 100 mL. (4) After electrophoresis, pry open the gel plate and remove the gel, then place it in a container containing 100 mL of fixative and fix it at room temperature for 30 min on a horizontal shaker. (5) After fixation, transfer the gel to a staining container containing 100 mL of staining solution and stain it at room temperature for 30 min on a horizontal shaker. (6) Remove the gel from the staining solution and place it in a container containing 100 mL of destaining solution and destain it at room temperature on a horizontal shaker. Change the destaining solution every 2 hours until the gel background is clear.

[0081] Experimental results are as follows Figure 3 As shown, a thin protein band appears at 130 kDa, which is larger than the molecular weight of recombinant human type III collagen COL3A1 (rhCOL3A1 protein) deduced from the DNA sequence (96.3 kDa). Since collagen is rich in proline, its migrating molecular weight in SDS-PAGE is more than 40% larger than its actual molecular weight, and this result is consistent with previous reports. Therefore, it can be confirmed that COL3A1 protein was successfully expressed in Pichia pastoris GS115, and the target band size is consistent with the theory.

[0082] 6. Western Blot Validation

[0083] SDS-PAGE gel electrophoresis was performed using the same method as in step 5, and the SDS-PAGE gel was used for Western blotting verification.

[0084] The steps for Western blotting are as follows:

[0085] (1) Wet transfer method for protein transfer: Prepare two sheets of filter paper the same size as the sponge, and cut out a 0.45μm PVDF membrane the same size as the gel. Activate the PVDF membrane with methanol, and immerse the filter paper, sponge and activated PVDF membrane in transfer solution. Assemble the transfer clamp, stacking them in the order of black electrode plate → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → red electrode plate, and fully remove air bubbles from each layer. Clamp the transfer clamp tightly and place it in the transfer fixation device in the manner of "black to black, red to red". Place it in the transfer tank and fill the transfer tank with transfer solution. Place the transfer tank in ice water. Turn on the power and maintain a constant voltage of 90V~100V for 1 hour for transfer.

[0086] (2) Blocking and antibody incubation: Turn off the power, take out the PVDF membrane and put it into the blocking solution, and block it on a shaker at room temperature for 30 min to 60 min; after blocking, recover the blocking solution and wash the PVDF membrane briefly with TBST for about 2 s; incubate the primary antibody (manufacturer: Santa Cruz, catalog number: sc-271249) at 4℃ overnight; recover the primary antibody incubation solution, wash the PVDF membrane 3 times with TBST for 5 min each time; incubate the secondary antibody (manufacturer: Tiangen Biotech Co., Ltd., catalog number: PA112-01) at room temperature on a shaker for 2 h; recover the secondary antibody incubation solution, and wash the PVDF membrane 3 times with TBST at room temperature on a shaker for 5 min each time.

[0087] (3) Development and exposure: The BeyoECL Plus kit (product number: P0018S) was used for development and exposure. The steps were as follows: 400mL of developer solution was prepared with solution A: solution B = 1:1 and stored in the dark. The PVDF membrane was placed on the plate in the developer and spread out. The developer solution was added to the membrane surface with a micropipette to drive out air bubbles and make the developer solution evenly cover the membrane. The developer door was closed for development. The exposure time was controlled between 10s and 25s.

[0088] The Western Blot results are shown in the figure below. Figure 4 As shown, the expression levels of recombinant human type III collagen COL3A1 at two time points are illustrated. The results showed that the protein expression level of strain CP2 was slightly higher than that of strain CP1, and strain CP2 had a higher yield of recombinant human type III collagen COL3A1 protein at 120 h.

[0089] 7. Protein purification

[0090] The recombinant protein was purified using the Ni-NTA 6FF His-tagged protein purification kit (purchased from Sangon Biotech, catalog number C600332-0001).

[0091] (1) Sample processing: Centrifuge the collected bacterial solution at 4℃, 5000×g, for 20min, discard the precipitate, collect the supernatant, and filter the supernatant using a 0.45μm filter.

[0092] (2) Nickel column pretreatment: Place the nickel column vertically, add 5 times the volume of deionized water to rinse, and add 10 times the volume of Binding Buffer to equilibrate the column (the Binding Buffer should be placed on ice, and the supernatant sample should also be placed on ice to minimize protein degradation loss).

[0093] (3) Binding of the target protein to the nickel column: Add the bacterial supernatant to the column and control the flow rate at 3-5 s / drop. Collect the flow-through liquid and pass it through the column again. Collect the first 4 mL of flow-through liquid when loading the sample for the second time.

[0094] (4) Nickel column washing: Add 15 column volumes of Binding Buffer to wash the column. The speed can be slightly faster (this step is to wash away impurities). Collect 1 mL of washing solution from each of the three time periods: before, during and after the washing.

[0095] (5) Elution of target protein: Add 10 column volumes of Elution Buffer to elute the protein at a slow rate of 3-5 seconds per drop. Collect 10 ml of the eluent.

[0096] (6) Nickel column preservation: Add 3 column volumes of Binding Buffer to wash the column; add 5 column volumes of deionized water to wash the column, the speed can be slightly faster; add 3 column volumes of 20% ethanol to preserve the column (it is advisable to let some ethanol flow out to completely immerse the packing).

[0097] Purification results are as follows Figure 5 As shown, the COL3A1 protein is mainly concentrated in the first 2 mL of the elution buffer, that is, the first 2 mL of the elution buffer is recombinant human type III collagen COL3A1.

[0098] 8. Biological Experiments

[0099] (1) Cytotoxicity assay of recombinant human type III collagen COL3A1. HaCaT cells were cultured at 5.0 × 10⁻⁶ cells / year. 4At a density of cells / mL, 100 μL was added to each well of a 96-well plate. After incubation at 37°C in a 5% CO2 incubator for 24 h, the original medium was replaced with medium containing different concentrations of recombinant human type III collagen COL3A1, and incubated at 37°C in a 5% CO2 incubator for 24 h. Then, the medium was replaced with medium containing 10% CCK-8 and incubated for 1 h. The absorbance at 450 nm was measured. HaCaT cells without recombinant human type III collagen COL3A1 served as a blank control group. The absorbance of CCK-8 in the blank control group was 100% activity, and each concentration was tested in triplicate.

[0100] The cytotoxic effects of different concentrations of collagen on HaCaT cells were detected using the CCK-8 colorimetric assay. The results are as follows: Figure 6 As shown in the figure. Cell viability analysis revealed that the number of cultured cells decreased with increasing concentration of recombinant human type III collagen COL3A1. There were no significant differences between the 7.8, 15.625, and 31.25 μg / mL treatment groups and the blank control group (P > 0.05). This result indicates that recombinant human type III collagen COL3A1 at concentrations of 7.8, 15.625, and 31.25 μg / mL had no effect on HaCaT activity.

[0101] (2) The effect of recombinant human type III collagen COL3A1 on UVB damage repair. HaCaT cells were 5.0 × 10 4 100 μL of the solution was added to each well of a 96-well plate at a density of 1 / mL. After incubation at 37°C in a 5% CO2 incubator for 24 h, the solution was further processed at 40 mJ / cm³. 2 Cells were either irradiated with UVB at a specific dose or left untreated (blank control group) and incubated for 24 h at 37°C in a 5% CO2 incubator with culture medium containing different concentrations of recombinant human type III collagen COL3A1. The medium was then replaced with a medium containing 10% CCK-8 and incubated for 1 h, with absorbance measured at 450 nm. The absorbance of CCK-8 in the blank control group was considered 100% activity. Three replicates were set up for each concentration.

[0102] The effect of different concentrations of collagen on the repair of HaCaT cells damaged by UVB was detected using the CCK-8 colorimetric method. The results are as follows: Figure 7 As shown in the figure, the cell viability of HaCaT cells after UVB irradiation was approximately 60%. Compared with the blank control group, cell viability was slightly improved in the treatment groups with recombinant human type III collagen COL3A1 at concentrations of 7.8 and 15.625 μg / mL, but the difference was not significant (P > 0.05). Compared with the blank control group, the treatment group with a concentration of 31.25 μg / mL showed a significant difference (P < 0.05).

[0103] 9. Identification of recombinant human type III collagen COL3A1

[0104] (1) Circular dichroism (CD) and mass spectrometry (MS) analysis. After separating recombinant human type III collagen COL3A1 by SDS-PAGE, the recombinant human type III collagen COL3A1 band with a molecular weight of approximately 130 kDa was cut off and sent to Shanghai Sangon Biotech Co., Ltd. for circular dichroism (CD) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0105] From the circular dichroism chromatogram Figure 8 The analysis shows that the sample solution exhibits strong negative absorption at 198 nm, followed by a gradual increase in the absorption peak. However, it fails to reach a positive absorption peak at 220 nm, and the absorption curve gradually flattens out. This indicates that the sample contains the characteristic structure of recombinant human type III collagen COL3A1, but has undergone partial denaturation, confirming that the sample is collagen. Furthermore, mass spectrometry analysis shows that the amino acid sequence of recombinant human type III collagen COL3A1 is shown in SEQ ID NO. 2.

[0106] (2) Ultraviolet full-wavelength scanning: The ReadMax 1200 microplate reader (Shanghai Flash Spectrum Biotechnology Co., Ltd., China) was used to perform ultraviolet full-wavelength scanning to detect the absorbance of 0.1125 mg / mL recombinant human type III collagen COL3A1 sample solution in the wavelength range of 190 nm to 500 nm.

[0107] The results are as follows Figure 9 As shown, the UV full-wavelength scan results of the 0.1125 mg / mL sample show that the sample exhibits a characteristic absorption peak of peptide bonds near 230 nm, and shows an absorption trend in the range of 230~280 nm that is consistent with the amino acid composition of recombinant human type III collagen COL3A1. This indicates that it has the typical molecular structure of recombinant human type III collagen COL3A1, and maintains good structural integrity and solution stability at this concentration.

[0108] This embodiment optimized the codons of the gene encoding human type III collagen and co-expressed it with viral proline hydroxylase in a yeast system. The induced protein expression results showed that the human type III collagen produced through the eukaryotic expression system had good safety and stable quality, eliminating the safety hazards associated with traditional extraction methods. While improving the hydrophilicity and immune rejection of collagen, it also increased the expression level and effective activity. The preparation method of this embodiment is of practical significance for the industrial production of high-quality recombinant human type III collagen. It not only solves the high risks and uncertainties associated with animal-derived collagen products but also addresses the difficulties in purification and low bioactivity of products from bacterial expression systems, achieving both high expression and high effective activity.

[0109] Example 2 Antioxidant peptide LR

[0110] 1. Screening of antioxidant peptides

[0111] Ten peptide sequences (SEQ ID NO. 7~SEQ ID NO. 16) derived from the skin mucus of the forest frog were screened by DPPH assay. Briefly, 150 μL of a peptide solution with a concentration of 1 mg / mL was mixed with 100 μL of DPPH solution (DPPH dissolved in 95% ethanol to a concentration of 0.1 mM). The mixture was incubated in the dark for 30 minutes and then centrifuged at 4000 × g for 5 minutes. A mixture of DPPH solution and 80% methanol solution (volume ratio of 2:3) was used as a blank control, and the absorbance was measured at 517 nm.

[0112] SL:SVVKGVLKAVGKNVAKNVGGSLLEQL, SEQ ID NO.7;

[0113] LK: LFSVVKGVLKGVGK, SEQ ID NO.8;

[0114] GK: GLFSVVKGVLKAVGKNVAK, SEQ ID NO.9;

[0115] KGK: KGVQKDINKAAAYYLLAGKNGHK, SEQ ID NO.10;

[0116] FK: FSVVKGVLKAVGKNVAK, SEQ ID NO.11;

[0117] KK: KVVNKAVNKAVNKAVNKVVNKAVNK, SEQ ID NO.12;

[0118] VE: VVKGVLKGVGKNVAGSLLE, SEQ ID NO.13;

[0119] CI: CPVLKGKPLCVPI, SEQ ID NO.14;

[0120] LR: LSGRGVARIADALLQLTCINCVR, SEQ ID NO.15;

[0121] GP: GILKGYIIRYCLAGLP, SEQ ID NO. 16.

[0122] The results are as follows Figure 10 As shown, LR has a DPPH radical scavenging rate of 76.25%, which is much higher than that of other sequences.

[0123] 2. In vitro antioxidant activity assay

[0124] (1) Total Antioxidant Capacity (T-AOC) Determination. The T-AOC of LR was determined using the iron reduction antioxidant (FRAP) assay. The FRAP reagent consisted of 30 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) solution, 20 mM FeCl3 solution, and deionized water. LR solutions of different concentrations were mixed with 1.5 mL of FRAP reagent and incubated at room temperature in the dark for 15 minutes. The absorbance was measured at 593 nm. Total antioxidant capacity was expressed as the molar concentration (μmol / mL) of Trolox, which was the same as the inhibition rate of the sample.

[0125] The results are as follows Figure 11 As shown in Figure A, the total antioxidant capacity of LR was concentration-dependent, reaching 0.58 μmol / mL at a concentration of 1024 μmol / L, which was significantly higher than the total antioxidant capacity of glutathione (GSH) at the same concentration (0.13 μmol / mL) (P<0.05). The results indicate that at the same concentration, the total antioxidant capacity of LR is significantly higher than that of GSH, demonstrating good antioxidant activity.

[0126] (2) DPPH scavenging rate determination. Briefly, 150 μL of peptide sample solutions of different concentrations were mixed with 100 μL of DPPH solution (DPPH dissolved in 95% ethanol to a concentration of 0.1 mM). The mixture was allowed to stand in the dark for 30 minutes and then centrifuged at 4000 × g for 5 minutes. The absorbance was measured at 517 nm using a mixture of DPPH solution and 80% methanol solution (volume ratio of DPPH solution to 80% methanol solution was 2:3) as a blank control.

[0127] The results are as follows Figure 11As shown in Figure B, LR and GSH exhibit certain antioxidant capabilities, and their DPPH scavenging ability increases with increasing concentration. When the LR concentration is 128 μmol / L, the DPPH scavenging rate reaches IC50. 50 The results showed that, at the same concentration, LR had a higher DPPH scavenging capacity than GSH, and the difference was significant up to 1024 μmol / L (P<0.05).

[0128] 3. Molecular docking analysis

[0129] The three-dimensional structures of DPPH and ABTS were obtained from the PubChem database. Molecular docking was performed using Autodock Vina, and the results were analyzed and visualized using Discovery Studio 2025.

[0130] This embodiment further elucidates the binding mechanism of LR with DPPH and ABTS using molecular docking technology. Figure 12 The results show that LR exhibits strong binding with DPPH and ABTS, with binding energies of -3.6 kcal / mol, respectively. Figure 12 (A in the text) and -4.3 kcal / mol ( Figure 12 (B in the text). The structure-antioxidant activity relationship of LR was analyzed. Figure 13 ).

[0131] 4. Biosafety Experiments

[0132] (1) Hemolysis test. Specific pathogen-free (SPF) Kunming mice (12 weeks old) were selected for this experiment. All animal experimental procedures were performed according to Chinese regulations. Blood was collected from the orbital cavity of the mice after anesthesia, and the mice were euthanized. The collected blood was washed three times with 100 mM PBS (pH 7.4). The sample was centrifuged at 4000 × g for 3 min to obtain fresh red blood cells. The red blood cells were diluted to a concentration of 4% using PBS, thoroughly mixed with an equal volume of LR solution of different concentrations, and incubated at 37°C for 30 min. The mixture was centrifuged at 10,000 × g for 5 min, and the absorbance of the supernatant was measured at 570 nm. Triton X-100 (1%) and 100 mM PBS were used as positive (PC) and negative (NC) controls, respectively.

[0133] The results are as follows Figure 14 As shown in Figure A, PBS and different concentrations of LR induced hemolysis of mouse erythrocytes at levels below 5%. In contrast, the positive control containing 1% Triton X-100 showed a hemolysis rate of 100%. This result indicates that LR is a non-hemolytic peptide and does not produce toxic effects on erythrocytes.

[0134] (2) Cytotoxicity assay. NIH-3T3 fibroblasts were seeded in 96-well flat-bottomed culture plates at a density of 5 × 10⁶ cells / well. 3 Cells were incubated in complete culture medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin / streptomycin and 10% FBS, and at 37°C in a 5% CO2 environment. After NIH-3T3 fibroblasts attached for 24 h, different concentrations of LR solution were introduced into the cells. After 24 h of incubation, the culture medium was removed, and the cells were washed three times with PBS. A blank control group (0) was used, with 10 μL of CCK8 and 90 μL of DMEM mixed and added to each well. The blank control group was also treated similarly. Cells were incubated at 37°C for 1 h, and absorbance was measured at 480 nm.

[0135] Cell viability results as follows Figure 14 As shown in B, the viability level of the 800 μmol / L LR treatment group was not significantly different from that of the blank control group (P>0.05). A cell viability level threshold greater than 80% is generally considered non-cytotoxic, and these findings indicate that LR did not exhibit cytotoxicity, but also did not promote NIH-3T3 cell proliferation.

[0136] 5. Stability of LR

[0137] (1) The antioxidant activity of LR solution was evaluated under different pH values, temperatures, times, and sodium chloride (NaCl) concentrations. In short, LR solutions with a concentration of 256 μmol / L were prepared in aqueous solutions with pH values ​​of 3, 5, 7, 9, and 11. The LR solutions were shaken at room temperature for 30 min to assess the effect of pH on the antioxidant stability of the peptides. Simultaneously, the 256 μmol / L LR solutions were placed in a water bath at different temperatures (25, 40, 60, 80, and 100 °C) for 30 min, and then rapidly cooled to room temperature on ice. This procedure was designed to evaluate the changes in the antioxidant activity of the peptides under different temperature conditions. Furthermore, the 256 μmol / L LR solutions were placed at 25 °C for 1, 3, 5, and 7 days to evaluate their time stability. Finally, in this example, LR solutions with a concentration of 256 μmol / L were prepared using different concentrations of NaCl solutions (0, 2, 4, 6, 8, and 10 mg / mL) as solvents and allowed to stand at room temperature for 30 min to evaluate the effect of salt concentration on their antioxidant activity. All of the above experiments were conducted in parallel using the same concentration of GSH as a positive control.

[0138] (2) Stability of antioxidant peptides in simulated gastric and intestinal juices. First, 0.2 g NaCl and 0.3 g pepsin were dissolved in 100 mL distilled water, and the pH was adjusted to 1.5 using HCl to simulate gastric juice. Then, 0.68 g potassium dihydrogen phosphate and 1 g trypsin were dissolved in 92.3 mL distilled water, followed by the addition of 7.7 mL NaOH (0.2 mol / L) to simulate intestinal juice. LR solutions with a concentration of 256 μmol / L were prepared using simulated gastric and intestinal juices as solvents, with GSH of the same concentration as a positive control. The solutions were incubated at 37 °C for 30 min, and the antioxidant activity after gastrointestinal digestion was assessed by measuring DPPH.

[0139] like Figure 15 As shown in Figure A, the antioxidant activity of LR showed no significant difference at 25℃, 80℃, and 100℃ (P>0.05), but decreased significantly at 40℃, and the DPPH scavenging rate was greater than 61.99% before and after heat treatment. These results indicate that LR possesses excellent heat treatment stability. Figure 15 As shown in Figure B, compared to a neutral environment, the DPPH scavenging rate of LR at pH=5 showed no significant difference. At pH=3, the free radical scavenging rate of LR decreased significantly (P<0.05), but remained above 60%. At pH=11, the DPPH free radical scavenging rate of LR decreased to 33.13%. This decrease may be due to acid-base triggered peptide modification, where peptides undergo physicochemical changes in an ionized state, leading to non-specific cleavage of some peptides. These results indicate that the bioactivity of LR is better maintained in neutral and slightly acidic environments. Figure 15 As shown in C, the DPPH scavenging rate of LR decreased significantly (P<0.05) when the NaCl concentration reached 6 mg / mL. This may be due to conformational changes in the peptide molecule caused by high ionic strength, which in turn led to shielding of the active site and a decrease in metal chelation ability. Time stability is as follows... Figure 15 As shown in Figure D, there was no significant difference in DPPH radical scavenging rate after 1, 3, and 5 days (P>0.05). The DPPH radical scavenging rate decreased slightly on day 7, but remained above 50%. In contrast, the DPPH radical scavenging rate of GSH decreased significantly on day 5 (P<0.05), indicating that LR has excellent temporal stability. Figure 15 As shown in Figure E, after 30 min of pepsin digestion, the DPPH free radical scavenging rate did not change significantly (P>0.05), while after 30 min of trypsin digestion, the DPPH free radical scavenging rate was significantly lower than that of the undigested group (P<0.05). However, LR consistently outperformed GSH.

[0140] 6. The protective effect of LR against H2O2-induced oxidative damage in NIH-3T3 fibroblasts.

[0141] (1) Establishment of the H2O2-induced oxidative damage model of NIH-3T3 cells. After resuscitating NIH-3T3 fibroblasts, they were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin) until the NIH-3T3 fibroblasts reached 80-90% confluence. Then, NIH-3T3 fibroblasts were treated with trypsin (0.25%) at 37°C for 5 min, and the cells were then divided into wells of 5 × 10⁶ cells each. 3 Cells were seeded at a density of 100 μmol / L in 96-well plates and cultured for 24 h. Afterward, NIH-3T3 fibroblasts were induced for 2 h with different doses (50, 100, 200, 400, 600, 800 μmol / L) of H2O2, labeled 50, 100, 200, 400, 600, and 800 respectively. After induction, the cells were washed three times with PBS. A control group (0) without H2O2 was added. 10 μL of CCK8 and 90 μL of DMEM were mixed and added to each well; the control and control groups were also treated similarly. Cells were incubated at 37°C for 1 h, and absorbance was measured at 480 nm. Subsequent experiments were performed when the NIH-3T3 fibroblast viability reached 50% with H2O2 concentration.

[0142] (2) Based on the above steps, NIH-3T3 fibroblasts were plated for oxidative damage protection experiments. NIH-3T3 fibroblasts were divided into two groups: the damage group (denoted as 0) was cultured in DMEM for 24 h and then induced with 600 μmol / L H2O2 for 2 h; the experimental group was cultured in DMEM containing different concentrations of LR for 24 h and then treated in the same way as the damage group. The concentrations of LR in the experimental group were set at 50, 100, 200, 400, 600, and 800 μmol / L, respectively, and denoted as 50, 100, 200, 400, 600, and 800. After the experiment, the culture medium was removed and the cells were washed with PBS. The activity of NIH-3T3 fibroblasts was evaluated according to the above method to explore the repair effect of LR on H2O2-induced damage to NIH-3T3 fibroblasts.

[0143] The viability of NIH-3T3 fibroblasts exposed to H2O2 is as follows: Figure 16 As shown in Figure A, under the condition of 2 h of treatment, cell viability gradually decreased with increasing H2O2 concentration, reaching approximately 53.55% after 2 h of treatment with 600 μmol / L H2O2. Therefore, an oxidative damage model was established by treating NIH-3T3 fibroblasts with 600 μmol / L H2O2 for 2 h. Figure 16As shown in Figure B, the repair effect of different concentrations of LR on H2O2-induced oxidative damage in NIH-3T3 fibroblasts was detected using the CCK-8 colorimetric assay. The cell viability of NIH-3T3 fibroblasts after H2O2 treatment was 47.16%, confirming successful model establishment. Compared with the damaged group, cell viability was slightly improved after treatment with 50 and 100 μmol / L LR, but not significantly (P > 0.05). Cell viability was significantly improved in all other groups (P < 0.05), with the highest cell viability (75.93%) observed after treatment with 600 μmol / L LR. These results indicate that LR has a significant protective effect against H2O2-induced oxidative damage in NIH-3T3 fibroblasts.

[0144] 7. Verification of in vitro anti-tyrosinase activity

[0145] (1) Sample preparation. A batch of apples with similar appearance and maturity were selected for testing. The apples were cut into small pieces of equal size (about 1g each). The apple pieces of the control group were soaked in distilled water at room temperature for 10min. The apple pieces of the positive control group and the sample treatment group were soaked in 1024μmol / L GSH and LR solutions, respectively, at room temperature for 10min. After drying, the samples were immediately aliquoted into individual petri dishes and stored at 25℃ for 1 day. The appearance of the apples was photographed every 6 hours under the same light source.

[0146] (2) Determination of browning index. The apple sample (2g) was homogenized in 10mL of 10% trichloroacetic acid and 40mL of distilled water, and allowed to stand at 35℃ for 2 hours, and then centrifuged at 4000g for 5 minutes. The absorbance of the supernatant was measured at 420nm on a UV-2100 spectrophotometer.

[0147] (3) Effect on tyrosinase activity in apples. Following the instructions of the tyrosinase activity assay kit, fresh apples were homogenized and centrifuged to collect the supernatant. Different concentrations of LR solutions were prepared using the supernatant. Finally, the absorbance was measured at 505 nm using a microplate reader. GSH was used as a positive control.

[0148] Figure 17 The letter A in the figure shows the browning index of apples at different times. Figure 18 The appearance of apple slices treated with different methods was described. The results showed that both LR and GSH treatments could inhibit browning of apple slices during storage to some extent compared with the control group soaked in water. After 3 hours of treatment, the control samples showed obvious browning, while the apple slices treated with LR and GSH showed no browning. With prolonged storage time, the browning index of apple slices in all groups increased, but was significantly lower than that in the control group (P<0.05). At all time points, the browning inhibition effect of LR at the same concentration was significantly better than that of the GSH group (P<0.05).

[0149] The effects of LR and GSH on tyrosinase activity in apples are as follows: Figure 17 As shown in Figure B, the inhibition rate of tyrosinase activity of each concentration of LR was significantly higher than that of the GSH group (P<0.05), further indicating that LR has an inhibitory effect on tyrosinase in apples.

[0150] This embodiment analyzed the biochemical indicators of a novel antioxidant peptide and its effect on preventing browning in apples. The results showed that the antioxidant peptide LR exhibited superior antioxidant activity and tyrosinase activity inhibition compared to glutathione (GSH) at the same concentration, and also demonstrated a protective effect against H2O2-induced oxidative damage in NIH-3T3 cells. This indicates that the novel antioxidant peptide LR possesses excellent antioxidant activity and tyrosinase activity inhibition, playing a protective role against cellular oxidative stress damage.

[0151] Example 3: Combination of antioxidant peptide LR (SEQ ID NO.15) and recombinant human type III collagen COL3A1 (SEQ ID NO.2)

[0152] 1. The combination of antioxidant peptide LR and recombinant human type III collagen COL3A1

[0153] The ratios of antioxidant peptide LR to recombinant human type III collagen COL3A1 are shown in Table 1. Antioxidant peptide LR and recombinant human type III collagen COL3A1 were dissolved in water or culture medium at mass ratios of 19:1, 13.9:1 and 9:1, respectively, for in vitro and cellular experiments.

[0154] Table 1. Ratio of antioxidant peptide LR to recombinant human type III collagen COL3A1

[0155]

[0156] 2. Biosafety of the compound

[0157] Hemolysis rate determination. Specific pathogen-free (SPF) Kunming mice (12 weeks old) were selected for this example. Mice were anesthetized, blood was collected from the orbital cavity, and the mice were euthanized. The blood was then washed three times with PBS (pH 7.4). Mouse blood was centrifuged at 4000×g for 3 min to obtain fresh red blood cells. The red blood cells were diluted to a concentration of 4% using 100 mM PBS to obtain a red blood cell solution. 100 μL of the red blood cell solution was thoroughly mixed with 100 μL of three different proportions of compounded mixtures and incubated at 37°C for 30 min. The mixtures were centrifuged at 10,000×g for 5 min, and the absorbance of the supernatant was measured at 570 nm. Triton X-100 (1%) and 100 mM PBS were used as positive (PC) and negative (NC) controls, respectively.

[0158] The results are as follows Figure 19 As shown, a hemolysis rate below 5% is considered a safe range, with the highest hemolysis rate in compound group 3 reaching 3.66%, which is less than 5%. This indicates that all groups have good biocompatibility.

[0159] 3. Screening for the optimal compound ratio

[0160] (1) DPPH scavenging rate determination. First, different proportions of the compound were dissolved in deionized water to form solutions with a total concentration of 1 mg / mL. Then, 150 μL of the above sample solution was mixed with 100 μL of DPPH radical solution (DPPH was dissolved in 95% ethanol to make the DPPH concentration 0.1 mM). The mixture was allowed to stand in the dark for 30 minutes and then centrifuged at 4000 × g for 5 minutes. A mixture of DPPH solution and 80% methanol solution (volume ratio of DPPH solution to 80% methanol solution was 2:3) was used as a blank control, and the absorbance was measured at 517 nm. Based on the above steps, the DPPH radical scavenging efficiency of the sample was calculated.

[0161] The results are as follows Figure 20 As shown in A, the DPPH clearance rate of compound group 1 was the highest, at 68.54%, which was significantly different from the other groups (P<0.05).

[0162] (2) ABTS scavenging rate determination. First, two stock solutions were prepared: a 7 mM ABTS solution and a 2.5 mM potassium persulfate solution. These two solutions were mixed in equal volumes and reacted at room temperature for 12 h under light-protected conditions to obtain the working solution. 800 μL of the working solution was mixed with 200 μL of a compound solution in different proportions, and after being protected from light for 10 min, the absorbance was measured at a wavelength of 734 nm. Deionized water without the compound was used as a blank control. Based on the above steps, the ABTS radical scavenging efficiency of the sample was calculated.

[0163] ABTS clearance rate results are as follows Figure 20 As shown in B, the ABTS clearance rate of compound group 1 was the highest, reaching 26.2%, which was significantly different from other groups (P<0.05).

[0164] (3) The repair effect of the compound on UVB-induced skin damage

[0165] After resuscitating HaCaT cells, they were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin) until the cells reached 80-90% confluence. Next, the cells were treated with trypsin (2.5%) at 37°C for 5 min, and then cultured at 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. Afterward, the cells were subjected to different doses (20, 30, 40 mJ / cm²). 2 The cells were treated with UVB radiation. During irradiation, the UVB lamp was placed 15 cm above the cells, and unirradiated wells were covered with aluminum foil. After irradiation, PBS was removed, and fresh complete culture medium was added. After culturing for 24 h, the culture medium was removed, and the cells were washed three times with PBS. 10 μL of CCK8 and 90 μL of DMEM were mixed and added to each well. The blank and control groups were also treated in the same way. The cells were incubated at 37 °C for 1 h, and the absorbance was measured at a wavelength of 480 nm. The results showed that when the UVB radiation dose reached 40 mJ / cm², the cells were effectively treated. 2 At that time, the HaCaT cell viability was 53.57%, reaching the median lethal dose, and this dose was used to establish the model in subsequent experiments.

[0166] Based on the above steps, cells were plated. Cells were divided into 5 groups: a blank control group cultured in DMEM only; a model group cultured in DMEM for 24 hours followed by treatment with 40 mJ / cm² water. 2 UVB light treatment; the compound groups (compound groups 1-3) underwent the same pretreatment as the model group, and were then cultured in DMEM containing different samples for 24 h. The total concentration of samples in each compound group (compound groups 1-3) was 1 mg / mL. After the experiment, the culture medium was removed and the cells were washed with PBS. Cell viability was assessed using the CCK8 assay to investigate the repair effect of different samples on UVB-induced HaCaT cell damage.

[0167] Figure 20 Figure C shows the repair effects of the three compound formulations on UVB-induced HaCaT cell damage. Compound formulation 1 showed the highest cell viability at 78.97%, which was not significantly different from compound formulation 2's 77.06%. Compound formulation 3, however, showed a significantly lower cell viability of 54.17%. These results indicate that compound formulations 1 and 2 were superior to compound formulation 3 in repairing UVB-induced HaCaT cell damage.

[0168] Based on the combined in vitro antioxidant activity and the effect on UVB-induced HaCaT cell damage, this invention selected compound group 1, with a mass ratio of antioxidant peptide LR to recombinant human type III collagen COL3A1 of 19:1, as the optimal compound group, and then conducted subsequent determination of intracellular antioxidant activity-related indicators to verify its effect.

[0169] 4. Assay of intracellular antioxidant activity of HaCaT cells

[0170] HaCaT cells were stored at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 mJ / cm² in 6-well plates and divided into 5 groups: a blank control group cultured in DMEM for 24 h; a model group cultured in DMEM for 24 h and then treated with 40 mJ / cm² water. 2 UVB light treatment; the COL3A1 group was cultured in DMEM for 24 hours and then received 40 mJ / cm² light. 2 UVB light treatment; the LR group was treated with 40 mJ / cm² light after 24 h of DMEM culture. 2 UVB light treatment; Group 1, after being cultured in DMEM for 24 hours, received 40 mJ / cm² light. 2 After UVB light treatment, the COL3A1 group (1 mg / mL), LR group (1 mg / mL), and compound 1 group underwent the same pretreatment as the model group, and were then cultured for 24 h in 1.5 mL of DMEM containing different samples (1 mg / mL recombinant human type III collagen COL3A1, 1 mg / mL antioxidant peptide LR, and compound 1). The blank control group and model group were cultured for another 24 h in 1.5 mL of DMEM medium. Subsequently, lysis was performed at 4 °C for 30 min using RIPA lysis buffer containing 1 mM PMSF, followed by centrifugation at 12000 × g for 5 min to obtain the supernatant. The obtained supernatant was used for biochemical analysis, and the levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured using commercially available assay kits.

[0171] MDA is considered a marker of lipid peroxidation, and fluctuations in its concentration reveal the severity of intracellular oxidative damage, such as at cellular levels... Figure 21 As shown in A in the figure. Except for the blank control group, the MDA level of compound group 1 was the lowest, at 112.54%, which was significantly lower than that of the LR group (136.17%, P<0.005) and the COL3A1 group (178.81%, P<0.0001). This indicates that the degree of oxidative damage to cells after treatment with compound group 1 was lower than that of the LR group and the COL3A1 group.

[0172] Intracellular protective enzyme systems include SOD and CAT. SOD is responsible for clearing O2. 2− It prevents cell damage; CAT decomposes H2O2 into water and oxygen, reducing oxidative damage. Through their synergistic effect, they can effectively prevent abnormal increases in intracellular reactive oxygen species levels. Figure 21 Figure B shows that the CAT level in compound group 1 was 96.99%, which was significantly different from the 89.59% in the LR group (P<0.05), and significantly better than the 78.03% in the COL3A1 group (P<0.005). Intracellular SOD levels were as follows... Figure 21As shown in C, the SOD levels in compound group 1 (102.37%) were significantly better than those in the LR group (86.77%) (P<0.05) and the COL3A1 group (80.71%) (P<0.05).

[0173] The above results indicate that the combined group 1 has a significantly better repair effect on oxidative damage in HaCaT cells than the groups using LR alone and the groups using COL3A1 alone.

[0174] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the composition in the preparation of products with antioxidant and skin-repairing effects, characterized in that, The composition comprises recombinant human type III collagen COL3A1 and antioxidant peptide LR; the amino acid sequence of the recombinant human type III collagen COL3A1 is shown in SEQ ID NO.2; The amino acid sequence of the antioxidant peptide LR is shown in SEQ ID NO.15; the mass ratio of the recombinant human type III collagen COL3A1 to the antioxidant peptide LR in the composition is 1:

19.

2. The application according to claim 1, characterized in that, The method for preparing the composition includes the step of mixing the recombinant human type III collagen COL3A1 and the antioxidant peptide LR.

3. A product with antioxidant and skin repair properties, characterized in that, The product is composed of recombinant human type III collagen COL3A1 and antioxidant peptide LR; the amino acid sequence of the recombinant human type III collagen COL3A1 is shown in SEQ ID NO.2; The amino acid sequence of the antioxidant peptide LR is shown in SEQ ID NO.15; the mass ratio of the recombinant human type III collagen COL3A1 to the antioxidant peptide LR in the composition is 1:

19.

4. The product according to claim 3, characterized in that, The product also includes auxiliary materials.

5. The product according to claim 4, characterized in that, The products include medical dressings.

6. The product according to claim 4, characterized in that, The dosage forms of the product include one or more of the following: aqueous solution, oil, emulsion, cream, ointment, and gel.

7. The application of antioxidant peptide LR in the preparation of products with antioxidant and skin repair effects, characterized in that, The amino acid sequence of the antioxidant peptide LR is shown in SEQ ID NO.15.

Images