Anti-photoaging damage peptide and preparation method thereof

Abstract

This invention belongs to the field of protein technology and relates to an anti-photoaging damage peptide and its preparation method. The amino acid sequence of the peptide is KSYELPDGQVITI, with the key active fragment being ELPDGQVIT. The peptide is obtained by enzymatic hydrolysis of defatted grain insect powder using alkaline protease, trypsin, papain, neutral protease, and flavor protease. This peptide can scavenge ROS free radicals after UVA irradiation, increase SOD activity, and inhibit MDA production, ultimately alleviating UVA-induced oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of protein technology and relates to an anti-photoaging damage peptide and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin influences the body's dynamic balance and protects the internal environment from harmful external factors. Studies have shown that photoaging caused by prolonged exposure to ultraviolet (UV) radiation is a significant cause of skin damage. It reduces the skin's defense capabilities, making it less resistant to harmful external stimuli. It can also trigger various skin diseases, including actinic keratosis, photoelastic fibrosis, melanoma, and basal cell carcinoma. This chronic skin damage not only affects appearance and causes psychological distress but also negatively impacts overall health.

[0003] The large amount of reactive oxygen species (ROS) free radicals induced by ultraviolet radiation have been proven to be the most critical factor causing photoaging of the skin. They can undergo peroxidation reactions with unsaturated fatty acids in cell membranes, producing malondialdehyde (MDA), which further damages macromolecules such as nucleic acids, proteins, and phospholipids, altering cell membrane permeability and inducing apoptosis. Simultaneously, ROS free radicals can also attack endogenous antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), and their related signaling pathways, such as Kelch-like ECH-associated protein 1 (Keap1) / Nuclear factor erythroid 2-related factor 2 (Nrf2), thereby inhibiting the activity and production of antioxidant enzymes. This leads to an imbalance in the body's oxidation / antioxidant defense system, resulting in oxidative stress and severe oxidative damage to the body.

[0004] Studies have shown that specific protein hydrolysates and peptides, such as milk peptides, Hepu pearl oyster peptides, and Zhanjiang isochoric algae peptides, have been proven to scavenge ROS free radicals, promote the synthesis and activity of antioxidant enzymes, and thus alleviate oxidative stress and inhibit photoaging of the skin. Therefore, in-depth research and development of peptides that can inhibit ROS free radicals and MDA production, enhance SOD activity, and activate the Keap1 / Nrf2 signaling pathway may provide new strategies for reducing oxidative stress, thereby helping to mitigate photoaging of the skin.

[0005] In clinical practice, drug therapy remains the primary approach to mitigating photoaging of the skin, mainly including topical application of retinoids, 5-fluorouracil creams, and ointments containing antioxidants or alpha-hydroxy acids. These drugs have proven effective in alleviating photoaging, but they also come with certain side effects, such as dry skin, peeling, erythema, itching, and increased photosensitivity. Long-term or improper use may exacerbate these adverse reactions, including skin irritation, pigmentation changes, telangiectasia, and an increased risk of secondary infections. Given the potential risks of drug and surgical treatments, nutritional intervention, as a complementary therapy strategy, is increasingly favored due to its lower side effects and higher patient compliance. In the field of nutritional intervention, food-derived bioactive peptides have become a focus of research due to their potential benefits in dietary supplements. *Hypericum perforatum* is a traditional Chinese medicine known for its ability to inhibit skin ulceration. However, research on its anti-photoaging effects is currently unclear, and the lack of effective preparation processes and screening methods to identify peptide compounds with anti-photoaging potential limits its application in improving photoaging after oral administration. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a peptide with anti-photoaging damage effect and a method for preparing the same, so as to provide an option for reducing skin photoaging.

[0007] On the one hand, the present invention provides a peptide that resists photoaging damage, the amino acid sequence of which is KSYELPDGQVITI.

[0008] In an embodiment of the present invention, the key active fragment of the anti-photoaging damage peptide is ELPDGQVIT.

[0009] On the other hand, the present invention provides an anti-photoaging damage composition comprising the anti-photoaging damage peptide KSYELPDGQVITI.

[0010] In a third aspect, the present invention provides the application of anti-photoaging damage peptides in the preparation of anti-photoaging damage drugs.

[0011] In a fourth aspect, the present invention provides a method for preparing the anti-photoaging damage peptide KSYELPDGQVITI, comprising the following steps:

[0012] 1) Dissolve defatted grain insect powder in water and incubate at 80-90℃ for 10-20 minutes;

[0013] 2) Adjust the pH of the defatted grain insect powder solution obtained in step 1) to 8.0, add alkaline protease and enzymatically hydrolyze at 45-55℃ for 1.5-2.5h to obtain the enzymatic hydrolysate;

[0014] 3) Adjust the pH of the enzymatic hydrolysate from step 2) to 8.0, add trypsin, and enzymatically hydrolyze at 35-42℃ for 1 hour; or adjust the pH of the enzymatic hydrolysate to 7.5, add papain, and enzymatically hydrolyze at 45-55℃ for 0.5-1.5 hours; or adjust the pH of the enzymatic hydrolysate to 7.0, add neutral protease, and enzymatically hydrolyze at 45-55℃ for 0.5-1.5 hours.

[0015] 4) Adjust the pH of the enzymatic hydrolysate obtained in step 3) to 7.5, add flavor protease, and enzymatically hydrolyze at 45-55℃ for 0.5-1.5h;

[0016] 5) The enzymatic hydrolysate obtained in step 4) is subjected to vacuum freeze-drying to obtain a powdered enzymatic hydrolysate. The peptides in the enzymatic hydrolysate are then identified to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

[0017] In step 1), the incubation temperature can be 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃ or 80℃, and the incubation time can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min.

[0018] In step 2), the enzymatic hydrolysis temperature can be 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃, and the enzymatic hydrolysis time can be 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h or 2.5h.

[0019] In step 3), for trypsin, the enzymatic hydrolysis temperature can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃ or 42℃; for papain or neutral protease, the enzymatic hydrolysis temperature can be 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃; and the enzymatic hydrolysis time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h.

[0020] In step 4), the enzymatic hydrolysis temperature can be 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃, and the enzymatic hydrolysis time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h.

[0021] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to alkaline protease is 50:1.

[0022] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to trypsin is 125:1.

[0023] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to papain is 390:1.

[0024] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to neutral protease is 50:1.

[0025] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to flavor protease is 10:1.

[0026] In embodiments of the present invention, after each enzymatic hydrolysis reaction is completed, a step may be included in which the enzymatic hydrolysate is heated in boiling water to terminate the reaction. In a specific embodiment, the enzymatic hydrolysis reaction can be terminated by boiling the enzymatic hydrolysate in boiling water.

[0027] The method of the fourth aspect of the present invention can be called the stepwise enzymatic hydrolysis method.

[0028] In a fifth aspect, the present invention provides a method for preparing peptides that resist photoaging damage, comprising the following steps:

[0029] 1) Dissolve defatted grain insect powder in water and incubate at 80-90℃ for 10-20 minutes;

[0030] 2) Adjust the pH of the solution obtained in step 1) to 7.5, add alkaline protease, trypsin, papain or neutral protease, and flavor protease, and enzymatically hydrolyze at 40-50℃ for 3-5 hours.

[0031] 3) The enzymatic hydrolysate obtained in step 2) is subjected to vacuum freeze-drying to obtain a powdered enzymatic hydrolysate. The peptides in the enzymatic hydrolysate are then identified to obtain the anti-photoaging damage peptide KSYELPDGQVITI.

[0032] In an embodiment of the present invention, in step 1), the incubation temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 80°C, and the incubation time can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0033] In an embodiment of the present invention, in step 2), the enzymatic hydrolysis temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃, and the enzymatic hydrolysis time can be 3h, 3.1h, 3.2h, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4.0h, 4.1h, 4.2h, 4.3h, 4.4h, 4.5h, 4.6h, 4.7h, 4.8h, 4.9h or 5.0h.

[0034] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to alkaline protease is 50:1, the mass ratios of defatted grain insect powder to trypsin, papain, and neutral protease are 125:1, 390:1, and 50:1, respectively, and the mass ratio of defatted grain insect powder to flavor protease is 10:1.

[0035] Alternatively, in an embodiment of the present invention, the mass ratio of alkaline protease:trypsin:flavor protease is 5:1:12.5, the mass ratio of alkaline protease:papain:flavor protease is 15.625:1:39.0625, and the mass ratio of alkaline protease:neutral protease:flavor protease is 2:1:5.

[0036] In an embodiment of the present invention, after step 2), a step may be included in which the enzymatic hydrolysate is heated in boiling water to terminate the reaction.

[0037] The method of the fifth aspect of the present invention can be called the simultaneous enzymatic hydrolysis method.

[0038] The peptide KSYELPDGQVITI of this invention has anti-photoaging ability that may originate from the active fragment ELPDGQVIT. Its main effects are that it can scavenge ROS free radicals in L929 cells after UVA irradiation, increase SOD activity and inhibit MDA production. At the same time, it can spontaneously bind to the Keap1 receptor to form a compact and stable conformation, which is beneficial to activating the Keap1 / Nrf2 signaling pathway, promoting SOD synthesis, and ultimately alleviating UVA-induced oxidative stress. Attached Figure Description

[0039] Figure 1 The process flow diagrams are for stepwise enzymatic hydrolysis (A) and simultaneous enzymatic hydrolysis (B).

[0040] Figure 2 The types and contents of amino acids that make up the anti-photoaging peptides of the five grain insect are shown. Figure 2 A represents the total content of different characteristic amino acids in the peptide chain; Figure 2 B represents the content of different characteristic amino acids at positions 1 and 2 at the C-terminus of the peptide chain and positions 1 and 2 at the N-terminus of the peptide chain; Figure 2C represents the content of 20 amino acid residues at positions 1 and 2 at the C-terminus of the peptide chain and positions 1 and 2 at the N-terminus of the peptide chain.

[0041] Figure 3 The affinity diagrams of different peptide segments docking with the Keap1 receptor molecule are shown.

[0042] Figure 4 The effects of anti-photoaging peptides (A), P1 peptide (B), and P2 peptide (C) from group A→T→F on the viability of L929 cells were shown.

[0043] Figure 5 The effects of the anti-photoaging peptides, P1 peptide, and P2 peptide from group A→T→F on intracellular ROS free radical levels, SOD activity, and MDA content in L929 cells were shown. Figure 5 A represents the intracellular ROS free radical content; Figure 5 B indicates intracellular SOD activity; Figure 5 C represents the intracellular MDA content.

[0044] Figure 6 The effects of P1 and P2 peptides on protein expression levels in the Keap1 / Nrf2 signaling pathway in L929 cells were shown. Figure 6 A represents a Western blotting image of the Keap1 / Nrf2 signaling pathway and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Figure 6 B represents the protein expression level of Keap1; Figure 6 C represents the protein expression level of Nrf2.

[0045] Figure 7 The results of molecular docking of P1 peptide (A) and P2 peptide (B) with Keap1 receptor protein are shown in the visualization.

[0046] Figure 8 The diagram shows the results of molecular dynamics simulations of the binding process between the P1 and P2 peptides and the Keap1 receptor protein. Figure 8 A represents the root mean square deviation of the Keap1 receptor protein, the Keap1-P1 and Keap1-P2 complex; Figure 8 B represents the root mean square fluctuation of the Keap1 receptor protein, the Keap1-P1 and Keap1-P2 complex; Figure 8 C represents the radius of gyration of the Keap1-P1 and Keap1-P2 complex; Figure 8 D represents the solvent-accessible surface area of ​​the Keap1-P1 and Keap1-P2 complexes. Detailed Implementation

[0047] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] The culture medium formulations used in the following examples are as follows:

[0050] 1) Basic culture medium: purchased from Gibco, USA.

[0051] 2) Complete culture medium: Thoroughly mix 10 mL of heat-inactivated horse serum, 5 mL of penicillin-streptomycin, 5 mL of non-essential amino acids, 5 mL of sodium pyruvate, and 475 mL of basal culture medium to obtain the complete culture medium. All reagents used in the complete culture medium were purchased from Gibco, USA.

[0052] 3) Culture medium containing the following groups of insect peptides: A→T→F, A→P→F, A→N→F, A+T+F, A+P+F, and A+N+F: Weigh 5 mg of insect peptide into a 10 mL centrifuge tube, add 5 mL of basal culture medium, and ensure the insect peptide is fully dissolved. This yields the culture medium containing the insect peptide.

[0053] 4) Culture medium containing peptides P1 and P2: Weigh 2 mg of peptides P1 and P2 into a 5 mL centrifuge tube, add 4 mL of basal culture medium and ensure that peptides P1 and P2 are fully dissolved. This yields a culture medium containing peptides P1 and P2.

[0054] Example 1. Preparation method of anti-photoaging damage peptide KSYELPDGQVITI (stepwise enzymatic hydrolysis method)

[0055] This embodiment utilizes a stepwise enzymatic hydrolysis method to prepare peptides that resist photoaging damage. The specific steps are as follows:

[0056] In the stepwise enzymatic hydrolysis method, the samples were divided into three groups. 3.4 g of defatted grain insect powder (containing approximately 2.5 g of protein) was weighed from each group, and 50 mL of distilled water was added. The solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 8.0, 0.025 g of alkaline protease was added, and the solution was incubated at 50°C for 2 h. After treatment, the hydrolysate was heated in boiling water for 10 min to terminate the enzymatic hydrolysis reaction. Next, the pH of the three solutions was adjusted to 8.0, 7.5, and 7.0, respectively, and 0.005 g of trypsin, 0.0016 g of papain, and 0.0125 g of neutral protease were added accordingly. Enzymatic hydrolysis was carried out at 37°C, 50°C, and 50°C for 1 h, respectively. Afterward, the hydrolysate was heated in boiling water for 10 min to terminate the reaction. Subsequently, the pH of the solution was adjusted to 7.5, 0.0625 g of flavor protease was added, and the reaction was carried out at 50°C for 1 h. After the reaction was completed, the enzymatic hydrolysate was heated in boiling water for 10 minutes to terminate the reaction. Finally, the enzymatic hydrolysate was placed at -40℃ and then freeze-dried under vacuum for 48 hours to obtain the five-grain insect peptides obtained by stepwise enzymatic hydrolysis. These were: Stepwise enzymatic hydrolysis group 1: alkaline protease → trypsin → flavor protease (A→T→F); Stepwise enzymatic hydrolysis group 2: alkaline protease → papain → flavor protease (A→P→F); Stepwise enzymatic hydrolysis group 3: alkaline protease → neutral protease → flavor protease (A→N→F). All proteases used in this method were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The enzyme activities of the five proteases are as follows: the enzyme activity of alkaline protease was 2×10⁻⁶. 5 U / g (S10154), trypsin enzyme activity is 2.5 × 10⁻⁶. 5 The enzyme activity of papain is 8 × 10 U / g (S10032). 5 U / g (S10011), the enzyme activity of neutral protease is 1×10⁻⁶. 5 U / g (S10013), the enzyme activity of the flavor protease is 2×10. 4 U / g(S10153).

[0057] The specific process is as follows: Figure 1 As shown in A in the diagram.

[0058] Example 2. Preparation method of anti-photoaging damage peptide KSYELPDGQVITI (simultaneous enzymatic hydrolysis method)

[0059] This embodiment utilizes a simultaneous enzymatic hydrolysis method to prepare peptides that resist photoaging damage. The specific operation is as follows:

[0060] In the simultaneous enzymatic hydrolysis method, the samples were divided into three groups. 3.4 g of defatted grain insect powder (containing approximately 2.5 g of protein) was weighed and added to 50 mL of distilled water. The solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 7.5. For each group, 0.025 g of alkaline protease, 0.005 g of trypsin, and 0.0625 g of flavor protease were added; 0.025 g of alkaline protease, 0.0016 g of papain, and 0.0625 g of flavor protease were added; and 0.025 g of alkaline protease, 0.0125 g of neutral protease, and 0.0625 g of flavor protease were added respectively. Enzymatic hydrolysis was carried out at 45°C for 4 h. After incubation, the hydrolysate was heated in boiling water for 10 min to terminate the reaction. Finally, the hydrolysate was placed at -40°C and then freeze-dried under vacuum for 48 h to obtain the grain insect peptides obtained by the simultaneous enzymatic hydrolysis method. The simultaneous enzymatic hydrolysis groups were: Group 1: alkaline protease + trypsin + flavor protease (A+T+F); Group 2: alkaline protease + papain + flavor protease (A+P+F); and Group 3: alkaline protease + neutral protease + flavor protease (A+N+F). All proteases used in this method were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The enzyme activities of the five proteases are as follows: the activity of alkaline protease was 2 × 10⁻⁶. 5 U / g (S10154), trypsin enzyme activity is 2.5 × 10⁻⁶. 5 The enzyme activity of papain is 8 × 10 U / g (S10032). 5 U / g (S10011), the enzyme activity of neutral protease is 1×10⁻⁶. 5 U / g (S10013), the enzyme activity of the flavor protease is 2×10. 4 U / g(S10153).

[0061] The specific process is as follows: Figure 1 As shown in B in the diagram.

[0062] Example 3. Screening of anti-photoaging peptides and key active fragments

[0063] In this embodiment, anti-photoaging peptides and their key active fragments were screened using a photoaging L929 cell model. The L929 mouse fibroblasts used in this embodiment were provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China).

[0064] In this embodiment, the process of screening anti-photoaging peptides is as follows:

[0065] First, refer to Ribeiro et al. [1] The effect of *Pteris vittatazoline* on the migration ability of photoaged cells was determined using a method that... L929 cells were cultured at 1.9 × 10⁻⁶... 5Cells were seeded at a density of [number] cells / mL in 6-well plates containing complete culture medium (containing 10% heat-inactivated horse serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% non-essential amino acids (Gibco, USA), 1% sodium pyruvate (Gibco, USA), and 87% basal medium (Gibco, USA)). After 24 hours of cell growth, the complete culture medium was removed, and 1 mL of phosphate-buffered saline (PBS) (Soluble Biotech, Beijing) was added. The plates were then irradiated with UVA for 50 min. Subsequent procedures were performed according to the literature.

[0066] Then, the effect of *Eriocaulon buergerianum* peptide on intracellular ROS free radical levels was determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beijing Gaoxinqiao Biotechnology Co., Ltd.). L929 cells were cultured at 1.8 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing *C. glutenin* peptides, respectively. The following groups were established: a control group (no UVA irradiation, basal culture medium added), a model group (UVA irradiation, basal culture medium added), and experimental groups (UVA irradiation, basal culture medium containing *C. glutenin* peptides from groups A→T→F, A→P→F, A→N→F, A+T+F, A+P+F, and A+N+F, respectively). After incubation, the solution was discarded and 1 mL of diluted DCFH-DA (DCFH-DA: basal culture medium = 1:1000) was added, and the cells were incubated for another 30 min. After completion, the cells were washed three times with basal medium, and then passaged with 0.05% trypsin at a passage ratio of 1:2. Next, the cells were resuspended in 1 mL PBS and added to a black 96-well plate. The excitation wavelength was set to 488 nm and the emission wavelength to 525 nm, and the fluorescence intensity of each group was measured.

[0067] Next, the effects of *Pteris vittatazoline* on intracellular SOD activity and MDA content were determined using an SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), respectively. L929 cells were cultured at 1.8 × 10⁻⁶ cells / year. 5Cells were seeded at a density of [number] cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing *Pseudomonas aeruginosa* peptide, respectively. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C was added, and cells were lysed at 4°C for 1 h. After lysis, the cells were centrifuged at 12000g for 5 min at 4°C, and the supernatant was collected. Finally, the protein concentration was determined using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and the SOD activity and MDA content were determined using an SOD assay kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.).

[0068] Finally, referring to Liu et al. [2] The effect of *Pteris vittatazoline* on collagen metabolism in L929 cells was determined using a method that... 5 Cells were seeded at a density of [number] cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing *Pseudomonas aeruginosa* peptide, respectively. Subsequently, the method described in the literature was followed. [2]Sample solutions were collected and protein concentrations were standardized. The proteins (25 μg) in the sample solutions were then separated on a 10% separating gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millibert & Co., USA). The PVDF membrane was blocked with 5% skim milk (BD-Difco, USA) at room temperature for 2 h, followed by overnight incubation at 4 °C with diluted primary specific antibodies, including matrix metalloproteinases (MMP) 1 / 3 / 9, type I collagen (COL1), and GAPDH (MMP1 and MMP9 rabbit polyclonal antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd.; MMP3, COL1A1, and GAPDH monoclonal antibodies were purchased from Shanghai Beyotime Biotechnology Co., Ltd.). The volume ratios of MMP1 to Western blotting primary antibody dilution buffer (Shanghai Beyotime Biotechnology Co., Ltd.) were 1:4000; MMP3 to Western blotting primary antibody dilution buffer was 1:2000; MMP9 to Western blotting primary antibody dilution buffer was 1:3000; COL1 to Western blotting primary antibody dilution buffer was 1:1000; and GAPDH to Western blotting primary antibody dilution buffer was 1:5000. The PVDF membrane was then incubated with the diluted horseradish peroxidase-labeled goat anti-rabbit (IgG(H+L)) secondary antibody at room temperature for 2 hours. The volume ratio of IgG(H+L) to Western blotting secondary antibody dilution buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was 1:2000. Finally, the method was followed according to the literature. [2] Determine the expression level of the target protein.

[0069] The results showed that the A→T→F group of the stepwise enzymatic digestion group had strong anti-photoaging potential. It exhibited the strongest effect in promoting L929 cell migration and scavenging ROS free radicals, and also showed strong effects in inhibiting MDA production, MMP1 / 3 / 9 expression, promoting COL1 expression, and enhancing SOD activity. Therefore, the A→N→F group was selected as a peptide for anti-photoaging peptide identification by *Strombus haematocephala*.

[0070] from Figure 2 As can be seen from AC, the C-terminus and N-terminus of the anti-photoaging peptides from *Polygonum multiflorum* in group A→N→F are enriched with hydrophobic and charged amino acids, respectively. Studies have shown... [3] This amino acid distribution characteristic is key to the anti-photoaging effect of bioactive peptides. Therefore, this structural feature of the anti-photoaging peptide from *Polygonum multiflorum* may also contribute to its high bioactivity.

[0071] Next, mass spectrometry was used to screen key active fragments from the anti-photoaging peptides of *Pteris vittata*. As shown in Table 1, a recurring parent peptide, ELPDGQVIT, was found among the identified peptides. Its amino acid composition and distribution characteristics conform to the structural features of anti-photoaging peptides. Therefore, it is believed that peptide ELPDGQVIT may be the key parent fragment for exerting anti-photoaging activity. The identified peptides were molecularly docked with the Keap1 receptor. The structure of Keap1 (PDB ID: 2FLU) was obtained from the RCSB PDB protein database (https: / / www.rcsb.org / ), and the structure of the peptide was obtained from the PEP-FOLD4 peptide structure prediction website (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ). AutoDockTools 1.5.6 was used to dehydrate and hydrogenate Keap1 and the peptides. The center coordinates of Keap1 were set as: x = 8.5, y = 10.1, z = 1.8; the docking box size was: Subsequently, molecular docking was performed using AutoDockVina to obtain the binding energies of different peptides to Keap1, as shown in the following results. Figure 3 As shown in Table 1, all 17 peptides exhibited negative affinity for the Keap1 receptor, indicating that they can bind spontaneously. Among them, YLPGSAPCR, GIPPAPR, VDSVLDVVRK, TEAPLNPK, SYELPDGQVITIG, and KSYELPDGQVITI showed the lowest affinity, at -7.4, -7.1, -6.8, -6.7, -6.7, and -6.5 kcal / mol, respectively. Simultaneously, the molecular weight, isoelectric point, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, and overall average hydrophilicity / hydrophobicity (GRAVY) of the peptides were analyzed using the ExpasyProtParam online database (https: / / web.expasy.org / protparam / ). Finally, based on the physicochemical properties, structural characteristics, affinity and content of peptides, peptide KSYELPDGQVITI (P1) and the repeatedly occurring potential active fragment ELPDGQVIT (P2) were selected for further validation.

[0072] Table 1. Physicochemical properties analysis of different peptide fragments

[0073]

[0074]

[0075] Example 4. Activity evaluation of the anti-photoaging damage peptide KSYELPDGQVITI

[0076] 4.1 Evaluation of the effects of anti-photoaging peptides and P1 and P2 peptides from the A→T→F group on the viability of L929 cells.

[0077] In this embodiment, the effects of the anti-photoaging peptides and P1 and P2 peptides of the A→T→F group of *C. tumefaciens* on the viability of L929 cells will be evaluated using the Cell Counting Kit-8 (CCK-8). CCK-8 was purchased from Beijing Gaoxinqiao Biotechnology Co., Ltd.

[0078] 100 μL of L929 cells (provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were loaded at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / mL in 96-well plates containing complete culture medium (containing 10% heat-inactivated horse serum, 1% penicillin-streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, and 87% basal medium) and incubated for 24 hours. Heat-inactivated horse serum, penicillin-streptomycin, non-essential amino acids, sodium pyruvate, and basal medium were all purchased from Gibco, USA. When the cell density reached 80%, the complete culture medium was discarded, and different concentrations of A→T→F group *Pteris vittata* anti-photoaging peptides (0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL) or P1 and P2 peptides (0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL) were added, and incubation continued for another 24 hours. After incubation, the basal culture medium containing the A→T→F group of *Gnaphalium affine* anti-photoaging peptides, P1 peptide, and P2 peptide was removed, and the cells were washed with PBS. 100 μL of 10% CCK-8 solution was added, and after incubation for 1 hour, the absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 4 As shown in Figures A and C, the anti-photoaging peptides from *Pteris vittatae* in the A→T→F group showed no significant damage to L929 cells within the concentration range of 0.1-10 mg / mL, with cell viability exceeding 80%. When the concentration of the anti-photoaging peptides in the A→T→F group was 1 mg / mL, cell viability was higher than that of the control group (P > 0.05), indicating that this concentration of anti-photoaging peptides had a certain promoting effect on cell proliferation. Therefore, the 1 mg / mL concentration of the anti-photoaging peptides in the A→T→F group was selected for subsequent experiments. For peptides P1 and P2, no toxicity was observed in L929 cells within the concentration range of 0.03-125 mg / mL. However, at a concentration of 0.5 mg / mL, the cell viability of both groups was higher than that of the control group (P < 0.05), indicating that this concentration promoted cell growth. Furthermore, the cell viability of each group was close to its maximum value at this concentration. Therefore, the 0.5 mg / mL concentration of peptides P1 and P2 was selected for subsequent experiments.

[0079] 4.2. The effects of three anti-photoaging peptides on UVA-induced intracellular ROS free radical levels in L929 cells were determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA).

[0080] L929 cells were fed at a concentration of 1.8 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing anti-photoaging peptides, respectively. The following groups were established: control group (no UVA irradiation, basal culture medium added), model group (UVA irradiation, basal culture medium added), A→T→F group (UVA irradiation, basal culture medium containing A→T→F group anti-photoaging peptides), P1 group (UVA irradiation, basal culture medium containing peptide P1), and P2 group (UVA irradiation, basal culture medium containing peptide P2). After incubation, the solution was discarded and 1 mL of diluted DCFH-DA (DCFH-DA:basal culture medium = 1:1000) was added, and the cells were incubated for another 30 min. After completion, the cells were washed three times with basal medium, followed by passage in 0.05% trypsin-EDTA (Gibco, USA) at a passage ratio of 1:2. Next, the cells were resuspended in PBS and added to black 96-well plates. The excitation wavelength was set to 488 nm and the emission wavelength to 525 nm, and the fluorescence intensity of each group was measured. The results are as follows: Figure 5 As shown in Figure A, compared with the control group, the ROS free radical content in the model group was significantly increased (P < 0.05), indicating that the cells had already suffered severe oxidative damage. After treatment with the anti-photoaging peptide, the ROS free radical levels in the cells of all three groups were significantly reduced (P < 0.05), with peptide P1 showing the strongest inhibitory activity, reducing the ROS free radical content by 43.11% compared with the model group. This indicates that the peptide KSYELPDGQVITI not only alleviated UVA-induced oxidative stress but also restored the intracellular ROS free radical content to the normal physiological range, confirming its strong free radical scavenging ability.

[0081] 4.3 The effects of three groups of anti-photoaging peptides on UVA-induced intracellular SOD activity and MDA content in L929 cells were determined using a SOD assay kit (WST-1 method) and an MDA assay kit (TBA method).

[0082] L929 cells were fed at a concentration of 1.8 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing anti-photoaging peptides, respectively. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C was added, and cells were lysed at 4°C for 1 h. Afterward, the cells were centrifuged at 12000g for 5 min at 4°C, and the supernatant was collected. Finally, the protein concentration was determined using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and the SOD activity and MDA content were determined using an SOD assay kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.). The results are as follows: Figure 5 As shown in Figure BC, after UVA irradiation, the SOD activity in L929 cells decreased significantly (P < 0.05), while the MDA content increased significantly (P < 0.05), indicating an imbalance between the intracellular antioxidant and oxidative systems, causing severe oxidative damage to the cells. After treatment with anti-photoaging peptides, the SOD activity and MDA content of all experimental groups were significantly improved. In terms of SOD activity, the P2 treatment group showed the highest activity at 13.41 ± 0.07 U / mg protein, an increase of 38.39% compared to the model group. This was followed by the A→T→F group and the P1 treatment group, with SOD activities of 12.78 ± 0.02 U / mg protein and 12.56 ± 0.07 U / mg protein, respectively. Although the SOD activities of the two groups were relatively similar, they still showed significant differences (P < 0.05). In terms of MDA content, the P2 treatment group also showed the best effect, reducing it by 74.99% compared to the model group, and showing no significant difference from the control group (P > 0.05). Secondly, the A→T→F group and the P1 treatment group showed no significant difference in MDA content between the two groups (P>0.05). This indicates that when the active fragment P2 is released through enzymatic hydrolysis, it possesses a stronger ability to enhance antioxidant enzyme activity. In conclusion, peptide KSYELPDGQVITI has a strong ability to scavenge ROS free radicals and enhance antioxidant enzyme activity, while peptide ELPDGQVIT, as the parent fragment, provides it with the ability to alleviate photoaging.

[0083] 4.4 The protein expression levels of Keap1 and Nrf2 were detected by Western Blot (WB) assay.

[0084] Refer to Liu et al. [2] Western blot (WB) experiments were performed using the method described above. L929 cells were cultured at a concentration of 1.8 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of [number] cells / mL in 6-well plates containing complete culture medium and incubated for 24 h. When the cell density reached 80%, the complete culture medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were incubated for another 24 h with basal culture medium and basal culture medium containing anti-photoaging peptides, respectively. After incubation, the solution was removed and RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C was added, and cells were lysed at 4°C for 1 h. After lysis, the cells were centrifuged at 12000g for 5 min at 4°C, and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and the protein concentration of the supernatant was then standardized to 2 mg / mL. The protein (25 μg) in the supernatant was separated on a 10% separating gel and then transferred to a PVDF membrane (Millipore, Inc., USA). PVDF membranes were blocked with 5% skim milk (BD-Difco, USA) for 2 hours at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibodies Keap1 (Chengdu Zhengneng Biotechnology Co., Ltd.), Nrf2 (Shanghai Beyotime Biotechnology Co., Ltd.), and GAPDH (Shanghai Beyotime Biotechnology Co., Ltd.). The volume ratio of Keap1 to Western blotting buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was 1:1000; the volume ratio of Nrf2 to Western blotting buffer was 1:2000; and the volume ratio of GAPDH to Western blotting buffer was 1:5000. The PVDF membranes were then incubated with diluted secondary antibody IgG (H+L) at room temperature for 2 hours. The volume ratio of IgG (H+L) to Western blotting buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was 1:2000. Finally, protein bands were detected using an ECL kit (Shanghai Beyotime Biotechnology Co., Ltd.), and the band grayscale was analyzed using ImageJ 1.8.0 software. The relative expression levels of the target protein were then calculated. The results are as follows: Figure 6As shown in Figures AC, the expression levels of Keap1 and Nrf2 in cells were normal before UVA irradiation. Upon UVA stimulation, the expression level of Nrf2 in the cell nucleus showed an increasing trend. This suggests that under continuous oxidative stress, cells respond to oxidative damage by releasing Nrf2 into the nucleus to activate antioxidant genes. After incubation with the anti-photoaging peptide, the Nrf2 level in the P1 treatment group was found to be further increased compared to the model group, indicating that the peptide KSYELPDGQVITI can further promote the synthesis of intracellular antioxidant enzymes, thereby alleviating damage caused by oxidative stress. Furthermore, an increased expression level of Keap1 was also observed in cells treated with UVA (P < 0.05). This directly inhibits the synthesis of endogenous antioxidant enzymes, reducing the cell's ability to scavenge ROS free radicals and exacerbating oxidative stress-induced cell damage. After treatment with P1 and P2 peptides, the expression level of Keap1 in cells was significantly lower than that in the model group and recovered to a level close to that of the normal group, indicating that both peptide KSYELPDGQVITI and active fragment ELPDGQVIT can restore the normal antioxidant function of cells.

[0085] 4.5 Molecular docking analysis was used to analyze the binding modes of peptides P1 and P2 to the Keap1 receptor protein.

[0086] The results of docking peptides P1 and P2 with the Keap1 receptor were visualized using Pymol, and the results are as follows: Figure 7As shown in A and B in the table. Subsequently, the complex formed by the two was uploaded to Protein-Ligand Interaction Profiler (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to analyze the interaction between the peptides and the Keap1 receptor protein (including binding mode, receptor protein binding site, etc.). The results are shown in Table 2. The affinity energies of P1 and P2 to the Kelch domain of Keap1 are all less than 0, indicating that peptides P1 and P2 can spontaneously and stably bind to Keap1. In addition, peptides P1 and P2 interact with the target protein through hydrophobic interactions, hydrogen bonds, π-π stacking, and salt bridges, among which hydrophobic interactions and hydrogen bonds are the two main binding modes. Among the intermolecular interactions, hydrogen bonds are one of the stronger non-covalent interaction types, and their number has an important influence on the stability of the complex. P1 forms the most hydrogen bonds with the Kelch domain (12), and P2 has 9 hydrogen bonds with Keap1. Therefore, P1 may have a stronger interaction with Keap1, thereby affecting the protein expression of Nrf2 in the cell nucleus. When peptides P1 and P2 interact with the domains of the Keap1 receptor protein, they can compete with Nrf2 for binding sites, thereby stimulating Nrf2 to dissociate from Keap1 and enter the cell nucleus to bind to ARE, ultimately stimulating the synthesis of antioxidant enzymes and promoting the dynamic balance of cellular redox.

[0087] Table 2. Interaction analysis of P1 and P2 with Keap1 receptor

[0088]

[0089] 4.6. Molecular dynamics simulations were used to model the binding process of peptides P1 and P2 to the Keap1 receptor protein.

[0090] Using Gromacs 2023, the P1-Keap1 complex, P2-Keap1 complex, and Keap1 receptor protein obtained through molecular docking were placed as the initial structures in a dodecahedral box, ensuring that the distance between the complex and the box boundary was at least 1 nm. Subsequently, water molecules were added to the box (solvation), and Na was added. + and Cl - The total charge of the system was set to zero. The Amber99SB force field and TIP3P water molecule model were chosen when constructing the simulation system. (Refer to An et al.) [4]The method employed a two-step energy minimization approach for the initial simulation system: the first step used the steepest descent method for 10,000 iterations, and the second step used the conjugate gradient method for 5,000 iterations. After energy optimization, 200ps NVT temperature-controlled simulations and NPT isobaric simulations were performed to balance the temperature and pressure to below 310K and 1 bar, respectively. Next, a 100ns formal simulation was conducted, with the conformation saved every 10ps. The temperature control algorithm used was V-rescale, and the pressure control algorithm was Parrinello-Rahman. Finally, Gromacs commands were used to analyze the simulation results. The stability of the system was assessed using root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA). Figure 8 As shown in Figure A, the RMSD values ​​fluctuated within the range of 0.12-0.30 within 100 ns, indicating that the complexes formed by different peptides with Keap1 reached a stable equilibrium. Furthermore, the average RMSD values ​​of Keap1, Keap1-P1, and Keap1-P2 were 0.267, 0.227, and 0.142, respectively. Among these, the average RMSD values ​​of the peptide-target protein complexes were all less than 0.267 (reference conformation), indicating that the binding of P1 and P2 enhanced the stability of the Keap1 receptor, with the Keap1-P2 complex exhibiting the best stability. Figure 8 In the figure, B represents the RMSF changes of Keap1 and the Keap1-peptide complex. The RMSF values ​​of key amino acid residues near the Keap1-P1 and Keap1-P2 binding sites (such as Arg380, Arg415, Ala556, Ser363, Ser555, Asn382, Tyr334, Ser602, Arg483, Ser508, and Gln530) show both increasing and decreasing trends. This indicates that the peptide binds directly to some sites of Keap1 through hydrophobic interactions or hydrogen bonds, thus restricting the degree of freedom in that region and leading to a more stable structure. At the same time, for indirect binding regions near the binding sites, it may be related to the allosteric effect caused by peptide binding—the peptide binding to Keap1 triggers a conformational change, leading to increased flexibility in that region. However, the overall fluctuation trends of the two complexes and Keap1 are similar, indicating that peptide binding does not significantly change the overall dynamic properties of Keap1, but improves the stability of the complex. Figure 8The value of C in the figure reflects the variation of Rg for the two complexes. The average Rg values ​​for the Keap1-P1 and Keap1-P2 complexes are 1.820 and 1.826, respectively. The Rg values ​​of both systems tend to stabilize after 60 ns, indicating that the complexes formed stable conformations during the simulation. The Keap1-P1 complex has a smaller Rg value, indicating that it has a more compact and stable structure. Figure 8 In the figure, D represents the change in surface area that can directly contact the solvent during the simulation of the two complexes. After 60 ns, the fluctuation of the SASA value of the complexes decreased, indicating that the conformation of the complexes tended to be stable at this time. The average SASA values ​​of Keap1-P1 and Keap1-P2 were 129.302 and 130.659, respectively, both in the range of 125-137 nm. 2 Within this range, the molecular surface contains moderately exposed flexible regions and rigid regions that bind stably to the peptides. This result is consistent with previous analyses of RMSF values. These results indicate that both anti-photoaging peptides can form stable complexes with Keap1, with Keap1-P1 exhibiting a more compact structure, while the Keap1-P2 conformation demonstrates the best stability. Ultimately, both peptides competitively inhibit the interaction between Nrf2 and Keap1, thereby maximally activating the Keap1 / Nrf2 signaling pathway.

[0091] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

[0092] References

[0093] [1]RIBEIRO FM,DE OLIVEIRA MM,SINGH S,et al.Ceria NanoparticlesDecreaseUVA-Induced Fibroblast Death Through Cell Redox Regulation Leading toCell Survival,Migration and Proliferation[J].Frontiers in Bioengineering andBiotechnology,2020,8.

[0094] [2]LIU W,YU S,HAN Y,et al.Systematic sequence characterization ofenzymatic-derivedsoybean peptides for precision enhancement of anti-inflammatory properties[J].FoodBioscience,2024,60.

[0095] [3]LIU R,HE L,CHEN L,et al.Skin’s New Shield:Food-Derived BioactivePeptides inCombating Photoaging-An Investigation Into Inhibitory Mechanismsand Structure-ActivityRelationships[J].JournalofFoodBiochemistry,2025,2025(1).

[0096] [4]AN J,WANG M,LIU W,et al.Unraveling amino acid sequence featuresthat confer highcalcium-binding capacity to soybean peptides and improveabsorption in caco-2cells[J].Journal of Functional Foods,2025,130.

Claims

1. Application of anti-photoaging damage peptide in the preparation of anti-photoaging damage drugs, wherein the amino acid sequence of the peptide is KSYELPDGQVITI.

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