A peptide to prevent collagen loss and its preparation method

Abstract

This invention belongs to the field of protein technology and relates to a peptide that prevents collagen loss and its preparation method. The amino acid sequence of the peptide is KSYELPDGQVITIG, 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 inhibit the expression of MMP1 and MMP9 in L929 cells after UVA irradiation, thus alleviating collagen loss.

Description

Technical Field

[0001] This invention belongs to the field of protein technology and relates to a peptide that prevents collagen loss 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] UV radiation can lead to the overexpression of matrix metalloproteinases (MMPs), resulting in collagen loss. This is a major histological characteristic of photoaged skin. Type I collagen (COL1) is the most abundant structural protein in the skin, playing a crucial role in maintaining skin elasticity, firmness, and moisture content. Among the MMP family, interstitial collagenase (MMP1) is a protease that specifically degrades COL1 and is one of the key MMPs leading to COL1 degradation. Gelatinase (MMP9), another key protease, not only degrades elastin, causing the breakage of elastic fibers in the skin, but also further enzymatically breaks down the products formed by MMP1's degradation of COL1 into smaller fragments, ultimately adversely affecting the collagen homeostasis of the skin.

[0004] Studies have shown that specific protein hydrolysates and peptides, such as silver carp peptides, tuna peptides, tilapia peptides, and *Isodon spp.* peptides, have been demonstrated to inhibit MMP expression and promote collagen synthesis. Therefore, further research and development of peptides that can downregulate MMP1 and MMP9 expression and upregulate COL1 expression may provide new strategies for mitigating collagen loss, thereby helping to reduce 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 medications 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. In particular, existing research suggests that *Gnaphalium affine* peptides may have a positive impact on photoaging by promoting directed cell migration to areas of skin damage. However, current research on the preparation process of Wugu insect peptides and their mechanism of action in alleviating collagen loss is not yet in-depth, and there is a lack of effective screening methods to identify peptide compounds with the potential to resist collagen loss, which limits their application in improving skin photoaging. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a peptide that prevents collagen loss and a method for preparing the same, thus providing an option for reducing skin photoaging.

[0007] On one hand, the present invention provides a peptide to prevent collagen loss, the amino acid sequence of which is KSYELPDGQVITIG.

[0008] In an embodiment of the present invention, the key active fragment of the peptide that prevents collagen loss is ELPDGQVIT.

[0009] On the other hand, the present invention provides a composition for preventing collagen loss, comprising the collagen loss-preventing peptide KSYELPDGQVITIG.

[0010] In a third aspect, the present invention provides the use of collagen-preventing peptides in the preparation of drugs for preventing collagen loss.

[0011] In a fourth aspect, the present invention provides a method for preparing the collagen-preventing peptide KSYELPDGQVITIG, 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) Identify the peptides in the enzymatic hydrolysate from step 4) to obtain the collagen-preventing peptide KSYELPDGQVITIG.

[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 prevent collagen loss, 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 peptides in the enzymatic hydrolysate obtained in step 2) were identified to obtain the peptide KSYELPDGQVITIG, which prevents collagen loss.

[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] 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 collagen-preventing peptide KSYELPDGQVITIG of this invention likely derives its ability to alleviate collagen loss from its active fragment ELPDGQVIT. Its main efficacy lies in inhibiting the expression of MMP1 and MMP9 in L929 cells after UVA irradiation and increasing intracellular COL1 levels. Simultaneously, the collagen-preventing peptide can spontaneously bind to the MMP1 receptor, altering its structural flexibility to make the MMP1 active pocket more open, thereby facilitating peptide docking with the active site, enhancing the peptide's inhibitory effect on MMP1 activity, and ultimately alleviating collagen loss. Attached Figure Description

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

[0040] Figure 2 The types and amounts of amino acids that make up the anti-collagen loss peptides 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 2 C 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 MMP1 receptor molecule are shown.

[0042] Figure 4 The effects of the five-grain insect peptide (A), P1 peptide (B), and P2 peptide (C) in the A→T→F group on the viability of L929 cells were shown.

[0043] Figure 5 The effects of the five-grain insect peptide, P1 peptide, and P2 peptide in the A→T→F group on the expression levels of MMP1, MMP9, and COL1 proteins in L929 cells were shown. Figure 5 A represents the Western blotting image of MMP1, MMP9, COL1, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Figure 5 B represents the protein expression level of MMP1; Figure 5 C represents the protein expression level of MMP9; Figure 5 D represents the protein expression level of COL1.

[0044] Figure 6 A visualization of the results of molecular docking of P1 peptide (A) and P2 peptide (B) with MMP1 receptor protein to alleviate collagen loss.

[0045] Figure 7 The diagram shows the results of molecular dynamics simulations of the binding process between the P1 and P2 peptides and the MMP1 receptor protein. Figure 7 A represents the root mean square deviation of the MMP1 receptor protein, the MMP1-P1 and MMP1-P2 complex; Figure 7 B represents the root mean square fluctuation of the MMP1 receptor protein, the MMP1-P1 and MMP1-P2 complex; Figure 7 C represents the radius of gyration of the MMP1-P1 and MMP1-P2 complex; Figure 7 D represents the solvent-accessible surface area of ​​the MMP1-P1 and MMP1-P2 complexes. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] The culture medium formulations used in the following examples are as follows:

[0049] 1) Basic culture medium: purchased from Gibco, USA.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Example 1: Preparation method of collagen loss prevention peptide KSYELPDGQVITIG (stepwise enzymatic hydrolysis method)

[0054] This embodiment uses a stepwise enzymatic hydrolysis method to prepare peptides that prevent collagen loss. The specific steps are as follows:

[0055] 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).

[0056] The specific process is as follows: Figure 1 As shown in A in the diagram.

[0057] Example 2. Preparation method of collagen loss prevention peptide KSYELPDGQVITIG (simultaneous enzymatic hydrolysis method)

[0058] This embodiment uses a simultaneous enzymatic hydrolysis method to prepare peptides that prevent collagen loss. The specific steps are as follows:

[0059] 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).

[0060] The specific process is as follows: Figure 1 As shown in B in the diagram.

[0061] Example 3. Screening of key active fragments of peptides for preventing collagen loss

[0062] In this embodiment, the anti-collagen loss peptides and their key active fragments from *Gnaphalium affine* were screened using a photo-aging 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).

[0063] In this embodiment, the process of screening anti-photoaging peptides is as follows:

[0064] 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.

[0065] Then, the effect of *Eriocaulon buergerianum* peptide on the level of intracellular reactive oxygen species (ROS) 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.

[0066] Next, the effects of *Pterocaryonium styracifolium* peptide on intracellular SOD activity and MDA content were determined using a superoxide dismutase (SOD) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and a malondialdehyde (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.).

[0067] 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, [following the literature...] [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., Inc., USA). The PVDF membrane was blocked with 5% skim milk (BD-Difco, Inc., USA) for 2 h at room temperature, 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 results were compared with the literature. [2] The method was used to determine the expression level of the target protein.

[0068] The results showed that the A→T→F group of the stepwise enzymatic digestion group had a strong potential to prevent collagen loss. 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 increasing SOD activity. Therefore, the A→N→F group was selected as a peptide for anti-collagen loss peptide identification in *Strombus haematocephala*.

[0069] 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 can greatly enhance the effect of bioactive peptides in alleviating collagen loss. Therefore, this structural feature of *Wugu Chongtai* peptides may also contribute to its high biological activity.

[0070] Next, mass spectrometry was used to screen key active fragments from the anti-collagen loss 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 are consistent with the structural features of peptides that alleviate collagen loss. Therefore, it is believed that peptide ELPDGQVIT may be the key parent fragment for alleviating collagen loss. The identified peptides were molecularly docked with the MMP1 receptor. The structure of MMP1 (PDB ID: 966C) 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 MMP1 and peptides. The center coordinates of MMP1 were set as: x = 6.6, y = -9.4, z = 38.5; the docking box size was: Subsequently, molecular docking was performed using AutoDockVina to obtain the binding energies of different peptides to MMP1, as shown in the following results. Figure 3 As shown in Table 1, all 17 peptides exhibited negative affinity for the MMP1 receptor, indicating spontaneous binding. Among them, GIPPAPR, YLPGSAPCR, GFAGDDAPR, GIGTVPVGR, KSYELPDGQVIT, EAPLNPK, KSYELPDGQVITI, and SYELPDGQVITIG showed the lowest affinity, at -7.8, -7.8, -7.6, -7.3, -7.2, -7.2, -7.0, and -7.0 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, the peptide KSYELPDGQVITIG (P1) and the repeatedly appearing potential active fragment ELPDGQVIT (P2) were selected for further validation.

[0071] Table 1. Physicochemical properties analysis of different peptide fragments

[0072]

[0073]

[0074] Example 4. Activity evaluation of the peptide KSYELPDGQVITIG for preventing collagen loss

[0075] 4.1 Evaluation of the effects of *Pteris vittatae* peptide and P1 and P2 peptides in group A→T→F on L929 cell viability

[0076] In this embodiment, the effects of the five-grain insect peptide and P1 and P2 peptides in the A→T→F group 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.

[0077] 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 basal medium containing A→T→F group of *Pteris vittataecarpa* 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 culture medium was removed, and the cells were washed with PBS. 100 μL of 10% CCK-8 solution was added, and after 1 hour of incubation, 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 A→T→F group of *Polygonum multiflorum* peptides showed no significant damage to L929 cells within the range of 0.1-10 mg / mL, with cell viability exceeding 80%. When the concentration of *Polygonum multiflorum* peptides in the A→T→F group was 1 mg / mL, cell viability was higher than that in the control group (P > 0.05), indicating that this concentration of *Polygonum multiflorum* peptides had a certain promoting effect on cell proliferation. Therefore, the 1 mg / mL A→T→F group of *Polygonum multiflorum* peptides was selected for subsequent experiments. For peptides P1 and P2, no toxicity was observed in the range of 0.03-1 mg / mL, while 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 at this concentration. Therefore, 0.5 mg / mL P1 and P2 peptides were selected for subsequent experiments.

[0078] 4.2 The protein expression levels of MMP1, MMP9, and COL1 were detected by Western Blot (WB) assay.

[0079] 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. 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 anti-collagen loss 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 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 MMP1 (Wuhan Sanying Biotechnology Co., Ltd.), MMP9 (Wuhan Sanying Biotechnology Co., Ltd.), COL1 (Shanghai Beyotime Biotechnology Co., Ltd.), and GAPDH (Shanghai Beyotime Biotechnology Co., Ltd.). The volume ratios of MMP1 to Western blotting buffer (Shanghai Beyotime Biotechnology Co., Ltd.) were 1:4000; MMP9 to Western blotting buffer 1:3000; COL1 to Western blotting buffer 1:1000; and GAPDH to Western blotting buffer 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, the 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 to calculate the relative expression level of the target protein.

[0080] The results are as follows Figure 5As shown in Figure AD, when L929 cells were exposed to UVA, the expression levels of MMP1 and MMP9 significantly increased, while the expression level of COL1 significantly decreased (P < 0.05), indicating that the cells had undergone significant photodamage. Nutritional intervention revealed that all peptides significantly inhibited MMP expression, thereby alleviating COL1 degradation. Peptide P2 showed the strongest inhibitory effect on MMP1, with its protein expression level decreasing by 22.2% compared to the model group, and showing no significant difference compared to the control group (P > 0.05), indicating that the peptide ELPDGQVIT can restore cellular collagen metabolism to normal levels. The next most effective peptides were P1 and the A→T→F group, but there was no significant difference in MMP1 expression levels between the two groups (P > 0.05). Regarding MMP9 and COL1 expression, the A→T→F group showed the best effect, followed by the P1 and P2 treatment groups. However, compared to the A→T→F group, the MMP9 and COL1 expression levels in the P2 treatment group were not significantly different (P > 0.05). MMP1 is the most critical protease in the MMP family in inducing collagen degradation in the skin. Therefore, the above results indicate that the peptide KSYELPDGQVITIG has a strong ability to inhibit COL1 degradation, while the peptide ELPDGQVIT, as the parent active fragment, provides the ability to alleviate collagen degradation.

[0081] 4.3 Molecular docking analysis was used to analyze the binding modes of peptides P1 and P2 to the MMP1 receptor protein.

[0082] The results of docking peptides P1 and P2 with the MMP1 receptor were visualized using Pymol, and the results are as follows: Figure 6As shown in AB. Subsequently, the complex formed by the two was uploaded to the Protein-Ligand Interaction profiler (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to analyze the interaction between the peptides and the MMP1 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 MMP1 are both less than 0, indicating that peptides P1 and P2 can spontaneously and stably bind to MMP1. In addition, peptides P1 and P2 interact with the target protein through hydrophobic interactions, hydrogen bonds, and salt bridges, among which hydrophobic interactions and hydrogen bonds are the two main binding modes. In 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. P2 forms the most hydrogen bonds with MMP1 (12), and P1 has 10 hydrogen bonds with MMP1. Therefore, P2 may have a stronger interaction with MMP1, thereby affecting the protein expression of MMP1 in L929 cells. This is consistent with the results of Western blotting. When peptides P1 and P2 interact with the active site of MMP1, they can inhibit the activity of MMP1, thus hindering the degradation of COL1.

[0083] Table 2. Interaction analysis of P1 and P2 with MMP1 receptor

[0084]

[0085]

[0086] 4.4 Molecular dynamics simulations were used to model the binding process of peptides P1 and P2 to the MMP1 receptor protein.

[0087] Using Gromacs 2023, the P1-MMP1 complex, P2-MMP1 complex, and MMP1 receptor protein obtained through molecular docking were placed as the initial structures in a dodecahedral box, ensuring a minimum distance of 1 nm between the complex and the box boundary. 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 7 As shown in A, the RMSD values ​​of MMP1-P1 and MMP1-P2 tend to stabilize after 60 ns, indicating that the conformations of the complexes formed by different peptides with MMP1 gradually reach a stable equilibrium. Furthermore, the average RMSD values ​​of MMP1 and the two complexes are 0.214, 0.214, and 0.306, respectively. The average RMSD value of the peptide-target protein complex is greater than 0.214 (reference conformation), suggesting that the binding of P1 and P2 to MMP1 leads to a looser structure and causes a slight conformational change in MMP1. Figure 7 Similarly, the changes in RMSF of MMP1 and the MMP1-peptide complex in B were observed. For both complexes, especially MMP1-P2, the RMSF value at residue position 171 was significantly increased. This may be due to the allosteric effect caused by peptide binding to MMP1, leading to increased residue variability at adjacent positions. Furthermore, the average RMSF value of both complexes was higher than that of MMP1, indicating that peptide binding increases the overall residue variability of the target protein, suggesting that the peptide can improve the conformational flexibility of MMP1. Figure 7 The C value in the figure reflects the changes in Rg for the two complexes. The results show that the curve fluctuations gradually stabilize after 60 ns, consistent with the RMSD values, further validating the conformational stability of the peptide-target protein during the simulation. Furthermore, the average Rg values ​​for the different complexes were 1.537 and 1.563, respectively, suggesting that P2 binding to MMP1 leads to a more porous overall structure, potentially inducing conformational expansion of the S1' and S3' substrate binding pockets, thereby increasing the peptide's accessibility to the MMP1 active site. Figure 7The changes in surface area between the two complexes of MMP1-P1 and the solvent indicate that the expansion of the complex structure mainly stems from enhanced solvent exposure in the binding region. Meanwhile, the average SASA values ​​of MMP1-P1 and MMP1-P2 are 91.82 nm. 2 and 93.67nm 2 All are in the 50-200nm range 2 Within this range, it is evident that the molecular surface contains moderately exposed flexible regions and rigid regions that stably bind to peptides. In summary, both peptides can stably bind to MMP1, and the resulting MMP1 structure is more porous. P1 and P2 expand the active pocket of MMP1 by altering its flexibility, thereby facilitating the docking of peptides with the active site and ultimately enhancing its inhibitory effect on MMP1 activity.

[0088] 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.

[0089] References

[0090] [1]RIBEIRO FM,DE OLIVEIRA MM,SINGH S,et al.Ceria NanoparticlesDecrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leadingto Cell Survival,

[0091] MigrationandProliferation[J].FrontiersinBioengineeringandBiotechnology,2020,8.

[0092] [2] LIU W, YU S, HAN Y, et al. Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties [J]. Food Bioscience, 2024, 60.

[0093] [3]LIU R,HE L,CHEN L,et al.Skin’s New Shield:Food-Derived BioactivePeptides in Combating Photoaging-An Investigation Into Inhibitory Mechanismsand Structure-Activity Relationships[J].JournalofFoodBiochemistry,2025,2025(1).

[0094] [4]AN J,WANG M,LIU W,et al.Unraveling amino acid sequence featuresthat confer high calcium-binding capacity to soybean peptides and improveabsorption in caco-2cells[J].

[0095] JournalofFunctionalFoods,2025,130。

Claims

1. The application of a peptide that prevents collagen loss in the preparation of a drug to reduce skin photoaging, wherein the amino acid sequence of the peptide is KSYELPDGQVITIG.

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