An anti-inflammatory active peptide derived from shell nacre and its application in preparing skin repair products

Abstract

The present invention discloses a shell nacre-derived anti-inflammatory active peptide and its application in the preparation of skin repair products. The active peptide GEIRYEYF provided by the present invention has a concentration-dependent inhibition rate on NO release, and at a concentration of 200 μg / mL, NO release decreases by 54.32 ± 2.13%; it can also significantly reduce the secretion of pro-inflammatory cytokines TNF‑α and IL‑6 of RAW264.7 macrophages, and increase the secretion of anti-inflammatory cytokine IL‑10, and it is dose-dependent, with the highest concentration affecting IL‑6 secretion by 47.76 ± 1.02%, TNF‑α secretion by 42.01 ± 4.64%, and IL‑10 secretion by 41.71 ± 1.67%, with good anti-inflammatory activity. In addition, the active peptide has a significant proliferative effect on L929 cells, indicating that it has the potential to promote skin wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, and in particular to an anti-inflammatory active peptide derived from shell nacre and application thereof in the preparation of skin repair products. Background Art

[0002] The skin is an important physical barrier that protects against external environmental influences. Factors such as surgery, accidental injuries, burns, microbial infections, skin diseases, or metabolic dysfunction can destroy this barrier, causing skin damage. Wound healing is a crucial process for the recovery and repair of tissue function after injury. Studies have shown that this process is mainly divided into four stages: hemostasis, inflammation, proliferation, and tissue remodeling. Stagnation at any stage will lead to potential functional impairments of the skin. Drugs on the market for skin wound repair all have various defects to a certain extent. For example, small molecule compounds derived from plants have poor stability and low activity, making it difficult to promote wound repair with stable and efficient activity. Epidermal growth factor proteins have defects such as harsh storage conditions and high transportation costs.

[0003] Therefore, it is particularly important and necessary to develop new drugs that promote skin wound repair. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an anti-inflammatory active peptide derived from the nacre layer of shells.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned shell nacre-derived anti-inflammatory active peptide.

[0006] Another object of the present invention is to provide an application of the above-mentioned shell nacre-derived anti-inflammatory active peptide.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An anti-inflammatory active peptide derived from shell nacre has the following amino acid sequence: Gly-Glu-Ile-Arg-Tyr-Glu-Tyr-Phe (GEIRYEYF).

[0009] Furthermore, the shell nacre-derived anti-inflammatory active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0010] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre-derived anti-inflammatory active peptides.

[0011] The invention relates to a solid phase synthesis product comprising the above-mentioned shell nacre-derived anti-inflammatory active peptide.

[0012] The above-mentioned anti-inflammatory active peptide derived from the nacre layer of shells is a cosmetically acceptable salt or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt includes a metal salt of a peptide, wherein the metal includes lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

[0014] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an organic base, and the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.

[0015] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide and an inorganic acid or an organic acid, wherein the organic acid includes: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0016] The use of the above-mentioned shell nacre-derived anti-inflammatory active peptides, shell nacre protein hydrolysates, solid-phase synthesis products, cosmetically acceptable salts or pharmaceutically acceptable salts thereof in the preparation of anti-inflammatory agents and / or skin wound repair products.

[0017] Furthermore, the anti-inflammatory agent has the effects of promoting macrophage proliferation, promoting macrophage NO release, promoting macrophage anti-inflammatory factor secretion and / or reducing pro-inflammatory factor secretion.

[0018] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0019] Furthermore, the active peptide is any one of an oral preparation and an external preparation.

[0020] The present invention has the following advantages and effects compared to the prior art:

[0021] This study isolated, purified, and identified multiple peptides from a protein hydrolyzate extracted from the nacreous layer of shells. Molecular docking was then used to screen a novel anti-inflammatory peptide, GEIRYEYF (1075.4974 Da). Molecular docking simulations revealed that the active peptide binds to the receptor proteins cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) through hydrogen bonds and hydrophobic interactions, thereby exerting anti-inflammatory activity. The active peptide effectively promoted the proliferation of RAW264.7 macrophages and inhibited lipopolysaccharide (LPS)-induced overproduction of nitric oxide (NO) and cytokines (TNF-α and IL-6) in RAW264.7 cells, while increasing the level of the anti-inflammatory cytokine IL-10, demonstrating significant anti-inflammatory activity. Furthermore, the active peptide exhibited a significant proliferative effect on L929 cells, suggesting its potential to promote skin wound healing.

[0022] The invention provides a theoretical basis for high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the research results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0024] Figure 2 This is a study result of the effect of active peptides on NO release in RAW264.7 macrophages (Note: different lowercase letters indicate significant differences (P<0.05)).

[0025] Figure 3 The graph shows the results of a study on the effects of active peptides on the cytokine release of RAW264.7 macrophages induced by LPS; A represents the effect on the release of cytokine TNF-α, B represents the effect on the release of cytokine IL-6, and C represents the effect on the release of cytokine IL-10 (Note: different lowercase letters indicate significant differences (P<0.05)).

[0026] Figure 4 This is a graph showing the research results on the effect of active peptides on the proliferation rate of L929 cells.

[0027] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "an", "the" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0030] Example 1

[0031] 1. Synthesis of anti-inflammatory peptides from nacre

[0032] The anti-inflammatory active peptide GEIRYEYF from the shell nacre layer screened by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0033] 2. Cell Culture

[0034] Mouse macrophage RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator and passaged when the cells reached the logarithmic growth phase.

[0035] 3. RAW264.7 Cytotoxicity Assay

[0036] RAW264.7 macrophages in the logarithmic growth phase were seeded into 96-well plates at a volume of 100 μL per well, with 5 × 10 cells per well. 4 After 24 hours of incubation, the original culture medium was discarded. A blank control group and sample groups were set up. The sample group received 100 μL of complete culture medium containing various concentrations of nacre anti-inflammatory peptides, while the blank control group received an equal amount of complete culture medium. After a further 24 hours of incubation, the original culture medium was discarded, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. The cells were then incubated in a cell culture incubator for 4 hours. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance at 490 nm was measured using a microplate reader.

[0037] 4. Determination of Inflammatory Factors in RAW264.7 Cells

[0038] RAW264.7 macrophages in the logarithmic growth phase were seeded into 24-well plates at a volume of 500 μL per well, with a cell count of 1 × 10 5After culturing for 24 hours, the original culture medium was discarded. A blank control group, a model group, a sample group, and a positive control group were set up. The sample group was added with 500 μL of complete culture medium containing different concentrations of anti-inflammatory active peptides from shell nacre. The blank control group and the model group were added with an equal amount of complete culture medium. The positive control group selected dexamethasone as a control. After a further 24 hours of culture, the original culture medium was discarded. The blank group was added with 500 μL of complete culture medium. The other groups were added with 500 μL of μg / mL LPS solution. The culture was continued for 24 hours. The supernatant was collected and the levels of NO, IL-10, IL-6, and TNF-α in the cell culture supernatant were detected using NO kit and ELISA kit.

[0039] 5. Data Analysis

[0040] All experiments were performed in triplicate, and data are presented as mean ± standard deviation. Statistical graphs were generated using Origin 2019 software, and one-way analysis of variance was performed on the group means using IBM SPSS Statistics 26. P < 0.05 was considered statistically significant.

[0041] result:

[0042] (1) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the activity of RAW264.7 macrophages

[0043] The effect of the anti-inflammatory active peptide GEIRYEYF derived from shell nacre on the viability of RAW264.7 macrophages at concentrations of 50-800 μg / mL was determined based on the MTT assay.

[0044] Depend on Figure 1 As shown, GEIRYEYF did not diminish cell viability in RAW264.7 macrophages after 24 hours of treatment. Furthermore, compared with the blank control group, the nacre anti-inflammatory peptide promoted the proliferation of RAW264.7 macrophages. Therefore, subsequent studies evaluated the anti-inflammatory activity of the peptide within a concentration range of 10–200 μg / mL.

[0045] (2) Effect of the anti-inflammatory peptide GEIRYEYF from shell nacre on NO release in RAW264.7 macrophages

[0046] In the present invention, LPS was used to induce the release of NO from macrophages RAW264.7 to construct a cellular inflammation model and explore the anti-inflammatory activity of the anti-inflammatory active peptide GEIRYEYF derived from the nacre layer of shells.

[0047] Depend on Figure 2As shown, LPS-stimulated RAW264.7 macrophages in the model group significantly increased NO release (P < 0.001). Compared with the model group, the nacre-derived anti-inflammatory peptide GEIRYEYF significantly inhibited NO release from RAW264.7 macrophages at various concentrations. Notably, the inhibition of NO release was concentration-dependent. At a concentration of 200 μg / mL, GEIRYEYF decreased NO release by 54.32 ± 2.13% (P < 0.001), significantly exceeding the positive control group (dexamethasone). These results demonstrate that GEIRYEYF effectively inhibits LPS-induced NO release from RAW264.7 macrophages, modulating the inflammatory response to a certain extent.

[0048] (3) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the release of inflammatory factors in RAW264.7 macrophages

[0049] Depend on Figure 3 As shown, compared with the blank control group, the release of inflammatory cytokines TNF-α (a), IL-6 (b), and IL-10 (c) by RAW264.7 macrophages in the model group under LPS stimulation significantly increased. Dexamethasone, a positive control drug with proven anti-inflammatory properties, significantly decreased the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by RAW264.7 macrophages after dexamethasone treatment (P < 0.001), demonstrating that the cellular inflammation model used in this invention is suitable for evaluating the activity of anti-inflammatory peptides from shell nacre. The GEIRYEYF treatment group not only significantly reduced the secretion of pro-inflammatory cytokines TNF-α and IL-6 in RAW264.7 macrophages, but also significantly increased the secretion of anti-inflammatory cytokine IL-10, and the effect was dose-dependent at concentrations of 10-200 μg / mL. At the highest concentration, the effect on IL-6 secretion was 47.76±1.02%; the effect on TNF-α secretion was 42.01±4.64%; and the effect on IL-10 secretion was 41.71±1.67%.

[0050] The above results indicate that the anti-inflammatory active peptide GEIRYEYF derived from shell nacre can effectively regulate the secretion of inflammatory factors in RAW264.7 macrophages and has good anti-inflammatory activity.

[0051] Example 2

[0052] 1. Synthesis of anti-inflammatory peptides from nacre

[0053] The anti-inflammatory active peptide GEIRYEYF from the shell nacre layer screened by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was determined by HPLC to be ≥98%.

[0054] 2. Cell Culture

[0055] Mouse fibroblast L929 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator and passaged when the cells reached the logarithmic growth phase.

[0056] 3. L929 Cytotoxicity Assay

[0057] Mouse fibroblast L929 cells in the logarithmic growth phase were seeded into 96-well plates at a volume of 100 μL per well, with a cell count of 5 × 10 cells per well. 4 After 24 hours of incubation, the original culture medium was discarded. A blank control group and sample groups were set up. The sample group received 100 μL of complete culture medium containing various concentrations of nacre anti-inflammatory peptides, while the blank control group received an equal amount of complete culture medium. After a further 24 hours of incubation, the original culture medium was discarded, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. The cells were then incubated in a cell culture incubator for 4 hours. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance at 490 nm was measured using a microplate reader.

[0058] 4. L929 cell scratch assay

[0059] Mouse fibroblast L929 cells were seeded into 6-well plates and incubated overnight in a 5% CO2, 37°C incubator. A midline was drawn across the bottom of the 6-well plate. Two scratches perpendicular to the midline were made across the confluent cell monolayer at three equal points using a 200 μL pipette tip. Each well was rinsed with PBS to remove cell debris. After removal, the experimental groups were incubated for 24 hours with varying concentrations of shell nacre active peptides. To minimize the effects of cell proliferation, the blank control group was incubated for an additional 24 hours in culture medium supplemented with 2% fetal bovine serum. After incubation, the cells were observed microscopically and photographed for comparison. The effects of the active peptides on cell migration were analyzed.

[0060] result:

[0061] (1) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the activity of skin fibroblast L929 cells

[0062] like Figure 4 As shown in the data, within the mass concentration range of 5 to 40 μg / mL, GEIRYEYF had no cytotoxicity to L929 cells. Compared with the blank control group, GEIRYEYF had a significant proliferative effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0063] (2) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the migration of skin fibroblasts L929

[0064] The effect of GEIRYEYF on the migration ability of L929 cells was investigated using an in vitro wound wound assay. ImageJ software was used to compare the changes in wound area at 0 h and 24 h.

[0065] like Figure 5 As shown in the results, compared with the blank control group, GEIRYEYF at concentrations of 5 to 20 μg / mL significantly increased L929 cell migration and significantly shortened the wound distance. In particular, treatment with 5 μg / mL GEIRYEYF resulted in almost complete wound healing. This suggests that GEIRYEYF significantly promotes L929 cell migration and fusion, demonstrating a promising skin repair effect.

[0066] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An anti-inflammatory active peptide derived from shell nacre, characterized by: The amino acid sequence is as follows: Gly-Glu-Ile-Arg-Tyr-Glu-Tyr-Phe.

2. The anti-inflammatory active peptide derived from shell nacre according to claim 1, characterized in that: The shell nacre-derived anti-inflammatory active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

3. A shell nacre protein hydrolyzate comprising the shell nacre-derived anti-inflammatory active peptide according to claim 1.

4. A solid phase synthesis product comprising the anti-inflammatory active peptide derived from the nacre layer of shells as claimed in claim 1.

5. The cosmetically acceptable salt or pharmaceutically acceptable salt of the anti-inflammatory active peptide derived from the nacre of shells as claimed in claim 1.

6. The cosmetically acceptable salt or pharmaceutically acceptable salt of the anti-inflammatory active peptide derived from the nacre of shells according to claim 5, characterized in that: The cosmetically acceptable salts or pharmaceutically acceptable salts include metal salts of peptides, wherein the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by the peptide and an organic base, wherein the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a peptide with an inorganic acid or an organic acid, wherein the organic acid includes: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

7. Use of the anti-inflammatory active peptide derived from shell nacre as claimed in claim 1, the shell nacre protein hydrolyzate as claimed in claim 3, the solid-phase synthesis product as claimed in claim 4, or the cosmetically acceptable salt or pharmaceutically acceptable salt thereof of the anti-inflammatory active peptide derived from shell nacre as claimed in any one of claims 5 to 6 in the preparation of anti-inflammatory agents and / or skin wound repair products.

8. The use according to claim 7, characterized in that: The anti-inflammatory agent has the effects of promoting macrophage proliferation, promoting macrophage NO release, promoting macrophage anti-inflammatory factor secretion and / or reducing pro-inflammatory factor secretion.

9. The use according to claim 7, characterized in that: The skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

10. The use according to any one of claims 7 to 9, characterized in that: The anti-inflammatory agent and / or skin wound repair product is an external preparation.

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