Shell pearl layer-derived anti-inflammatory active peptide and application thereof in preparation of skin repair products

Abstract

The invention discloses a shell nacre-derived anti-inflammatory active peptide and application thereof in preparation of a skin repair product. The inhibition rate of the active peptide GEIRYEYF provided by the invention on the NO release amount is concentration-dependent, and the NO release amount is reduced by 54.32 + / -2.13% under the concentration of 200 mu g / mL; in addition, secretion of RAW264.7 macrophage pro-inflammatory cytokines TNF-alpha and IL-6 can be remarkably reduced, secretion of anti-inflammatory cytokines IL-10 can be improved, dose dependence is achieved, under the highest concentration, the influence on IL-6 secretion is 47.76 + / -1.02%, the influence on TNF-alpha secretion is 42.01 + / -4.64%, the influence on IL-10 secretion is 41.71 + / -1.67%, and good anti-inflammatory activity is achieved. In addition, the bioactive peptide has a remarkable proliferation effect on L929 cells, and it is indicated that the bioactive peptide has the potential of promoting skin wound healing.

Description

Technical Field

[0001] The 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 preparing skin repair products. Background Art

[0002] The skin is an important physical barrier that can resist the influence of the external environment. Factors such as surgery, accidental injuries, burns, microbial infections, skin diseases or metabolic dysfunctions can destroy this barrier, causing skin damage. Wound healing is an important 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 in any stage will lead to potential functional disorders of the skin. To a certain extent, the drugs on the market for skin trauma repair have different defects. For example, small molecule compounds from plant sources 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 peptides.

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

[0007] The purpose of the present invention is achieved through the following technical solutions: An anti-inflammatory active peptide derived from shell nacre has the following amino acid sequence: Gly-Glu-Ile-Arg-Tyr-Glu-Tyr-Phe (GEIRYEYF).

[0008] Furthermore, the shell nacre-derived anti-inflammatory active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0009] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre-derived anti-inflammatory active peptides.

[0010] The invention relates to a solid phase synthesis product comprising the above-mentioned shell nacre-derived anti-inflammatory active peptide.

[0011] The above-mentioned anti-inflammatory active peptide derived from the pearl layer of shells is a cosmetically acceptable salt or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the cosmetically acceptable salt or pharmaceutically acceptable salt comprises a metal salt of a peptide, wherein the metal comprises lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

[0013] 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.

[0014] 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; and the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0015] 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.

[0016] 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.

[0017] Furthermore, the skin wound repair product has the effect of promoting the proliferation and / or migration of fibroblasts.

[0018] Furthermore, the active peptide is any one of an oral preparation and an external preparation.

[0019] Compared with the prior art, the present invention has the following advantages and effects: The present invention separates, purifies and identifies multiple peptides from the protein hydrolysate extracted from the pearl layer of shells, and then screens out a new anti-inflammatory peptide GEIRYEYF (1075.4974 Da) through molecular docking. Molecular docking simulation shows that the active peptide can bind to the receptor protein cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) through hydrogen bonds and hydrophobic interactions, thereby exerting anti-inflammatory activity. The active peptide can effectively promote the proliferation of macrophage RAW264.7 cells, and can inhibit the excessive production of nitric oxide (NO) and cytokines (TNF-α, IL-6) in RAW264.7 cells induced by lipopolysaccharide (LPS), while increasing the level of anti-inflammatory cytokines (IL-10), showing significant anti-inflammatory activity. In addition, the active peptide has a significant proliferation effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0020] The invention provides a theoretical basis for high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the study results on the effect of active peptides on the viability of RAW264.7 macrophages.

[0022] Figure 2 This is a study result of the effect of active peptides on NO release from RAW264.7 macrophages (Note: different lowercase letters indicate significant differences (P<0.05)).

[0023] Figure 3 This is a study result of the effect of active peptides on the cytokine release of RAW264.7 macrophages induced by LPS; A is the effect on the release of cytokine TNF-α, B is the effect on the release of cytokine IL-6, and C is the effect on the release of cytokine IL-10 (Note: different lowercase letters indicate significant differences (P<0.05)).

[0024] Figure 4 This is a graph showing the study results on the effect of active peptides on the proliferation rate of L929 cells.

[0025] Figure 5 This is a graph showing the study results on the effect of active peptides on L929 cell migration. DETAILED DESCRIPTION

[0026] 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 used to limit the present invention.

[0027] 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", "said" 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 other meanings.

[0028] Example 1 1. Synthesis of anti-inflammatory active peptides from shell nacre The anti-inflammatory active peptide GEIRYEYF derived from the nacre layer of shells screened out by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was ≥98% as determined by HPLC.

[0029] 2. Cell Culture 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 the cells were passaged when they grew to the logarithmic growth phase.

[0030] 3. RAW264.7 Cell Cytotoxicity Assay 100 μL of RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, with 5×10 cells per well. 4 After culturing for 24 h, the original culture medium was discarded. A blank control group and a sample group were set up. The sample group was added with 100 μL of complete culture medium containing different concentrations of shell nacre anti-inflammatory peptides, and the blank control group was added with an equal amount of complete culture medium. After continuing to culture for 24 h, the original culture medium was discarded, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and then placed in a cell culture incubator for 4 h. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and after shaking for 10 min, the absorbance value at a wavelength of 490 nm was measured using an enzyme marker.

[0031] 4. Determination of inflammatory factors in RAW264.7 cells 500 μL of RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 24-well plate, with a cell number of 1×10 5 After culturing for 24 h, 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, and the positive control group selected dexamethasone as a control. After continuing to culture for 24 h, the original culture medium was discarded, 500 μL of complete culture medium was added to the blank group, and 500 μL of μg / mL LPS solution was added to the other groups. After continuing to culture for 24 h, the supernatant was taken, and the levels of NO, IL-10, IL-6, and TNF-α in the cell culture supernatant were detected using NO kits and ELISA kits.

[0032] 5. Data Analysis All experiments were set up in three parallel groups, and the final data were expressed as mean ± standard deviation. Statistical graphs were completed in Origin 2019 software, and the experimental results were analyzed by one-way analysis of variance for the means of each group using IBM SPSS Statistics 26 software. P < 0.05 was considered statistically significant.

[0033] result: (1) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the activity of RAW264.7 macrophages The MTT method was used to determine the effect of the anti-inflammatory active peptide GEIRYEYF derived from shell nacre on the viability of RAW264.7 macrophages at a concentration of 50-800 μg / mL.

[0034] Depend on Figure 1 It can be seen that after GEIRYEYF treated RAW264.7 macrophages for 24 hours, the cell viability did not weaken, and compared with the blank control group, the shell nacre anti-inflammatory peptide promoted the proliferation of RAW264.7 macrophages. Therefore, the subsequent study chose to evaluate the anti-inflammatory activity in the concentration range of 10-200 μg / mL.

[0035] (2) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on NO release in RAW264.7 macrophages The present invention constructs a cellular inflammation model by inducing the release of NO from macrophages RAW264.7 using LPS, and explores the anti-inflammatory activity of the anti-inflammatory active peptide GEIRYEYF derived from the nacre layer of shells.

[0036] Depend on Figure 2 It can be seen that the NO release of RAW264.7 macrophages in the model group increased significantly after LPS stimulation (P<0.001). Compared with the model group, the sample group of GEIRYEYF, an anti-inflammatory active peptide derived from shell nacre, can significantly inhibit the release of NO from RAW264.7 macrophages at different concentrations. It is worth noting that its inhibition rate on NO release is concentration-dependent. At a concentration of 200 μg / mL, the NO release of GEIRYEYF decreased by 54.32±2.13% (P<0.001), and was higher than that of the positive control group dexamethasone. The experimental results show that GEIRYEYF can effectively inhibit the release of NO from RAW264.7 macrophages induced by LPS, and can regulate the inflammatory response to a certain extent.

[0037] (3) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the release of inflammatory factors in RAW264.7 macrophages Depend on Figure 3It can be seen that compared with the blank control group, the release of inflammatory factors TNF-α (a), IL-6 (b) and IL-10 (c) in the model group RAW264.7 macrophages under LPS stimulation increased significantly. The positive control dexamethasone is a drug that has been proven to have anti-inflammatory effects. After dexamethasone treatment, the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by RAW264.7 macrophages was significantly reduced (P<0.001), indicating that the cell inflammation model used in the present invention can be applied to the activity evaluation 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 showed a dose-dependent effect 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%.

[0038] 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.

[0039] Example 2 1. Synthesis of anti-inflammatory active peptides from shell nacre The anti-inflammatory active peptide GEIRYEYF derived from the nacre layer of shells screened out by molecular docking was synthesized by solid phase synthesis method, and the purity of the synthetic peptide was ≥98% as determined by HPLC.

[0040] 2. Cell Culture Mouse fibroblast L929 cells were cultured in DMEM high-glucose medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator, and the cells were passaged when they grew to the logarithmic growth phase.

[0041] 3. L929 cytotoxicity assay Mouse fibroblast L929 cells in logarithmic growth phase were inoculated into 96-well plates at 100 μL per well, with 5×10 cells per well. 4After culturing for 24 h, the original culture medium was discarded. A blank control group and a sample group were set up. The sample group was added with 100 μL of complete culture medium containing different concentrations of shell nacre anti-inflammatory peptides, and the blank control group was added with an equal amount of complete culture medium. After continuing to culture for 24 h, the original culture medium was discarded, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and then placed in a cell culture incubator for 4 h. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and after shaking for 10 min, the absorbance value at a wavelength of 490 nm was measured using an enzyme marker.

[0042] 4. L929 cell scratch assay Mouse fibroblast L929 cells were inoculated into 6-well plates and incubated overnight in a 5% CO2, 37°C incubator. A midline was drawn on the bottom of the six-well plate, and two scratches perpendicular to the midline were made on the monolayer of fused cells at the three equal points with a 200 μL pipette tip. Each well was rinsed with PBS solution to remove cell debris. After removal, the experimental group was incubated for 24 h with different concentrations of shell nacre active peptides. In order to avoid the influence of cell proliferation, the blank control group was incubated for 24 h with a medium containing 2% fetal bovine serum. After the incubation, the cell status was observed under a microscope and photographed and recorded for comparison. The effect of active peptides on cell migration ability was analyzed.

[0043] result: (1) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the activity of skin fibroblast L929 cells like Figure 4 As shown in the data, within the mass concentration range of 5 to 40 ug / mL, GEIRYEYF had no cytotoxicity to L929 cells. Compared with the blank control group, GEIRYEYF had a significant proliferation effect on L929 cells, indicating that it has the potential to promote skin wound healing.

[0044] (2) Effect of the anti-inflammatory active peptide GEIRYEYF from shell nacre on the migration of skin fibroblasts L929 In vitro scratch test was used to investigate the effect of GEIRYEYF on the migration ability of L929 cells. ImageJ software was used to process and compare the changes in scratch area at 0 h and 24 h.

[0045] like Figure 5 As shown in the figure, compared with the blank control group, 5-20 μg / mL GEIRYEYF can significantly increase the migration of L929 cells, and the scratch distance is significantly shortened. In particular, after treatment with 5 μg / mL GEIRYEYF, the wound was almost completely healed. It can be seen that GEIRYEYF can significantly promote the migration and fusion of L929 cells and has a good skin repair effect.

[0046] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design 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 all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-inflammatory active peptide derived from shell nacre, characterized in that: It has the following amino acid sequence: Gly-Glu-Ile-Arg-Tyr-Glu-Tyr-Phe.

2. The anti-inflammatory active peptide derived from the pearl layer of shells 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 as claimed in claim 1.

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

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

6. The cosmetically acceptable salt or pharmaceutically acceptable salt thereof of the anti-inflammatory active peptide derived from the pearl layer of shells according to claim 5, characterized in that: 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; The cosmetically acceptable salt or pharmaceutically acceptable salt includes a salt formed by a 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 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.

7. Use of the anti-inflammatory active peptide derived from shell nacre as claimed in claim 1, or the shell nacre protein hydrolyzate as claimed in claim 3, or 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 function 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 any one of an oral preparation and an external preparation.

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