Antimicrobial peptide promoter and use thereof

Abstract

The present application relates to the technical field of antimicrobial care, and in particular to an antimicrobial peptide promoter and a use thereof. The antimicrobial peptide promoter disclosed in the present application comprises gamma-aminobutyric acid, at least one first hyaluronic acid or a salt thereof having a molecular weight of greater than 5000 Da and less than 10000 Da, and at least one second hyaluronic acid or a salt thereof having a molecular weight between 379 Da and 2000 Da. The three can produce a synergistic effect, and can effectively promote the expression of antimicrobial peptide by epithelial cells. The promoter can be widely applied in the preparation of a formulation for promoting the formation of antimicrobial peptide, and has huge market application value and economic benefits.

Description

An antimicrobial peptide promoter and its application

[0001] This application claims priority to Chinese Patent Application No. 202411971685.4, filed on December 30, 2024, entitled “An Antimicrobial Peptide Promoter and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of antimicrobial care technology, and in particular to an antimicrobial peptide promoter and its application. Background Technology

[0003] In daily life, people are exposed to potential hazards from the external environment, such as pathogenic microorganisms, chemical agents, and allergens, which can infect the skin, mouth, intestines, urinary tract, and respiratory tract. Therefore, regulating the cellular immune defense mechanism to inhibit pathogen invasion is of great significance. Antimicrobial peptides, also known as antimicrobial peptides or polypeptide antibiotics, are a class of small molecule peptides with broad-spectrum resistance. Defensins and cathelicidin are the main members of the skin antimicrobial peptide class. Other skin peptides, such as protease inhibitors, chemokines, glucanoids, and neuropeptides, also exhibit antimicrobial activity. The epidermis establishes a barrier against microbial infection by producing antimicrobial peptides. Furthermore, these peptides can regulate local inflammatory responses, activate cellular immunity and adaptive immunity, playing a crucial role in immune defense and are an important component of innate immunity.

[0004] Human β-defensin 2 (hBD2) is a major inducible peptide that plays an important role in host defense. hBD2 can kill or inhibit a variety of pathogenic microorganisms, including bacteria, fungi, and viruses, and can also regulate the body's immune defense, maintaining the stability and diversity of the human microecology.

[0005] Although some active ingredients have been tested to promote the expression of antimicrobial peptides, further research is needed to determine how to further enhance the expression of antimicrobial peptides and improve their barrier function against microbial infections. Summary of the Invention

[0006] To address the aforementioned problems, this application discloses an antimicrobial peptide promoter and its application. Through experimental research, the inventors discovered that when γ-aminobutyric acid (GABA) is used in combination with at least one first hyaluronic acid or its salt with a molecular weight between 5000 and 10000 Da and at least one second hyaluronic acid or its salt with a molecular weight between 379 and 2000 Da, it can synergistically promote the expression of antimicrobial peptides. The effect is significantly better than using the aforementioned substances alone or in combination.

[0007] In a first aspect, this application provides an antimicrobial peptide promoter, said promoter comprising, in a physiologically acceptable medium:

[0008] a) γ-aminobutyric acid

[0009] b) At least one first hyaluronic acid or its salt with a molecular weight between 5000 Da and less than 10000 Da, and

[0010] c) At least one second hyaluronic acid or its salt with a molecular weight between 379 and 2000 Da.

[0011] Further, the mass ratio of components a), b), and c) in the accelerator is (1–20):(1–10):(1–10). For example, (1–20) can be any value or range between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the values ​​between them, and (1–10) can be any value or range between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the values ​​between them.

[0012] Further, component b) may be a first hyaluronic acid or a salt thereof with a molecular weight of 5000 Da, 5500 Da, 6000 Da, 6500 Da, 7000 Da, 7500 Da, 8000 Da, 8500 Da, 9000 Da, 9500 Da, 9900 Da, 9990 Da, 9999 Da or any value and range thereof.

[0013] Further, component c) may be a second hyaluronic acid or a salt thereof with a molecular weight of 379 Da, 380 Da, 390 Da, 400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650 Da, 700 Da, 750 Da, 800 Da, 850 Da, 900 Da, 950 Da, 1000 Da, 1100 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1600 Da, 1700 Da, 1800 Da, 1900 Da, 2000 Da or any value and range thereof.

[0014] Furthermore, the reducing end of component c) includes a uronic acid group.

[0015] Furthermore, the antimicrobial peptide is preferably human β-defensin-2.

[0016] Furthermore, the administration of the promoter includes in vivo and / or in vitro methods. Specifically, when the promoter is administered in vitro, it can be applied topically to the human body surface, such as the face, mouth, scalp, etc.; when the promoter is administered in vivo, it can be taken orally, and the promoter exerts its effect of promoting the expression of intestinal antimicrobial peptides after reaching the intestine.

[0017] The antimicrobial peptide promoter of this application can be used alone to promote the expression of antimicrobial peptides, or it can include a component for promoting the expression of antimicrobial peptides. Depending on the intended use, the type of physiologically acceptable medium in the antimicrobial peptide promoter can be adjusted.

[0018] In this application, physiologically acceptable media include UV filters, antioxidants, emulsifiers, fillers, colorants, preservatives, fragrances, and active agents such as moisturizers, anti-aging agents, whitening agents, and mixtures thereof.

[0019] As UV filters, they include benzophenone, 4-butylmethoxydibenzoylmethane, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, phenylbenzimidazole sulfonic acid, or homosalyl ester.

[0020] As antioxidants, for example, tocopherol acetate, BHT and BHA, etc.

[0021] As emulsifiers, such as anionic emulsifiers, cationic emulsifiers, amphoteric emulsifiers, and nonionic emulsifiers.

[0022] As fillers, such as oxides of silica, mica, kaolin, zinc and titanium from natural or synthetic sources; calcium carbonate, magnesium carbonate and magnesium bicarbonate; zinc stearate, magnesium stearate or lithium stearate, zinc laurate, magnesium myristate, etc.

[0023] As colorants, such as iron oxide, carbon black, titanium dioxide, zinc oxide, carmine, carotene, chlorophyll, etc.

[0024] As preservatives, such as phenoxyethanol, benzyl alcohol, benzoic acid, sorbic acid, imidazolidinyl urea, etc.

[0025] As a fragrance ingredient, it can be used in various ways, such as citronellol and tricyclodecenyl acetate.

[0026] As a moisturizer, it can be found in mannitol, sodium hyaluronate, lactic acid, glycerin, urea, propylene glycol, etc.

[0027] As anti-aging agents, such as retinol, phytosterols, and ceramides.

[0028] As skin whitening agents, such as arbutin, phenylethyl resorcinol, ascorbic acid and its derivatives.

[0029] A second aspect of the present invention provides the use of the antimicrobial peptide promoter in the preparation of products that promote the formation of antimicrobial peptides.

[0030] Optionally, the product includes topical and oral products. It should be noted that this application does not specifically limit the type of product. Those skilled in the art will understand that other products that, when combined with the antimicrobial peptide promoter described in this application, achieve the same effect as promoting the formation of antimicrobial peptides described in this application, should also be within the scope of protection of this application.

[0031] On the other hand, this application provides the application of the aforementioned antimicrobial peptide promoter in regulating the human microecology.

[0032] Preferably, the regulation of the human microecology includes inhibiting the growth and proliferation of microorganisms harmful to the human body.

[0033] Preferably, the human microecology includes at least one of oral microecology, skin microecology, intestinal microecology, and reproductive tract microecology.

[0034] Preferably, the microorganisms harmful to the human body include bacteria, fungi, or viruses that are harmful to the human body.

[0035] On the other hand, this application provides a method for promoting the formation of antimicrobial peptides in the human body, the method comprising the step of using the antimicrobial peptide promoter described in this application to promote the expression of antimicrobial peptides in the human body.

[0036] Preferably, the antimicrobial peptide is human β-defensin-2.

[0037] On the other hand, this application provides the use of γ-aminobutyric acid as an antimicrobial peptide promoter.

[0038] Preferably, the antimicrobial peptide is human β-defensin-2.

[0039] On the other hand, this application provides the use of hyaluronic acid or its salts with a molecular weight between 5000 Da and less than 10000 Da as antimicrobial peptide promoters.

[0040] Preferably, the antimicrobial peptide is human β-defensin-2.

[0041] The beneficial effects of this application include, but are not limited to:

[0042] This application discovers that the combined use of γ-aminobutyric acid with at least one first hyaluronic acid or its salt with a molecular weight between 5000 Da and less than 10000 Da, and at least one second hyaluronic acid or its salt with a molecular weight between 379 and 2000 Da, can produce a synergistic effect on the expression of antimicrobial peptides, providing a new direction and combination for promoting the generation of antimicrobial peptides. Detailed Implementation

[0043] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0044] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0045] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0046] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0047] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0048] Experiment 1 verifies the effect of different compositions on hBD2 production in Ca9-22 cells.

[0049] This experiment tested the effects of different ratios of promoters on the generation of relevant biomarkers in human oral epithelial cancer cells (Ca9-22, purchased from Shanghai Yiyan Biotechnology Co., Ltd.) under different stimuli.

[0050] 1. Experimental Materials

[0051] γ-Aminobutyric acid (hereinafter referred to as GABA, Bloomage Biotechnology), first hyaluronic acid (Nano HA, Bloomage Biotechnology, molecular weight 8000 Da, batch number 2004721), second hyaluronic acid (Hybloom) TM Micro-True Hyaluronic Acid (from Bloomage Biotechnology, molecular weight <1000 Da, batch number 22071401), other hyaluronic acid (low molecular weight hyaluronic acid, HA-TLM, purchased from Bloomage Biotechnology, molecular weight 888 kDa, batch number 20071451).

[0052] 2. Experimental Methods

[0053] The control group and blank group were prepared with complete Ca9-22 cell culture medium (containing 10% FBS, 1% penicillin and streptomycin, DMEM).

[0054] Sample preparation: Weigh each raw material according to the concentration recorded in Table 1 and dissolve it in Ca9-22 complete cell culture medium to obtain a stock solution. Dilute the stock solution by 2 times and prepare a Ca9-22 complete cell culture medium containing 50% of the composition for cell experiments.

[0055] The specific proportions of each component in the mother liquor are as follows:

[0056] In Example 1 (T1), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 1:1:1.

[0057] In Example 2 (T2), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 1:1:10.

[0058] In Example 3 (T3), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 1:10:1;

[0059] In Example 4 (T4), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid was 1:10:10.

[0060] In Example 5 (T5), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid was 20:1:1.

[0061] In Example 6 (T6), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 20:1:10.

[0062] In Example 7 (T7), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 20:10:1.

[0063] In Example 8 (T8), the mass ratio of GABA, first hyaluronic acid, and second hyaluronic acid was 2:1:1.

[0064] Comparative Example 1 (C1) contains only GABA;

[0065] Comparative Example 2 (C2) contains only hyaluronic acid;

[0066] Comparative Example 3 (C3) contains only the second type of hyaluronic acid;

[0067] In Comparative Example 4 (C4), the mass ratio of GABA to hyaluronic acid was 1:1.

[0068] In Comparative Example 5 (C5), the mass ratio of GABA to the second hyaluronic acid was 1:1;

[0069] In Comparative Example 6 (C6), the mass ratio of the first hyaluronic acid to the second hyaluronic acid was 1:1;

[0070] In Comparative Example 7 (C7), the mass ratio of GABA, primary hyaluronic acid, and other hyaluronic acids was 1:1:1.

[0071] In Comparative Example 8 (C8), the mass ratio of the first hyaluronic acid to the other hyaluronic acid was 1:1.

[0072] In Comparative Example 9 (C9), the mass ratio of GABA to other hyaluronic acid was 1:1.

[0073] In Comparative Example 10 (C10), the mass ratio of GABA, first hyaluronic acid, and other hyaluronic acids was 20:1:1.

[0074] CA9-22 cells were cultured in T75 culture flasks using complete CA9-22 cell culture medium until the cell density reached approximately 80%. The cells were then seeded into 24-well plates with coverslips and cultured at 37°C and 5% CO2. After 24 hours of cell attachment, the components listed in Table 1 were added, and the plates were incubated at 37°C and 5% CO2 for another 72 hours. After incubation, the supernatant was aspirated, and the cells were washed twice. The cells were then fixed with ice-cold methanol at -20°C, followed by incubation with hBD2 primary antibody at 4°C overnight. The cells were washed three times with PBS the next day, and then incubated with the appropriate secondary antibody at room temperature for 1.5 hours in the dark. The cells were then stained with DAPI and mounted. The images were then observed and photographed under a fluorescence microscope, and the fluorescence data from the images were semi-quantitatively analyzed using ImageJ software.

[0075] The data were analyzed using Graphpad Prism statistical software. Pairwise comparisons were performed using t-tests. *P<0.05 indicated a statistically significant difference, and **P<0.01 indicated a statistically significant difference. In this application, a p<0.05 was considered statistically significant. Based on meeting the statistical significance requirement, a higher hBD2 expression rate indicates a stronger antimicrobial effect of the composition. The test results are shown in Table 2.

[0076] Table 1

[0077] Table 2. Relative expression of hBD2

[0078] The results of the embodiments and comparative examples of this application showed significant differences compared with the control group, indicating that the compositions of the embodiments and comparative examples of this application have the effect of promoting hBD-2 expression.

[0079] Compared with Comparative Examples 1-3, Comparative Examples 4-6 showed comparable or worse effects in promoting hBD-2 expression, indicating that the combination of GABA, the first hyaluronic acid, and the second hyaluronic acid did not produce a synergistic effect on hBD-2 expression or even produced an antagonistic effect.

[0080] A significant difference analysis was performed between Examples 1-8 and Comparative Example 3, which showed the best effect among Comparative Examples 1-6. Examples 1-8 significantly increased the expression of hBD-2 compared with Comparative Example 3, indicating that the combination of GABA, the first hyaluronic acid, and the second hyaluronic acid has a synergistic effect in promoting the expression of hBD2.

[0081] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. An antimicrobial peptide promoter, characterized in that, Physiologically acceptable media include: a) γ-aminobutyric acid b) At least one first hyaluronic acid or its salt with a molecular weight between 5000 Da and less than 10000 Da, and c) At least one second hyaluronic acid or its salt with a molecular weight between 379 and 2000 Da.

2. The accelerator according to claim 1, characterized in that, The mass ratio of components a), b), and c) is (1-20):(1-10):(1-10).

3. The accelerator according to claim 1 or 2, characterized in that, The antimicrobial peptide includes human β-defensin-2.

4. The accelerator according to any one of claims 1-3, characterized in that, The accelerator can be administered in vivo or in vitro.

5. The use of the promoter according to any one of claims 1-4 in the preparation of products that promote the formation of antimicrobial peptides.

6. The application of the promoter according to any one of claims 1-4 in regulating the human microecology.

7. The application according to claim 6, characterized in that, The regulation of the human microecology includes inhibiting the growth and proliferation of microorganisms harmful to the human body. Preferably, the human microecology includes at least one of oral microecology, skin microecology, intestinal microecology, and reproductive tract microecology.

8. A method for promoting the formation of antimicrobial peptides, characterized in that, Use the promoter according to any one of claims 1-4 to promote the expression of antimicrobial peptides in humans.

9. Application of γ-aminobutyric acid as an antimicrobial peptide promoter.

10. Application of hyaluronic acid or its salts with a molecular weight between 5000 Da and less than 10000 Da as antimicrobial peptide promoters.

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