Antibacterial peptide accelerant and application thereof

Abstract

The invention relates to the technical field of antibacterial nursing, in particular to an antibacterial peptide accelerant and application thereof. The antibacterial peptide accelerant disclosed by the invention comprises gamma-aminobutyric acid, at least one first hyaluronic acid or salt thereof with the molecular weight of more than 5000Da and less than 10000Da, and at least one second hyaluronic acid or salt thereof with the molecular weight of 379-2000Da, the three can generate a synergistic effect, and epithelial cells can be effectively promoted to express the antibacterial peptide. The accelerant disclosed by the invention can be widely applied to preparation of a preparation for promoting the formation of the antibacterial peptide, and has huge market application value and economic benefit.

Description

Technical Field

[0001] The present application relates to the field of antimicrobial care technology, and in particular to an antimicrobial peptide promoter and its application. Background Art

[0002] In daily life, people are affected by some potential hazards in the external environment, such as pathogenic microorganisms, chemicals, allergens, etc. in the environment, which can infect people's skin, oral cavity, intestines, urinary tract and respiratory tract. Therefore, it is of great significance to regulate the immune defense ability of the cell background to inhibit the invasion of pathogenic bacteria. Antimicrobial peptides, also known as antimicrobial peptides and polypeptide antibiotics, are a class of small molecule peptides with broad-spectrum resistance. Defensins and cathelicidins are the main members of skin antimicrobial peptides. In addition, other skin peptides, such as protease inhibitors, chemokines, granulin family and neuropeptides, also show antimicrobial activity. The epidermis establishes a barrier against microbial infection by producing antimicrobial peptides. In addition, these peptides can also regulate local inflammatory responses, activate cellular immunity and adaptive immunity, play an important role in immune defense, and are an important component of biological innate immunity.

[0003] 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 and maintain the stability and diversity of the human microecology.

[0004] Although some active substances have been tested to promote the expression of antimicrobial peptides, further research is needed on how to further enhance the expression of antimicrobial peptides and improve the barrier effect against microbial infection. Summary of the invention

[0005] In order to solve the above problems, the present application discloses an antimicrobial peptide promoter and its application. The inventors found through experimental research that when γ-aminobutyric acid is used in combination with at least one first hyaluronic acid or its salt with a molecular weight of more than 5000 Da and less than 10000 Da and at least one second hyaluronic acid or its salt with a molecular weight of 379-2000 Da, it can produce a synergistic effect of promoting the expression of antimicrobial peptides, and its effect is significantly better than the case of using the above substances alone or in combination.

[0006] In a first aspect, the present application provides an antimicrobial peptide promoter, the promoter comprising in a physiologically acceptable medium:

[0007] a) γ-aminobutyric acid,

[0008] b) at least one first hyaluronic acid or a salt thereof having a molecular weight of more than 5000 Da and less than 10000 Da, and

[0009] c) at least one second hyaluronic acid or a salt thereof having a molecular weight of 379-2000 Da.

[0010] Furthermore, 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any value and range therebetween, and (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any value and range therebetween.

[0011] Further, the component b) can be a first hyaluronic acid or a salt thereof having a molecular weight of 5000Da, 5500Da, 6000Da, 6500Da, 7000Da, 7500Da, 8000Da, 8500Da, 9000Da, 9500Da, 9900Da, 9990Da, 9999Da or any value and range therebetween.

[0012] Further, the component c) can be a second hyaluronic acid or a salt thereof having 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 therebetween.

[0013] Furthermore, the reducing end of the component c) includes a uronic acid group.

[0014] Furthermore, the antimicrobial peptide is preferably human β-defensin-2.

[0015] Furthermore, the administration of the enhancer includes in vivo and / or in vitro. Specifically, when the enhancer is administered in vitro, it can be applied topically to the human body surface, such as topically to the face, oral cavity, scalp and other parts; when the enhancer is administered in vivo, it can be administered orally. After reaching the intestine, the enhancer plays a role in promoting the expression of intestinal antimicrobial peptides.

[0016] The antimicrobial peptide promoter of the present application can be used alone to promote the expression of antimicrobial peptides, or can be used to promote the expression of antimicrobial peptides. According to different purposes of use, the type of physiologically acceptable medium in the antimicrobial peptide promoter can be adjusted.

[0017] In the present application, the physiologically acceptable medium is such as UV filters, antioxidants, emulsifiers, fillers, colorants, preservatives, fragrances, active agents such as moisturizers, anti-aging agents, whitening agents and mixtures thereof.

[0018] As UV filters, for example, benzophenone, 4-butylmethoxydibenzoylmethane, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, phenylbenzimidazole sulfonic acid or homosalate may be used.

[0019] As the antioxidant, for example, tocopherol acetate, BHT, BHA and the like are mentioned.

[0020] Examples of the emulsifier include anionic emulsifiers, cationic emulsifiers, amphoteric emulsifiers, and nonionic emulsifiers.

[0021] As fillers, for example, silicon dioxide, mica, kaolin, oxides of zinc and titanium of natural or synthetic origin; calcium carbonate, magnesium carbonate and magnesium hydrogen carbonate; zinc stearate, magnesium stearate or lithium stearate, zinc laurate, magnesium myristate and the like.

[0022] Examples of the coloring agent include iron oxide, carbon black, titanium dioxide, zinc oxide, cochineal, carotene, and chlorophyll.

[0023] Examples of the preservative include phenoxyethanol, benzyl alcohol, benzoic acid, sorbic acid, and imidazolidinyl urea.

[0024] Examples of the flavor include citronellol and tricyclodecenyl acetate.

[0025] Examples of the moisturizer include mannitol, sodium hyaluronate, lactic acid, glycerin, urea, and propylene glycol.

[0026] As the anti-aging agent, there are, for example, retinol, phytosterols, ceramide and the like.

[0027] As whitening agents, there are arbutin, phenethylresorcinol, ascorbic acid and its derivatives and the like.

[0028] The second aspect of the present invention provides the use of the antimicrobial peptide promoter in the preparation of a product that promotes the formation of antimicrobial peptides.

[0029] Optionally, the product includes a topical product and an oral product. It should be noted that the present application does not specifically limit the type of product, and those skilled in the art can understand that other products that add the antimicrobial peptide promoter described in the present application to play the same role as promoting the formation of antimicrobial peptides in the present application should also be within the scope of protection of the present application.

[0030] On the other hand, the present application provides the use of the antimicrobial peptide promoter in regulating human microecology.

[0031] Preferably, the regulating human microecology includes inhibiting the growth and proliferation of microorganisms that are harmful to the human body.

[0032] Preferably, the human microecology includes at least one of oral microecology, skin microecology, intestinal microecology, and reproductive tract microecology.

[0033] Preferably, the microorganisms harmful to the human body include bacteria, fungi or viruses harmful to the human body.

[0034] On the other hand, the present 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 the present application to promote the expression of antimicrobial peptides in the human body.

[0035] Preferably, the antimicrobial peptide is human β-defensin-2.

[0036] On the other hand, the present application provides the use of γ-aminobutyric acid as an antimicrobial peptide promoter.

[0037] Preferably, the antimicrobial peptide is human β-defensin-2.

[0038] On the other hand, the present application provides the use of hyaluronic acid or a salt thereof having a molecular weight of greater than 5000 Da and less than 10000 Da as an antimicrobial peptide promoter.

[0039] Preferably, the antimicrobial peptide is human β-defensin-2.

[0040] The beneficial effects of this application include but are not limited to:

[0041] The present application discovered that the combination of γ-aminobutyric acid with at least one first hyaluronic acid or its salt having a molecular weight of greater than 5000Da and less than 10000Da, and at least one second hyaluronic acid or its salt having a molecular weight of 379-2000Da, can produce a synergistic effect on the expression of antimicrobial peptides, providing a new direction and combination for promoting the production of antimicrobial peptides. DETAILED DESCRIPTION

[0042] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. The present application generally and / or specifically describes the materials and test methods used in the test. In the following examples, if there is no other special description, % represents wt%, i.e. weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0043] Experimental Example 1 Verification of the effects of different compositions on the production of hBD2 in Ca9-22 cells

[0044] This experimental example 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.

[0045] 1. Experimental Materials

[0046] γ-aminobutyric acid (hereinafter referred to as GABA, Bloomage Biotechnology), first hyaluronic acid (nano HA, Bloomage Biotechnology, molecular weight 8000Da, batch number 2004721), second hyaluronic acid (Hybloom TM Micro-real, Bloomage Biotechnology, molecular weight <1000Da, batch number 22071401), other hyaluronic acid (low molecular weight hyaluronic acid, HA-TLM, purchased from Bloomage Biotechnology, molecular weight 888kDa, batch number 20071451).

[0047] 2. Experimental Methods

[0048] The control group and blank group were incubated with complete culture medium of Ca9-22 cells (containing 10% FBS, 1% penicillin-streptomycin, DMEM).

[0049] Sample group preparation: Weigh each raw material according to the concentration recorded in Table 1 and dissolve it in Ca9-22 cell complete culture medium to obtain a mother solution, dilute the mother solution 2 times, and prepare a Ca9-22 cell complete culture medium containing 50% of the composition for cell experiments.

[0050] The specific proportions of each composition in the mother liquor are as follows:

[0051] In Example 1 (T1), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 1:1:1;

[0052] In Example 2 (T2), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 1:1:10;

[0053] In Example 3 (T3), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 1:10:1;

[0054] In Example 4 (T4), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 1:10:10;

[0055] In Example 5 (T5), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 20:1:1;

[0056] In Example 6 (T6), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 20:1:10;

[0057] In Example 7 (T7), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 20:10:1;

[0058] In Example 8 (T8), the mass ratio of GABA, the first hyaluronic acid, and the second hyaluronic acid is 2:1:1;

[0059] Comparative Example 1 (C1) contained only GABA;

[0060] Comparative Example 2 (C2) contains only the first hyaluronic acid;

[0061] Comparative Example 3 (C3) contains only the second hyaluronic acid;

[0062] In Comparative Example 4 (C4), the mass ratio of GABA to the first hyaluronic acid is 1:1;

[0063] In comparative example 5 (C5), the mass ratio of GABA to the second hyaluronic acid is 1:1;

[0064] In Comparative Example 6 (C6), the mass ratio of the first hyaluronic acid to the second hyaluronic acid is 1:1;

[0065] In Comparative Example 7 (C7), the mass ratio of GABA, the first hyaluronic acid, and the other hyaluronic acids is 1:1:1;

[0066] In Comparative Example 8 (C8), the mass ratio of the first hyaluronic acid to the other hyaluronic acid is 1:1;

[0067] In Comparative Example 9 (C9), the mass ratio of GABA to other hyaluronic acid is 1:1;

[0068] In Comparative Example 10 (C10), the mass ratio of GABA, the first hyaluronic acid, and other hyaluronic acids is 20:1:1.

[0069] CA9-22 cells were cultured in a T75 culture flask with complete CA9-22 cell culture medium. When the cell density was about 80%, they were inoculated in a 24-well plate with a coverslip and cultured at 37°C and 5% CO2. After 24 hours of cell attachment, the components in Table 1 were added respectively and placed in a 37°C, 5% CO2 incubator for further incubation for 72 hours. After the incubation, the supernatant was aspirated, the cells were washed twice, and then the cells were fixed with ice methanol at -20°C, followed by the addition of hBD2 primary antibody and incubation at 4°C overnight. The next day, the cells were washed three times with PBS, and the corresponding secondary antibody was added and incubated at room temperature in the dark for 1.5 hours. The nuclear seal was stained with DAPI, and then the photos were observed and taken under a fluorescence microscope. Finally, the fluorescence data of the photos were semi-quantitatively analyzed using Image J software.

[0070] Graphpad Prism statistical software was used for analysis and processing. Pairwise comparisons were performed using t-tests, *P<0.05 indicated statistical differences, and **P<0.01 indicated significant statistical differences. In this application, as long as P<0.05, there was a significant difference. On the basis of meeting the significant difference requirement, the higher the hBD2 expression rate, the stronger the anti-microbial ability of the composition. The test results are shown in Table 2.

[0071] Table 1

[0072]

[0073] Table 2 Relative expression of hBD2

[0074]

[0075] The results of the examples and comparative examples of the present application are significantly different from those of the control group, indicating that the compositions of the examples and comparative examples of the present application have the effect of promoting the expression of hBD-2.

[0076] Compared with Comparative Examples 1-3, Comparative Examples 4-6 have the same or worse effects on promoting hBD-2 expression than Comparative Examples 1-3, indicating that the combination of GABA, the first hyaluronic acid, and the second hyaluronic acid in pairs does not produce synergistic effects on hBD-2 expression, or even produces antagonistic effects.

[0077] A significant difference analysis was performed between Examples 1-8 and Comparative Example 3, which had the best effect among Comparative Examples 1-6. Examples 1-8 significantly increased the expression of hBD-2 relative to 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.

[0078] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. An antimicrobial peptide promoter, characterized in that: Included in a physiologically acceptable medium are: a) γ-aminobutyric acid, b) at least one first hyaluronic acid or a salt thereof having a molecular weight of more than 5000 Da and less than 10000 Da, and c) at least one second hyaluronic acid or a salt thereof having a molecular weight of 379-2000 Da.

2. The accelerator according to claim 1, characterized in that The mass ratio of the 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 peptides include human β-defensin-2.

4. The accelerator according to any one of claims 1 to 3, characterized in that The administration of the enhancer includes in vivo and / or in vitro.

5. Use of the promoter according to any one of claims 1 to 4 in the preparation of a product for promoting the formation of antimicrobial peptides.

6. Use of the promoter described in any one of claims 1 to 4 in regulating human microecology.

7. The use according to claim 6, characterized in that: The regulation of human microecology includes inhibiting the growth and proliferation of microorganisms that are 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: The promoter described in any one of claims 1 to 4 is used to promote the expression of antimicrobial peptides in the human body.

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

10. Use of hyaluronic acid or its salt with a molecular weight of more than 5000Da and less than 10000Da as an antimicrobial peptide promoter.

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