Applications and products of sialic acid in promoting the formation of antimicrobial peptides

Abstract

This application relates to the application and products of sialic acid in promoting the formation of antimicrobial peptides, belonging to the field of sialic acid application technology. The inventors have discovered and verified through experimental research that N-acetylneuraminic acid can increase the expression levels of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7 genes, thereby promoting the formation of antimicrobial peptides. The promoting effect of sialic acid on antimicrobial peptide formation has not been disclosed in existing technologies. The disclosure of this application is of great significance for the application of sialic acid in promoting the formation of antimicrobial peptides in food, feed, nutritional supplements, skin care products, or pharmaceuticals, and has enormous market application value and economic benefits.

Description

Technical Field

[0001] This application relates to the application and products of sialic acid in promoting the formation of antimicrobial peptides, and belongs to the field of sialic acid application technology. Background Technology

[0002] Antimicrobial peptides are a class of peptides with diverse activities produced by the body's biological defense system. They can resist the invasion of foreign pathogens and are an important component of the body's own immunity. Initially, it was only discovered that these active polypeptides could exert a broad inhibitory effect on bacteria. Later, it was found that some antimicrobial peptides could also exhibit killing effects against fungi, protozoa, tumor cells, and viruses. Antimicrobial peptides have a broad antibacterial spectrum and multiple targets, making it difficult for microorganisms to develop resistance. Therefore, antimicrobial peptides hold promise as alternatives to traditional antibiotics. In addition to resisting foreign pathogens, the immunomodulatory function of antimicrobial peptides is receiving increasing attention. Antimicrobial peptides have small molecular weights and weak immunogenicity, making them important mediators of innate immunity. Antimicrobial peptides can inhibit pro-inflammatory cytokines, prevent endotoxemia caused by bacterial products, and inhibit the activity of certain enzymes that inhibit wound healing, thereby preventing wound deterioration. Antimicrobial peptides share common structural characteristics. They are typically composed of about 20 to 80 amino acid residues, are all cationic peptides with an isoelectric point greater than 7, and all have an amphiphilic structure that allows them to insert into the lipid bilayer of cells.

[0003] The U.S. Food and Drug Administration (FDA) has already officially approved the bacterial peptide nisin as a food additive. Researchers have successfully used genetic engineering methods to transfer genes encoding antimicrobial peptides into crops, directly enhancing their antiviral capabilities. For example, introducing the Shiva 21 gene into tobacco and potatoes has resulted in new varieties resistant to leaf blight. Furthermore, we will also attempt to directly transfer antimicrobial peptide genes into animal chromosomes to create new transgenic animal breeds resistant to pathogenic infections, which represents a new avenue for livestock development.

[0004] Nearly 2,000 different antimicrobial peptides have been discovered and studied. In animals, antimicrobial peptides are expressed at the highest levels in sites of high environmental contact. Human antimicrobial peptides can be classified into the following categories: 1) Defensins: Defensins are extremely important and widely studied endogenous antimicrobial peptides in humans. α-defensins include HNP-1–4, HD5, and HD6; all α-defensins are constitutively expressed. β-defensins (HEFB) include dozens of types, most of which can be induced to express; 2) Cathepsin inhibitors: In humans, only one member of this class of antimicrobial peptides, LL-37, has been found, and its corresponding gene is called the S100A7 gene, which can be induced to express; 3) Histidine-rich proteins: These proteins mainly function locally in the oral cavity; 4) Glycine-rich proteins: These proteins include CM12, etc. Most antimicrobial peptides can be induced to express; therefore, providing a specific environment to promote the expression of endogenous antimicrobial peptides is a feasible strategy that warrants further investigation.

[0005] Numerous studies have explored methods to increase the expression of endogenous antimicrobial peptides. Adding various amino acids to pig feed can increase the expression of intestinal antimicrobial peptides. Zinc oxide, fatty acids, probiotics, amino acids, vitamins, and other components have all been found to enhance the function of antimicrobial peptides in the liver, lungs, gastrointestinal tract, and other organs of pigs, chickens, and mice, with synergistic effects between different inducers. Some traditional Chinese medicines and their active ingredients can induce the expression of antimicrobial peptides, with similar results observed in in vitro cell experiments and in vivo studies in pigs, mice, and humans.

[0006] Sialic acid is a collective term for many structurally similar compounds. It contains the core structure 3-deoxy-5-aminononyl glycosylate, also known as neuraminic acid. More than 60 naturally occurring sialic acids have been discovered, the most common being N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid, N-acyl-O-acetylneuraminic acid, and their respective glycosidic derivatives. Sialic acid was initially isolated from submandibular gland mucin, representing a class of compounds with similar structural characteristics, hence the name sialic acid. Sialic acid is a neurotransmitter of gangliosides and a component of the brain. The sialic acid content in the gray matter of the brain is 15 times that of internal organs such as the liver and lungs. The main dietary source of sialic acid is breast milk, and it is also found in cow's milk, eggs, and cheese. Sialic acid may play an important role in the normal neurological development of infants.

[0007] Whether sialic acid promotes the production of antimicrobial peptides has not yet been studied. Summary of the Invention

[0008] To address the aforementioned issues, this study provides applications and products of sialic acid in promoting the formation of antimicrobial peptides. Through experimental research, the inventors discovered and verified that N-acetylneuraminic acid can promote the expression of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7 genes, thereby promoting the generation of antimicrobial peptides. This has significant implications for the application of sialic acid in promoting the formation of antimicrobial peptides in food, feed, nutritional supplements, skincare products, or pharmaceuticals, and possesses enormous market application value and economic benefits.

[0009] According to one aspect of this application, an antimicrobial peptide formation promoter is provided, which includes sialic acid as an active ingredient.

[0010] Optionally, the antimicrobial peptide gene includes at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7.

[0011] Optionally, it can be administered via in vivo and / or in vitro methods.

[0012] In some embodiments, the antimicrobial peptide formation promoter is administered via in vivo and / or in vitro application. When administered in vitro, such as by smearing or applying topically, the concentration of sialic acid at the time of application can be 0.0001 wt% to 60 wt%, for example, 0.0001 wt%, 0.0002 wt%, 0.0003 wt%, 0.0004 wt%, 0.0005 wt%, 0.0006 wt%, 0.0007 wt%, 0.0008 wt%, 0.0009 wt%, 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.004 wt%, 0.005 wt%, 0.006 wt%, 0.007 wt%, 0.008 wt%, 0.009 wt%, 0.01 wt%, 0.02 wt%, 0. 03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt% ,0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt %, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, 36wt%, 37wt%, 38wt%, 39wt%, 40wt%, 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, 49wt%, 50wt%, 51wt%, 52wt%, 53wt%, 54wt%, 55wt%, 56wt%, 57wt%, 58wt%, 59wt%, 60wt%, etc., and any values ​​between these values. The concentration range for sialic acid when administered internally, such as orally, can be 0.0001 wt% to 60 wt%, for example, 0.0001 wt%, 0.0002 wt%, 0.0003 wt%, 0.0004 wt%, 0.0005 wt%, 0.0006 wt%, 0.0007 wt%, 0.0008 wt%, 0.0009 wt%, 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.004 wt%, 0.005 wt%, 0.006 wt%, 0.007 wt%, 0.008 wt%, 0.009 wt%, 0.01 wt%, 0. ...03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt% , 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7 wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32w t%, 33wt%, 34wt%, 35wt%, 36wt%, 37wt%, 38wt%, 39wt%, 40wt%, 41wt%, 42wt%, 43wt%, 44wt% , 45wt%, 46wt%, 47wt%, 48wt%, 49wt%, 50wt%, 51wt%, 52wt%, 53wt%, 54wt%, 55wt%, 56wt%, 5 7wt%, 58wt%, 59wt%, 60wt%, etc., and any range between these values; for example, when injected, the concentration of sialic acid can be 10μg / L-50g / L, such as 10μg / L, 100μg / L, 1000μg / L, 1g / L, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, etc., and any range between these values.

[0013] According to another aspect of this application, the use of sialic acid in the preparation of formulations that promote the formation of antimicrobial peptides is provided.

[0014] Optionally, the antimicrobial peptide gene includes at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7.

[0015] Optionally, the sialic acid may be administered in vivo and / or in vitro.

[0016] Optionally, the formulation may be a food, feed, nutritional supplement, skin care product, or pharmaceutical that promotes the formation of antimicrobial peptides. 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 add sialic acid to achieve the same effect as promoting the formation of antimicrobial peptides in this application should also be within the scope of protection of this application.

[0017] Optionally, the formulation may be a solid dosage form, a cream, a colloidal formulation, an emulsion, or an aqueous solution. It should be noted that the formulation types listed herein are only common types; other formulation types that, with the addition of sialic acid, achieve the same effect as promoting the formation of antimicrobial peptides in this application should also be within the scope of protection of this application.

[0018] Optionally, the formulation is a solid dosage form, which includes N-acetylneuraminic acid, starch, and sucrose.

[0019] Optionally, the method for preparing the solid dosage form includes the step of fully mixing the fine powders of each raw material and then compressing them into tablets.

[0020] Optionally, the solid dosage form contains 60±10% N-acetylneuraminic acid, 35±5% starch, and 5±5% sucrose.

[0021] Optionally, the solid dosage form contains 60% N-acetylneuraminic acid, 35% starch, and 5% sucrose.

[0022] Optionally, the formulation is a paste formulation, which includes N-acetylneuraminic acid, hawthorn fruit puree, sodium benzoate, and potassium benzoate.

[0023] Optionally, the preparation method of the paste includes the steps of crushing hawthorn fruit into a paste, adding the remaining raw materials and water, and then crushing and mixing them evenly.

[0024] Optionally, the ointment preparation contains 0.001±0.0005% N-acetylneuraminic acid, 80±10% hawthorn fruit puree, 0.1±0.05% sodium benzoate, and 0.1±0.05% potassium benzoate.

[0025] Optionally, the ointment formulation contains 0.001% N-acetylneuraminic acid, 80% hawthorn fruit puree, 0.1% sodium benzoate, and 0.1% potassium benzoate.

[0026] Optionally, the formulation is an aqueous solution comprising N-acetylneuraminic acid, 1,2-hexanediol, 1,2-pentanediol, and p-hydroxyacetophenone.

[0027] Optionally, the preparation method of the aqueous agent includes the step of stirring and dissolving the raw materials in water.

[0028] Optionally, the aqueous solution contains 5±1% N-acetylneuraminic acid, 1±0.5% 1,2-hexanediol, 1±0.5% 1,2-pentanediol, and 0.5±0.2% p-hydroxyacetophenone.

[0029] Optionally, the aqueous solution contains 5% N-acetylneuraminic acid, 1% 1,2-hexanediol, 1% 1,2-pentanediol, and 0.5% p-hydroxyacetophenone.

[0030] According to another aspect of this application, a method for promoting the formation of antimicrobial peptides is provided, the method using sialic acid as a promoter for the formation of antimicrobial peptides.

[0031] Optionally, the antimicrobial peptide gene includes at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7.

[0032] Optionally, the sialic acid may be administered in vivo and / or in vitro.

[0033] Optionally, the sialic acid is selected from one or more of 3-deoxy-5-aminononyl glycosylate, N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid, and N-acyl-O-acetylneuraminic acid.

[0034] Optionally, the sialic acid is N-acetylneuraminic acid.

[0035] The beneficial effects of this application include, but are not limited to:

[0036] 1. Based on the application of sialic acid in the preparation of products that promote the formation of antimicrobial peptides according to this application, the inventors, through experimental design verification, gene expression detection and data analysis, found that 0.3% sialic acid has a significant promoting effect on the expression of DEFB1, DEFB4A, DEFB103B and DEFB104B genes, with promotion rates of 57%, 42%, 36% and 39%, respectively; 0.1% sialic acid has a significant promoting effect on the expression of DEFB1, DEFB103B and S100A7 genes, with promotion rates of 38%, 22% and 27%, respectively; and 0.03% sialic acid has a significant promoting effect on all DEFB1, DEFB4A, DEFB103B, DEFB104B and S100A7 genes, with promotion rates of 276%, 61%, 72%, 84% and 180%, respectively, showing the largest promotion effect.

[0037] 2. The application of sialic acid in the preparation of products that promote the formation of antimicrobial peptides, as described in this application, is of great significance for promoting the formation of antimicrobial peptides in food, feed, nutritional supplements, skin care products, or pharmaceuticals, and has enormous market application value and economic benefits. It also provides a new direction for experiments and research related to antimicrobial peptide formation, and is of great importance to research on antimicrobial peptide formation. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0039] Figure 1 This is a graph showing the effect of sialic acid samples on the expression of antimicrobial peptide genes in human primary epidermal keratinocytes, as described in Example 1 of this application. Detailed Implementation

[0040] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0041] In this application, the inventors used human primary epidermal keratinocytes as the cell material for efficacy testing. Through experiments, testing, and data processing, they discovered that N-acetylneuraminic acid promotes the formation of antimicrobial peptides. The sialic acid-promoting effect on antimicrobial peptides verified by the inventors in this application is of great significance for promoting the formation of antimicrobial peptides in food, feed, nutritional supplements, skin care products, or pharmaceuticals, and has enormous market application value and economic benefits.

[0042] It should be noted that the experimental materials used in the specific embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the specific embodiments of this invention, they are generally performed under conventional conditions, such as those described in J. Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual* (3rd Edition), Science Press, 2002; D.L. Spector et al., eds., *Cellular Laboratory Manual*, Science Press, 2001; or according to the conditions recommended by the manufacturer.

[0043] The present application solution will be specifically described below through specific embodiments.

[0044] Example 1: The promoting effect of N-acetylneuraminic acid on antimicrobial peptide expression

[0045] The experimental reagents used in this embodiment include: human primary skin keratinocytes (P3-P6) and culture medium, which were purchased from [source missing]. Cell Technology; CCK reagent was purchased from Tongren Biotechnology (catalog number 40203ES80); 12-well and 96-well cell culture plates were purchased from Thermo; RNA extraction kit was purchased from Qiagen (catalog number 714104), and reverse transcription and quantitative PCR kits were purchased from Takara (catalog numbers RR037A and RR420A). HBD-1 ELISA kit was purchased from CUSABIO (catalog number CSB-E14186h), and HBD-2 ELISA kit was purchased from CUSABIO (catalog number CSB-E13201h).

[0046] The experimental methods in this embodiment include:

[0047] 1) Antimicrobial peptide gene expression experiment: Human primary epidermal keratinocytes were cultured in an incubator at 37℃, containing 5% CO2 and saturated humidity. After the cells reached more than 80% confluence, they were digested and seeded into 12-well plates and cultured for another 48 hours for testing. On the day of the experiment, keratinocytes were treated with an appropriate sample concentration for 24 hours, the supernatant was discarded, and each well was washed twice with 1 mL of pre-cooled phosphate-buffered saline (PBS). Cell lysis buffer was then prepared using RNA extraction reagent. After collecting all the lysis buffer samples, total RNA was extracted from the cells, reverse transcribed into cDNA, and the expression level of each antimicrobial peptide gene was detected by real-time quantitative PCR. The results were expressed as follows: -ΔΔCt Gene expression levels in each cell group were obtained by analyzing and quantifying the data. The primer sequences for the PCR test are shown in Table 1.

[0048] Table 1 Primer sequences for PCR testing

[0049]

[0050] 2) Antimicrobial peptide protein expression experiment: Human primary epidermal keratinocytes were cultured in an incubator at 37℃ with 5% CO2 and saturated humidity. After the cells reached more than 80% confluence, they were digested and seeded into 96-well plates, and cultured for another 48 hours for testing. On the day of the experiment, keratinocytes were treated with appropriate sample concentrations for 48 hours, and the supernatant was collected. The concentrations of HBD-1 and HBD-2 were detected using HBD-1 ELISA kits and HBD-2 ELISA kits, respectively.

[0051] 3) Statistical methods: All results are expressed as Mean ± SD. Differences in measurement data were compared using the unpaired t-test. A p-value < 0.05 was considered statistically significant. In figures and tables, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0052] In the experiment investigating the effect of samples on the expression of antimicrobial peptide genes in human primary epidermal keratinocytes, keratinocytes were treated with sialic acid at concentrations of 0.3%, 0.1%, and 0.03% for 24 hours. Total RNA was then collected, and the expression levels of antimicrobial peptide genes were detected using qPCR. The expression levels and significance results are as follows: Figure 1 As shown in the experimental results, 0.3% sialic acid significantly promoted the growth of DEFB1, DEFB4A, DEFB103B, and DEFB104B, with promotion rates of 57% (p<0.001), 42% (p<0.01), 36% (p<0.01), and 39% (p<0.01), respectively; 0.1% sialic acid significantly promoted the growth of DEFB1, DEFB103B, and S100A7, with promotion rates of 3... 8% (p<0.05), 22% (p<0.05), 27% (p<0.05); 0.03% sialic acid significantly promoted DEFB1, DEFB4A, DEFB103B, DEFB104B and S100A7, with promotion rates of 276% (p<0.001), 61% (p<0.01), 72% (p<0.05), 84% (p<0.01) and 180% (p<0.001), respectively.

[0053] Experimental data showed that N-acetylneuraminic acid (N-acetylneuraminic acid) at a concentration of 0.3% promoted the expression levels of DEFB1, DEFB4A, DEFB103B, and DEFB104B genes; at a concentration of 0.1%, N-acetylneuraminic acid promoted the expression levels of DEFB1, DEFB103B, and S100A7 genes; and at a concentration of 0.03%, N-acetylneuraminic acid promoted the expression levels of all three genes more significantly.

[0054] Example 2: Solid Formulation of Composition for Promoting Antimicrobial Peptide Formulation

[0055] The components of the solid dosage form in this embodiment are shown in Table 2 below. The preparation method includes the step of thoroughly mixing the fine powders of each raw material and then pressing them into tablets using a tablet press.

[0056] Table 2 Components of Solid Dosage Forms

[0057]

[0058] Example 3: Ointment Formulation of Composition for Promoting Antimicrobial Peptide Formulation

[0059] The components of the ointment preparation in this embodiment are shown in Table 3 below. The preparation method includes the steps of peeling and pitting hawthorn fruit, crushing it into a paste, adding the remaining raw materials, and then crushing and mixing them thoroughly.

[0060] Table 3 Components of Ointment Formulations

[0061]

[0062] Example 4: Aqueous composition for promoting antimicrobial peptide formation

[0063] The components of the aqueous solution in this embodiment are shown in Table 4 below. The preparation method includes the step of thoroughly stirring each raw material in water until it is completely dissolved.

[0064] Table 4 Aqueous Components

[0065]

[0066] In this application, the inventors experimentally verified the role of sialic acid in promoting the formation of antimicrobial peptides. Human primary epidermal keratinocytes were used, and the expression levels of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7 genes were detected. Through experimental design and operation, gene expression level detection, and data analysis, the application of sialic acid in promoting the formation of antimicrobial peptides provided in this application is of great significance for promoting the formation of antimicrobial peptides in food, feed, nutritional supplements, skincare products, or pharmaceuticals, and has enormous market application value and economic benefits.

[0067] 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 formation promoter, characterized in that, It includes sialic acid as an active ingredient, and the antimicrobial peptide includes at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B and S100A7.

2. The antimicrobial peptide formation promoter according to claim 1, wherein the administration method includes in vivo and / or in vitro.

3. The application of sialic acid in the preparation of formulations that promote the formation of antimicrobial peptides, characterized in that, The antimicrobial peptides include at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B, and S100A7.

4. The application according to claim 3, wherein the sialic acid is administered via in vivo and / or in vitro methods.

5. A method for promoting the formation of antimicrobial peptides, characterized in that, The method uses sialic acid as a promoter to promote the formation of antimicrobial peptides, which include at least one of DEFB1, DEFB4A, DEFB103B, DEFB104B and S100A7, and the method is not intended for the diagnosis or treatment of a disease.

6. The method of claim 5, wherein the sialic acid is administered via in vivo and / or in vitro methods.

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