An antibacterial peptide for maintaining oral flora balance and a preparation method and application thereof

Abstract

This invention relates to the field of antimicrobial peptide technology, specifically to an antimicrobial peptide for maintaining oral flora balance, its preparation method, and its application. The antimicrobial peptide for maintaining oral flora balance according to this invention includes any one of the following polypeptides (a)-(e): (a) a polypeptide whose amino acid sequence includes the amino acid sequence shown in SEQ ID NO. 1; (b) an amino acid sequence composed of amino acid residues shown in SEQ ID NO. 1; (c) a polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequence defined in (a) or (b); (d) a polypeptide having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in (a) or (b); (e) a polypeptide obtained by modifying the N-terminus and / or C-terminus of any one of the polypeptides defined in (a)-(d). The antimicrobial peptides of this invention, used to maintain oral flora balance, exhibit antibacterial and bactericidal activity against oral pathogens while not affecting the normal growth of beneficial bacteria. This balance is maintained, protecting gum health and freshening breath. These antimicrobial peptides are suitable for preparing oral antibacterial agents and related oral care products, regulating the balance of the oral microecology, and thus used to prevent and treat oral diseases, especially dental caries, periodontitis, oral mucosal diseases, and oral cancer. The antimicrobial peptides of this invention are relatively short, making chemical synthesis easier and allowing for direct synthesis into high-purity products. They also exhibit clear effects and have wide applications.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide for maintaining oral flora balance, its preparation method, and its application. Background Technology

[0002] In interpersonal communication, the whiteness of gums and the odor of breath affect the conversation and the impression made on others. Gum diseases (such as tooth decay and periodontal disease) and bad breath can also cause social difficulties, sleep disorders, and psychological problems, impacting people's normal lives. Oral pathogens and dental plaque play a direct role in the occurrence and development of gum diseases. Adjunctive treatments for gum diseases mainly rely on traditional antibiotics, but long-term use can easily lead to bacterial resistance and oral flora imbalance. Therefore, there is an urgent need to develop new drugs to avoid the risk of bacterial resistance.

[0003] Antimicrobial peptides, as novel antimicrobial agents, are widely available and possess broad-spectrum antimicrobial activity. Compared to traditional antibiotics, most antimicrobial peptides kill bacteria primarily by disrupting their cell membranes, making them less likely to induce antibiotic resistance and thus considered a good alternative. In 1980, Boman et al. successfully isolated and purified cephalosporin from silkworm pupae, marking the first discovery of an antimicrobial peptide. Subsequently, antimicrobial peptides have been discovered in bacteria, fungi, amphibians, higher plants, mammals, and humans. Currently, more than 40 antimicrobial peptides have been isolated from human oral epithelial cells, saliva, and periodontal tissues. Some antimicrobial peptides have shown antimicrobial activity against periodontal pathogens. For example, LL-37 has a cytotoxic effect against *Actinobacillus actinomycetii* and *Prevotella intermedius*; β-defensin has a cytotoxic effect against *Actinobacillus actinomycetii*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Prevotella intermedius*; and lactoferrin has a cytotoxic effect against *Porphyromonas gingivalis* and *Prevotella intermedius*.

[0004] Natural antimicrobial peptides suffer from high production costs, low antimicrobial activity, and poor stability, failing to meet practical application needs. With ongoing in-depth research into the properties, structures, and mechanisms of action of existing natural antimicrobial peptides, increasing research focuses on developing synthetically produced antimicrobial peptides. Researchers have successfully utilized various modern biotechnologies to molecularly modify or synthesize novel antimicrobial peptides. Therefore, developing antimicrobial peptides with simple structures and strong antimicrobial activity is of great significance. Summary of the Invention

[0005] To address the problems in the prior art, the first aspect of the present invention provides an antimicrobial peptide for maintaining oral flora balance, wherein the antimicrobial peptide is any one of the following polypeptides (a)-(e):

[0006] (a) A polypeptide whose amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1;

[0007] (b) The amino acid sequence consists of the amino acid residues shown in SEQ ID NO.1;

[0008] (c) A polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence defined in (a) or (b);

[0009] (d) A polypeptide having 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in (a) or (b);

[0010] (e) A polypeptide obtained by modifying the N-terminus and / or C-terminus of any of the polypeptides defined in (a)-(d).

[0011] More preferably, the antimicrobial peptide is P14, and its amino acid sequence is shown in SEQ ID NO.1.

[0012] Preferably, the antimicrobial peptide is P14S, and its amino acid sequence is shown in SEQ ID NO.2.

[0013] A second aspect of the present invention provides a method for preparing an antimicrobial peptide for maintaining oral flora balance, wherein the preparation method is a solid-phase synthesis method.

[0014] A third aspect of the present invention provides an application of an antimicrobial peptide for maintaining oral flora balance, used in the preparation of oral health care, cosmetic or pharmaceutically acceptable salts.

[0015] Preferably, the oral health, cosmetic, or pharmaceutically acceptable salt is a salt formed from an antimicrobial peptide and an organic base.

[0016] Preferably, the oral health, cosmetic or pharmaceutically acceptable salt is a salt formed by antimicrobial peptides and inorganic or organic acids.

[0017] Preferably, the use of the antimicrobial peptide for maintaining oral flora balance, and the use of the oral health care, cosmetic or pharmaceutically acceptable salt in oral pathogen inhibitors.

[0018] Preferably, the oral pathogenic bacteria include one or more of Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, and Stenotrophomonas maltophilia.

[0019] Preferably, the application of the antimicrobial peptide for maintaining oral flora balance and the oral health care, cosmetic or pharmaceutically acceptable salt in oral care products, wherein the oral care products are one or more of toothpaste, mouthwash, dental nerve devitalizer, and anti-infective drugs.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention designs and synthesizes an antimicrobial peptide for maintaining oral flora balance. It exhibits antibacterial and bactericidal activity against oral pathogens while not affecting the normal growth of beneficial bacteria, thus maintaining oral flora balance, protecting gum health, and freshening breath. This antimicrobial peptide is suitable for preparing oral antibacterial agents and related oral care products, regulating the balance of the oral microecology, and thereby preventing and treating oral diseases, especially dental caries, periodontitis, oral mucosal diseases, or oral cancer. The antimicrobial peptide of this invention has a short length, is easy to chemically synthesize, can be directly synthesized into a high-purity product, and has a clear effect and wide range of applications. Attached Figure Description

[0022] Figure 1A and Figure 1B The images show the antimicrobial effects of the antimicrobial peptide described in Example 1 after incubation in Streptococcus mutans ATCC35668 for 24 h and 40 h, respectively.

[0023] Figure 2A and Figure 2B The figures show the antibacterial results of the antimicrobial peptide in Example 1 after incubation in Lactobacillus salivarius ATCC11741 for 24 h and 48 h, respectively. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] The first aspect of this invention provides an antimicrobial peptide for maintaining oral flora balance, wherein the antimicrobial peptide is any one of the following polypeptides (a)-(e):

[0026] (a) A polypeptide whose amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1;

[0027] (b) The amino acid sequence consists of the amino acid residues shown in SEQ ID NO.1;

[0028] (c) A polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence defined in (a) or (b);

[0029] (d) A polypeptide having 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in (a) or (b);

[0030] (e) A polypeptide obtained by modifying the N-terminus and / or C-terminus of any of the polypeptides defined in (a)-(d).

[0031] More preferably, the antimicrobial peptide is P14, and its amino acid sequence is shown in SEQ ID NO.1.

[0032] Preferably, the antimicrobial peptide is P14S, and its amino acid sequence is shown in SEQ ID NO.2.

[0033] A second aspect of the present invention provides a method for preparing an antimicrobial peptide for maintaining oral flora balance, wherein the preparation method is a solid-phase synthesis method.

[0034] A third aspect of the present invention provides an application of an antimicrobial peptide for maintaining oral flora balance, used in the preparation of oral health care, cosmetic or pharmaceutically acceptable salts.

[0035] Preferably, the oral health, cosmetic, or pharmaceutically acceptable salt is a salt formed from an antimicrobial peptide and an organic base.

[0036] Preferably, the oral health, cosmetic or pharmaceutically acceptable salt is a salt formed by antimicrobial peptides and inorganic or organic acids.

[0037] Preferably, the use of the antimicrobial peptide for maintaining oral flora balance, and the use of the oral health care, cosmetic or pharmaceutically acceptable salt in oral pathogen inhibitors.

[0038] Preferably, the oral pathogenic bacteria include one or more of Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, and Stenotrophomonas maltophilia.

[0039] Preferably, the application of the antimicrobial peptide for maintaining oral flora balance and the oral health care, cosmetic or pharmaceutically acceptable salt in oral care products, wherein the oral care products are one or more of toothpaste, mouthwash, dental nerve devitalizer, and anti-infective drugs.

[0040] The above technical solution will be described in detail below with reference to specific embodiments.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Example 1

[0043] This embodiment provides an antimicrobial peptide P14 for maintaining oral flora balance, with the amino acid sequence RKVLRKLLVCKS, as shown in SEQ ID NO.1.

[0044] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:

[0045] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.

[0046] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.

[0047] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% ​​(V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% ​​(V / V) piperidine-DMF solvent, and wash for 20 minutes.

[0048] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.

[0049] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).

[0050] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.

[0051] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.

[0052] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0053] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.1 from right to left.

[0054] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.

[0055] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.

[0056] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.

[0057] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure RKVLRKLLVCKS.

[0058] The detection reagent is ninhydrin.

[0059] Example 2

[0060] This embodiment provides an antimicrobial peptide P14S for maintaining oral flora balance, with the amino acid sequence RKVLRKLLVSKS, as shown in SEQ ID NO.2.

[0061] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:

[0062] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.

[0063] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.

[0064] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% ​​(V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% ​​(V / V) piperidine-DMF solvent, and wash for 20 minutes.

[0065] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.

[0066] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).

[0067] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.

[0068] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.

[0069] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0070] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.2 from right to left.

[0071] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.

[0072] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.

[0073] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.

[0074] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure RKVLRKLLVSKS.

[0075] The detection reagent is ninhydrin.

[0076] Experimental Example

[0077] 1. Antibacterial test:

[0078] The antimicrobial assay of the antimicrobial peptide P14 described in Example 1 was performed on liquid culture medium, with an inoculum concentration of 10 μL for Streptococcus mutans. 3 CFU, Lactobacillus salivarius inoculation concentration 5×10 5 CFU.

[0079] The bacteria mentioned were Streptococcus mutans ATCC35668 and Lactobacillus salivarius ATCC11741, with chloramphenicol as the control group. All of them were commercially available.

[0080] 2. Experimental Results

[0081] Results of antibacterial experiments against Streptococcus mutans are as follows: Figure 1A and Figure 1BAs shown, after incubation with 10 μg / mL of antimicrobial peptide for 24 hours, the inhibition rate against Streptococcus mutans reached 100%; after continuous incubation for 40 hours, absorbance measurement showed that the 100% antibacterial effect was still present, thus demonstrating that the antimicrobial peptide described in this invention has an inhibitory effect on Streptococcus mutans.

[0082] The results of the antibacterial experiment against Lactobacillus salivarius are as follows: Figure 2A and Figure 2B As shown, after incubation with 40 μg / mL of antimicrobial peptide for 24 hours, there was no inhibitory effect on Lactobacillus salivarius; after continuous incubation for 40 hours, absorbance measurements showed that Lactobacillus salivarius was still not inhibited, thus indicating that the antimicrobial peptide did not have an inhibitory effect on Lactobacillus salivarius.

[0083] This invention designs and synthesizes an antimicrobial peptide for maintaining oral flora balance. It exhibits antibacterial and bactericidal activity against oral pathogens (such as Streptococcus mutans) while not affecting the normal growth of beneficial bacteria (such as Lactobacillus salivarius), thus maintaining oral flora balance, protecting gingival health, and freshening breath. This antimicrobial peptide is suitable for preparing oral antibacterial agents and related oral care products, regulating the balance of the oral microecology, and subsequently used to prevent and treat oral diseases, especially dental caries, periodontitis, oral mucosal diseases, or oral cancer. The antimicrobial peptide of this invention has a short length, is easy to chemically synthesize, can be directly synthesized into a high-purity product, and has a clear effect and wide range of applications.

[0084] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An antibacterial peptide for maintaining oral flora balance, characterized by, The antimicrobial peptide is P14, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing the antibacterial peptide for maintaining the balance of oral cavity flora according to claim 1, characterized in that, The preparation method is a solid-phase synthesis method.

3. The use of the antimicrobial peptide of claim 1 in the preparation of oral care products or in pharmaceutically acceptable salts.

4. Use according to claim 3, characterized in that, The salt that is acceptable for oral care products or pharmaceutical use is the salt formed by the antimicrobial peptide and an organic base.

5. Use according to claim 3, characterized in that, The salt that is acceptable for oral care products or pharmaceutical use is the salt formed by the antimicrobial peptide and an inorganic or organic acid.

6. Use of the antibacterial peptide for maintaining the balance of oral cavity flora according to claim 1, or the pharmaceutically acceptable salt according to any one of claims 3 to 5 on an oral care product or a pharmaceutical preparation for the preparation of an agent for inhibiting oral cavity pathogenic bacteria, characterized in that, The oral pathogen is Streptococcus mutans.

7. The use of the antimicrobial peptide for maintaining oral flora balance as described in claim 1, and the pharmaceutically acceptable salt for oral products as described in any one of claims 3-5, in the preparation of oral products for treating oral pathogens, characterized in that... The oral care products are one or both of toothpaste and mouthwash; the oral pathogens are Streptococcus mutans.

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