Antibacterial peptide, preparation method and application of antibacterial peptide in prevention and treatment of oral cavity problems

Abstract

The invention discloses an antibacterial peptide, a preparation method and application of the antibacterial peptide in preventing and treating oral problems, and belongs to the technical field of antibacterial peptides. The amino acid sequence of the antibacterial peptide is FIRLLKSAGKAIHKLIRRGH, and the amino acid sequence of the antibacterial peptide is shown in the description. In-vitro experiments show that the compound has broad-spectrum antibacterial activity on oral pathogens such as fusobacterium nucleatum, porphyromonas gingivalis and streptococcus mutans, the minimum inhibitory concentration is 31.25-125 mu g / mL, no obvious hemolysis effect exists, the therapeutic index reaches 50.79, the cell selectivity is high, and certain protection and anti-inflammatory effects are achieved in a Pg-induced human gingival fibroblast in-vitro inflammatory model. The traditional Chinese medicine composition has a certain potential effect of inhibiting oral gingivitis. The invention provides an important research and development thought for the development of novel oral disease treatment medicines and nursing products, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide, its preparation method, and its application in the prevention and treatment of oral problems. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Antimicrobial peptides, as small molecule polypeptides with broad-spectrum antimicrobial activity and unique membrane action mechanisms, are novel drug candidates for overcoming antibiotic resistance. Their cationic and amphiphilic characteristics enable them to target and disrupt negatively charged bacterial cell membranes, showing significant potential in the treatment of oral infections and other diseases. Chrysophsin3, derived from red sea bream, is a histidine-rich α-helical antimicrobial peptide. Its unique C-terminal RRRH domain endows it with significant antimicrobial activity against a variety of oral pathogens. However, natural Chrysophsin3 induces approximately 10% hemolytic activity even at low concentrations (about 1 μM). While its hemolytic toxicity is lower than that of melilotinib but significantly higher than that of dermalinib 2, it severely limits the safety of clinical application. Existing research has attempted to reduce cytotoxicity by removing or modifying the C-terminal RRRH sequence, but how to simultaneously reduce hemolytic toxicity while enhancing antimicrobial activity remains a key challenge. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an antimicrobial peptide, its preparation method, and its application in the prevention and treatment of oral problems.

[0005] To overcome the bottlenecks of existing technologies and improve the antibacterial efficacy of antimicrobial peptides, this invention, based on a systematic analysis of the amino acid sequence and spatial structure of the red sea bream antimicrobial peptide Chrysophsin3, designs an antimicrobial peptide Chrysophsin3-2 containing 20 amino acid residues, providing a new theoretical basis and technical pathway for the development of drugs for the treatment of oral diseases. The main design ideas are as follows: For antimicrobial peptides, physicochemical parameters such as net positive charge, amphiphilic α-helix degree, and hydrophobic moment jointly determine their membrane lysis efficiency. Increasing the net positive charge can significantly enhance electrostatic adsorption with the negatively charged bacterial outer membrane; moderately increasing the hydrophobic moment makes it easier for the peptide chain to vertically insert when contacting the phospholipid bilayer, forming stable transmembrane channels; optimizing the helix degree promotes the coordinated transformation of the peptide chain from random coiling to α-helix on the membrane surface by reducing conformational entropy loss, thereby enhancing the synergistic effect of membrane perturbation and lipid arrangement disorder. Therefore, rationally controlling the balance window of the above parameters can lay the molecular design foundation for constructing highly active antimicrobial peptides.

[0006] The antimicrobial peptide Chrysophsin3-2 described in this invention significantly enhances the comprehensive inhibition and killing ability against oral pathogens while retaining the basic antibacterial activity of the template peptide. At the same time, by optimizing the amino acid sequence, it greatly reduces the hemolytic toxicity to mammalian erythrocytes, exhibiting better cell selectivity and safety, and providing a higher-quality candidate molecule for the treatment of oral diseases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0008] This invention provides a novel antimicrobial peptide, Chrysophsin3-2, obtained through precise modification of Chrysophsin3, an antimicrobial peptide derived from red sea bream. Compared to the template peptide, this peptide significantly enhances the overall inhibitory and bactericidal ability against oral pathogens, particularly showing an 8-fold increase in antibacterial and bactericidal activity against *Porphyromonas gingivalis* (Pg) and a 4-fold increase in antibacterial and bactericidal activity against *Streptococcus mutans* (Sm). Simultaneously, this peptide effectively inhibits the formation of oral pathogen biofilms, significantly inhibiting Sm biofilm formation at a concentration of 31.25 μg / mL, significantly inhibiting biofilm formation of *Fusobacterium nucleatum* (Fn) and Pg at a concentration of 250 μg / mL, and significantly inhibiting the formation of mixed bacterial (Pg+Sm+Fn) biofilms at a concentration of 500 μg / mL. In terms of safety, this peptide exhibits extremely low hemolytic toxicity, with a hemolysis rate of only 2.91% at concentrations up to 2000 μg / mL and less than 1% at concentrations ≤1000 μg / mL, demonstrating a therapeutic index as high as 50.79 and excellent cell selectivity. Furthermore, this peptide can significantly downregulate the expression of Pg-stimulated inflammatory genes (IL-6, IL-8, IL-1β) by inhibiting the pathogenic protective effect of Pg on human gingival fibroblasts (HGF), showcasing anti-inflammatory effects and potential for the prevention or treatment of gingivitis and periodontitis. In conclusion, the antimicrobial peptide Chrysophsin3-2, while retaining the basic activity of the template peptide, significantly enhances antimicrobial efficacy and optimizes safety, providing a superior candidate molecule for the treatment of oral diseases.

[0009] In some embodiments of the present invention, the antimicrobial peptide has an α-helical structure, comprising 20 amino acids, a molecular weight of 2314.82 Da, a net charge of +6.2, and a hydrophobic residue ratio of 45%.

[0010] In some embodiments of the present invention, the molecular formula of the antimicrobial peptide is C0. 106 H 184 N 36 O 22 .

[0011] A second aspect of the present invention provides a method for preparing the antimicrobial peptide described in the first aspect, comprising: The antimicrobial peptide was synthesized and purified according to the amino acid sequence shown in SEQ ID NO.1 using a solid-phase synthesis method.

[0012] The antimicrobial peptide described in this invention was obtained by optimizing the design based on the sequence structure analysis of the natural antimicrobial peptide Chrysophsin3. The specific optimized sites are: G→R at position 3, I→K at position 6, D→K at position 14, and R→G at position 19.

[0013] The antimicrobial peptide Chrysophsin3-2 provided by this invention has antibacterial and bactericidal activities against oral periodontal pathogens. The minimum inhibitory concentration (MIC) against Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans ranges from 31.25 to 125 μg / mL, and the minimum bactericidal concentration (MIC) ranges from 125 to 500 μg / mL. Compared with the template antimicrobial peptide Chrysophsin3, Chrysophsin3-2 has an 8-fold increase in both antimicrobial and bactericidal activity against Porphyromonas gingivalis and a 4-fold increase in both antimicrobial and bactericidal activity against Streptococcus mutans. For Fusobacterium nucleatum, the antimicrobial activity of the two peptides is basically the same.

[0014] This peptide effectively inhibits the formation of oral pathogenic bacterial biofilms. At a concentration of 31.25 μg / mL, it significantly inhibits the formation of Sm biofilms; at a concentration of 250 μg / mL, it significantly inhibits the formation of Fn and Pg biofilms; at concentrations ≥250 μg / mL, the inhibition rate of biofilms against all three bacteria is greater than 50%; and at a concentration of 500 μg / mL, it significantly inhibits the formation of mixed bacterial biofilms. Therefore, the antimicrobial peptide Chrysophsin3-2 provided by this invention can inhibit common oral pathogenic bacteria and effectively inhibit the formation of oral pathogenic bacterial biofilms.

[0015] Therefore, a third aspect of the present invention provides the use of the antimicrobial peptide described in the first aspect in the preparation of products for the prevention and treatment of oral problems.

[0016] In some embodiments of the present invention, the oral problems include any one or more of bacterial infection, dental plaque, gingivitis, and periodontitis.

[0017] In some embodiments of the present invention, the bacteria are selected from any one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans.

[0018] In some embodiments of the present invention, the dosage form of the product includes any one or more of the following: spray, aerosol, mouthwash, ointment, film, and effervescent agent.

[0019] In some embodiments of the present invention, the product includes any one or more of pharmaceuticals and oral care products.

[0020] In some embodiments of the present invention, the oral care product includes any one or more of toothpaste, mouthwash, effervescent tablets, and oral care liquid.

[0021] A fourth aspect of the present invention provides a product comprising the antimicrobial peptide described in the first aspect.

[0022] In some embodiments of the present invention, the product includes any one or more of antibacterial drugs and oral care products; The bacteria are any one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans.

[0023] The beneficial effects of this invention are as follows: This invention precisely modifies the antimicrobial peptide Chrysophsin3, derived from red sea bream, to design a novel antimicrobial peptide, Chrysophsin3-2, composed of 20 amino acids. The antimicrobial peptide Chrysophsin3-2 provided by this invention significantly enhances the comprehensive inhibitory and bactericidal ability against oral pathogens while retaining the basic antibacterial activity of the template peptide. Simultaneously, by optimizing the amino acid sequence, it substantially reduces hemolytic toxicity to mammalian erythrocytes, exhibiting superior cell selectivity and safety, thus providing a higher-quality candidate molecule for the treatment of oral diseases.

[0024] Testing revealed that, compared to the natural template antimicrobial peptide Chrysophsin3, the antimicrobial peptide Chrysophsin3-2 exhibits stronger overall antimicrobial activity against oral pathogens, particularly showing an 8-fold increase in antimicrobial and bactericidal activity against *Porphyromonas gingivalis* and a 4-fold increase in antimicrobial and bactericidal activity against *Streptococcus mutans*. This peptide effectively inhibits oral biofilm formation. At a concentration of 31.25 μg / mL, it significantly inhibits *Sm* biofilm formation; at 250 μg / mL, it significantly inhibits *Fn* and *Pg* biofilm formation; and at 500 μg / mL, it significantly inhibits mixed bacterial biofilm formation. This peptide has no significant toxic side effects. At a concentration of 2000 μg / mL, the hemolysis rate is only 2.91%, and at concentrations ≤1000 μg / mL, the hemolysis rate is less than 1%, indicating negligible hemolytic activity. Its therapeutic index is as high as 50.79, demonstrating high cell selectivity and better safety. This peptide can protect human gingival fibroblasts by inhibiting the pathogenicity of *Porphyromonas gingivalis*. The expression levels of inflammatory genes (IL-6, IL-8, IL-1β) induced by peptide-treated Pg were significantly downregulated compared to the Pg-stimulated group alone, indicating that the antimicrobial peptide Chrysophsin3-2 plays a certain protective and anti-inflammatory role in the Pg-induced HGF cell inflammation model, and has the potential to prevent or treat clinical gingivitis and periodontitis. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a predicted three-dimensional structure of the antimicrobial peptide Chrysophsin3-2.

[0027] Figure 2 This is a spiral projection diagram of the antimicrobial peptide Chrysophsin3-2.

[0028] Figure 3 This is a high-performance liquid chromatogram of the antimicrobial peptide Chrysophsin3-2.

[0029] Figure 4 This is the mass spectrum of the antimicrobial peptide Chrysophsin3-2.

[0030] Figure 5 These are the results of experiments on the minimum inhibitory concentrations of the antimicrobial peptides Chrysophsin3 and Chrysophsin3-2 against oral pathogens.

[0031] Figure 6These are the experimental results of the minimum bactericidal concentrations of the antimicrobial peptides Chrysophsin3 and Chrysophsin3-2 against oral pathogens.

[0032] Figure 7 These are experimental results regarding the inhibitory effect of the antimicrobial peptide Chrysophsin3-2 on biofilm formation by oral pathogenic bacteria. In the figures, A represents the OD values ​​of different concentrations of the antimicrobial peptide on *Fusobacterium nucleatum*, B represents the inhibition rate of different concentrations of the antimicrobial peptide on *Fusobacterium nucleatum*, C represents the OD values ​​of different concentrations of the antimicrobial peptide on *Porphyromonas gingivalis*, D represents the inhibition rate of different concentrations of the antimicrobial peptide on *Porphyromonas gingivalis*, E represents the OD values ​​of different concentrations of the antimicrobial peptide on *Streptococcus mutans*, F represents the inhibition rate of different concentrations of the antimicrobial peptide on *Streptococcus mutans*, G represents the OD values ​​of different concentrations of the antimicrobial peptide on mixed bacteria (*Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Streptococcus mutans*), and H represents the inhibition rate of different concentrations of the antimicrobial peptide on mixed bacteria (*Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Streptococcus mutans*).

[0033] Figure 8 This is a bar chart showing the hemolysis rates of the antimicrobial peptides Chrysophsin3 and Chrysophsin3-2.

[0034] Figure 9 The results are from the hemolysis test of the antimicrobial peptides Chrysophsin3 and Chrysophsin3-2.

[0035] Figure 10 The effect of the antimicrobial peptide Chrysophsin3-2 on the mRNA expression levels of inflammatory factors in Pg-induced HGF cells was investigated. A represents IL-6, B represents IL-8, and C represents IL-1β. Detailed Implementation

[0036] Terminology Explanation: In this invention, the therapeutic index (TI) is the ratio of the minimum hemolytic concentration (HC10, defined as the concentration that just causes more than 10% hemolysis of red blood cells; when 2000 μg / mL still does not cause 10% hemolysis of red blood cells, 4000 μg / mL is considered as HC10) of the antimicrobial peptide to the geometric mean (GM) of the MIC of the test bacteria.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] The raw materials used in the following examples are all commercially available products that can be purchased.

[0039] Among them, Fusobacterium nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277) and Streptococcus mutans (ATCC 25175) were purchased from Beijing BioBio Biotechnology Co., Ltd.

[0040] Example 1: Design of antimicrobial peptides Table 1. Amino acid sequence and physicochemical parameters of antimicrobial peptides

[0041] Antimicrobial peptide design concept: For antimicrobial peptides, physicochemical parameters such as net positive charge, amphiphilic α-helix degree, and hydrophobic moment jointly determine their membrane lysis efficiency. Increasing the net positive charge can significantly enhance electrostatic adsorption with the negatively charged bacterial outer membrane; moderately increasing the hydrophobic moment makes it easier for the peptide chain to vertically insert when contacting the phospholipid bilayer, forming stable transmembrane channels; optimizing the helix degree promotes the coordinated transformation of the peptide chain from random coiling to α-helix on the membrane surface by reducing conformational entropy loss, thereby enhancing the synergistic effect of membrane perturbation and lipid arrangement disorder. Therefore, rationally controlling the balance window of the above parameters can lay the molecular design foundation for constructing highly active antimicrobial peptides.

[0042] Based on the above principles, this invention designs the antimicrobial peptide Chrysophsin3-2 by replacing amino acids in the natural antimicrobial peptide Chrysophsin3. Specifically, the substitutions are: G→R at position 3; I→K at position 6; D→K at position 14; and R→G at position 19. The antimicrobial peptide Chrysophsin3-2 moderately increases the positive charge and hydrophobic moment, and optimizes the helicity.

[0043] The physicochemical parameters of Chrysophsin3-2 were predicted using the Expasy online website and https: / / dbaasp.org / home, and the results are shown in Table 1. The three-dimensional structure of the antimicrobial peptide was visualized using the Alphafold 3 protein structure prediction software; the predicted three-dimensional structure of Chrysophsin3-2 is shown in [Figure 1]. Figure 1 ,from Figure 1 As can be seen, Chrysophsin3-2 exhibits a typical α-helix structure. The helical projection diagram of the antimicrobial peptide Chrysophsin3-2, predicted by the HeliQuest program, is shown below. Figure 2 As shown, the spool Figure 2 The prediction results indicate that the hydrophilic and hydrophobic amino acids are arranged on both sides of the helical structure, which is consistent with the classic model of antimicrobial peptides exerting their effects through membrane perturbation mechanisms.

[0044] Example 2 Synthesis and Identification of Antimicrobial Peptides The antimicrobial peptide Chrysophsin3-2 of this invention was synthesized by Nanjing Taopu Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method. The peptide was purified to a purity of 97.26% by reversed-phase high-performance liquid chromatography (RP-HPLC) desalting, and further confirmed by mass spectrometry. The HPLC chromatogram of the antimicrobial peptide Chrysophsin3-2 prepared in this embodiment is shown below. Figure 3 As shown, the peak results are shown in Table 2, and the mass spectrum is shown in... Figure 4 As shown.

[0045] Table 2 Peak results of high-performance liquid chromatography (HPLC) chromatogram of the antimicrobial peptide Chrysophsin 3-2

[0046] Example 3: Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides The periodontal pathogenic bacteria involved in this embodiment are: *Fusobacterium nucleatum* (ATCC 25586), *Porphyromonas gingivalis* (ATCC 33277), and *Streptococcus mutans* (ATCC 25175). The methods for determining MIC and MBC are as follows: (1) MIC refers to the lowest concentration at which a drug can inhibit the growth of microorganisms. An 8 mg / mL stock solution of antimicrobial peptide powder was prepared using sterile water, and the MIC value was determined using the micro-broth dilution method. Glycerol-containing bacteria stored at -80℃ were streaked onto BHI blood agar solid medium and incubated statically in a 37℃ anaerobic incubator. Once colonies reached a suitable size, single colonies were randomly selected and cultured in BHI liquid medium at 37℃ until the logarithmic growth phase (Streptococcus mutans was cultured in a conventional incubator at 37℃ and 200 rpm). The logarithmic growth phase bacterial solution was centrifuged at 5000 rpm for 5 min, and the bacterial cells were adjusted to a final concentration of 1×10⁻⁶ using BHI liquid medium. 7 CFU / mL. In 96-well plates, the antimicrobial peptide stock solution was serially diluted half-fold with BHI liquid medium. Then, 100 μL of bacterial suspension was added to each well. Wells with 200 μL of culture medium or 200 μL of bacterial suspension served as the blank test group and negative control group, respectively. The 96-well plates were incubated in a 37°C anaerobic incubator or a regular constant temperature incubator for 24–48 h. The degree of turbidity of the culture medium in each well was observed visually, and the drug concentration corresponding to the completely clear well was taken as the MIC value of the test sample.

[0047] (2) MBC refers to the minimum concentration at which a drug can kill microorganisms. Take 10 μL of bacterial solution from each well corresponding to the concentration at or above the MIC value, streak it on the surface of a BHI blood agar plate, invert it and incubate it in an anaerobic incubator at 37℃ or a regular constant temperature incubator for 3-4 days. Observe the minimum drug concentration at which no colonies grow on the surface of the plate, which is the MBC value.

[0048] Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides Chrysophsin3 and Chrysophsin3-2

[0049] Through Table 3, Figure 5 and Figure 6 The results showed that the minimum inhibitory concentration (MIC) of the antimicrobial peptide Chrysophsin3 against the three tested bacteria ranged from 125 to 1000 μg / mL, and the minimum bactericidal concentration (MBC) ranged from 125 to 4000 μg / mL, with a slightly weaker inhibitory effect against *Porphyromonas gingivalis*. The designed antimicrobial peptide Chrysophsin3-2 had a MIC range of 31.25 to 125 μg / mL and a MBC range of 125 to 500 μg / mL against the three tested bacteria. Compared to the template antimicrobial peptide Chrysophsin3, Chrysophsin3-2 showed an 8-fold increase in both inhibitory and bactericidal activity against *Porphyromonas gingivalis*, and a 4-fold increase in both inhibitory and bactericidal activity against *Streptococcus mutans*. For *Fusobacterium nucleatum*, the inhibitory activity of both peptides was essentially the same. The geometric mean (GM, the root of the product of the MIC values ​​of all tested bacteria) of Chrysophsin 3 was 250 μg / mL, while that of Chrysophsin 3-2 was 78.75 μg / mL. Overall, Chrysophsin 3-2 showed better antibacterial activity than Chrysophsin 3, possibly due to the increased net charge of Chrysophsin 3-2. Based on the principle that an MBC / MIC ≤ 4 indicates bactericidal activity, Chrysophsin 3-2 can be considered effective bactericidal agents against all three periodontal pathogens.

[0050] Example 4: Inhibitory effect of antimicrobial peptide Chrysophsin3-2 on bacterial biofilm formation The inhibitory effect of the antimicrobial peptide Chrysophsin 3-2 on the formation of oral pathogenic bacteria biofilm was evaluated using crystal violet staining. The specific steps are as follows: (1) Prepare oral pathogenic bacteria solution according to the method of Example 3.

[0051] (2) Take a 96-well plate and add 50 μL of bacterial culture containing periodontal pathogens and 50 μL of antimicrobial peptide solutions of different concentrations to each well, so that the final concentrations of the antimicrobial peptide Chrysophsin3-2 in the well plate are 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL. At the same time, a positive control (oral pathogens and culture medium) and a negative control (culture medium only) are set up, and each group is set up in triplicate.

[0052] (3) Place the 96-well plate in an anaerobic incubator or a regular constant temperature incubator at 37°C and incubate for 24 h.

[0053] (4) After culturing, discard the supernatant, rinse slowly three times with 200 μL of PBS buffer to remove airborne oral pathogens, and dry at room temperature.

[0054] (5) Add 100 μL of methanol to each well, fix for 15 min, remove the methanol, and air dry.

[0055] (6) Add 100 μL of 0.1% crystal violet staining agent to each well to stain the bacterial biofilm at room temperature for 20 min.

[0056] (7) After staining, remove the crystal violet staining agent, rinse off the excess staining agent with PBS, and air dry.

[0057] (8) Then add 100 μL of 30% acetic acid to each well, gently shake to decolorize for about 10 min at room temperature, and record the OD value at 595 nm using a microplate reader. The results are as follows. Figure 7 As shown. Using the formula: Inhibition rate (%) = [1 - (OD)] 测试 / OD 对照 )]×100%, calculate the biofilm formation inhibition rate.

[0058] like Figure 7 As shown, compared with the control group, the antimicrobial peptide Chrysophsin 3-2 significantly inhibited the formation of biofilms from Fn, Pg, Sm, and the mixed biofilms of the three bacteria. At a concentration of 31.25 μg / mL, Chrysophsin 3-2 significantly inhibited Sm biofilm formation; at 250 μg / mL, it significantly inhibited Fn and Pg biofilm formation; at concentrations ≥250 μg / mL, the inhibition rate of biofilms from all three bacteria was greater than 50%; and at 500 μg / mL, it significantly inhibited the formation of mixed bacterial biofilms. These data indicate that the antimicrobial peptide Chrysophsin 3-2 can effectively inhibit the formation of oral pathogenic bacterial biofilms.

[0059] Example 5: Hemolytic activity of antimicrobial peptides Sterile defibrinated sheep blood samples were collected in centrifuge tubes and centrifuged at 3000 rpm for 5 min to remove impurities. Cells were then washed three times with sterile 10 mM PBS (pH 7.4), and red blood cells were collected and incubated with different concentrations of antimicrobial peptides at 37°C for 1 h. Finally, the cells were centrifuged at 3000 rpm for 5 min at room temperature. The absorbance of the supernatant at 570 nm was read using a microplate reader. Red blood cells were separately suspended in sterile PBS and 1% Triton X-100 as negative and positive controls, respectively, with three replicates for each group. The hemolysis rate was calculated as: Hemolysis rate (%) = (AT-AO) / (AC-AO) × 100%.

[0060] Wherein, AT represents the absorbance of the test samples treated with different concentrations of antimicrobial peptides at 570 nm, and AC and AO refer to the absorbance of the positive control and negative control at 570 nm, respectively.

[0061] The therapeutic index (TI) below is the ratio of the minimum hemolytic concentration (HC10, defined as the concentration that just causes more than 10% hemolysis of red blood cells; 4000 μg / mL is considered HC10 when 2000 μg / mL does not cause 10% hemolysis of red blood cells) of the antimicrobial peptide to the geometric mean (GM) of the MIC of the test bacteria.

[0062] Table 4. Hemolytic activity of antimicrobial peptides at different concentrations

[0063] The natural linear cationic peptide melitrix venom peptide can induce hemolysis of more than 10% of erythrocytes at a concentration of 2.219 μg / mL. Therefore, the lack of sufficient cell selectivity is one of the main bottlenecks in the widespread application of antimicrobial peptides. Table 4 shows that the antimicrobial peptide Chrysophsin3 exhibits strong hemolytic activity within the MIC concentration range of the three tested bacteria. At a concentration of 125 μg / mL, the hemolysis rate is 16.68%, and at a concentration of 500 μg / mL, the hemolysis rate is as high as 96.39%, almost complete hemolysis. In contrast, the antimicrobial peptide Chrysophsin3-2 has a hemolysis rate of only 2.91% at 2000 μg / mL, and at concentrations less than or equal to 1000 μg / mL, the hemolysis rate is less than 1%, and the hemolytic effect is negligible.

[0064] Table 5. Calculation of the therapeutic index of antimicrobial peptides

[0065] The therapeutic index of antimicrobial peptides can comprehensively reflect their selective toxicity to eukaryotic cells and bacteria, and is a commonly used indicator for the comprehensive evaluation of antimicrobial peptides. As shown in Table 5, the antimicrobial peptide Chrysophsin 3-2 has no significant hemolytic toxicity, and its therapeutic index is as high as 50.79, indicating high cell selectivity. Therefore, the antimicrobial peptide Chrysophsin 3-2 designed in this invention can inhibit common oral pathogens and has good application prospects in the preparation of highly effective and low-toxicity antibacterial drugs.

[0066] Example 6: Inhibitory effect of antimicrobial peptides on the inflammatory response of *Porphyromonas gingivalis*-induced human gingival fibroblasts. (1) Cell culture: Primary human gingival fibroblasts (HGF) were obtained from clinically collected isolated gingival tissue. HGF was passaged to 5-8 generations for experiments. After trypsin digestion, 250,000 cells / well were seeded into 6-well plates and allowed to adhere overnight.

[0067] (2) Bacterial treatment: Porphyromonas gingivalis Pg in the logarithmic growth phase was centrifuged at 5000 rpm for 5 min, and the bacterial pellet was resuspended in DMEM until OD=1 (10⁻¹⁰). 9 The concentration of antimicrobial peptide (CFU / ml) was determined by dispensing 1 mL of DMEM and 6.25 μL of bacterial culture (MOI = 25) into 1.5 mL sterile EP tubes. Different concentrations of antimicrobial peptide were added to achieve a final concentration of 3.9–125 μg / mL of Chrysophsin 3-2 in the EP tubes. The tubes were then incubated in an anaerobic incubator at 37°C for 3 h.

[0068] (3) Stimulate cells: Discard the culture medium in each well, wash twice with PBS, add all the bacterial suspension containing antimicrobial peptides in the EP tube into the cells, and incubate at 37°C for 3 h.

[0069] (4) RT-qPCR detection: Total RNA was extracted from cells using a kit and reverse transcribed into cDNA. GAPDH was used as an internal reference gene to detect the expression of inflammatory genes in cells after co-culturing with HGF and antimicrobial peptide-treated Pg.

[0070] (5) Calculate relative gene expression levels: 2 -( Ct) The value represents the expression level of the target gene. The primer sequences are as follows.

[0071] Table 6. Reaction Primer Sequences

[0072] *Porphyromonas gingivalis* can stimulate the host immune response through lipopolysaccharide and the inflammatory cytokines it produces. Among these, the pro-inflammatory cytokines interleukins IL-6, IL-8, and IL-1β have been shown to be important cytokines leading to periodontal tissue destruction. Figure 10 As shown, compared with the control group, only the Pg stimulation group significantly increased the mRNA expression levels of IL-6, IL-8, and IL-1β in HGF. Compared with the Pg stimulation group alone, the expression levels of inflammatory genes (IL-6, IL-8, and IL-1β) induced by Pg treatment with the antimicrobial peptide Chrysophsin3-2 were significantly downregulated, indicating that Chrysophsin3-2 plays a certain protective and anti-inflammatory role in the Pg-induced HGF cell inflammation model.

[0073] Based on the above test results, the antimicrobial peptide Chrysophsin3-2 designed in this invention can inhibit common oral pathogens. It exhibits broad-spectrum antibacterial activity against oral pathogens such as *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Streptococcus mutans*, with a minimum inhibitory concentration (MIC) of 31.25–125 μg / mL. It also shows no significant hemolytic effect, a therapeutic index of 50.79, and high cell selectivity. In a Pg-induced in vitro inflammatory model of human gingival fibroblasts, it demonstrates certain protective and anti-inflammatory effects, and has the potential to inhibit oral gingivitis. This invention provides an important research and development approach for the development of novel oral disease treatment drugs and care products, and has promising application prospects.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

2. A method for preparing the antimicrobial peptide according to claim 1, characterized in that, include: The antimicrobial peptide was synthesized and purified according to the amino acid sequence shown in SEQ ID NO.1 using a solid-phase synthesis method.

3. The use of the antimicrobial peptide according to claim 1 in the preparation of products for the prevention and treatment of oral problems.

4. The application as described in claim 3, characterized in that, The oral problems mentioned include any one or more of bacterial infections, dental plaque, gingivitis, and periodontitis.

5. The application as described in claim 4, characterized in that, The bacteria are selected from any one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans.

6. The application as described in claim 3, characterized in that, The dosage forms of the product include any one or more of the following: spray, aerosol, mouthwash, ointment, film, and effervescent tablet.

7. The application as described in claim 3, characterized in that, The products include any one or more of pharmaceuticals and oral care products.

8. The application as described in claim 7, characterized in that, The oral care products include any one or more of toothpaste, mouthwash, effervescent tablets, and oral care solutions.

9. A product characterized in that, Includes the antimicrobial peptide as described in claim 1.

10. The product as described in claim 9, characterized in that, The products include any one or more of antibacterial drugs and oral care products; The bacteria are any one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans.

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