Helicobacter pylori-resistant mouthwash as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly discloses anti-helicobacter pylori mouth wash, which is prepared from small molecule peptides of SEQ NO: 1-Trp-Cys-Glu-Arg-Met-His-Ile and SEQ NO: 2-Phe-Asp-Val-Lys-Tyr-Gln-Leu according to a specific proportion, 5-(2-aminopropyl)-1, 3, 4-thiadiazole-2-mercaptan (APTT), and auxiliaries such as purified water, ethanol and the like. The SEQ NO: 1 is targeted to helicobacter pylori cell membrane transporter, and cooperates with SEQ NO: 2 acting on DNA repair enzyme for bacteriostasis, APTT synergism is achieved, and biological peptide is stabilized. During preparation of the mouthwash, all the components are synthesized and purified firstly and then are mixed and stirred. The product has the advantages of high helicobacter pylori inhibition rate, long action time and good stability, can be used for daily oral care and prevention and adjuvant therapy of helicobacter pylori infection related diseases, has good biocompatibility, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical pharmaceutical products, and in particular is an anti-Helicobacter pylori mouthwash and a preparation method and application thereof. Background Art

[0002] At present, there are relatively few oral care products targeting Helicobacter pylori on the market, and there are many limitations. Traditional mouthwashes mainly focus on cleaning the mouth, removing odors and preventing tooth decay, and their inhibitory effects on Helicobacter pylori are relatively limited. Although some mouthwashes containing chemical antibacterial agents can kill bacteria to a certain extent, long-term use may disrupt the balance of oral microecology and cause other oral health problems, such as oral mucosal inflammation and tooth discoloration. In addition, some antibacterial mouthwashes are not specific to Helicobacter pylori. While inhibiting Helicobacter pylori, they will also cause significant damage to the beneficial bacteria in the mouth, which is not conducive to the long-term maintenance of oral health.

[0003] As people's understanding of oral health and the hazards of Helicobacter pylori infection continues to deepen, the development of an anti-Helicobacter pylori mouthwash that is efficient, safe, highly specific and can maintain the balance of oral microecology has extremely important practical significance and broad market prospects. Summary of the invention

[0004] The present invention provides an anti-Helicobacter pylori mouthwash comprising the following ingredients:

[0005] SEQ NO:1Trp-Cys-Glu-Arg-Met-His-Ile small molecule peptide.

[0006] Furthermore, the following ingredients are also included:

[0007] SEQ NO: 2 (Phe-Asp-Val-Lys-Tyr-Gln-Leu) small molecule peptide.

[0008] Furthermore, the following ingredients are also included:

[0009] 5-(2-Aminopropyl)-1,3,4-thiadiazole-2-thiol.

[0010] Further, an anti-Helicobacter pylori mouthwash comprises the following ingredients in percentage by weight:

[0011] SEQ NO: 1 (Trp-Cys-Glu-Arg-Met-His-Ile) small molecule peptide 0.08-0.12 parts;

[0012] SEQ NO: 2 (Phe-Asp-Val-Lys-Tyr-Gln-Leu) small molecule peptide 0.06-0.09 parts; 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol (APTT) 1.2-2.0 parts;

[0013] The solvent is a mixture of 70-80 parts of purified water and 10-20 parts of ethanol;

[0014] The moisturizing agent is 6-9 parts of propylene glycol and 2-4 parts of trehalose;

[0015] The surfactant is 1-2 parts of cocamidopropyl hydroxysultaine;

[0016] The flavoring agent is 0.1-0.2 parts of menthol and 0.1-0.2 parts of blueberry flavor;

[0017] The preservative is 0.1 to 0.3 parts of potassium sorbate.

[0018] Furthermore, the small molecule peptides of SEQ NO: 1 and SEQ NO: 2 are prepared by chemical synthesis, purified and identified by high performance liquid chromatography, and the purity is above 95%.

[0019] Furthermore, the APTT is prepared by organic synthesis reaction and purified by recrystallization and column chromatography, with a purity of more than 95%.

[0020] Further, the preparation method thereof comprises the following steps:

[0021] S1: Dissolve SEQ NO: 1, SEQ NO: 2 small molecule peptides and APTT in a small amount of solvent respectively;

[0022] S2: adding a moisturizer, a surfactant, a flavoring agent, and a preservative in sequence, and stirring in a stirrer at a speed of 300-500 rpm for 30-60 minutes until a uniform solution is formed;

[0023] S3: Put the mouthwash into a sealed bottle.

[0024] Furthermore, the application of anti-Helicobacter pylori mouthwash in oral care is used to inhibit the growth of Helicobacter pylori in the oral cavity, and to prevent and assist in the treatment of oral and gastric diseases related to Helicobacter pylori infection.

[0025] Furthermore, the biological peptide combination of the anti-Helicobacter pylori mouthwash includes SEQ NO: 1 and SEQ NO: 2 small molecule peptides, and the weight ratio of the two is 4:3.

[0026] Furthermore, the synergist of the anti-Helicobacter pylori mouthwash is 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol (APTT), which is added in an amount of 1.2 to 2.0 parts by weight to the total weight of the mouthwash to enhance the inhibitory effect of the biological peptide on Helicobacter pylori and prolong the action time.

[0027] Beneficial effects of the invention:

[0028] 1. Highly effective and long-lasting anti-Helicobacter pylori performance. The novel biological peptide SEQ NO: 1 and its combination with SEQ NO: 2 of the present invention have significant antibacterial effects on Helicobacter pylori, and the addition of 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol (APTT) further enhances the antibacterial effect and prolongs the duration of action. Compared with traditional mouthwashes, it can still maintain a high antibacterial rate at a lower biological peptide concentration, providing stronger support for the prevention and adjuvant treatment of Helicobacter pylori-related diseases.

[0029] 2. Good biocompatibility and safety. Biopeptides are composed of natural amino acids, and the APTT structure is relatively mild. Ingredients such as solvents, moisturizers, surfactants, flavoring agents, and preservatives also have good biocompatibility. The entire mouthwash has minimal irritation to the oral mucosa and teeth. Unlike traditional mouthwashes containing antibiotics or irritating chemical antibacterial ingredients, the product of the present invention reduces the potential risk of oral mucosal damage, allergies, etc. caused by ingredient stimulation, and is suitable for long-term use. It can also provide a safe and reliable oral care option for special groups such as children, pregnant women, and people with oral sensitivity, and effectively inhibit Helicobacter pylori while protecting the health of users.

[0030] 3. Optimized product formula and performance. In addition to biopeptides and APTT, mouthwash also contains a variety of carefully selected solvents, moisturizers, surfactants, flavoring agents and preservatives. Solvents ensure the dissolution and dispersion of ingredients, moisturizers maintain oral moisture, surfactants improve contact effects, flavoring agents improve taste, and preservatives ensure quality and safety. The synergy of various ingredients makes mouthwash not only excellent in antibacterial performance, but also has advantages in convenience of use, taste and oral health maintenance. It has strong competitiveness in the oral care product market and provides a comprehensive and excellent performance solution for oral care for Helicobacter pylori infection.

[0031] 4. The mouthwash of the present invention has dual effects of prevention and auxiliary treatment. It is used for daily oral care, can effectively inhibit the growth and reproduction of Helicobacter pylori in the oral cavity, prevent it from infecting the stomach, and reduce the infection rate. For patients who have been infected with Helicobacter pylori, using it in conjunction with clinical treatment can reduce the amount of oral colonization, reduce the risk of re-infection of the stomach, help improve the overall treatment effect, shorten the treatment cycle, and has important application value in the prevention and auxiliary treatment of Helicobacter pylori infection. DETAILED DESCRIPTION

[0032] Example 1

[0033] (I) Novel biological peptide sequences

[0034] 1. Design a new biological peptide sequence SEQ NO:1-Trp-Cys-Glu-Arg-Met-His-Ile (WCERMHI). Design principle: This biological peptide mainly targets specific transport proteins on the cell membrane of Helicobacter pylori. These transport proteins play a key role in the bacterial uptake of nutrients, excretion of metabolic wastes, and maintenance of intracellular environmental stability. The Cys residue in the peptide segment of SEQ NO:1 can form a disulfide bond with the cysteine ​​residue in the transport protein to achieve specific binding. The aromatic ring structure of the Trp residue enhances the hydrophobic interaction and π-π stacking effect with the transport protein. The Glu and Arg residues carry negative and positive charges, respectively, and can interact with the charged groups on the transport protein through electrostatic attraction, further stabilizing the binding of the biological peptide to the transport protein. The Met and Ile residues provide certain hydrophobicity and structural support, and the imidazole group of the His residue can participate in the formation of hydrogen bonds. Multiple interaction modes jointly change the conformation of the transport protein, thereby interfering with the material transport process of Helicobacter pylori and inhibiting its growth and reproduction.

[0035] (II) New organic compounds

[0036] An unreported organic compound named 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol (APTT for short) is used as a synergist and stabilizer of biological peptides.

[0037] Design principle: The amino group in the APTT molecule can react with the carboxyl group of the biological peptide to form a chemical bond or ion pair, thereby protecting the biological peptide from degradation by oral enzymes and improving its stability. Functional groups such as thiadiazole rings and thiol groups may have antibacterial activity, which can interfere with the cell membrane structure or metabolic pathway of Helicobacter pylori, synergistically work with biological peptides, and enhance the inhibitory effect on Helicobacter pylori. In addition, the combination of APTT and biological peptides can gradually dissociate in the oral environment, achieving the slow release of biological peptides and prolonging their action time.

[0038] (III) Antibacterial synergy of single sequence, combined sequence and organic compounds

[0039] 1. Antibacterial effect of single sequence

[0040] Experiments show that the small molecule peptide of SEQ NO: 1 has an inhibition rate of about 36% on Helicobacter pylori at a concentration of 0.3 mg / mL. It mainly inhibits bacterial growth by binding to the transport protein on the cell membrane of Helicobacter pylori, interfering with the bacterial substance transport.

[0041] 2. Synergistic effect of combined sequence (without APTT)

[0042] When SEQ NO:1 is combined with another biological peptide SEQ NO:2-Phe-Asp-Val-Lys-Tyr-Gln-Leu (FDVKYQL) in a ratio of 4:3, the inhibition rate of Helicobacter pylori can be increased to about 68 parts at a total concentration of 0.35 mg / mL. SEQ NO:2 mainly acts on the DNA repair enzyme of Helicobacter pylori, interfering with the DNA repair process of bacteria. When the two are combined, SEQ NO:1 destroys the material transport of bacteria, causing the bacteria to lack nutrients and energy supply, and the activity of DNA repair enzymes is inhibited, thereby making the inhibitory effect of SEQ NO:2 on DNA repair enzymes more significant; and the inhibition of DNA repair enzymes by SEQ NO:2 will affect the synthesis and function of transport proteins on bacterial cell membranes, further enhancing the antibacterial effect of SEQ NO:1, and the two synergistically enhance the antibacterial effect.

[0043] 3. Synergistic effect of combined sequences containing organic compounds

[0044] When APTT (the added amount is 1.2-2.0 parts by weight of the total mouthwash) is added to the combination system of SEQ NO: 1 and SEQ NO: 2, the inhibition rate of Helicobacter pylori can reach more than 80 parts when the total peptide concentration is reduced to 0.2 mg / mL, and the antibacterial effect time is extended by about 42 parts. APTT slowly releases the biological peptide, ensuring that there is a sufficient concentration of biological peptide in the oral cavity to exert an antibacterial effect for a long time. At the same time, its own possible antibacterial activity cooperates with the biological peptide, further improving the antibacterial effect.

[0045] (IV) Composition of mouthwash

[0046] The mouthwash of the present invention contains the following ingredients in addition to the novel biological peptide and organic compound:

[0047] 1. Solvent: A mixed solvent of purified water and ethanol is used. Purified water is used as the main solvent to ensure the mildness and biocompatibility of the mouthwash. Ethanol helps to dissolve and disperse other ingredients, and has a certain bactericidal effect, which can inhibit the growth of some bacteria in the oral cavity in a short period of time.

[0048] 2. Moisturizer: Add propylene glycol and trehalose. Propylene glycol can absorb and retain moisture in the mouth, preventing the mouth from being too dry after using mouthwash. Trehalose can form a protective film on the surface of the oral mucosa to reduce irritation. At the same time, it helps maintain the stability of biological peptides and other active ingredients and prolong the action time of the product.

[0049] 3. Surfactant: Choose cocamidopropyl hydroxysulfonyl betaine. It has mild surface activity, can reduce the surface tension of mouthwash, make it easier for mouthwash to diffuse and penetrate in the oral cavity, increase the contact opportunity between biological peptides and Helicobacter pylori, enhance the antibacterial effect, and has minimal irritation to the oral mucosa, suitable for long-term use.

[0050] 4. Flavoring: Add menthol and fruit flavors (such as blueberry flavor). Menthol gives the mouthwash a cool taste and effectively improves oral odor, while fruit flavors provide a pleasant fruity aroma, enriching the smell of the mouthwash, improving the user's acceptance and usage experience, making the mouthwash easier for users to accept during the oral care process.

[0051] 5. Preservatives: Potassium sorbate is used. Potassium sorbate is a commonly used food-grade preservative with high antibacterial properties. It can inhibit the growth and reproduction of microorganisms during the production, storage and use of mouthwash, ensure the quality and safety of the product, extend the shelf life of the product, and has low toxicity to the human body, meeting the safety requirements of oral care products.

[0052] (V) Preparation method

[0053] 1. Synthesis and purification of biological peptides

[0054] For SEQ NO:1 and SEQ NO:2 small molecule peptides, both were prepared by chemical synthesis. First, a suitable solid phase synthetic resin, such as Wang resin, was selected, and the corresponding amino acids were modified with protective groups, and then the amino acids were coupled to the resin in sequence. During the coupling process, the temperature was strictly controlled at 20-26°C, the pH value was 6-7, and the coupling time of each amino acid was 1-

[0055] 1.5 hours. After the peptide chain is synthesized, the protective groups are removed and cleaved, and then purified and identified by high performance liquid chromatography (HPLC) to ensure that the purity of the biological peptide reaches more than 95%.

[0056] 2. Synthesis and purification of APTT

[0057] APTT is prepared by organic synthesis reaction. 2-aminopropanol and carbon disulfide are used as starting materials, and react in the presence of appropriate solvents and catalysts to generate intermediate products, which are then reacted with hydrazine. After a series of reaction steps, crude 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol is obtained. The crude product is purified by multiple recrystallization and column chromatography to obtain a high-purity APTT product with a purity of more than 95%.

[0058] 3. Preparation of Mouthwash

[0059] The purified SEQ NO: 1 small molecule peptide is mixed in a suitable container at a weight ratio of 0.08-0.12 parts, the SEQ NO: 2 small molecule peptide is mixed in a weight ratio of 0.06-0.09 parts, APTT (1.2-2.0 parts by weight), solvent (70-80 parts by weight of purified water, 10-20 parts by weight of ethanol), moisturizer (6-9 parts by weight of propylene glycol, 2-4 parts by weight of trehalose), surfactant (1-2 parts by weight of cocamidopropyl hydroxysulfobetaine), flavoring agent (0.1-0.2 parts by weight of menthol, 0.1-0.2 parts by weight of blueberry flavor), preservative (0.1-0.3 parts by weight of potassium sorbate) and other ingredients. First, the biological peptide and APTT are dissolved in a small amount of solvent, and then other ingredients are added in sequence, and stirred in a stirrer at a speed of 300-500 rpm for 30-60 minutes until a uniform solution is formed. Finally, put the mouthwash into a sealable bottle.

[0060] (VI) Application

[0061] The anti-Helicobacter pylori mouthwash of the present invention can be applied to daily oral care, especially oral care and auxiliary treatment for high-risk groups of Helicobacter pylori infection or infected patients. The user can take 10-20mL of mouthwash and gargle in the oral cavity for 30-60 seconds every morning and evening or after a meal, so that the mouthwash fully contacts the teeth, gums, oral mucosa and tongue fur and other parts, and then spits it out. Through long-term use, the growth and reproduction of Helicobacter pylori in the oral cavity can be effectively inhibited, the amount of colonization in the oral cavity can be reduced, the risk of spreading to the stomach and infecting the stomach again can be reduced, and the oral cavity can also be cleaned, other common oral diseases such as caries, periodontitis, etc. can be prevented, and the oral health status can be improved.

[0062] (I) Synthesis and identification of biological peptides

[0063] 1. According to the above-mentioned biological peptide synthesis method, SEQ NO: 1 and SEQ NO: 2 small molecule peptides were synthesized respectively.

[0064] Taking the synthesis of SEQ NO:1 as an example, after the Wang resin is swollen in the reaction vessel, the first amino acid Trp is modified with the Fmoc protecting group, and then coupled to the resin in the presence of a coupling reagent. The reaction temperature is controlled at 23°C, the pH value is 6.5, and the coupling time is 1.3 hours. Then, the other amino acids are coupled in sequence. After each amino acid is coupled, washing and detection are performed to ensure that the coupling reaction is complete. After the peptide chain is synthesized, a deprotecting agent is used to remove the Fmoc protecting group, and then a cutting agent is used to cut the peptide chain from the resin to obtain a crude SEQ NO:1 peptide.

[0065] The crude peptide was purified by high performance liquid chromatography (HPLC). A reverse phase chromatographic column was used, mobile phase A was 0.1 parts of trifluoroacetic acid aqueous solution, mobile phase B was acetonitrile, and separation and purification were performed by gradient elution. The target peak components were collected and freeze-dried to obtain the purified SEQ NO: 1 small molecule peptide, which was identified to have a purity of 96%.

[0066] The same method was used to synthesize and purify the small molecule peptide of SEQ NO: 2, with a purity of 95.5%.

[0067] (II) Synthesis and identification of APTT

[0068] 1. Synthesis steps

[0069] Add 2-aminopropanol and carbon disulfide to a three-necked flask, slowly drop an appropriate amount of sodium hydroxide solution under stirring, control the reaction temperature at 10-20°C, react for 2-3 hours, and generate an intermediate product. Then heat the reaction system to 60-80°C, add an appropriate amount of hydrazine, and react for 4-6 hours. During the reaction, monitor the reaction progress by thin layer chromatography (TLC). When the raw material point basically disappears, the reaction is over.

[0070] After the reaction is completed, the mixture is cooled to room temperature and the pH value is adjusted to neutral with hydrochloric acid solution. The reaction product is then extracted with ethyl acetate for 3-5 times. The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and ethyl acetate is removed by distillation under reduced pressure to obtain a crude product 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol.

[0071] 2. Purification and Identification

[0072] The crude product was purified by a combination of recrystallization and column chromatography. The crude product was first dissolved in a small amount of hot ethanol, then slowly cooled and crystallized, and filtered to obtain a preliminarily purified product. The preliminarily purified product was subjected to column chromatography on a silica gel column, with petroleum ether-ethyl acetate as the eluent, and gradient elution was performed. The target product fractions were collected to obtain a purified APTT product. After testing, the purity reached more than 96 parts. The structure of APTT was identified by analytical methods such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), and the results were consistent with the expected structure.

[0073] (III) Preparation and performance testing of mouthwash

[0074] The purified SEQ NO: 1 small molecule peptide 0.1 parts by weight, SEQ NO: 2 small molecule peptide 0.075 parts by weight and APTT 1.6 parts by weight, solvent (purified water 75 parts by weight, ethanol 15 parts by weight), moisturizer (propylene glycol 7 parts by weight, trehalose 3 parts by weight), surfactant (cocamidopropyl hydroxysulfo betaine 1.5 parts by weight), flavoring agent (menthol 0.15 parts by weight, blueberry flavor 0.15 parts by weight), preservative (potassium sorbate 0.2 parts by weight) and other ingredients are mixed in an appropriate container. First, the biological peptide and APTT are dissolved in 40mL solvent and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then add other ingredients in sequence and stir at 400 rpm in a stirrer for 40 minutes until a uniform solution is formed. Finally, the mouthwash is put into a sealed bottle.

[0075] The antibacterial effect of mouthwash on Helicobacter pylori was tested by test tube dilution method. Mouthwash of different concentrations was mixed with Helicobacter pylori suspension, and after 24 hours at 37°C in a microaerobic environment (5 parts O2, 10 parts CO2, 85 parts N2), an appropriate amount of the mixed solution was inoculated on a Helicobacter pylori special culture medium plate, and the number of colonies was counted after 72 hours of culture to calculate the antibacterial rate. The test results show that the mouthwash prepared by the present invention has an antibacterial rate of more than 80 parts on Helicobacter pylori when the total peptide concentration is 0.2 mg / mL, indicating that it has a good antibacterial effect.

[0076] The mouthwash was placed under high temperature (40℃), low temperature (4℃) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the contents of biopeptides and APTT were detected by high performance liquid chromatography (HPLC) to evaluate the stability of the product.

[0077] The test results show that under the above conditions, the content of biopeptide and APTT remained above 90 parts of the initial content within 16 weeks, indicating that the product has good stability and can meet the storage and use requirements.

[0078] Example 2

[0079] (I) Novel biological peptide sequences

[0080] 1. Design a new biopeptide sequence SEQ NO:3-Tyr-Cys-Arg-Gln-Leu-His-Asp (YCRQLHD). Design principle: This biopeptide mainly acts on the cell surface adhesin of Helicobacter pylori. Helicobacter pylori relies on adhesin to attach to the oral and gastric mucosal surfaces, thereby colonizing and causing infection. The Cys residue in the SEQ NO:3 peptide can form a disulfide bond with the cysteine ​​residue in the adhesin to achieve specific binding. The Tyr and Arg residues enhance the hydrophobic interaction and electrostatic attraction with the adhesin through the aromatic ring structure and the positively charged guanidine group, respectively. The Gln, Leu and Asp residues provide different chemical environments and structural supports, which help the biopeptide to bind tightly to the adhesin. The imidazole group of the His residue can participate in the formation of hydrogen bonds, further stabilizing the biopeptide-adhesin complex, and inhibiting the colonization and infection ability of Helicobacter pylori in the oral cavity by preventing its adhesion.

[0081] (II) New organic compounds

[0082] An unreported organic compound named 3-(2-aminoethyl)-1,2,4-oxadiazole-5-carboxylic acid methyl ester (AEMOC for short) was used as a protective and synergist of biological peptides.

[0083] Design principle: The amino group in the AEMOC molecule can react chemically with the carboxyl group of the biopeptide to form a chemical bond or ion pair, thereby protecting the biopeptide from degradation by oral enzymes and improving its stability. Functional groups such as oxadiazole rings and ester groups may have antibacterial activity, which can interfere with the cell membrane structure or metabolic pathway of Helicobacter pylori, synergistically work with biopeptides, and enhance the inhibitory effect on Helicobacter pylori. In addition, the combination of AEMOC and biopeptides can gradually dissociate in the oral environment, achieving the slow release of biopeptides and prolonging their action time.

[0084] (III) Antibacterial synergy of single sequence, combined sequence and organic compounds

[0085] 1. The single sequence antibacterial effect experiment showed that the small molecule peptide SEQ NO: 3 had an inhibition rate of about 38% against Helicobacter pylori at a concentration of 0.3 mg / mL. It mainly inhibits bacterial growth by binding to the cell surface adhesin of Helicobacter pylori to prevent bacterial adhesion.

[0086] 2. Synergistic effect of combined sequence (without AEMOC)

[0087] When SEQ NO:3 is combined with another biological peptide SEQ NO:4-Ser-Phe-Val-Trp-Lys-Glu-Ile (SFVWKEI) in a ratio of 3:2, the inhibition rate of Helicobacter pylori can be increased to about 70% at a total concentration of 0.35 mg / mL. SEQ NO:4 mainly acts on the cell wall synthesis-related enzyme system of Helicobacter pylori, interfering with the construction of the cell wall. When the two are combined, SEQ NO:3 inhibits bacterial adhesion, making it difficult for bacteria to colonize in the oral mucosa, and the role of the cell wall synthesis-related enzyme system is limited, thereby making the inhibitory effect of SEQ NO:4 on cell wall synthase more significant; and the inhibition of cell wall synthesis by SEQ NO:4 will affect the expression and function of bacterial surface adhesins, further enhancing the antibacterial effect of SEQ NO:3, and the two synergistically enhance the antibacterial effect.

[0088] 3. Synergistic effect of combined sequences containing organic compounds

[0089] When AEMOC (the amount added is 1-2 parts by weight of the total mouthwash) is added to the combination system of SEQ NO:3 and SEQ NO:4, the inhibition rate of Helicobacter pylori can reach more than 80% when the total peptide concentration is reduced to 0.2 mg / mL, and the antibacterial effect time is extended by about 45%. AEMOC releases the biological peptide slowly, ensuring that there is a sufficient concentration of biological peptide in the oral cavity to exert the antibacterial effect for a long time. At the same time, its own possible antibacterial activity cooperates with the biological peptide, further improving the antibacterial effect.

[0090] (IV) Composition of mouthwash

[0091] The mouthwash of the present invention contains the following ingredients in addition to the novel biological peptide and organic compound:

[0092] 1. Solvent: A mixed solvent of deionized water and glycerin is used. Deionized water ensures the mildness and biocompatibility of the mouthwash. Glycerin has good moisturizing and solubility, which can help other ingredients dissolve better in the solution. At the same time, it can form a moisturizing film on the surface of the oral mucosa, reducing the dryness after using the mouthwash. It also has a certain antibacterial effect, and synergizes with other ingredients to enhance the antibacterial effect of the mouthwash.

[0093] 2. Moisturizer: Add sorbitol and sodium hyaluronate. Sorbitol can absorb and retain moisture in the mouth, prevent oral dryness, and help maintain the stability of biological peptides and other active ingredients. Sodium hyaluronate can form a protective film on the surface of the oral mucosa to reduce irritation, and to a certain extent can protect biological peptides from degradation by oral enzymes and prolong their action time.

[0094] 3. Surfactant: Sodium laureth sulfate is used. It has excellent foaming and detergency capabilities, can reduce the surface tension of mouthwash, make it easier for mouthwash to diffuse and penetrate in the oral cavity, increase the contact opportunity between biological peptides and Helicobacter pylori, enhance the antibacterial effect, and at the same time have less irritation to the oral mucosa.

[0095] 4. Flavoring: Add menthol and fruit flavors (such as strawberry flavor). Menthol gives the mouthwash a cool taste and effectively improves oral odor. Fruit flavors provide a pleasant fruity aroma, enrich the smell of the mouthwash, improve the user's acceptance and usage experience, and make the mouthwash easier for users to accept during the oral care process.

[0096] 5. Preservative: Sodium benzoate is used. It has antibacterial and preservative effects, which can prevent the growth and reproduction of microorganisms during the production, storage and use of mouthwash, ensure the quality and safety of the product, extend the shelf life of the product, and has low toxicity to the human body, meeting the safety requirements of oral care products.

[0097] (V) Preparation method

[0098] 1. Synthesis and purification of biological peptides

[0099] For SEQ NO:3 and SEQ NO:4 small molecule peptides, both are prepared by chemical synthesis. First, select a suitable solid phase synthetic resin, such as 2-chlorotrityl chloride resin, modify the corresponding amino acids with protective groups, and then couple the amino acids to the resin in sequence. During the coupling process, the temperature is strictly controlled at 20-25°C, the pH value is 6-7.5, and the coupling time for each amino acid is 1-1.5 hours. After the peptide chain is synthesized, the protective group is removed and cut, and then purified and identified by high performance liquid chromatography (HPLC) to ensure that the purity of the biological peptide reaches more than 95%.

[0100] 2. Synthesis and purification of AEMOC

[0101] AEMOC is prepared by organic synthesis reaction. 2-aminoacetonitrile and methyl chloroformate are used as starting materials, and the reaction is carried out in the presence of appropriate solvents and catalysts. After the reaction is completed, a series of purification steps such as extraction, distillation, and recrystallization are performed to obtain a high-purity AEMOC product with a purity of more than 95%.

[0102] 3. Preparation of Mouthwash

[0103] The purified SEQ NO:3 small molecule peptide is mixed in a suitable container at a weight ratio of 0.08-0.12 parts, the SEQ NO:4 small molecule peptide is mixed in a weight ratio of 0.053-0.08 parts, AEMOC (1-2 parts by weight), solvent (70-80 parts by weight of deionized water, 10-20 parts by weight of glycerol), moisturizer (5-8 parts by weight of sorbitol, 0.5-1 parts by weight of sodium hyaluronate), surfactant (1-2 parts by weight of sodium laureth sulfate), flavoring agent (0.1-0.2 parts by weight of menthol, 0.1-0.2 parts by weight of strawberry flavor), preservative (0.1-0.3 parts by weight of sodium benzoate) and the like. First, the biological peptide and AEMOC are dissolved in a small amount of solvent, and then other ingredients are added in sequence, and stirred in a stirrer at a speed of 200-400 rpm for 30-60 minutes until a uniform solution is formed. Finally, put the mouthwash into a sealable bottle.

[0104] (VI) Application

[0105] The anti-Helicobacter pylori mouthwash of the present invention can be applied to daily oral care, especially oral care and auxiliary treatment for high-risk groups of Helicobacter pylori infection or infected patients. The user can take 10-20mL of mouthwash and gargle in the oral cavity for 30-60 seconds every morning and evening or after a meal, so that the mouthwash fully contacts the teeth, gums, oral mucosa and tongue fur and other parts, and then spits it out. Through long-term use, the growth and reproduction of Helicobacter pylori in the oral cavity can be effectively inhibited, the amount of colonization in the oral cavity can be reduced, the risk of spreading to the stomach and infecting the stomach again can be reduced, and the oral cavity can also be cleaned, other common oral diseases such as caries, periodontitis, etc. can be prevented, and the oral health status can be improved.

[0106] 4. Beneficial Effects

[0107] 1. Highly effective and long-lasting anti-Helicobacter pylori performance

[0108] The novel biological peptide SEQ NO:3 and its combination with SEQ NO:4 of the present invention have significant antibacterial effect on Helicobacter pylori, and the addition of 3-(2-aminoethyl)-1,2,4-oxadiazole-5-carboxylic acid methyl ester (AEMOC) further enhances the antibacterial effect and prolongs the duration of action. Compared with traditional mouthwashes, it can still maintain a high antibacterial rate at a lower biological peptide concentration, providing stronger support for the prevention and adjuvant treatment of Helicobacter pylori-related diseases.

[0109] 2. Good biocompatibility and safety

[0110] Biopeptides are composed of natural amino acids, the AEMOC structure is relatively mild, and the ingredients such as solvents, moisturizers, surfactants, flavoring agents and preservatives also have good biocompatibility. The entire mouthwash has minimal irritation to the oral mucosa and teeth. Unlike traditional mouthwashes containing antibiotics or irritating chemical antibacterial ingredients, the product of the present invention reduces the potential risk of oral mucosal damage, allergies and other hazards caused by ingredient stimulation, is suitable for long-term use, and can also provide a safe and reliable oral care option for special groups such as children, pregnant women and people with oral sensitivity, while effectively inhibiting Helicobacter pylori and protecting the health of users.

[0111] 3. Optimized product formula and performance

[0112] In addition to biopeptides and AEMOC, mouthwash also contains a variety of carefully selected solvents, moisturizers, surfactants, flavoring agents and preservatives. Solvents ensure that the ingredients are dissolved and dispersed, moisturizers maintain oral moisture, surfactants improve contact effects, flavoring agents improve taste, and preservatives ensure quality and safety. The various ingredients work together to make mouthwash not only perform well in antibacterial properties, but also have advantages in terms of ease of use, taste, and oral health maintenance. It has strong competitiveness in the oral care product market and provides a comprehensive and excellent solution for oral care for Helicobacter pylori infection.

[0113] 4. Dual effects of prevention and auxiliary treatment

[0114] The mouthwash of the present invention is used for daily oral care, can effectively inhibit the growth and reproduction of Helicobacter pylori in the oral cavity, prevent it from infecting the stomach, and reduce the infection rate. For patients who have been infected with Helicobacter pylori, the use of the mouthwash in conjunction with clinical treatment can reduce the amount of oral colonization, reduce the risk of re-infection of the stomach, help improve the overall treatment effect, shorten the treatment cycle, and has important application value in the prevention and auxiliary treatment of Helicobacter pylori infection.

[0115] In summary, the anti-Helicobacter pylori mouthwash of the present invention has important application value and broad market prospects in the field of oral care for Helicobacter pylori infection through innovative biopeptide design, application of new organic compounds, reasonable formula composition and scientific preparation method.

[0116] (I) Synthesis and identification of biological peptides

[0117] 1. According to the above-mentioned biological peptide synthesis method, SEQ NO: 3 and SEQ NO: 4 small molecule peptides were synthesized respectively.

[0118] Taking the synthesis of SEQ NO:3 as an example, after swelling the 2-chlorotrityl chloride resin in the reaction vessel, the first amino acid Tyr is modified with the Fmoc protecting group, and then coupled to the resin in the presence of a coupling reagent. The reaction temperature is controlled at 23°C, the pH value is 7, and the coupling time is 1.2 hours. Then, the other amino acids are coupled in sequence, and after each amino acid is coupled, washing and detection are performed to ensure that the coupling reaction is complete. After the peptide chain is synthesized, a deprotecting agent is used to remove the Fmoc protecting group, and then the peptide chain is cut from the resin with a cutting reagent to obtain a crude SEQ NO:3 peptide.

[0119] The crude peptide was purified by high performance liquid chromatography (HPLC). A reverse phase column was used, mobile phase A was 0.1 parts of trifluoroacetic acid aqueous solution, mobile phase B was acetonitrile, and separation and purification were performed by gradient elution. The target peak components were collected and freeze-dried to obtain the purified SEQ NO: 3 small molecule peptide, which was identified to have a purity of 96%.

[0120] The small molecule peptide of SEQ NO: 4 was synthesized and purified by the same method, and the purity reached 95.5%.

[0121] (II) Synthesis and identification of AEMOC

[0122] 1. Synthesis steps

[0123] Add 2-aminoacetonitrile, methyl chloroformate and an appropriate amount of potassium carbonate powder into a three-necked flask, then add an appropriate amount of organic solvent (such as acetone), heat to 40-60°C under stirring, and react for 3-5 hours. During the reaction, monitor the reaction progress by thin layer chromatography (TLC). When the raw material point basically disappears, the reaction is completed.

[0124] After the reaction is completed, the mixture is cooled to room temperature, solid impurities are filtered out, and the organic solvent is removed by distillation under reduced pressure. The residue is dissolved in ethyl acetate, washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and water in sequence, and then dried over anhydrous sodium sulfate, filtered, and ethyl acetate is removed by distillation under reduced pressure to obtain a crude product of 3-(2-aminoethyl)-1,2,4-oxadiazole-5-carboxylic acid methyl ester.

[0125] 2. Purification and Identification

[0126] The crude product was purified by column chromatography. A silica gel column was selected, petroleum ether-ethyl acetate was used as the eluent, gradient elution was performed, and the target product fractions were collected to obtain the purified AEMOC product. After testing, its purity reached more than 96%. The structure of AEMOC was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) and other analytical methods, and the results were consistent with the expected structure.

[0127] (III) Preparation and performance testing of mouthwash

[0128] 1. Preparation of Mouthwash

[0129] The purified SEQ NO:3 small molecule peptide 0.1 weight ratio, SEQ NO:4 small molecule peptide 0.067 weight ratio and AEMOC 1.5 weight ratio, solvent (deionized water 75 weight ratio, glycerol 15 weight ratio), moisturizer (sorbitol 6 weight ratio, sodium hyaluronate 0.8 weight ratio), surfactant (sodium laureth sulfate 1.5 weight ratio), flavoring agent (menthol 0.15 weight ratio, strawberry flavor 0.15 weight ratio), preservative (sodium benzoate 0.2 weight ratio) and other ingredients are mixed in an appropriate container. First, the biological peptide and AEMOC are dissolved in 50mL solvent and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then other ingredients are added in sequence and stirred at a speed of 300 rpm for 40 minutes in a stirrer until a uniform solution is formed. Finally, the mouthwash is placed in a sealed bottle.

[0130] 2. Antibacterial performance test

[0131] The antibacterial effect of mouthwash on Helicobacter pylori was tested by paper diffusion method. A circular filter paper (6 mm in diameter) was soaked in the mouthwash sample, then taken out and placed on a plate of Helicobacter pylori culture medium, with 3 soaked filter paper pieces placed on each plate. A sterile cotton swab was dipped in an appropriate amount of Helicobacter pylori suspension (concentration of 1×10 7 CFU / mL), spread evenly on the surface of the plate, and culture at 37°C for 72 hours in a microaerobic environment (5 parts O2, 10 parts CO2, 85 parts N2). Measure the diameter of the inhibition zone, repeat 3 times for each sample, and take the average value.

[0132] The test results show that when the total peptide concentration of the mouthwash prepared by the present invention is 0.2 mg / mL, the average diameter of the antibacterial zone against Helicobacter pylori is 20.5 mm, indicating that it has a good antibacterial effect.

[0133] 3. Stability test

[0134] The mouthwash was placed under high temperature (40℃), low temperature (4℃) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the content of biopeptides and AEMOC were detected by high performance liquid chromatography (HPLC) to evaluate the stability of the product.

[0135] The test results showed that under the above conditions, the content of biopeptides and AEMOC remained above 90% of the initial content within 16 weeks, indicating that the product has good stability and can meet storage and use requirements.

[0136] The above examples further verify that the preparation method of the anti-Helicobacter pylori mouthwash of the present invention is feasible, the product has good antibacterial performance and stability, and can meet the needs of oral care against Helicobacter pylori.

Claims

1. An anti-Helicobacter pylori mouthwash, characterized in that: Contains the following ingredients: SEQ NO:1Trp-Cys-Glu-Arg-Met-His-Ile small molecule peptide.

2. The anti-Helicobacter pylori mouthwash according to claim 1, characterized in that Also contains the following ingredients: SEQ NO:2Phe-Asp-Val-Lys-Tyr-Gln-Leu small molecule peptide.

3. The anti-Helicobacter pylori mouthwash according to claim 1, characterized in that Also contains the following ingredients: 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol.

4. An anti-Helicobacter pylori mouthwash, characterized in that: Contains the following ingredients by weight: SEQ NO:1Trp-Cys-Glu-Arg-Met-His-Ile small molecule peptide 0.08 parts to 0.12 parts; SEQ NO:2 Phe-Asp-Val-Lys-Tyr-Gln-Leu small molecule peptide 0.06-0.09 parts; 1.2 to 2.0 parts of 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol; The solvent is a mixture of 70-80 parts of purified water and 10-20 parts of ethanol; The moisturizing agent is 6-9 parts of propylene glycol and 2-4 parts of trehalose; The surfactant is 1-2 parts of cocamidopropyl hydroxysultaine; The flavoring agent is 0.1-0.2 parts of menthol and 0.1-0.2 parts of blueberry flavor; The preservative is 0.1 to 0.3 parts of potassium sorbate.

5. The anti-Helicobacter pylori mouthwash according to claim 1, characterized in that The small molecule peptides of SEQ NO: 1 and SEQ NO: 2 are prepared by chemical synthesis, purified and identified by high performance liquid chromatography, and the purity reaches more than 95%.

6. The anti-Helicobacter pylori mouthwash according to claim 1, characterized in that The APTT is prepared by organic synthesis reaction and purified by recrystallization and column chromatography, with a purity of more than 95%.

7. The anti-Helicobacter pylori mouthwash according to claim 1, wherein the preparation method comprises the following steps: S1: Dissolve SEQ NO: 1, SEQ NO: 2 small molecule peptides and APTT in a small amount of solvent respectively; S2: adding a moisturizer, a surfactant, a flavoring agent, and a preservative in sequence, and stirring in a stirrer at a speed of 300-500 rpm for 30-60 minutes until a uniform solution is formed; S3: Put the mouthwash into a sealed bottle.

8. Use of the anti-Helicobacter pylori mouthwash according to claim 1 in oral care, for inhibiting the growth of Helicobacter pylori in the oral cavity, and preventing and assisting in the treatment of oral and gastric diseases related to Helicobacter pylori infection.

9. A biological peptide combination for an anti-Helicobacter pylori mouthwash, characterized in that: It contains SEQ NO: 1 and SEQ NO: 2 small molecule peptides, and the weight ratio of the two is 4:

3.

10. A synergist for an anti-Helicobacter pylori mouthwash, characterized in that: It is 5-(2-aminopropyl)-1,3,4-thiadiazole-2-thiol (APTT), and its added amount in the total weight of the mouthwash is 1.2 parts to 2.0 parts, which is used to enhance the inhibitory effect of biological peptides on Helicobacter pylori and prolong the action time.