PH helicobacter pylori antibacterial biological peptide oral care product and application

Abstract

The invention belongs to the technical field of biochemical environmental protection, and particularly discloses a PH helicobacter pylori antibacterial biological peptide oral care product and application. The product comprises small molecule peptides shown as SEQ NO: 1 and SEQ NO: 2 and a 4-(2-ethoxyl)-1, 3-thiazolidine-2-keto-5-carboxylic acid ethyl ester (ETHC) sustained release agent in a specific proportion, and also comprises auxiliary agents such as propylene glycol. The SEQ NO: 1 aims at helicobacter pylori urease and cooperates with the SEQ NO: 2 for bacteriostasis, and ETHC interacts with biological peptide to achieve slow release and possibly have bacteriostatic activity. Tests show that the composition containing the sustained-release agent has a good helicobacter pylori inhibition effect and long action time, is superior to a traditional product, has good biocompatibility of biological peptide and ETHC, can prevent and assist in treating helicobacter pylori infection related diseases, and has great significance and wide prospects in the field of oral care.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical pharmaceutical products, and specifically is a biopeptide oral care product which has an antibacterial effect on Helicobacter pylori and contains an organic compound sustained-release agent. Background Art

[0002] Helicobacter pylori (Hp) is closely related to a variety of gastric diseases, such as chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer. As a potential reservoir of H. pylori, its colonization and spread in the oral cavity cannot be ignored. Traditional treatments for H. pylori infection have many drawbacks, the problem of antibiotic resistance is becoming increasingly serious, and is often accompanied by adverse reactions. Existing oral care products are insufficient in their targeted inhibition of H. pylori or have adverse effects on the balance of oral microecology. Although biopeptides have potential, their stability and long-term effectiveness in products need to be improved. In this context, it is crucial to develop an oral care product that is efficient, safe and long-lasting against H. pylori. Summary of the invention

[0003] The present invention provides a PH Helicobacter pylori antibacterial biological peptide oral care product, comprising the following ingredients:

[0004] SEQ NO: 1Gly-Leu-Cys-His-Asp-Arg-Pro small molecule peptide.

[0005] Furthermore, the following ingredients are also included:

[0006] SEQ NO: 2Lys-Tyr-Thr-Val-Glu-Ile-Met small molecule peptide.

[0007] The following ingredients are also included: Extended release agent: ethyl 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylate.

[0008] Furthermore, a PH Helicobacter pylori antibacterial biological peptide oral care product comprises the following ingredients in weight ratio:

[0009] SEQ NO: 1 (Gly-Leu-Cys-His-Asp-Arg-Pro) small molecule peptide 0.1-0.3 parts; SEQ NO: 2 (Lys-Tyr-Thr-Val-Glu-Ile-Met) small molecule peptide 0.025-0.075 parts; sustained release agent: 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylic acid ethyl ester (ETHC) 0.5-1.5 parts; moisturizer: propylene glycol 10-15 parts, trehalose 3-6 parts; surfactant: sodium lauroyl sarcosinate 2-4 parts; flavoring agent: sweet orange oil 0.1-0.3 parts, sucralose 0.5-1.0 parts; preservative: benzyl alcohol 0.1-0.3 parts; buffer: sodium bicarbonate 0.3-0.6 parts, potassium dihydrogen phosphate 0.2-0.4 parts.

[0010] 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 have a purity of more than 95%, and the ETHC is prepared and purified by organic synthesis.

[0011] Furthermore, the product preparation method comprises the following steps:

[0012] S1 Dissolve the small molecule peptides SEQ NO: 1 and SEQ NO: 2 and ETHC in a small amount of deionized water respectively;

[0013] S2 adds a humectant, a surfactant, a flavoring agent, a preservative and a buffer in sequence, and stirs in a stirrer at a speed of 200-400 rpm for 30-60 minutes until a uniform solution is formed;

[0014] S3: filtering the mixed solution with a 0.22 μm microporous membrane to remove insoluble impurities;

[0015] S4 performs sterile packaging.

[0016] Furthermore, the PH Helicobacter pylori antibacterial biological peptide oral care product is used in oral care 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.

[0017] Beneficial effects of the invention:

[0018] The novel biological peptide SEQ NO: 1 of the present invention and its combination with SEQ NO: 2 have significant antibacterial effect on Helicobacter pylori. After adding ETHC sustained-release agent, not only the antibacterial rate is high, but also the high antibacterial efficiency can be maintained at a lower biological peptide concentration, and the action time is greatly extended. Compared with traditional oral care products, the inhibitory effect on Helicobacter pylori is significantly superior, providing stronger support for the prevention and adjuvant treatment of Helicobacter pylori-related diseases. The biological peptide is composed of natural amino acids, and the ETHC structure is relatively mild. The entire product has minimal irritation to the oral mucosa and teeth. Unlike traditional oral care products containing antibiotics or irritating chemical antibacterial ingredients, it reduces the potential risks of oral mucosal damage, allergies, etc. caused by ingredient stimulation, is suitable for long-term use, and is safe and reliable for special groups such as children, pregnant women and people with oral sensitivity. It effectively inhibits bacteria while protecting the health of users.

[0019] Oral care products contain biological peptides, sustained-release agents, and a variety of carefully selected adjuvants. Moisturizers keep the mouth moist and the product stable; surfactants improve the cleaning and antibacterial effects; flavoring agents improve the taste; preservatives ensure quality and safety; buffers maintain a suitable pH value to ensure the activity of biological peptides and oral health. The synergy of various ingredients makes the product excellent in antibacterial properties, comprehensively improves oral health and comfort, has strong competitiveness in the oral care product market, and provides a comprehensive performance solution for oral care for Helicobacter pylori infection. The product of the present invention is used for daily oral care, can inhibit the growth and reproduction of Helicobacter pylori in the oral cavity, prevent it from infecting the stomach, and reduce the infection rate. For infected patients, using it in conjunction with clinical treatment can reduce the amount of oral colonization, reduce the risk of reinfection of the stomach, improve the overall treatment effect, shorten the treatment cycle, and has high application value in the prevention and auxiliary treatment of Helicobacter pylori infection. DETAILED DESCRIPTION

[0020] Example 1

[0021] The present invention designs a novel biological peptide sequence SEQ NO: 1: Gly-Leu-Cys-His-Asp-Arg-Pro (GLCHDRP). Design principle: The biological peptide mainly targets urease on the surface of Helicobacter pylori. Helicobacter pylori relies on urease to decompose urea to produce ammonia to survive in an acidic environment and colonize on the surface of gastric mucosa and oral mucosa. The SEQ NO: 1 peptide segment can specifically bind to the active center of urease, the sulfur atoms and imidazole groups of Cys and His residues coordinate with the metal ions in the active center of urease, the Asp and Arg residues interact with the charged groups at the active site of the enzyme through electrostatic attraction, the Gly and Leu residues participate in the construction of the peptide segment structure and hydrophobic interaction, and the Pro residue increases the structural stability, thereby changing the conformation of urease, inhibiting its activity, and destroying the living environment of Helicobacter pylori to achieve antibacterial effect.

[0022] The present invention uses an unreported organic compound as a sustained-release agent, and its chemical name is 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylic acid ethyl ester (abbreviated as ETHC). Design principle: ETHC has a special chemical structure. The ester group (-COO-), carbonyl group (C=O) and thiazolidine ring structure in its molecule enable it to interact with biological peptides and other ingredients in oral care products. The ester group can interact with certain amino acid residues of biological peptides through hydrogen bonds, while the carbonyl group can also participate in the formation of weak hydrogen bonds, and the thiazolidine ring provides a certain spatial structural stability and hydrophobic environment. This interaction can wrap or adsorb the biological peptide around it to form a slow-release system. In the oral environment, with the action of factors such as moisture and salivary enzymes during rinsing or brushing, the combination of ETHC and biological peptides gradually dissociates, allowing the biological peptides to be slowly released, thereby prolonging the action time of the biological peptides in the oral cavity and continuously inhibiting Helicobacter pylori. In addition, ETHC itself may have certain antibacterial activity, and the heteroatoms (N, S, O) and functional groups in its molecular structure may interfere with the metabolic process or cell membrane integrity of Helicobacter pylori, and synergistically enhance the antibacterial effect with biological peptides.

[0023] Antibacterial enhancement of single sequence, combined sequence and sustained release

[0024] 1. Antibacterial effect of single sequence

[0025] Experiments show that the small molecule peptide of SEQ NO: 1 can inhibit Helicobacter pylori by about 45% at a concentration of 0.25 mg / mL. It mainly inhibits the urease activity on the surface of Helicobacter pylori, reduces the production of ammonia, and makes it difficult for bacteria to survive in an acidic environment, thereby inhibiting bacterial growth.

[0026] 2. Synergistic effect of combined sequence (without sustained-release agent)

[0027] When SEQ NO: 1 and SEQ NO: 2: Lys-Tyr-Thr-Val-Glu-Ile-Met (KYTVEIM) are combined in a ratio of 4: 1, the inhibition rate of Helicobacter pylori can be increased to more than 80% at a total concentration of only 0.3 mg / mL. SEQ NO: 2 mainly binds to specific proteins on the cell membrane of Helicobacter pylori, destroys the integrity of the cell membrane, causes leakage of cell contents, and inhibits bacterial growth. When the two are combined, SEQ NO: 1 inhibits urease activity, weakens the survival ability of Helicobacter pylori, and makes the bacterial cell membrane more vulnerable to attack by SEQ NO: 2; while SEQ NO: 2's destruction of the cell membrane affects urease synthesis and transport, enhancing the inhibitory effect of SEQ NO: 1, and synergistically enhances the antibacterial effect.

[0028] 3. Synergistic effect of combination sequence containing sustained-release agents

[0029] When ETHC is added to the combination system of SEQ NO: 1 and SEQ NO: 2 (the addition amount is 0.5%-1.5% of the total weight of the oral care product), the inhibition rate of Helicobacter pylori can be maintained at more than 85% when the total peptide concentration is reduced to 0.2 mg / mL, and the antibacterial effect time is extended by about 50%. ETHC 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.

[0030] The PH Helicobacter pylori antibacterial biological peptide oral care product of the present invention can be a toothpaste or a mouthwash. Taking the mouthwash as an example, in addition to containing the above-mentioned novel biological peptide and sustained-release agent, it also includes the following ingredients:

[0031] 1. Moisturizers: such as propylene glycol and trehalose. Propylene glycol can absorb and retain oral moisture, help dissolve other ingredients, and make the mouthwash texture uniform. Trehalose is moisturizing and stable, and can form a film on the surface of the oral mucosa, reduce irritation, protect the activity of biological peptides, and maintain product stability.

[0032] 2. Surfactant: Sodium lauroyl sarcosinate is used. It is mild and efficient, can reduce surface tension, help clean the oral cavity, enhance the contact between biological peptides and Helicobacter pylori, improve the antibacterial effect, and has little irritation to the oral mucosa.

[0033] 3. Flavoring: Add sweet orange oil and sucralose. Sweet orange oil gives a fresh fruity aroma and improves the taste. Sucralose is high in sweetness and low in calories, provides sweetness, masks bad taste, and prevents tooth decay.

[0034] 4. Preservative: Use benzyl alcohol. It is antibacterial and preservative, ensuring product quality and safety, extending shelf life, and low toxicity.

[0035] 5. Buffer: Add sodium bicarbonate and potassium dihydrogen phosphate. The buffer maintains the pH value of the mouthwash at 6.0-7.5, which is beneficial to the activity and stability of biological peptides, close to the physiological pH value of the oral cavity, and avoids acid-base stimulation. Sodium bicarbonate neutralizes acidic substances, and potassium dihydrogen phosphate buffers and regulates.

[0036] Preparation method:

[0037] 1. For the small molecule peptides of SEQ NO: 1 and SEQ NO: 2, both were prepared by chemical synthesis. First, a suitable solid phase synthetic resin was selected according to each sequence, and the amino acids were modified with protective groups. Then, the amino acids were coupled to the resin in sequence, and the temperature was strictly controlled at 20°C, pH 6, and the coupling time was 1 hour / amino acid. After the peptide chain was synthesized, the protective groups were removed and cleaved, and it was purified and identified by high performance liquid chromatography (HPLC) to ensure that the purity was more than 95%. ETHC was prepared by organic synthesis and was purified for use.

[0038] 2. The purified SEQ NO: 1 small molecule peptide was mixed with ETHC (0.5 weight ratio) at a weight ratio of 0.1 parts, SEQ NO: 2 small molecule peptide was mixed with ETHC (0.5 weight ratio) at a weight ratio of 0.025 parts, moisturizer (propylene glycol 10 weight ratio, trehalose 3 weight ratio), surfactant (sodium lauroyl sarcosinate 2 weight ratio), flavoring agent (orange oil 0.1 weight ratio, sucralose 0.5 weight ratio), preservative (benzyl alcohol 0.1 weight ratio) and buffer (sodium bicarbonate 0.3 weight ratio, potassium dihydrogen phosphate 0.2 weight ratio) in an appropriate amount of deionized water. First, the small molecule peptide and ETHC were dissolved in a small amount of deionized water, and then the other ingredients were added in sequence, and the mixture was slowly stirred at 200 rpm in a stirrer for 30 minutes until a uniform solution was formed.

[0039] 3. Finally, the mixed solution is filtered using a 0.22 μm microporous filter membrane to remove insoluble impurities to obtain the PH Helicobacter pylori antibacterial bio-peptide mouthwash product, which is then aseptically packaged to ensure the hygiene and quality of the product during storage and use.

[0040] The PH Helicobacter pylori antibacterial biological peptide oral care product of the present invention can be applied to daily oral care, especially for oral care and auxiliary treatment of Helicobacter pylori infection high-risk population or infected patients. The user can use mouthwash every morning and evening or after a meal, take an appropriate amount (generally 10-20mL) and gargle in the oral cavity for 30-60 seconds, so that the mouthwash fully contacts the positions such as teeth, gums, oral mucosa and tongue fur, and then spits out. For toothpaste products, it can be used according to conventional brushing methods, brush teeth every morning and evening, and each brushing time is not less than 2 minutes. Through long-term use, the growth and reproduction of Helicobacter pylori in the oral cavity can be effectively suppressed, the colonization amount of Helicobacter pylori in the oral cavity is reduced, and the risk of its transmission to the stomach and re-infection of the stomach is reduced, while the oral cavity can also be cleaned, other common oral diseases can be prevented, and oral health is improved.

[0041] Example 2

[0042] An example of a synthetic preparation process of ethyl 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylate (ETHC): 1. Synthetic route: ETHC is synthesized using L-cysteine ​​hydrochloride and ethyl acrylate as starting materials through addition reaction, cyclization reaction and esterification reaction.

[0043] 2. Specific steps

[0044] (I) Addition reaction

[0045] 1. In a three-necked flask equipped with a stirrer, a thermometer and a reflux condenser, add an appropriate amount of L-cysteine ​​hydrochloride (molar ratio of 1:1.2-1:1.5 relative to ethyl acrylate) and deionized water, and stir to dissolve.

[0046] 2. Slowly add ethyl acrylate, control the speed of addition, and keep the temperature of the reaction system at 20-30° C. After the addition is completed, continue stirring and reacting at this temperature for 2-3 hours.

[0047] 3. After the reaction is completed, the intermediate product N-(2-carboxyethyl)-L-cysteine ​​ethyl ester hydrochloride solution is obtained and directly used in the next step reaction without separation.

[0048] (II) Cyclization reaction

[0049] 1. Add an appropriate amount of sodium hydroxide solution (concentration of 5-10 mol / L) to the above reaction system and adjust the pH value to 9.

[0050] -10, and then heated to 60-70°C for cyclization reaction.

[0051] 2. The reaction progress was monitored by thin layer chromatography (TLC). After about 3-4 hours of reaction, the raw material spot basically disappeared, indicating that the cyclization reaction was completed.

[0052] 3. After the reaction is completed, cool to room temperature and adjust the pH value to 2-3 with hydrochloric acid solution (concentration of 3-6 mol / L). At this time, a white solid precipitates and is filtered to obtain a crude product of 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylic acid.

[0053] (III) Esterification reaction

[0054] 1. Add the above crude product into a three-necked flask with a stirrer, a thermometer and a water separator, and add an appropriate amount of ethanol (molar ratio is 1:5-1:8 relative to the crude product) and concentrated sulfuric acid (as a catalyst, the amount is 2-5 parts of the crude product quality).

[0055] 2. Heat to reflux reaction, separate the water generated by the reaction through a water separator, the reaction time is 4-6 hours.

[0056] 3. After the reaction is completed, cool to room temperature, pour the reaction solution into ice water, adjust the pH value to 7-8 with saturated sodium carbonate solution, and then extract with ethyl acetate (extraction 3-5 times).

[0057] 4. Combine the organic phases, dry over anhydrous sodium sulfate, filter, and remove ethyl acetate by distillation under reduced pressure to obtain a crude product of ethyl 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylate (ETHC).

[0058] 5. Finally, the crude product is purified by vacuum distillation or column chromatography to obtain a high-purity ETHC product, and the purity can reach more than 95% after testing.

[0059] Through the above synthesis process, the 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylic acid ethyl ester (ETHC) required by the present invention can be prepared and used in PH Helicobacter pylori antibacterial biological peptide oral care products to play the role of a sustained-release agent and synergistically improve the product's antibacterial effect and action time on Helicobacter pylori.

[0060] Example 3

[0061] (I) Novel biological peptide sequences

[0062] 1. A novel biological peptide sequence SEQ NO:3-Ala-Met-Phe-Cys-Gln-Arg-Lys (AMFCQRK) was designed. Design principle: The biological peptide targets the outer membrane protein of Helicobacter pylori. The outer membrane protein of Helicobacter pylori plays a key role in maintaining the structural integrity of bacteria, material transport, and interaction with host cells. The Cys residue in the peptide segment of SEQ NO:3 can form a disulfide bond with the cysteine ​​residue in the outer membrane protein, thereby specifically anchoring to the bacterial outer membrane. The Ala and Met residues provide certain hydrophobicity and structural support, and the aromatic ring structure of the Phe residue can enhance the hydrophobic interaction and π-π stacking effect with the outer membrane protein. The Gln, Arg and Lys residues are polar and positively charged, and bind to the negatively charged region on the outer membrane protein through electrostatic interaction, changing the conformation and function of the outer membrane protein, resulting in increased permeability of the bacterial outer membrane, leakage of intracellular substances, and ultimately inhibiting the growth of Helicobacter pylori.

[0063] (II) New sustained-release carrier materials

[0064] 1. A self-synthesized organic-inorganic hybrid material was used as a sustained-release carrier, named silicon-based polylactic acid-co-glycolic acid-chitosan composite (Silica-PLGA-CS).

[0065] Design principle: This complex has a unique structure and properties. The silicon-based component provides good stability and mechanical strength, which helps to maintain the shape and integrity of the gel in the oral environment. PLGA is a biodegradable polymer with good sustained-release properties. Its degradation process is slow and controllable, and it can gradually release the biological peptides and other active ingredients encapsulated therein. Chitosan has antibacterial and biocompatible properties. On the one hand, it can enhance the antibacterial activity of the entire complex and synergize with biological peptides to inhibit Helicobacter pylori; on the other hand, its amino group can electrostatically interact with the carboxyl or other charged groups of the biological peptide, further promoting the adsorption and sustained release of the biological peptide in the carrier. At the same time, chitosan can also interact with the negative charge on the surface of the oral mucosa, so that the gel can better adhere to the oral mucosa and prolong the action time.

[0066] (III) Antibacterial enhancement of single sequence, combined sequence and sustained-release system

[0067] 1. Antibacterial effect of single sequence

[0068] Experiments show that the small molecule peptide of SEQ NO: 3 has an inhibition rate of about 40% on Helicobacter pylori at a concentration of 0.3 mg / mL, mainly by interacting with the outer membrane protein of Helicobacter pylori, destroying the outer membrane structure and function, and inhibiting bacterial growth.

[0069] 2. Synergistic effect of combined sequence (without sustained-release carrier)

[0070] When SEQ NO:3 is combined with another biological peptide SEQ NO:4-Ile-Ser-Trp-His-Pro-Asp-Glu (ISTHPDE) 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 inner membrane protein of Helicobacter pylori, interfering with the energy metabolism and material synthesis process of the bacteria. When the two are combined, SEQ NO:3 destroys the outer membrane, making the inner membrane more susceptible to the action of SEQ NO:4, and the effect of SEQ NO:4 on the inner membrane in turn weakens the repair and maintenance ability of the outer membrane, synergizing with each other to enhance the antibacterial effect.

[0071] 3. Synergistic effect of combined sequence containing sustained-release carrier

[0072] When the combined peptide system of SEQ NO:3 and SEQ NO:4 is encapsulated in the Silica-PLGA-CS sustained-release carrier (the encapsulation rate of the peptide is about 60%-70%, and the content of the carrier in the oral care gel is 10%-15%), 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 60% compared with the combined peptide system without a sustained-release carrier. The Silica-PLGA-CS sustained-release carrier slowly releases the biological peptide, ensuring that there is a sufficient concentration of biological peptides in the oral cavity for a long time to exert an antibacterial effect. At the same time, its own antibacterial property and synergistic effect with the biological peptide further enhance the antibacterial effect.

[0073] (IV) Composition of oral care gel

[0074] The oral care gel of the present invention contains the following ingredients in addition to the novel biopeptide and sustained-release carrier material:

[0075] 1. Moisturizers: such as glycerin and sodium hyaluronate. Glycerin can absorb and retain moisture in the mouth, prevent the gel from drying out, and help to evenly disperse other ingredients. Sodium hyaluronate can form a moisturizing and lubricating film on the surface of the oral mucosa, reduce the irritation of the gel to the oral mucosa, and help maintain the health of the oral mucosa.

[0076] 2. Thickener: Carbomer is used. Carbomer has good thickening properties, which can make the gel have suitable consistency and rheological properties, making it easy to apply and adhere in the mouth without affecting the activity and release of other ingredients.

[0077] 3. Flavoring: Add menthol and stevioside. Menthol gives the gel a cool taste, which can effectively improve oral odor and give users a refreshing feeling. Stevioside, as a natural sweetener, can provide moderate sweetness, improve the taste and acceptability of the product, and will not cause tooth decay.

[0078] 4. Preservative: Use ethyl paraben. It has strong antibacterial and preservative capabilities, and can inhibit the growth and reproduction of microorganisms during the production, storage and use of the gel, ensuring the quality and safety of the product and extending the shelf life of the product.

[0079] (V) Preparation method

[0080] 1. Synthesis and purification of biological peptides

[0081] For SEQ NO:3 and SEQ NO:4 small molecule peptides, chemical synthesis was used to prepare them. First, a suitable solid phase synthetic 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 reaction, the temperature was strictly controlled at 22-25°C, the pH value was 6-7, and the coupling time for each amino acid was 1-1.5 hours. After the peptide chain was synthesized, the protective group was removed and cut, and then purified and identified by high performance liquid chromatography (HPLC) to ensure that the purity of the biological peptide reached more than 95%.

[0082] 2. Preparation of sustained-release carrier

[0083] First, prepare the silicon-based material, and hydrolyze and condense tetraethyl orthosilicate under acidic conditions to form silica sol. Then dissolve polylactic acid-co-glycolic acid (PLGA) in an organic solvent, add an appropriate amount of chitosan solution, stir evenly, and then add silica sol dropwise, continue stirring to react, so that the components are fully mixed and reacted, and form a silicon-based polylactic acid-co-glycolic acid-chitosan complex (Silica-PLGA-CS) precipitate. After centrifugation, washing and drying, a purified sustained-release carrier material is obtained.

[0084] 3. Preparation of Oral Care Gel

[0085] The purified SEQ NO:3 small molecule peptide is mixed in a weight ratio of 0.1-0.2 parts, the SEQ NO:4 small molecule peptide is mixed in a weight ratio of 0.05-0.1 parts, and the silica-PLGA-CS sustained-release carrier (10-15 parts by weight), moisturizer (glycerol 8-12 parts by weight, sodium hyaluronate 0.2-0.4 parts by weight), thickener (carbomer 1.5-2.5 parts by weight), flavoring agent (menthol 0.1-0.2 parts by weight, stevioside 0.3-0.5 parts by weight), preservative (ethyl paraben 0.05-0.1 parts by weight) and other ingredients in an appropriate amount of deionized water. First, the biological peptide and the sustained-release carrier are dispersed in a small amount of deionized water, and then other ingredients are added in sequence, and stirred in a stirrer at a speed of 300-500 rpm for 40-60 minutes until a uniform gel product is formed.

[0086] (VI) Application

[0087] The novel anti-Helicobacter pylori oral care gel of the present invention can be applied to daily oral care, especially oral care and auxiliary treatment for Helicobacter pylori infection high-risk groups or infected patients. The user can take an appropriate amount of gel (about 2-3 grams) and apply it to the teeth, gums, oral mucosa and tongue fur every morning and evening or after meals, massage gently for 1-2 minutes, keep for a period of time (about 3-5 minutes), and then rinse the mouth with water or swallow it directly (a small amount of swallowing is harmless to the body). Through long-term use, the growth and reproduction of Helicobacter pylori in the oral cavity can be effectively suppressed, 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 can be prevented, and the oral health status can be improved.

[0088] 4. Beneficial Effects

[0089] 1. Highly effective and long-lasting anti-Helicobacter pylori performance

[0090] 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 application of the silicon-based polylactic acid-co-glycolic acid-chitosan complex (Silica-PLGA-CS) sustained-release carrier further enhances the antibacterial effect and significantly prolongs the duration of action. Compared with traditional oral care products, 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.

[0091] 2. Good biocompatibility and safety

[0092] The biological peptide is composed of natural amino acids, and the Silica-PLGA-CS sustained-release carrier material has good biocompatibility. The entire oral care gel has minimal irritation to the oral mucosa and teeth. Unlike traditional oral care products 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.

[0093] 3. Optimized product formula and performance

[0094] In addition to biological peptides and sustained-release carriers, oral care gels also contain a variety of carefully selected adjuvants. Moisturizers keep the mouth moist, thickeners give the appropriate consistency for easy use, flavoring agents improve the taste, and preservatives ensure product quality and safety. The various ingredients work together to make the gel not only perform well in antibacterial performance, but also have advantages in 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.

[0095] 4. Dual effects of prevention and auxiliary treatment

[0096] The oral care gel 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 gel 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 adjuvant treatment of Helicobacter pylori infection.

[0097] In summary, the novel anti-Helicobacter pylori oral care gel 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, unique sustained-release carrier material, reasonable formula composition and scientific preparation method.

[0098] (I) Synthesis and identification of biological peptides

[0099] 1. According to the above-mentioned biological peptide synthesis method, SEQ NO: 3 and SEQ NO: 4 small molecule peptides were synthesized respectively.

[0100] Taking the synthesis of SEQ NO:3 as an example, Wang resin was selected as the solid phase synthesis resin. The first amino acid Ala was first modified with the Fmoc protecting group and then coupled to the resin. During the coupling process, the temperature of the reaction system was controlled at 23°C, the pH value was 6.5, and the coupling time was 1.2 hours. Then the other amino acids were coupled in sequence, and each step of coupling was washed and tested to ensure that the coupling reaction was complete. After the peptide chain was synthesized, a deprotecting agent was used to remove the Fmoc protecting group, and then a cutting reagent was used to cut the peptide chain from the resin to obtain a crude SEQ NO:3 peptide.

[0101] 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%.

[0102] The small molecule peptide of SEQ NO: 4 was synthesized and purified by the same method, and the purity reached 95.5%.

[0103] (II) Preparation and characterization of sustained-release carriers

[0104] 1. Preparation of sustained-release carrier

[0105] Preparation of silica sol: Add 100 mL of ethanol, 30 mL of deionized water and 3 mL of hydrochloric acid into a three-necked flask, stir evenly, then slowly drop 10 mL of ethyl orthosilicate, stir and react at room temperature for 2 hours to obtain silica sol.

[0106] Preparation of Silica-PLGA-CS complex: 5g PLGA was dissolved in 50mL dichloromethane solution, 2g chitosan was dissolved in 20mL acetic acid solution (1% concentration) was added, and after stirring evenly, the silica sol prepared above was added dropwise, and the stirring reaction was continued for 4 hours. After the reaction was completed, the reaction solution was poured into a large amount of anhydrous ethanol to precipitate the complex. The precipitate was collected by centrifugation, washed with anhydrous ethanol 3 times, and then dried in a vacuum drying oven for 24 hours to obtain a Silica-PLGA-CS sustained-release carrier material.

[0107] 2. Characterization of sustained-release carriers

[0108] Scanning electron microscopy (SEM) was used to characterize the microstructure of the Silica-PLGA-CS sustained-release carrier, and the results showed that it had a porous structure with an average pore size of about 50-100 nm. This porous structure is conducive to the encapsulation and release of biological peptides.

[0109] Through Fourier transform infrared spectroscopy (FT-IR) analysis, the superposition of the characteristic peaks of silica, PLGA and chitosan can be observed in the infrared spectrum of the composite, indicating that the Silica-PLGA-CS composite was successfully synthesized.

[0110] (III) Example 3: Preparation and performance testing of oral care gel

[0111] 1. Preparation of Oral Care Gel

[0112] The purified SEQ NO: 3 small molecule peptide 0.15 parts, SEQ NO: 4 small molecule peptide 0.075 parts and Silica-PLGA-CS sustained-release carrier 12 parts, moisturizer (glycerol 10 parts, sodium hyaluronate 0.3 parts), thickener (carbomer 2 parts), flavoring agent (menthol 0.15 parts, stevioside 0.4 parts), preservative (ethyl paraben 0.075 parts) and other ingredients were mixed in an appropriate amount of deionized water. The biological peptide and the sustained-release carrier were first dispersed in 20mL of deionized water and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then the other ingredients were added in sequence and stirred in a stirrer at a speed of 400 rpm for 50 minutes until a uniform gel product was formed.

[0113] 2. Antibacterial performance test

[0114] The agar plate diffusion method was used to test the antibacterial effect of oral care gel on Helicobacter pylori. Prepare a special culture medium plate for Helicobacter pylori and divide the plate evenly into several areas. Take 10 μL of oral care gel samples of different concentrations and drop them on the culture medium surface of the corresponding area. Place an Oxford cup (diameter 8 mm) to allow the sample to spread evenly. Use a sterile cotton swab to dip an appropriate amount of Helicobacter pylori suspension (concentration 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.

[0115] The test results show that when the total peptide concentration of the oral care gel prepared by the present invention is 0.2 mg / mL, the average diameter of the inhibition zone against Helicobacter pylori is 20.5 mm, indicating that it has a good antibacterial effect.

[0116] 3. Sustained release performance test

[0117] The sustained release performance of biological peptides in oral care gel was tested by dialysis bag method. The oral care gel sample containing biological peptides was placed in a dialysis bag (molecular weight cutoff of 8000-10000Da), and then the dialysis bag was placed in a container containing 500mL phosphate buffer solution (pH 7.2) and oscillated at a constant temperature at 37°C. 5mL of dialysate was taken out at regular intervals (1, 2, 4, 6, 8, 12, 24 hours), and the content of biological peptides in the dialysate was determined by ultraviolet spectrophotometry, and the cumulative release rate was calculated.

[0118] The test results show that the release of biological peptides in oral care gel presents a slow and continuous process, with a cumulative release rate of about 70 parts within 24 hours, indicating that the Silica-PLGA-CS sustained-release carrier can effectively control the release rate of biological peptides and achieve long-term antibacterial effect.

[0119] The above examples further verify that the preparation method of the novel anti-Helicobacter pylori oral care gel of the present invention is feasible, the product has good antibacterial performance and sustained-release performance, and can meet the needs of oral care against Helicobacter pylori.

Claims

1. A PH Helicobacter pylori antibacterial biological peptide oral care product, characterized in that: Contains the following ingredients: SEQ NO: 1Gly-Leu-Cys-His-Asp-Arg-Pro small molecule peptide.

2. A PH Helicobacter pylori antibacterial biological peptide oral care product according to claim 1, characterized in that: Also contains the following ingredients: SEQ NO: 2Lys-Tyr-Thr-Val-Glu-Ile-Met small molecule peptide.

3. A PH Helicobacter pylori antibacterial biological peptide oral care product according to claim 1, characterized in that: Also contains the following ingredients: Sustained release agent: ethyl 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylate.

4. A PH Helicobacter pylori antibacterial biological peptide oral care product, characterized in that: Contains the following ingredients in weight ratio: SEQ NO: 1Gly-Leu-Cys-His-Asp-Arg-Pro small molecule peptide 0.1-0.3 parts; SEQ NO: 2Lys-Tyr-Thr-Val-Glu-Ile-Met small molecule peptide 0.025-0.075 parts; sustained release agent: 4-(2-hydroxyethyl)-1,3-thiazolidine-2-one-5-carboxylic acid ethyl ester (ETHC) 0.5-1.5 parts; moisturizer: propylene glycol 10-15 parts, trehalose 3-6 parts; surfactant: sodium lauroyl sarcosinate 2-4 parts; Flavoring agent: 0.1-0.3 parts of sweet orange oil, 0.5-1.0 parts of sucralose; preservative: 0.1-0.3 parts of benzyl alcohol; Buffer: 0.3-0.6 parts of sodium bicarbonate, 0.2-0.4 parts of potassium dihydrogen phosphate.

5. The PH Helicobacter pylori antibacterial biological peptide oral care product 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 have a purity of more than 95%. The ETHC is prepared and purified by organic synthesis.

6. The PH Helicobacter pylori antibacterial biological peptide oral care product according to claim 1, wherein the preparation method comprises the following steps: S1 Dissolve the small molecule peptides SEQ NO: 1 and SEQ NO: 2 and ETHC in a small amount of deionized water respectively; S2 adds a humectant, a surfactant, a flavoring agent, a preservative and a buffer in sequence, and stirs in a stirrer at a speed of 200-400 rpm for 30-60 minutes until a uniform solution is formed; S3: filtering the mixed solution with a 0.22 μm microporous membrane to remove insoluble impurities; S4 performs sterile packaging.

7. Use of the PH Helicobacter pylori antibacterial biological peptide oral care product as claimed in any one of claims 1 to 3 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.