Search

CN-121975017-A - Mouthwash capable of effectively inhibiting helicobacter pylori and preparation method thereof

CN121975017ACN 121975017 ACN121975017 ACN 121975017ACN-121975017-A

Abstract

The invention discloses a helicobacter pylori resistant mouthwash and preparation and application thereof. The mouthwash is prepared by taking an anti-helicobacter pylori urease alpha subunit single domain antibody and 3,4, 5-tri-O-methyl gallic acid as core active ingredients and adding hydroxypropyl-beta-cyclodextrin, glycerol, phenoxyethanol and citric acid-sodium citrate buffer solution as auxiliary materials. The single domain antibody can block the catalytic activity of urease and the adhesion function of bacteria, and 3,4, 5-tri-O-methyl gallic acid can penetrate the bacterial biomembrane and destroy the cell membrane. The inhibition rate of the two synergistic agents on the bacteria adhesion gastric epithelial cells reaches 90.8 percent. Mouse experiments show that the mouthwash can reduce the bacterial load of gastric mucosa by 99.8%, reduce inflammation and repair mucous membrane injury, and has no skin and oral mucous membrane irritation. The mouthwash has high stability, and the retention rate of active ingredients exceeds 91% after being stored for 6 months at 25 ℃, thereby providing a safe and efficient oral care product for preventing and treating helicobacter pylori infection.

Inventors

  • LIU YU

Assignees

  • 广州诺捷生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260210

Claims (10)

  1. 1. The anti-helicobacter pylori urease alpha subunit single domain antibody is characterized in that the single domain antibody is VHH-3A4, and the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 3.
  2. 2. The single domain antibody of claim 1, wherein the gene sequence of the single domain antibody is shown in SEQ ID No. 4.
  3. 3. The single domain antibody of claim 1, wherein the immunogen for preparing the single domain antibody is rUreA 1-150 , the amino acid sequence of which is shown in SEQ ID NO. 1, and the corresponding nucleotide sequence of which is shown in SEQ ID NO. 2.
  4. 4. A composition for combating helicobacter pylori, said composition comprising an effective amount of an anti-helicobacter pylori urease alpha subunit single domain antibody according to claim 1 and an effective amount of 3,4, 5-tri-O-methyl gallic acid.
  5. 5. The composition according to claim 4, wherein the 3,4, 5-tri-O-methyl gallic acid has a log P value of 2.8, a minimum inhibitory concentration of 0.5. Mu.g/mL for the standard strain of helicobacter pylori ATCC 43504, and a minimum inhibitory concentration of 1.0. Mu.g/mL for the resistant strain of helicobacter pylori containing the blaOXA-181 resistance gene.
  6. 6. The composition according to claim 5, wherein the preparation process of 3,4, 5-tri-O-methyl gallic acid comprises extracting and purifying gallic acid from gallnut, subjecting gallic acid and methyl iodide to methylation reaction in anhydrous N, N-dimethylformamide, extracting with ethyl acetate, washing with sodium thiosulfate solution, and purifying by silica gel column chromatography to obtain 3,4, 5-tri-O-methyl gallic acid with purity not lower than 98.9%.
  7. 7. The composition of claim 6, wherein the molar ratio of gallic acid to methyl iodide in the methylation reaction is 1:3, the reaction temperature is room temperature, the reaction time is 24 hours, the progress of the reaction is monitored by thin layer chromatography, the developing agent is petroleum ether-ethyl acetate=3:1, and the color-developing agent is 5% ferric trichloride ethanol solution until gallic acid spots completely disappear.
  8. 8. The anti-helicobacter pylori mouthwash is characterized by comprising, by mass, 0.04% of the anti-helicobacter pylori urease alpha subunit single domain antibody according to claim 1, 0.2% of the 3,4, 5-tri-O-methyl gallic acid according to claim 5, 3% of hydroxypropyl-beta-cyclodextrin, 2% of glycerol, 0.4% of phenoxyethanol, 0.1M citric acid-sodium citrate buffer solution and purified water, wherein the pH value of the mouthwash is adjusted to 6.8 by the 0.1M citric acid-sodium citrate buffer solution, and the balance of the purified water.
  9. 9. A method for preparing the anti-helicobacter pylori mouthwash according to claim 8, which is characterized by comprising the steps of heating purified water to 50 ℃, adding hydroxypropyl-beta-cyclodextrin, stirring at 300rpm for 15 minutes until the hydroxypropyl-beta-cyclodextrin is dissolved, cooling to room temperature, adding glycerol and phenoxyethanol, stirring uniformly, dripping 0.1M citric acid-sodium citrate buffer solution, regulating the pH value to 6.8, adding an anti-helicobacter pylori urease alpha subunit single-domain antibody and 3,4, 5-tri-O-methyl gallic acid, stirring at 200rpm for 30 minutes until the anti-helicobacter pylori urease alpha subunit single-domain antibody and 3,4, 5-tri-O-methyl gallic acid are dissolved, adding purified water to a certain volume, filtering and sterilizing by a 0.22 mu M polyether sulfone filter membrane, and filling the mixture into a brown light-resistant bottle in a sterile manner.
  10. 10. Use of a composition according to claim 4 for the preparation of an oral care product for the prevention or adjuvant treatment of helicobacter pylori infection.

Description

Mouthwash capable of effectively inhibiting helicobacter pylori and preparation method thereof Technical Field The invention belongs to the technical field of oral care and antibacterial preparations, and particularly relates to mouthwash for targeted inhibition of helicobacter pylori (Helicobacter pylori, hp) and a preparation method thereof. Background Helicobacter pylori (Helicobacter pylori, hp) is one of the most common bacterial pathogens worldwide. The strain can be planted in gastric mucosa for a long time to induce chronic gastritis and peptic ulcer. Clinically, hp eradication mainly relies on a bismuth agent quadruple scheme consisting of a proton pump inhibitor, a bismuth agent and two antibiotics. However, as the drug resistance rate continues to rise, the efficacy is significantly reduced. In addition to gastric infections, the oral cavity is considered an important reservoir for Hp. Research shows that the detection rate of Hp in dental plaque biomembrane, tongue fur and oral mucosa fold is as high as 75% -85%. Because conventional antibiotics are difficult to access effectively to the oral microenvironment by systemic administration, oral colonization strains often survive gastric eradication therapy and become a key source of recurrence. Clinical studies have shown that oral Hp positive individuals can have stomach recurrence rates as high as 40%/year, while negative individuals are less than 5%. The prior measures for clearing oral Hp still have obvious defects. Conventional mouthwashes (e.g., chlorhexidine) only reduce bacterial load for a short period of time, but lack specificity for Hp, long term application may also cause bacterial imbalance, staining of teeth, and fungal secondary infections. Specific agents such as anti-Urease antibodies, while inhibiting some enzymatic activities, have limited specificity and persistence due to cross-reactivity with other oral Urease positive bacteria. Although natural active molecules such as polyphenols have a certain antibacterial effect, the natural active molecules have the problems of low solubility, difficulty in penetrating biological membranes, high effective concentration and the like. In general, the existing scheme can not form comprehensive intervention aiming at a biological membrane barrier-adhesion field planting mechanism of oral cavity Hp, and is difficult to realize the synergistic effects of targeted identification, barrier penetration, effective bacteriostasis and re-field planting prevention. Therefore, the development of the innovative mouthwash which has high specificity, good biological film penetrability, low drug resistance risk and can block reinfection has important significance for improving Hp eradication rate, reducing recurrence and improving clinical prognosis. Disclosure of Invention In order to solve the technical problems, the invention provides the mouthwash capable of effectively inhibiting helicobacter pylori and the preparation method thereof. Therefore, on the one hand, the invention discloses an anti-helicobacter pylori urease alpha subunit single domain antibody, which is VHH-3A4, the amino acid sequence of which is shown in SEQ ID NO. 3, the gene sequence of which is shown in SEQ ID NO. 4, preferably, the immunogen for preparing the single domain antibody is rUreA 1-150, the amino acid sequence of which is shown in SEQ ID NO. 1, and the corresponding nucleotide sequence of which is shown in SEQ ID NO. 2. In one aspect, the invention also discloses a composition for resisting helicobacter pylori, which comprises an effective amount of the helicobacter pylori resistant urease alpha subunit single domain antibody and an effective amount of 3,4, 5-tri-O-methyl gallic acid. Wherein the 3,4, 5-tri-O-methyl gallic acid has a log P value of 2.8, a minimum inhibitory concentration of 0.5 mug/mL for a standard strain of helicobacter pylori ATCC 43504, and a minimum inhibitory concentration of 1.0 mug/mL for a resistant strain of helicobacter pylori containing the blaOXA-181 resistant gene. The preparation method of the 3,4, 5-tri-O-methyl gallic acid comprises the steps of extracting and purifying gallic acid from gallnuts, carrying out methylation reaction on the gallic acid and methyl iodide in anhydrous N, N-dimethylformamide, extracting by ethyl acetate, washing by sodium thiosulfate solution, and purifying by silica gel column chromatography to obtain the 3,4, 5-tri-O-methyl gallic acid with the purity of not less than 98.9%. The molar ratio of gallic acid to methyl iodide is 1:3 during methylation reaction, the reaction temperature is room temperature, the reaction time is 24 hours, the reaction progress is monitored through thin layer chromatography, the developing agent is petroleum ether-ethyl acetate=3:1, and the color developing agent is 5% ferric trichloride ethanol solution until gallic acid spots completely disappear. In one aspect, the invention also discloses a helicobacter pylori resistant mouthwas