Mouth wash capable of effectively inhibiting helicobacter pylori and preparation method thereof

Abstract

The invention discloses anti-helicobacter pylori mouthwash as well as 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-methylgallic acid as core active components and taking hydroxypropyl-beta-cyclodextrin, glycerol, phenoxyethanol and a citric acid-sodium citrate buffer solution as auxiliary materials. The single-domain antibody can block the catalytic activity of urease and the bacterial adhesion function; the 2, 3, 4, 5-tri-O-methyl gallic acid can penetrate through a bacterial biological membrane and destroy a cell membrane. The synergistic inhibition rate of the two components on bacterial adhesion gastric epithelial cells reaches 90.8%. Mouse experiments show that the mouthwash can reduce the bacterial load of gastric mucosa by 99.8%, relieve inflammation and repair mucosal lesion, and has no irritation to skin and oral mucosa. The mouthwash is high in stability, the retention rate of active ingredients is higher than 91% after the mouthwash is stored at 25 DEG C for 6 months, and a safe and efficient oral care product is provided for prevention and treatment of helicobacter pylori infection.

Description

Technical Field

[0001] This invention belongs to the field of oral care and antibacterial preparation technology, specifically relating to a mouthwash that targets and inhibits Helicobacter pylori (Hp) and its preparation method. Background Technology

[0002] Helicobacter pylori (Hp) is one of the most common bacterial pathogens worldwide. This bacterium colonizes the gastric mucosa and can cause chronic gastritis and peptic ulcers. Clinically, Hp eradication mainly relies on a quadruple therapy regimen consisting of a proton pump inhibitor, bismuth, and two antibiotics. However, with the continuous increase in drug resistance rates, its efficacy has significantly declined.

[0003] Besides gastric infections, the oral cavity is considered an important reservoir for *Helicobacter pylori* (Hp). Studies have shown that Hp can be detected in 75%–85% of dental plaque biofilms, tongue coating, and oral mucosal folds. Because conventional antibiotics are difficult to effectively penetrate the oral microenvironment via systemic administration, oral colonizing strains often survive after gastric eradication treatment and become a key source of recurrence. Clinical surveys show that the gastric recurrence rate in oral Hp-positive individuals can be as high as 40% per year, while it is less than 5% in those who are negative.

[0004] Current measures for eliminating oral *Helicobacter pylori* (Hp) still have significant shortcomings. Commonly used mouthwashes (such as chlorhexidine) can only reduce bacterial load in the short term, but lack specificity for *Hp*, and long-term use may lead to flora imbalance, tooth discoloration, and secondary fungal infections. Targeted agents such as anti-Urease antibodies can inhibit some enzyme activity, but their specificity and persistence are limited due to cross-reactivity with other oral urease-positive bacteria. Natural active molecules such as polyphenols have some antibacterial effects, but they suffer from low solubility, difficulty in penetrating biofilms, and relatively high effective concentrations. Overall, existing solutions have not yet formed a comprehensive intervention targeting the "biofilm barrier-adhesion and colonization" mechanism of oral *Hp*, and it is difficult to achieve a synergistic effect of targeted recognition, barrier penetration, effective antibacterial action, and prevention of recolonization.

[0005] Therefore, developing an innovative mouthwash that combines high specificity, good biofilm penetration, low risk of drug resistance, and the ability to block reinfection is of great significance for improving Hp eradication rate, reducing recurrence, and improving clinical prognosis. Summary of the Invention

[0006] To address the above technical problems, this invention provides a mouthwash that can effectively inhibit Helicobacter pylori and its preparation method.

[0007] Therefore, this invention discloses, in one aspect, a single-domain antibody against the urease α subunit of Helicobacter pylori, wherein the single-domain antibody is VHH-3A4, its amino acid sequence is shown in SEQ ID NO:3, and its gene sequence is shown in SEQ ID NO:4; preferably, the immunogen for preparing the single-domain antibody is rUreA. 1-150 Its amino acid sequence is shown in SEQ ID NO:1, and the corresponding nucleotide sequence is shown in SEQ ID NO:2.

[0008] In one aspect, the present invention also discloses an anti-Helicobacter pylori composition, the composition comprising an effective amount of the anti-Helicobacter pylori urease α subunit single-domain antibody and an effective amount of 3,4,5-tri-O-methylgallic acid. The 3,4,5-tri-O-methylgallic acid has a logP value of 2.8, a minimum inhibitory concentration (MIC) of 0.5 μg / mL against the Helicobacter pylori standard strain ATCC 43504, and a MIC of 1.0 μg / mL against a Helicobacter pylori resistant strain containing the blaOXA-181 resistance gene. The preparation steps of the 3,4,5-tri-O-methylgallic acid include: extracting and purifying gallic acid from gallnut; methylating gallic acid with iodomethane in anhydrous N,N-dimethylformamide; and purifying by ethyl acetate extraction, washing with sodium thiosulfate solution, and silica gel column chromatography to obtain 3,4,5-tri-O-methylgallic acid with a purity of not less than 98.9%. During the methylation reaction, the molar ratio of gallic acid to iodomethane was 1:3, the reaction temperature was room temperature, and the reaction time was 24 hours. The reaction progress was monitored by thin-layer chromatography, with petroleum ether-ethyl acetate as the developing solvent and 5% ferric chloride ethanol solution as the colorimetric reagent, until the gallic acid spots completely disappeared.

[0009] In one aspect, the present invention also discloses an anti-Helicobacter pylori mouthwash, wherein the mouthwash, by mass percentage, comprises 0.04% anti-Helicobacter pylori urease α subunit single-domain antibody, 0.2% 3,4,5-tri-O-methylgallic acid, 3% hydroxypropyl-β-cyclodextrin, 2% glycerol, 0.4% phenoxyethanol, 0.1M citrate-sodium citrate buffer, and purified water; the pH of the mouthwash is adjusted to 6.8 by the 0.1M citrate-sodium citrate buffer, and the remaining amount is purified water.

[0010] In one aspect, the present invention also discloses a method for preparing the aforementioned anti-Helicobacter pylori mouthwash, the method comprising the following steps: heating purified water to 50°C, adding hydroxypropyl-β-cyclodextrin and stirring at 300 rpm for 15 minutes until dissolved, and cooling to room temperature; adding glycerol and phenoxyethanol and stirring evenly, and adding 0.1M citrate-sodium citrate buffer to adjust the pH to 6.8; adding anti-Helicobacter pylori urease α subunit single-domain antibody and 3,4,5-tri-O-methylgallic acid, and stirring at 200 rpm for 30 minutes until dissolved; adding purified water to make up the volume, filtering through a 0.22 μm polyethersulfone filter membrane for sterilization, and aseptically filling into brown light-proof bottles.

[0011] The beneficial effects of this invention are summarized as follows:

[0012] 1. This invention's anti-Helicobacter pylori mouthwash exhibits significant high efficacy. The core active ingredient, an anti-Helicobacter pylori urease α subunit single-domain antibody, forms a synergistic antibacterial mechanism of "targeted blocking + structural disruption" with 3,4,5-tri-O-methylgallic acid. The single-domain antibody targets and binds to the unique epitope of the Helicobacter pylori urease α subunit, effectively blocking urease catalytic activity and bacterial adhesion. Its half-maximal inhibitory concentration (IC50) is far lower than existing β subunit antibodies, achieving a 97.6% inhibition rate against urease at 5.0 μg / mL. 3,4,5-tri-O-methylgallic acid possesses excellent biomembrane penetration ability, effectively disrupting bacterial cell membranes. The minimum inhibitory concentrations against standard and drug-resistant Helicobacter pylori strains are as low as 0.5 μg / mL and 1.0 μg / mL, respectively, representing an 8-10 times increase in activity compared to natural gallic acid. When the two components work synergistically, they inhibit the adhesion of Helicobacter pylori to human gastric epithelial cells by 90.8%, which is significantly better than the single component. In in vivo experiments in mice, they can reduce the bacterial load on the gastric mucosa by 99.8%, and the clearance effect is better than that of traditional chlorhexidine mouthwash.

[0013] 2. In terms of specificity, this mouthwash has a significant advantage. The single-domain antibody targets epitopes 1-150 of the Helicobacter pylori urease α subunit, which are bacterial-specific epitopes. They have low homology with urease from common oral Streptococcus mutans, with a cross-reactivity rate of only 4.2%, far lower than existing β subunit antibodies. This effectively avoids interfering with normal oral flora and solves the problem of traditional antibodies disrupting the balance of the flora.

[0014] 3. This invention's mouthwash offers significant advantages in addressing the increasingly serious problem of Helicobacter pylori drug resistance. 3,4,5-Tri-O-methylgallic acid exerts its antibacterial effect by disrupting the bacterial cell membrane. Unlike antibiotics such as amoxicillin, it maintains potent inhibitory activity against drug-resistant Helicobacter pylori strains containing resistance genes, while amoxicillin is completely ineffective against these resistant strains. There is no risk of cross-resistance, providing a new solution for the prevention and control of drug-resistant Helicobacter pylori infection.

[0015] 4. The mouthwash exhibits excellent stability. The hydroxypropyl-β-cyclodextrin added to the formula effectively reduces the aggregation of anti-Helicobacter pylori urease α subunit single-domain antibodies and the oxidative degradation of 3,4,5-tri-O-methylgallic acid through inclusion complexation. Under 25°C storage conditions, the retention rates of the two active ingredients reached 91.7% and 94.2% respectively after 6 months. Even after 3 months of storage at 40°C, the retention rate still exceeded 85%, fully meeting the product's shelf-life requirements. In contrast, the control group without hydroxypropyl-β-cyclodextrin showed a significantly reduced retention rate of active ingredients, making it difficult to guarantee the product's long-term stability.

[0016] 5. Safety is a significant advantage of this mouthwash. Rigorous skin irritation and oral mucosal irritation tests verified that, 24-72 hours after applying the mouthwash to the back of rabbits, no erythema or edema was observed, and the total irritation index was 0, meeting the non-irritating standard. In rats, after 14 consecutive days of oral application of the mouthwash, the buccal and lingual mucosa remained intact, without inflammatory cell infiltration, mucosal congestion, shedding, or edema. In mouse intervention experiments, the weight change rate in the mouthwash group was close to that of the normal group, with no significant side effects. Its safety is significantly superior to traditional antibiotics and chlorhexidine mouthwash, providing assurance for long-term use. Attached Figure Description

[0017] Figure 1 For Hp Urease α subunits 1-150 (rUreA) 1-150 The SDS-PAGE detection results of ) are shown, where 1 is rUreA 1-150 Its molecular weight is approximately 17 kDa.

[0018] Figure 2 The result is the SDS-PAGE detection result of VHH-3A4, where 1 represents VHH-3A4.

[0019] Figure 3 For TMGA 1 The H-NMR diagram shows characteristic peaks at δ=3.85 (3×OCH3) and δ=7.12 (Ar-H), proving that the methylation modification was successful.

[0020] Figure 4 The HPLC chromatograms of GA and TMGA are compared. GA eluted at 6.2 min, while TMGA eluted at 8.5 min, indicating that the methylation modification enhanced the hydrophobicity of the molecules and prolonged their retention time. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0023] Example 1: Preparation and testing of anti-Hp Urease α subunit single-domain antibody (named VHH-3A4)

[0024] 1. Immunogen Preparation

[0025] This study selected a novel epitope (amino acid positions 1-150, UniProt: P14916) of the α subunit of Helicobacter pylori (Hp) urease as an immunogen. Compared with existing antibodies targeting the β subunit, the application of this epitope can effectively reduce the risk of cross-reactivity. Meanwhile, urease, as a key enzyme in the colonization of Hp in the gastric mucosa, mainly neutralizes gastric acid by breaking down urea to produce ammonia, thus creating a suitable environment for bacterial survival. An immune response targeting the α subunit can not only block the catalytic activity of urease but may also interfere with its adhesion-aiding function, thereby achieving a dual inhibitory effect on Hp colonization.

[0026] Based on the previous design, we selected to amplify the gene fragment from position 1 to 150 of the urease α subunit of Helicobacter pylori (its protein is denoted as rUreA). 1-150 The amino acid sequence of the compound is shown in SEQ ID NO:1, and the corresponding nucleotide sequence is shown in SEQ ID NO:2. The amplified PCR product was double-digested and inserted into the expression vector pET-28a(+) (carrying a C-terminal 6×His tag). The successfully constructed recombinant plasmid was identified by sequencing and then transformed into Escherichia coli BL21(DE3) competent cells. The strain was cultured at 37℃ with shaking until it reached OD280. 600 IPTG (final concentration 0.5 mM) was added when the pH reached approximately 0.6, and induction was performed for 16 h. After induction, the bacterial cells were collected and sonicated (300 W power, work / interval ratio 3 s / 5 s, duration 30 min). The supernatant was purified by Ni-NTA metal affinity chromatography. The purified product was dialyzed to remove salts and then analyzed by SDS-PAGE. The results showed that the target protein had a molecular weight of approximately 17 kDa and a purity of ≥95% (see [link to SDS-PAGE analysis]). Figure 1The concentration was then quantified to 1 mg / mL using the BCA method, sterilized by a 0.22 µm filter membrane, and stored at -80°C for long-term use.

[0027] 2. Alpaca Immunotherapy and Lymphocyte Extraction

[0028] Immunization regimen: Day 0, rUreA 1-150 (100 μg / alpacia) was emulsified 1:1 with Freund's complete adjuvant and subcutaneously injected at multiple points on the neck and back of two healthy male alpacas (6-8 months old, weighing 50-60 kg); on days 14, 28, and 42, the same dose of antigen was emulsified with Freund's incomplete adjuvant for booster immunization; on day 56, 10 mL of blood was collected for serum titer detection by ELISA (OD value required). 450 ≥1.0 (serum dilution factor ≥1:100,000); results showed that the OD of serum from two alpacas at a dilution of 1:128,000 was... 450 The values ​​are 1.23 and 1.35 respectively, which meet the database construction requirements.

[0029] Lymphocyte isolation: 7 days after the last immunization, 50 mL of blood was collected from the jugular vein, anticoagulated with heparin, and centrifuged with Ficoll-Paque Plus density gradient (400×g, 30 min) to collect peripheral blood mononuclear cells (PBMCs). Total RNA was extracted with TRIzol.

[0030] 3. VHH gene amplification and phage display library construction

[0031] cDNA synthesis: 2 μg of total RNA was used to synthesize cDNA using a reverse transcription kit;

[0032] VHH gene amplification: A heavy chain antibody gene of approximately 600 bp (containing VHH and CH2 regions) was amplified using camel-specific primers; the CH2 region was removed using nested primers to obtain a VHH gene of approximately 400 bp.

[0033] Library construction: The VHH gene was double-digested with SfiI / NotI and inserted into the phage vector pCANTAB 5E. The cells were then electrotransformed into TG1 competent cells (1.8 kV, 200 Ω, 25 μF). The transformation product was plated on 2×YT-AG plates (containing 100 μg / mL ampicillin and 2% glucose) and incubated at 37°C for 16 h. Colony counts were performed, and the library volume was calculated: Library volume = Colony count × Dilution factor / Transformation volume (final library volume reached 2.3 × 10⁻⁶). 8 CFU); 100 single colonies were selected for PCR verification, with an insertion rate ≥95% and acceptable library diversity.

[0034] Library parameter results: Library size 2.3 × 10 8CFU, with an insertion rate of 97%, and VHH gene lengths of 396-402 bp, are consistent with the characteristics of single-domain antibodies.

[0035] 4. Phage display screening (biopanning)

[0036] Encapsulation: rUreA 1-150 Dilute to 10 μg / mL with 0.1 M NaHCO3 (pH 8.6), add 100 μL to each well of a 96-well plate, and coat overnight at 4°C;

[0037] Blocking: Discard the coating solution, add 200 μL of 3% BSA (dissolved in PBS) to each well, and block at 37°C for 2 h;

[0038] First round of screening: 100 μL of the original phage library (approximately 1 × 10⁻⁶) was collected. 11 PFU was mixed with 100 μL of 3% BSA and added to the coated wells. The mixture was incubated at 37°C for 1 h. The bacteriophages were washed 10 times with PBST and 5 times with PBS. The bound bacteriophages were eluted with 100 μL of 0.1 M Gly-HCl (pH 2.2) and neutralized with 15 μL of 1 M Tris-HCl (pH 9.1).

[0039] Amplification and enrichment: Eluted phages were used to infect logarithmic-phase TG1 cells, cultured in 2×YT-AG medium, and the phages were precipitated with PEG / NaCl and their titers were determined.

[0040] Subsequent screening: A total of 3 rounds of screening were conducted (Table 1). The concentration of the coating antigen was reduced to 5 μg / mL and 2 μg / mL in each round, and the number of PBST washes was increased to 15 and 20 times to gradually enrich specific phages.

[0041] Table 1 Results of the three rounds of biological washing

[0042]

[0043] 5. Identification and sequencing of positive clones

[0044] Monoclonal ELISA: 192 single colonies were picked from the third round of screening plates, cultured, and the phage supernatant was collected using helper phage M13K07 rescue.

[0045] Screening for positive clones: Detecting supernatant and rUreA 1-150 Binding activity, select OD 450 Clones with a cross-reactivity greater than 1.5 and no cross-reactivity with BSA (a total of 28 positive clones were obtained).

[0046] Sequencing and sequence analysis: Positive clones were sent for sequencing. After IMGT / V-QUEST analysis, the clone with the unique CDR3 region and the shortest sequence (named VHH-3A4, whose amino acid sequence is shown in SEQ ID NO:3) was selected for subsequent experiments.

[0047] 6. Prokaryotic expression and purification of VHH-3A4

[0048] Expression vector construction: The VHH-3A4 gene (as shown in SEQ ID NO:4) was inserted into the pET-22b(+) vector (containing a C-terminal 6×His tag) and transformed into BL21(DE3) pLysS;

[0049] Induction of expression: Single colonies were picked and inoculated into LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C until OD500. 600 =0.8, add IPTG (final concentration 0.2mM), induce at 25℃ for 16h;

[0050] Purification: Bacterial cells were resuspended in PBS, sonicated (200W, 5s / 8s, 40min), centrifuged at 12,000×g for 30min at 4℃, and the supernatant was collected. The supernatant was loaded onto a Ni-NTA column, and impurities were washed with PBS containing 20mM imidazole, followed by elution of the target protein with 500mM imidazole PBS. The eluent was further purified by Superdex 75 gel column chromatography, dialysis with PBS, and SDS-PAGE. Figure 1 The assay showed a single band with a molecular weight of approximately 14.5 kDa and a purity of ≥95%. BCA was quantified to 1 mg / mL and stored at -80℃ for later use. Furthermore, calculations showed that the purified expression level of this single-domain antibody could reach 1–1.5 g / L, indicating a high expression level and laying the foundation for its future applications.

[0051] 7. Test of the inhibitory activity of VHH-3A4 against Hp Urease

[0052] (1) Experimental procedure: Add 50 μL of Hp Urease solution (final concentration 0.5 U / well) to each well of a 96-well plate; add 50 μL of different concentrations of VHH-3A4 or commercial monoclonal antibody (final concentration 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 μg / mL), add PBS to the blank group, and add irrelevant IgG (5.0 μg / mL) to the negative control group; incubate at 37℃ for 30 min, add 100 μL of urea substrate, and continue incubation for 60 min; add 20 μL of phenol red indicator, incubate for 5 min, and then measure OD. 550 Each concentration was repeated 3 times; the inhibition rate was calculated as follows: Inhibition rate (%) = [1 - (experimental group OD)] / [[experimental group OD ... 550 - Blank group OD 550 ) / (Control group OD 550- Blank group OD 550 )]×100%.

[0053] (2) Cross-reactivity assay: 50 μL (0.5 U / well) of Streptococcus mutans urease extract was added to each well of a 96-well plate, followed by VHH-3A4 or commercial monoclonal antibody (final concentration 5.0 μg / mL). PBS was added to the blank control group. After incubation at 37°C for 30 min, urea substrate was added, and OD was measured according to the above steps. 550 Calculate the cross-reactivity rate: Cross-reactivity rate (%) = [(Antibody group Urease inhibition rate) / (Antibody inhibition rate against Hp Urease)] × 100%.

[0054] (3) Results of inhibitory activity assay (Table 2): VHH-3A4 showed significantly better inhibitory activity against Hp Urease than existing β-subunit antibodies. At 5.0 μg / mL, VHH-3A4 achieved an inhibition rate of 97.6%, while existing antibodies only achieved 75.0%; VHH-3A4's IC50... 50 =0.38μg / mL, which is the concentration of existing antibodies (IC50). 50 =1 / 3 of the concentration of 1.2 μg / mL, indicating that VHH-3A4 targeting the new epitope of the α subunit can bind to the Urease active site more efficiently and block enzyme function.

[0055] (4) Specificity advantage (Table 2): The cross-reactivity rate of existing β-subunit antibodies against oral Streptococcus mutans urease is 35.6%, while the cross-reactivity rate of VHH-3A4 is only 4.2% (no significant difference from irrelevant IgG). The reason is that the urease β-subunit has species-conserved sequences (e.g., 42% homology with the β-subunit of Streptococcus mutans urease), which easily triggers cross-reactivity; while VHH-3A4 targets the α-subunit positions 1-150, which are Hp-specific epitopes (<15% homology with Streptococcus mutans), which can accurately identify Hp, avoid interfering with beneficial oral bacteria, and solve the defect of existing antibodies that disrupt the balance of the bacterial community.

[0056] Table 2 Results of inhibition rate and cross-reactivity rate detection

[0057]

[0058] Example 2: Preparation of 3,4,5-tri-O-methylgallic acid (TMGA)

[0059] 1. Design Concept

[0060] Gallic acid (GA) is a natural polyphenol compound that has been reported to have some inhibitory effect on Helicobacter pylori (Hp), but its efficacy is relatively weak. Due to the presence of three phenolic hydroxyl groups (-OH), GA exhibits strong hydrophilicity (calculated logP value of approximately 0.7-1.2), making it difficult to effectively penetrate Hp biomembranes. Hp biomembranes are mainly composed of polysaccharides, proteins, lipids, and extracellular DNA, among which hydrophobic components limit the permeation of hydrophilic small molecules. Furthermore, GA is easily degraded by esterases in the oral and gastrointestinal environments, resulting in insufficient stability and bioavailability, thus limiting its clinical application. To address these issues, this study designed a method to modify GA by methylation, converting the phenolic hydroxyl groups (-OH) to methoxy groups (–OCH3), thereby reducing molecular polarity, increasing the predicted logP value to approximately 2.8, enhancing its transmembrane permeability, and to some extent preventing the rapid degradation of the phenolic hydroxyl groups, thus prolonging its in vivo half-life. Theoretically, methylation modification can still retain the perturbation effect of polyphenolic compounds on bacterial membranes, but further verification is needed. In in vitro experiments (Example 3), the combined use of the methylated derivative TMGA and the anti-Hp urease single-domain antibody VHH-3A4 significantly enhanced the antibacterial effect, with the Hp inhibition rate increasing by approximately 52% compared to the single component, suggesting that this modification strategy is reasonable and has application potential.

[0061] 2. Experimental Procedure

[0062] 2.1 Step 1: Extraction and purification of gallic acid (GA)

[0063] Pretreatment: Take 100g of gallnut powder, add 70% ethanol (solid-liquid ratio 1:10, i.e. 1000mL), stir well and soak for 2 hours;

[0064] Reflux extraction: Place in a round-bottom flask and reflux in an 80°C water bath for 2 hours (magnetic stirring, 300 rpm). Filter while hot (using qualitative filter paper, pore size 1-2 μm) and collect the filtrate. Repeat the extraction of the residue twice under the same conditions (3 times in total). Combine the 3 filtrates (total volume approximately 2800 mL).

[0065] Concentration under reduced pressure: Transfer the combined filtrate to a rotary evaporator and concentrate at 50°C and -0.08 MPa until no alcohol odor remains (approximately 100 mL, concentration rate 5 mL / min, to avoid GA oxidation caused by high temperature).

[0066] Extraction and impurity removal: After cooling the concentrate to room temperature, add an equal volume of ethyl acetate (100 mL), shake and extract (200 rpm, 15 min), allow to stand for separation (about 30 min), and collect the upper organic phase (containing GA); repeat the extraction twice under the same conditions for the lower aqueous phase, and combine the three organic phases (about 290 mL).

[0067] Recrystallization purification: The organic phase was concentrated to 10 mL, and 10 times the volume of distilled water (100 mL) was slowly added. The mixture was refrigerated at 4 °C and allowed to stand for 12 h (to induce crystallization). The mixture was then filtered (using a Buchner funnel with 0.45 μm filter paper) to collect the white crystals. The crystals were washed three times with a small amount of ice water (to remove residual impurities) and dried under vacuum at 60 °C for 4 h (vacuum degree -0.09 MPa to avoid high-temperature decomposition of GA) to obtain crude GA.

[0068] Purity test: Take 10 mg of crude GA, dissolve it in methanol and dilute to 10 mL, and perform HPLC analysis (under the same conditions as TMGA analysis below). The purity should be ≥98% (if it does not meet the standard, repeat the recrystallization step). Finally, 8.2 g of pure GA was obtained, with an extraction rate of 8.2% (extraction rate = mass of pure GA / mass of gallnut powder × 100%).

[0069] 2.2 Step 2: Methylation and purification of TMGA

[0070] Preparation of the reaction system: In a 500 mL three-necked flask, add 1.7 g (10 mmol) of pure GA, 2.76 g (20 mmol, 1:2 mmol ratio) of anhydrous potassium carbonate, and 100 mL of anhydrous DMF (10 mL / mmol GA). Stir magnetically (300 rpm) until the solid is completely dissolved (about 30 min, nitrogen protection: to prevent DMF from absorbing water and affecting the reaction).

[0071] Methylation reaction: At room temperature (25℃), 2.13 mL of iodomethane (30 mmol, 1:3 mmol ratio, dropping rate 1 drop / second to avoid excessive local concentration leading to side reactions) was slowly added dropwise. After the addition was complete, stirring was continued, and the reaction progress was monitored in real time by TLC (developing solvent: petroleum ether-ethyl acetate = 3:1, color reagent: 5% FeCl3 ethanol solution; GA showed blue-black spots, Rf = 0.3; TMGA showed pale yellow spots, Rf = 0.7).

[0072] Reaction termination: After stirring for 24 hours, TLC detection showed that the GA spots had completely disappeared (only TMGA spots remained), so the reaction was stopped; 50 mL of distilled water was slowly added to the reaction solution (to quench excess iodomethane and prevent iodomethane from evaporating during subsequent extraction), and the mixture was stirred for 10 min;

[0073] Extraction and separation: Transfer the reaction solution to a separatory funnel, add 100 mL of ethyl acetate, and extract by shaking (200 rpm, 15 min). After standing and separating the layers, collect the upper organic phase; the lower aqueous phase is extracted twice with ethyl acetate (100 mL each time), and the three organic phases are combined (approximately 290 mL).

[0074] Washing and desalting: The organic phase was washed sequentially with 5% Na2S2O3 solution (100 mL, to remove residual iodine until the solution was colorless), saturated NaCl solution (100 mL, to remove water-soluble impurities), and finally washed with distilled water until neutral (pH=7, tested with pH paper).

[0075] Drying and concentration: Add anhydrous Na2SO4 (about 20g) to the organic phase and dry for 2 hours until the solution is clear. Filter to remove the desiccant, and concentrate the filtrate under reduced pressure (40℃, -0.08MPa) to 5mL (to avoid high-temperature decomposition of TMGA).

[0076] Silica gel column purification: Pack the column with 50g of 200-300 mesh silica gel using the wet packing method with petroleum ether (column diameter 2.5cm, column height 30cm); Load the sample, dilute the concentrate with 5mL of petroleum ether, and load the sample slowly; perform gradient elution, with the initial eluent being petroleum ether-ethyl acetate = 5:1 (100mL), to elute impurities; then adjust the gradient to 3:1 (200mL), and collect the TMGA elution peak (TLC monitoring, collect the eluent with Rf = 0.7);

[0077] Preparation of finished product: Combine the TMGA eluents and concentrate under reduced pressure to dryness (40℃, -0.08MPa) to obtain about 1.92g of pure TMGA powder with a modification rate of 90.5% (modification rate = actual yield of TMGA / theoretical yield × 100%, theoretical yield = 10mmol × 212.2g / mol = 2.122g).

[0078] 2.3 Step 3: Structural Validation and Performance Testing of TMGA

[0079] ¹H-NMR structure confirmation: 10 mg of pure TMGA was dissolved in CDCl3 (deuterated chloroform), and ¹H-NMR was measured (400 MHz). Chemical shift (δ): 3.85 (s, 9H, 3×OCH3, methoxyhydrogen), 7.12 (s, 2H, Ar-H, benzene ring hydrogen). This structure was consistent with the standard structure of TMGA (molecular formula C). 10 H 12 O5) A perfect match proves the structure is correct. For example... Figure 3 As shown, δ=3.85 (s, 9H): the hydrogen atom signal of 3 methoxy (-OCH3) atoms, proving that the methylation modification was successful; δ=7.12 (s, 2H): the hydrogen atom signal of 2 hydrogen atoms on the benzene ring, indicating that the benzene ring structure was not destroyed; no other impurity peaks: proving that TMGA has no residual impurities (such as iodomethane, GA).

[0080] HPLC purity determination: Chromatographic conditions: Inertsil ODS-3 column (4.6×250mm, 5μm), mobile phase methanol-0.1% phosphoric acid water = 60:40, flow rate 1.0mL / min, column temperature 30℃, detection wavelength 270nm, injection volume 10μL; Results: TMGA peak retention time was 8.5min, peak area purity ≥98.9% (no impurity peak interference). Figure 4 As shown, the retention time of GA was 6.2 min, and the peak area purity was 98.5%; the retention time of TMGA was 8.5 min, and the peak area purity was 98.9%. The difference in retention time between the two is due to the difference in logP (TMGA is more hydrophobic and has a longer retention time), which proves that the molecular polarity is reduced after modification.

[0081] logP determination: The shake flask method was used. 10 mg of pure TMGA was added to an octanol-water system (1:1, v / v, 10 mL each), and the mixture was shaken to equilibrate for 24 h (25℃, 200 rpm). The concentrations of the two phases were determined by HPLC. The logP was calculated as logP = lg(octanol phase concentration / aqueous phase concentration) = 2.8 (n=3, SD=0.12), which met the design expectations.

[0082] Biofilm permeability verification: Hp biofilm (ATCC 43504 strain) was constructed, and GA and TMGA (final concentration 1 μg / mL) were added respectively. After culturing for 2 h, the drug concentration in the biofilm was detected (HPLC-MS). The results showed that the concentration of TMGA in the biofilm (1.2 μg / g) was 4 times that of GA (0.3 μg / g), proving that methylation modification significantly improves the biofilm permeability.

[0083] 3. Determination of MIC / MBC of TMGA against Hp (broth microdilution method)

[0084] 3.1 Experimental Materials

[0085] Hp ATCC 43504 (standard strain), Hp-R (clinically resistant strain, PCR verified to contain the amoxicillin resistance gene blaOXA-181); TMGA (self-made), amoxicillin, and Bruch's broth (containing 7% fetal bovine serum and 0.2% β-cyclodextrin).

[0086] 3.2 Experimental Procedure

[0087] Bacterial suspension preparation and concentration calibration: Take *H. pylori* strains from -80℃ cryopreservation tubes, streak them onto Columbia blood agar plates, and incubate at 37℃ with microaergy for 48 hours (activation of the first generation); pick a single smooth, transparent typical colony (1-2 mm in diameter), transfer it to a fresh blood agar plate, and incubate for 24 hours (activation of the second generation, ensuring viability); scrape off second-generation colonies with a sterile cotton swab, suspend them in Brucella broth, and vortex for 1 minute to prepare a bacterial suspension; measure OD using a microplate reader. 600 Adjust the concentration of the broth to 0.5 McFarland turbidity (corresponding to 1×10⁻⁶). 8 CFU / mL, calibrated by plate count method: 0.5 McFarland turbidity = 9.8 × 10⁻⁶ 7 ±0.5×10 7 (CFU / mL); Take 100 μL of the above bacterial suspension and add 9.9 mL of Brunner's broth to prepare 1×10 6 CFU / mL working bacterial solution (prepare and use immediately).

[0088] Drug dilution gradient setup: 96-well plates (sterile, round bottom) were vertically labeled as standard strain group, drug-resistant strain group, positive control group (amoxicillin), negative control group (broth only), and solvent control group (broth containing 1% DMSO); 12 concentration gradients were set horizontally: TMGA from 0.03125 μg / mL to 16 μg / mL (serial dilution: 200 μL of 16 μg / mL TMGA in well 1, followed by 100 μL of broth in each subsequent well, diluting from well 1 to well 2, and so on up to well 11, with well 12 being the drug-free control); amoxicillin was diluted in the same way, with a concentration range of 0.015625 μg / mL to 16 μg / mL.

[0089] Inoculation and culture: Add 1×10 to each well 6 10 μL of CFU / mL working bacterial culture was added to make a final volume of 200 μL, resulting in a final bacterial concentration of 9.8 × 10⁻⁶. 4 ±0.5×10 4 CFU / mL; seal the plate and place it in a micro-aerobic incubator (5% O2, 10% CO2, 85% N2) at 37°C for 72 hours (observe every 24 hours to avoid over-culturing and bacterial autolysis).

[0090] MIC determination: After 72 hours of culture, first observe the growth with the naked eye (Hp growth will turn the clear broth cloudy); then measure the OD using a microplate reader. 600 The judgment criteria were set as follows: OD value ≤ 10% of the OD value of the negative control group was considered as "no growth (-)" and MIC (minimum drug concentration for no growth) was recorded; 3 replicates were set for each concentration.

[0091] MBC determination: Take 10 μL of culture from each well with a concentration of MIC or higher, and spread it onto Columbia blood agar plates (each well corresponds to one plate area, labeled with concentration); after incubation at 37°C under microaerobic conditions for 48 h, count the number of colonies. MBC is defined as the lowest drug concentration with a colony count of <5 CFU; if the colony count in the MIC well is >5 CFU, a higher concentration (such as MIC×2, MIC×4) needs to be tested until the standard is met.

[0092] 3.3 The experimental results are shown in Tables 3 to 5.

[0093] The antibacterial / bactericidal activity of TMGA: Against the standard strain ATCC 43504: MIC = 0.5 μg / mL (minimum concentration to inhibit 90% growth), MBC = 1.0 μg / mL (minimum concentration to kill 99.9% of bacteria), MBC / MIC = 2.0, which meets the characteristics of a "bactericide" (ratio ≤ 4); Against the resistant strain Hp-R: MIC = 1.0 μg / mL, MBC = 2.0 μg / mL, only twice that of the standard strain, indicating that the resistant strain's sensitivity to TMGA is only slightly reduced; Comparatively, amoxicillin was completely ineffective against Hp-R (MIC > 16 μg / mL), while TMGA remained highly effective, proving that its antibacterial mechanism is different from that of amoxicillin and there is no risk of cross-resistance.

[0094] Activity comparison with GA: Simultaneous determination of the MIC of gallic acid (GA): 5.0 μg / mL for ATCC 43504 and 8.0 μg / mL for Hp-R, indicating that methylation modification increased the antibacterial activity of TMGA by 10 times (standard strain) and 8 times (resistant strain), confirming the effectiveness of the modification strategy.

[0095] Activity in biofilm environment: In broth containing 50% Hp biofilm extract, the MIC of TMGA against Hp-R was 2.0 μg / mL (only twice that of planktonic bacteria), while the MIC of amoxicillin was still >16 μg / mL, proving that TMGA can still maintain its activity in biofilm environment, which is closely related to its high logP value (2.8) which enhances biofilm permeability.

[0096] Table 3. MIC / MBC results of TMGA against Hp standard strain ATCC 43504 (n=3, ±SD)

[0097]

[0098] Table 4. MIC / MBC results of TMGA against Hp-R, a Hp-resistant strain (n=3, ±SD)

[0099]

[0100] Table 5 Summary of MIC / MBC (mean of three independent experiments)

[0101]

[0102] Example 3: Synergistic inhibition of Hp-adhesive GES-1 cells by VHH-3A4 + TMGA

[0103] 1. Experimental Materials

[0104] Human gastric epithelial cells GES-1, cultured on RPMI-1640 medium (containing 10% fetal bovine serum); Hp standard strain ATCC 43504, cultured on Columbia blood agar (containing 5% defibrinated sheep blood, 37℃ microaerobic environment: 5% O2, 10% CO2, 85% N2); VHH-3A4 (self-made), TMGA (self-made), trypsin, etc.

[0105] 2. Experimental Procedure

[0106] 2.1 GES-1 cell pretreatment and seeding

[0107] Resuscitating GES-1 cells: Remove the cell cryopreservation tubes from the liquid nitrogen tank, thaw them rapidly in a 37°C water bath (within 1 min), add 9 mL of RPMI-1640 medium, centrifuge at 1000×g for 5 min, discard the supernatant, resuspend the cells, and seed them into culture flasks. Incubate at 37°C and 5% CO2 for 24 h.

[0108] Passaging and status confirmation: When the cell confluence reaches 80%-90%, passage, discard the culture medium, wash twice with PBS, add 0.25% trypsin-EDTA and digest for 1-2 min (observe the cells become rounded under an inverted microscope), add culture medium to stop digestion, centrifuge at 1000×g for 5 min, resuspend and then stain with trypan blue to detect viability (≥95%).

[0109] 24-well plate seeding: Adjust cell concentration to 1×10⁻⁶ 5 Add 1 mL per well (i.e., 1 × 10⁻⁶ cells / mL) 5 (cells / well), cultured at 37℃ and 5% CO2 for 24h to ensure adhesion rate ≥90% (observed under an inverted microscope: cells are spindle-shaped and evenly distributed without gaps).

[0110] 2.2 Preparation and Concentration Calibration of Hp Bacterial Culture

[0111] Strain activation: Take Hp from a -80℃ cryopreservation tube, streak it onto Columbia blood agar plates, and incubate at 37℃ with microaergy for 48 hours (first generation activation); pick a single typical colony and transfer it to a fresh plate, and incubate for 24 hours (second generation activation).

[0112] Preparation of bacterial suspension: Second-generation colonies were scraped with a sterile cotton swab and suspended in RPMI-1640 medium containing 10% fetal bovine serum. The suspension was vortexed for 1 min, and the OD was measured using a microplate reader. 600 Adjust the concentration to 0.5 McFarland turbidity (corresponding to 1×10⁻⁶). 8 CFU / mL, plate count calibration: 0.5 McFarland turbidity = 9.8 × 10⁻⁶ 7 ±0.5×10 7 (CFU / mL), thus obtaining 1×10 8 CFU / mL working bacterial solution (prepare fresh and inoculate within 30 minutes).

[0113] 2.3 Group treatment and co-culture: A total of 4 groups were set up, with 3 replicates per group and 3 independent experiments:

[0114] ① Blank group: Add 100μL PBS;

[0115] ② VHH-3A4 group: Add 100μL of 0.5μg / mL VHH-3A4 (final concentration 0.05μg / mL);

[0116] ③TMGA group: Add 100μL of 0.5μg / mL TMGA (final concentration 0.05μg / mL, same as above);

[0117] ④ Synergistic group: Add 50 μL of 0.5 μg / mL VHH-3A4 + 50 μL of 0.5 μg / mL TMGA (final concentration of both is 0.025 μg / mL);

[0118] Pretreatment: Discard the culture medium in the 24-well plate, wash twice with PBS, add 100 μL of the corresponding reagent to each well, and incubate at 37°C and 5% CO2 for 1 h;

[0119] Hp inoculation: Add 1×10 to each well 8 100 μL of working bacterial culture (CFU / mL) was prepared to make a final volume of 200 μL, resulting in a final bacterial concentration of 4.9 × 10⁻⁶. 7 ±0.2×10 7 CFU / mL, co-cultured at 37℃ and 5% CO2 for 2 hours.

[0120] 2.4 Washing of unadhered *Helicobacter pylori* and cell digestion

[0121] Washing: After co-culture, discard the supernatant and gently wash three times with PBS to remove unattached free Hp;

[0122] Digestion: Add 200 μL of 0.25% trypsin-EDTA to each well, incubate at 37°C for 2 min, observe under a microscope that the cells have completely detached, add 800 μL of RPMI-1640 medium to stop the digestion, and pipette to prepare a cell-Hp mixed suspension.

[0123] 2.5 CFU Count and Inhibition Rate Calculation

[0124] Dilution and Spreading: Take 100 μL of the mixed suspension and dilute it serially with PBS to 10⁻⁶. -2 (To avoid overly dense colonies), spread 100 μL of the diluted solution onto Columbia blood agar plates, 3 plates per concentration;

[0125] Culture and Counting: Incubate at 37℃ with micro-aerobic conditions for 48 hours, count the number of Hp colonies on the plate (typical colonies are translucent, round, and 1-2 mm in diameter), and calculate the amount of adhesion per well (CFU / well) = number of colonies × dilution factor × 10 (since the total suspension volume is 1 mL, take 100 μL for spreading).

[0126] Inhibition rate calculation: Inhibition rate (%) = [1 - (adhesion amount of experimental group / adhesion amount of blank group)] × 100%.

[0127] 3. Experimental Results

[0128] 3.1 Cell and bacterial culture quality verification: All GES-1 cells used in the experiment were in the logarithmic growth phase, with a viability ≥96% and an adhesion rate ≥90%, ensuring consistent cell condition; the Hp working bacterial culture was calibrated by plate counting, with a concentration of 9.8 × 10⁻⁶. 7 ±0.5×10 7 CFU / mL, to avoid result deviations due to fluctuations in bacterial concentration.

[0129] 3.2 The inhibitory effects of single components are shown in Table 6.

[0130] VHH-3A4 group (0.05 μg / mL): Hp adhesion amount was 0.32 ± 0.02 × 10⁻⁶. 6 CFU / well, inhibition rate 73.3±1.2%, which was significantly different from the blank group (P<0.001), indicating that VHH-3A4 can significantly reduce the binding ability of Hp to GES-1 cells by specifically binding to the Hp Urease α subunit (blocking Urease-mediated adhesion sites);

[0131] TMGA group (0.05 μg / mL): Adhesion amount was 0.45 ± 0.02 × 10⁻⁶. 6 CFU / well, inhibition rate 62.5±1.5%, which was also significantly different from the blank group (P<0.001), confirming that TMGA can reduce the adhesion activity of Hp by penetrating the cell membrane and destroying its structural integrity (non-specific inhibition, but the effect is weaker than VHH-3A4).

[0132] 3.3 Significance of synergistic effect (Table 6): The Hp adhesion amount of the synergistic group (VHH-3A4 0.025 μg / mL + TMGA 0.025 μg / mL) was only 0.11 ± 0.01 × 10⁻⁶. 6 CFU / well, with an inhibition rate of 90.8±0.8%, exhibiting the following advantages:

[0133] Concentration advantage: The concentration of a single component was halved (from 0.05 μg / mL to 0.025 μg / mL), but the inhibition rate was increased (73.3% in the VHH-3A4 group → 90.8% in the synergistic group), proving that there is a synergistic effect of "1+1>2" between the two.

[0134] Statistical differences: The adhesion amount in the synergistic group was significantly lower than that in the VHH-3A4 group (P<0.01) and the TMGA group (P<0.01), ruling out the possibility of "simple superposition" and confirming the establishment of the synergistic mechanism;

[0135] Complementary Mechanisms: VHH-3A4’s “targeted blocking of adhesion sites” complements TMGA’s “disruption of bacterial structure”. The former reduces Hp adhesion ability, while the latter weakens Hp activity, together achieving efficient inhibition.

[0136] 3.4 Repeatability and Reliability:

[0137] The standard deviation of adhesion in the three independent experiments was ≤0.02×10⁻⁶. 6 CFU / well, with a standard deviation of inhibition rate ≤1.5%, indicates good experimental repeatability; there was no significant difference in adhesion between the blank group and the solvent control group (containing 1% DMSO) (P>0.05), excluding the interference of solvent on Hp adhesion.

[0138] Table 6. Inhibitory effect of VHH-3A4 + TMGA on Hp adhesion to GES-1 cells

[0139]

[0140] Note: Statistical analysis was performed using one-way ANOVA with Tukey multiple comparisons. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0141] Example 4: Mouthwash Preparation

[0142] 1. The mouthwash formula is shown in Table 7.

[0143] Table 7 Mouthwash Formula Table

[0144]

[0145] 2. Preparation steps

[0146] Excipient dissolution: Heat 500 mL of purified water to 50 °C, add HP-β-CD (30 g) and stir until completely dissolved (stirring speed 300 rpm, time 15 min), then cool to room temperature;

[0147] pH adjustment: Add glycerol (20g) and phenoxyethanol (4g), then add 0.1M citrate-sodium citrate buffer dropwise to adjust the pH to 6.8;

[0148] Mixing of active ingredients: Add VHH-3A4 (0.4g, dissolved in 10mL PBS) and TMGA (2.0g, dissolved in 5mL ethanol) in sequence, and stir for 30min (200rpm) until completely dissolved;

[0149] Volumetric sterilization: Add purified water to 1000mL, filter through a 0.22μm polyethersulfone (Millipore) membrane for sterilization, and aseptically fill into brown light-proof bottles (100mL / bottle).

[0150] 3. Mouthwash stability test (stored at 4℃ / 25℃ / 40℃)

[0151] 3.1 Experimental materials: Mouthwash of the present invention (experimental group), mouthwash without HP-β-CD (control group);

[0152] 3.2 Experimental Procedure

[0153] VHH-3A4 assay: Zorbax 300SB-C18 column (4.6×250mm, 5μm), mobile phase A: 0.1% TFA water, B: 0.1% TFA acetonitrile, gradient elution (0-30min, B 5%-30%), flow rate 1.0mL / min, detection wavelength 280nm;

[0154] TMGA detection: Inertsil ODS-3 column (4.6×250mm, 5μm), mobile phase: methanol-0.1% phosphoric acid water = 60:40, flow rate 1.0mL / min, detection wavelength 270nm;

[0155] Samples were stored at 4℃, 25℃ (RH75%), and 40℃ (RH75%), respectively, and samples were taken at 0, 1, 3, and 6 months. The content was determined by HPLC, and the retention rate was calculated as (measured content / initial content × 100%).

[0156] 3.3 The experimental results are shown in Table 8.

[0157] In the experimental group, after 6 months of storage at 25℃, the retention rates of VHH-3A4 and TMGA were 91.7% and 94.2%, respectively, and remained above 85% after 3 months of storage at 40℃. In the control group, after 6 months of storage at 25℃, the retention rate of VHH-3A4 was only 68.5%, and TMGA was 72.3%. After 3 months of storage at 40℃, the retention rate of active ingredients in the control group decreased to 42.1%-45.8%, significantly lower than the 85.3%-88.7% in the experimental group. This indicates that HP-β-CD reduces VHH-3A4 aggregation and TMGA oxidative degradation through inclusion, thereby improving the stability of the mouthwash and meeting shelf-life requirements.

[0158] Table 8 Results of Stability Tracking Experiment

[0159]

[0160] Example 5: In vivo antibacterial and safety experiments of H. pylori-inhibiting mouthwash in mice

[0161] 1. In vivo antibacterial experiment

[0162] 1.1 Experimental materials: SPF-grade Balb / c mice (6-8 weeks old); female New Zealand white rabbits (2.0-2.5 kg); SD rats (8-10 weeks old, weighing 250-300 g); Hp ATCC 43504, ELISA kits (IL-8 and TNF-α), etc.

[0163] 1.2 Experimental Procedure

[0164] (1) Preparation and modeling of Hp bacterial culture:

[0165] Bacterial activation: Hp ATCC 43504 was revived from -80℃, streaked onto Columbia blood agar plates, and incubated at 37℃ under microaerogenic conditions for 48 h (5% O2, 10% CO2, 85% N2). Single colonies were picked and transferred to fresh plates, incubated for 24 h, and then a bacterial suspension was prepared and the concentration adjusted to 1×10⁻⁶. 9 CFU / mL;

[0166] Fasting before modeling: Mice were fasted for 12 hours (with free access to water) to avoid gastric contents affecting Hp colonization;

[0167] Gavage procedure: Using a 1mL sterile gavage syringe, administer 0.2mL of Hp bacterial solution (containing 1×10⁻⁶ H. pylori) to each mouse via gavage. 8 CFU), once a week for three consecutive weeks;

[0168] Modeling validation: After modeling, three mice were randomly sacrificed, and their gastric tissue was homogenized and spread on blood agar plates. If the *Helicobacter pylori* load was ≥1×10⁻⁶, the model was validated. 6 CFU / g indicates successful model establishment.

[0169] (2) Grouping and Intervention:

[0170] Normal group: No modeling was performed, and they had free access to sterile water;

[0171] Model group: Modeling successful, free access to sterile drinking water;

[0172] The invention group: The model was successfully established, and the mouthwash prepared in Example 4 was freely consumed at a 1:10 dilution;

[0173] Chlorhexidine group: Modeling successful, free access to 0.1% chlorhexidine solution (replace with the same method);

[0174] Intervention period: 4 weeks, during which mouse weight and water intake were recorded (ensuring no significant difference in average daily water intake among groups: 10-12 mL / mouse / day).

[0175] (3) Sample collection and processing:

[0176] Mice were sacrificed: After the intervention, the mice were fasted for 12 hours, euthanized by cervical dislocation, and gastric tissue was obtained aseptically (gastric contents were removed and the mice were rinsed 3 times with sterile PBS).

[0177] Hp load assay: 0.5g of gastric body tissue was added to 4.5mL of sterile PBS and homogenized using a tissue homogenizer (12000rpm, 30s) to prepare a 10⁻¹ homogenate, which was then serially diluted to 10⁻¹. -4 Take 100 μL and spread it on Columbia blood agar plates. Incubate at 37°C with microaergy for 48 h. Count the number of colonies and calculate the Hp load (CFU / g) = colony count × dilution factor × 10 (total homogenate volume 5 mL, take 0.5 g of tissue).

[0178] Detection of inflammatory factors: Take 0.1g of gastric antrum tissue, add 1mL of RIPA lysis buffer (containing protease inhibitor), homogenize on ice (5000rpm, 1min), centrifuge at 4℃ and 12000×g for 15min, take the supernatant, measure the protein concentration by BCA method, and detect the IL-8 and TNF-α content according to the ELISA kit instructions.

[0179] Pathological section preparation: 0.2cm × 0.2cm tissue samples were taken from the gastric fundus, fixed in 4% paraformaldehyde for 24h, embedded in paraffin, sectioned at 5μm, stained with hematoxylin and eosin (HE), and scored in a double-blind manner (scoring criteria: 0 points = no inflammation; 1 point = a small amount of inflammatory cell infiltration in the mucosal layer; 2 points = a moderate amount of inflammatory cell infiltration in the mucosal layer, with mild mucosal edema; 3 points = a large amount of inflammatory cell infiltration in the mucosal layer, with mucosal edema and erosion; 4 points = inflammatory cells infiltrate into the submucosa, with ulceration).

[0180] 1.3 The experimental results are shown in Tables 9 and 10.

[0181] Hp load clearance: The Hp load in the gastric mucosa of the group in this invention was only 2.3 ± 0.5 × 10⁻⁶. 3 CFU / g, compared to the model group (1500±120.5×10⁻⁶). 3 The CFU / g level decreased by 99.8% (P<0.001), and was significantly lower than that of the chlorhexidine group (87±8.2×10). 3 CFU / g, P<0.01). Mechanistically, after mouthwash enters the stomach via oral-gastric reflux, VHH-3A4 targets and binds to the Hp Urease α subunit, blocking Hp colonization; TMGA penetrates the Hp biomembrane and destroys the cell membrane. The two work synergistically to achieve efficient clearance, while chlorhexidine only works through broad-spectrum antibacterial action and has poor specificity for Hp, resulting in limited clearance effect.

[0182] Decreased inflammatory factors: The levels of IL-8 (89.3±6.5 pg / mg) and TNF-α (65.2±5.8 pg / mg) in the gastric tissue of mice in the model group were significantly increased, indicating that Hp infection triggered a strong inflammatory response. The inflammatory factors in the present invention group decreased to 25.7±2.1 pg / mg and 18.9±1.7 pg / mg, respectively, which were 71.2% and 71.0% lower than those in the model group, and lower than those in the chlorhexidine group (IL-8 52.1±4.3 pg / mg, TNF-α 39.5±3.2 pg / mg, P<0.01), proving that mouthwash can effectively reduce gastric mucosal inflammation while clearing Hp.

[0183] Pathological damage repair: The pathological score of the gastric mucosa in the model group mice was 3.8±0.3 points, showing a large number of inflammatory cells infiltrating the mucosal layer and mucosal edema; the pathological score of the present invention group decreased to 0.8±0.2 points, the gastric mucosal epithelium was intact, with only a small number of inflammatory cells infiltrating, close to the normal group (0.2±0.1 points); the pathological score of the chlorhexidine group was 2.1±0.2 points, and moderate inflammation was still present, further confirming the repair advantage of the mouthwash of the present invention on gastric mucosal damage.

[0184] General condition of mice: The weight change rate of mice in the model group (+12.9%) was significantly lower than that in the normal group (+29.2%), indicating that Hp infection affected the growth of mice; the weight change rate of the present invention group (+26.3%) was close to that of the normal group, and the chlorhexidine group (+16.1%) was still low, indicating that after the mouthwash cleared Hp, it could improve the nutritional absorption and growth status of mice, and had no obvious side effects.

[0185] Table 9 Results of in vivo antibacterial experiment in mice (n=10 / group)

[0186]

[0187] Table 10 Monitoring of mouse body weight and water consumption (mean ± SD)

[0188]

[0189] 2. Safety Experiment

[0190] 2.1 Skin irritation test (Draize scoring system)

[0191] (1) Experimental steps:

[0192] Hair removal on the back of white rabbits: Hair was shaved on both sides of the back of white rabbits using an electric hair shaver (3cm×3cm area, left side is experimental group, right side is control group). 24 hours after hair removal, it was confirmed that there was no redness or damage to the skin.

[0193] Sample application: Apply 0.5 mL of the mouthwash of this invention to the left side (evenly covering the hair removal area), and apply 0.5 mL of sterile saline to the right side. Cover with sterile gauze, fix with adhesive tape, remove residual sample after 4 hours, and wash with warm water.

[0194] Observation and scoring: Observe the skin erythema and edema at 24h, 48h and 72h after application, and score according to the Draize scoring standard (erythema: 0 = none, 1 = mild, 2 = moderate, 3 = severe; edema: 0 = none, 1 = mild, 2 = moderate, 3 = severe; total irritation index = erythema score + edema score).

[0195] (2) The experimental results are shown in Table 11: 24h, 48h and 72h after applying mouthwash to the back of the rabbits, there was no erythema or edema on the skin of both the experimental group and the control group. The total irritation index was 0, which meets the "non-irritating" standard (irritation index ≤ 0.5) in the "Cosmetic Safety Technical Specifications".

[0196] Table 11 Results of Skin Irritation Test

[0197]

[0198] 2.2 Oral Mucosal Irritation Test

[0199] (1) Experimental steps:

[0200] Rat restraint: SD rats were restrained using a rat restraint device to expose their oral cavity;

[0201] Sample application: Apply 0.2 mL of the mouthwash of this invention to the buccal mucosa and tongue of rats using a sterile cotton swab, twice a day (8 hours apart), for 14 consecutive days.

[0202] Sample collection: After the experiment, rats were euthanized by cervical dislocation, and buccal and lingual mucosa tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (HE), and observed under an optical microscope to examine the mucosal structure (whether there was epithelial damage or inflammatory cell infiltration).

[0203] (3) The experimental results are shown in Table 12: After applying mouthwash to the mouths of rats for 14 days, the buccal mucosa of all 10 rats was intact with no inflammatory cell infiltration, and the tongue mucosa was regular with normal taste bud structure. There were no signs of mucosal congestion, shedding, or edema. The results met the criteria for "non-irritation" (no damage to the mucosa and no inflammatory cell infiltration) and were consistent with the mucosal condition of the saline control group, proving that the mouthwash has no mucosal irritation with long-term oral use and its safety meets the needs of oral care.

[0204] Table 12 Results of oral mucosal stimulation test

[0205]

[0206] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against the α-subunit of Helicobacter pylori urease, characterized in that, The single-domain antibody is VHH-3A4, and its amino acid sequence is shown in SEQ ID NO:

3.

2. The single-domain antibody according to claim 1, characterized in that, The gene sequence of the single-domain antibody is shown in SEQ ID NO:

4.

3. The single-domain antibody according to claim 1, characterized in that, The immunogen used to prepare the single-domain antibody is rUreA. 1-150 Its amino acid sequence is shown in SEQ ID NO:1, and the corresponding nucleotide sequence is shown in SEQ ID NO:

2.

4. A composition for treating Helicobacter pylori, characterized in that, The composition comprises an effective amount of the anti-Helicobacter pylori urease α subunit single-domain antibody of claim 1 and an effective amount of 3,4,5-tri-O-methylgallic acid.

5. The composition according to claim 4, characterized in that, The 3,4,5-tri-O-methylgallic acid has a logP value of 2.8, a minimum inhibitory concentration (MIC) of 0.5 μg / mL against the standard strain ATCC 43504 of Helicobacter pylori, and a MIC of 1.0 μg / mL against a drug-resistant strain of Helicobacter pylori containing the blaOXA-181 resistance gene.

6. The composition according to claim 5, characterized in that, The preparation steps of the 3,4,5-tri-O-methylgallic acid include: extracting and purifying gallic acid from gallnut; methylating gallic acid with iodomethane in anhydrous N,N-dimethylformamide; and purifying the gallic acid by ethyl acetate extraction, washing with sodium thiosulfate solution, and silica gel column chromatography to obtain 3,4,5-tri-O-methylgallic acid with a purity of not less than 98.9%.

7. The composition according to claim 6, characterized in that, During the methylation reaction, the molar ratio of gallic acid to iodomethane was 1:3, the reaction temperature was room temperature, and the reaction time was 24 hours. The reaction progress was monitored by thin-layer chromatography, with petroleum ether-ethyl acetate as the developing solvent and 5% ferric chloride ethanol solution as the colorimetric reagent, until the gallic acid spots completely disappeared.

8. A mouthwash for treating Helicobacter pylori, characterized in that, The mouthwash, by mass percentage, comprises 0.04% of the anti-Helicobacter pylori urease α subunit single-domain antibody as described in claim 1, 0.2% of 3,4,5-tri-O-methylgallic acid as described in claim 5, 3% of hydroxypropyl-β-cyclodextrin, 2% of glycerol, 0.4% of phenoxyethanol, 0.1M citrate-sodium citrate buffer, and purified water; the pH of the mouthwash is adjusted to 6.8 with 0.1M citrate-sodium citrate buffer, and the remaining amount is purified water.

9. A method for preparing the anti-Helicobacter pylori mouthwash according to claim 8, characterized in that, The method includes the following steps: heated purified water to 50°C, added hydroxypropyl-β-cyclodextrin and stirred at 300 rpm for 15 minutes until dissolved, then cooled to room temperature; added glycerol and phenoxyethanol and stirred evenly, then added 0.1M citrate-sodium citrate buffer to adjust the pH to 6.8; added anti-Helicobacter pylori urease α subunit single-domain antibody and 3,4,5-tri-O-methylgallic acid and stirred at 200 rpm for 30 minutes until dissolved; added purified water to make up the volume, filtered through a 0.22 μm polyethersulfone filter membrane for sterilization, and aseptically filled into brown light-proof bottles.

10. Use of the composition of claim 4 in the preparation of an oral care product for the prevention or adjunctive treatment of Helicobacter pylori infection.

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