Oral cleaning solution having antibacterial and Anti-inflammatory effects, preparation method therefor, and use thereof

Abstract

An oral cleaning solution, comprising the following components: 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, cetylpyridinium chloride, potassium nitrate, sodium bicarbonate, and a herbal antibacterial ingredient. Use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof in preventing and / or treating oral diseases.

Description

An oral cleaning solution with antibacterial and anti-inflammatory effects, its preparation method and uses. Technical Field

[0001] This invention relates to the field of oral care, and in particular to an oral cleaning solution with antibacterial and anti-inflammatory effects, its preparation method and uses. Background Technology

[0002] With the improvement of socioeconomic level and people's quality of life, oral hygiene and oral health care have received increasing attention from the general public. In fact, oral health is closely related to overall health. When oral hygiene is poor, various oral pathogens, such as Porphyromonas gingivalis and their related pathogenic factors, enter tissues and blood, thus affecting overall health. Studies have reported that many serious chronic diseases, such as Alzheimer's disease, diabetes, and cardiovascular disease, are related to oral hygiene.

[0003] Currently, brushing is the primary method of oral hygiene in my country. However, even with correct brushing techniques and frequency, it's difficult to remove dirt and debris from the interdental spaces and pits / fissures of teeth. Furthermore, brushing presents several problems. For instance, the standard Bass brushing technique requires considerable skill from the user, making it inconvenient for the elderly, children, or bedridden patients to brush, and leaving insufficient time for some students and working professionals to brush their teeth. Due to these combined factors, a significant number of people in my country brush incorrectly, infrequently, or even not at all, making brushing alone insufficient to meet current oral hygiene needs.

[0004] In addition, smoking and chewing betel nut are common and prevalent habits. Nicotine in cigarette smoke irritates the oral mucosa, induces apoptosis of oral soft tissue cells, affects periodontal health, and increases the accumulation of oral pathogens. Chewing betel nut is directly related to the development of various oral malignant or potentially malignant lesions, such as oral submucosal fibrosis, oral leukoplakia, and oral squamous cell carcinoma, seriously endangering public health. Summary of the Invention

[0005] To better meet the needs of oral hygiene, the inventors of this application have developed a novel multifunctional oral hygiene cleaning solution, which can be used as a mouthwash or in conjunction with a water flosser or other water jet oral cleaning devices. The oral cleaning solution provided in this application has rapid antibacterial, anti-inflammatory, and tooth sensitivity-reducing effects, and can, to a certain extent, prevent tooth decay and periodontitis, and reduce the harm of smoking and betel nut chewing to oral soft tissues. The oral cleaning solution provided in this application uses a compound antibacterial formula combining cetylpyridinium chloride, sodium bicarbonate, and herbal ingredients, and does not contain alcohol or other irritating substances, thus avoiding the oral flora imbalance caused by long-term use of traditional medicated oral cleaning solutions. Therefore, this application provides the following invention:

[0006] In one aspect, this application provides an oral cleaning solution comprising the following ingredients: 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, cetylpyridinium chloride, potassium nitrate, sodium bicarbonate, and herbal antibacterial ingredients.

[0007] 3-Hydroxybutyric acid (3HB), also known as β-hydroxybutyric acid, is a very important metabolite found in animals, bacteria, and plants. In animals, 3HB is a product of normal fatty acid oxidation metabolism, and therefore can serve as an energy source in cases of glucose deficiency. Furthermore, 3HB is not only an intermediate metabolite but also an important regulatory molecule that can affect gene expression, lipid metabolism, neuronal function, and overall metabolic rate.

[0008] The antibacterial and anti-inflammatory oral cleansing solution of this invention contains 3HB, which has immunomodulatory properties and is beneficial for restoring high-level inflammation in existing inflamed tissues to normal. The 3HB in the oral cleansing solution can enhance mitochondrial function, induce excitation of oral soft tissue cells, and increase the resistance of normal tissues to infection and inflammatory diseases. In particular, 3HB can improve oral soft tissue discomfort caused by long-term smoking and betel nut chewing, and can reduce the incidence of oral mucosal diseases and oral submucosal fibrosis caused by long-term smoking and betel nut chewing.

[0009] 3-Hydroxybutyric acid can form pharmaceutically acceptable salts with suitable inorganic or organic cations, including but not limited to alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; other metal salts such as aluminum, iron, zinc, copper, nickel, and cobalt salts; and inorganic alkaline salts such as ammonium salts.

[0010] Cetylpyridinium chloride is a cationic quaternary ammonium compound that has been shown to effectively reduce dental plaque and alleviate gingivitis. Sodium bicarbonate, acting as a pH buffer, also affects the stability of the plaque biofilm. Since plaque formation depends on an overall acidic environment, sodium bicarbonate can effectively increase the oral pH, neutralize the acidic environment, and disrupt mature plaque, thus inhibiting its formation. It also has some preventative and therapeutic effects on caries and periodontal disease, and can enhance the plaque removal efficiency of various methods such as brushing, water flossing, and mouthwash.

[0011] Tooth sensitivity caused by factors such as horizontal brushing and teeth grinding at night has an extremely high incidence rate in my country. The high water flow rate and low water temperature of oral rinsing / medication devices such as water flossers easily trigger discomfort caused by tooth sensitivity. The oral cleaning solution of this invention contains potassium nitrate, which can seal dentinal tubules and alleviate tooth sensitivity symptoms.

[0012] The antibacterial and anti-inflammatory oral cleansing solution of this invention contains herbal antibacterial ingredients, such as extracts of honeysuckle, peppermint, chamomile, rosemary, purslane, tea tree oil, resveratrol, and other herbal plants with antibacterial effects. The extracts may be in the form of extracts, pastes, powders, crystals, etc. These extracts can be obtained commercially.

[0013] In some embodiments, the herbal antibacterial ingredient is honeysuckle extract, whose active ingredients are mainly chlorogenic acid, flavonoids, volatile oils and organic acids.

[0014] In some embodiments, the concentration of the 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof in the oral cleaning solution is 1 mM-100 mM, more preferably 1 mM-50 mM, and even more preferably 5 mM-20 mM.

[0015] In some embodiments, the cetylpyridinium chloride has a mass fraction of 0.5%-10% in the oral cleaning solution, more preferably 1%-8%, and even more preferably 2%-5%.

[0016] In some embodiments, the potassium nitrate in the oral cleaning solution has a mass fraction of 0.5%-10%, more preferably 0.8%-8%, and even more preferably 1%-5%.

[0017] In some embodiments, the sodium bicarbonate in the oral cleaning solution has a mass fraction of 1%-10%, more preferably 2%-8%, and even more preferably 2.5%-5%.

[0018] In some embodiments, the herbal antibacterial ingredient has a mass fraction of 5%-35% in the oral cleaning solution, more preferably 8%-30%, and even more preferably 10%-20%.

[0019] The oral cleaning solution has a mild pH to reduce irritation to the oral cavity; in some embodiments, the oral cleaning solution has a pH of 6.0-7.0.

[0020] The oral cleaning solution of the present invention can be used as an adjunct treatment for oral diseases, and can also be used for long-term daily oral care and oral cleaning, without easily causing oral flora imbalance.

[0021] In some embodiments, the oral cleaning solution is a mouthwash. As a mouthwash, the oral cleaning solution may contain excipients for use in mouthwashes, including but not limited to one or more of solvents (e.g., water), solubilizers, lubricants, humectants, flavorings, and sweeteners.

[0022] In some embodiments, the excipients include one or more of glycerin, flavoring agents, xylitol, and sodium lauryl sulfate.

[0023] The oral cleaning solution of the present invention still has a good inhibitory effect on the growth of Porphyromonas gingivalis even when diluted up to 1000 times. After dilution, it can be used as cleaning water in conjunction with oral rinsing / medication devices such as water flossers, which is beneficial to achieving better oral hygiene and disease prevention / treatment.

[0024] Therefore, in some embodiments, the oral cleaning solution of the present invention can be a regular product that can be used directly, or it can be a concentrated product that is diluted according to the recommended ratio in the product instructions before use.

[0025] In some embodiments, the oral cleaning solution is cleaning water for an oral rinsing / drug administration device, preferably concentrated cleaning water for an oral rinsing / drug administration device, which can be diluted 1-1000 times before use.

[0026] In one aspect, this application provides a method for preparing any of the above-mentioned oral cleaning solutions, the method comprising: dissolving 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, cetylpyridinium chloride, potassium nitrate, sodium bicarbonate, herbal antibacterial ingredients, and optional excipients for mouthwash, in a solvent, and adjusting the pH.

[0027] The solvent used to prepare the oral cleaning solution of the present invention is preferably water to reduce irritation to the oral cavity, and is more preferably ultrapure water.

[0028] Potassium nitrate and sodium bicarbonate used in preparing the oral cleaning solution of the present invention are preferably food-grade potassium nitrate and food-grade sodium bicarbonate.

[0029] In some implementations, the method includes the following steps:

[0030] (1) Dissolve the excipients for mouthwash in a solvent to obtain an excipient solution;

[0031] (2) Add herbal antibacterial ingredients, cetylpyridinium chloride, 3-hydroxybutyric acid or its pharmaceutically acceptable salt, potassium nitrate, and sodium bicarbonate to the excipient solution in sequence, stirring thoroughly after each addition until the raw materials are completely dissolved.

[0032] (3) Measure and adjust the pH value of the solution at room temperature (e.g., 25°C), and add solvent to the total volume of the solution to obtain the oral cleaning solution.

[0033] In some implementations, the method includes the following steps:

[0034] 1) Dissolve common mouthwash excipients in ultrapure water, which accounts for 50% of the total solution volume, to obtain an excipient solution.

[0035] 2) Add honeysuckle extract, a herbal antibacterial ingredient, to the above solution at a mass fraction of 10%-20% (e.g., 10%-15% or 15%-20%) of the total prepared solution, and stir thoroughly until the raw material is completely dissolved.

[0036] 3) Add ultrapure water to the above solution until it reaches 80%-95% of the total prepared solution volume (e.g., 80%-85%, 85%-90% or 90%-95%), stir and add cetylpyridinium chloride and stir thoroughly until the raw material is completely dissolved.

[0037] 4) Add 3HB to the above solution to make its concentration reach 5mM-20mM (e.g., 5mM-10mM, 10mM-15mM or 15mM-20mM), and stir thoroughly until completely dissolved.

[0038] 5) Add edible potassium nitrate to the above solution to achieve a mass fraction of 1%-5% (e.g., 1%-3% or 3%-5%), and stir thoroughly until completely dissolved.

[0039] 6) Add edible sodium bicarbonate to the above solution to make its mass fraction reach 2.5%-5%, and stir thoroughly until completely dissolved.

[0040] 7) Measure and adjust the pH value of the solution at 25°C, and add ultrapure water to the total volume of the solution to obtain the oral cleaning solution.

[0041] The preparation method of this invention is simple to operate, operates under mild conditions, and does not require high temperatures, which helps to protect the activity of the loaded drug. Furthermore, the oral cleaning solution of this invention can also be loaded with various water-soluble drugs other than 3HB; the specific preparation process depends on the specific drug components and the application concentration.

[0042] The oral cleaning solution of the present invention contains honeysuckle extract and sodium bicarbonate, which can inhibit the growth of pathogenic bacteria in the oral cavity and improve the efficiency of plaque removal; the bioactive ingredient 3HB can reduce the harm of smoking or chewing betel nut to oral soft tissues, reduce oral soft tissue inflammation, improve oral soft tissue cell activity, and accelerate oral wound healing; the potassium nitrate can prevent / alleviate tooth sensitivity.

[0043] Therefore, in one aspect, this application provides the use of the oral cleaning solution of the present invention, the use of which is selected from inhibiting the growth of pathogenic bacteria in the oral cavity, improving plaque removal efficiency, reducing the harm of smoking or chewing betel nut to oral soft tissues, reducing oral soft tissue inflammation, improving oral soft tissue cell activity, accelerating oral wound healing, and preventing / relieving tooth sensitivity.

[0044] In one aspect, this application provides the use of the oral cleaning solution of the present invention in the preparation of a medicament for the prevention and / or treatment of oral diseases in patients, including but not limited to mild / severe periodontitis, dental caries, oral candidiasis, and other fungal infections.

[0045] In some implementations, the patient is a patient in the wound healing period after intraoral surgery, a person who has difficulty brushing their teeth, a long-term smoker, or a person who chews betel nut for a long time.

[0046] The inventors discovered that 3-hydroxybutyric acid can downregulate RNA expression in gingival fibroblasts in periodontal environments and in the presence of arecoline or nicotine, thus providing good protection for oral soft tissue cells.

[0047] Therefore, in one aspect, this application provides the use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention and / or treatment of oral diseases, including but not limited to mild / severe periodontitis, oral submucosal fibrosis, and oral mucosal diseases. In some embodiments, the oral diseases are associated with smoking and / or chewing betel nut.

[0048] In one aspect, this application provides the use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof for improving oral soft tissue discomfort caused by smoking and / or chewing areca nut.

[0049] In one aspect, this application provides the use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof for the preparation of formulations for downregulating RNA expression in gingival fibroblasts. Attached Figure Description

[0050] Figure 1 shows the weight change curve of mice that were given oral oral cleaning solution in Example 2.

[0051] Figure 2 is a statistical graph showing the growth curve of Porphyromonas gingivalis under the action of oral cleaning solution and its diluent in Example 3.

[0052] Figure 3 shows the colony growth of *Porphyromonas gingivalis* in Example 4 after continuous action of oral cleaning solution for 30-300 seconds.

[0053] Figure 4 shows the downregulated genes in gingival fibroblasts of the in vitro simulated 3HB periodontitis treatment group compared to the simulated periodontitis group in Example 5.

[0054] Figure 5 shows the upregulated genes in gingival fibroblasts of the in vitro simulated 3HB periodontitis treatment group compared to the simulated periodontitis group in Example 5.

[0055] Figure 6 shows the downregulated genes in gingival fibroblasts of the in vitro simulated 3HB-treated smoking group compared to the simulated smoking group in Example 6.

[0056] Figure 7 shows the upregulated genes in gingival fibroblasts of the in vitro simulated 3HB-treated smoking group compared to the simulated smoking group in Example 6.

[0057] Figure 8 shows the downregulated genes in gingival fibroblasts of the in vitro simulated 3HB treatment group compared to the simulated areca nut chewing group in Example 7.

[0058] Figure 9 shows the upregulated genes in gingival fibroblasts of the in vitro simulated 3HB treatment group compared to the simulated areca nut chewing group in Example 7. Beneficial effects

[0059] The oral cleaning solution of this invention, through the compounding of 3-hydroxybutyric acid (3HB) or its salt, cetylpyridinium chloride, potassium nitrate, sodium bicarbonate, and herbal antibacterial ingredients, exhibits good cell compatibility, biocompatibility, antibacterial effect, and anti-inflammatory activity. This oral cleaning solution can be used as a mouthwash or, after dilution 1-1000 times, as cleaning water in oral rinsing / medication devices such as water flossers. This oral cleaning solution can inhibit the growth of pathogenic bacteria in the oral cavity, improve plaque removal efficiency, reduce the harm of smoking and betel nut chewing to oral soft tissues, reduce oral soft tissue inflammation, enhance oral soft tissue cell activity, accelerate oral wound healing, and prevent tooth sensitivity.

[0060] The oral cleaning solution of this invention addresses the problems of existing oral cleaning solutions, such as the tendency for long-term use to cause oral flora imbalance, lack of anti-inflammatory and immunomodulatory effects, and the tendency for water flow mechanical stimulation from oral rinsing / drug administration devices like water flossers to cause tooth sensitivity. Furthermore, for patients who smoke or chew betel nut, the oral cleaning solution of this invention provides some protection for oral soft tissues. Detailed Implementation

[0061] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0062] Unless otherwise specified, the experiments and methods described in the examples were generally performed according to conventional methods well known in the art and described in various references. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0063] Example 1: Preparation of oral cleaning solution

[0064] In this embodiment, the oral cleaning solution is prepared through the following steps:

[0065] (1) Dissolve the commonly used mouthwash excipients in ultrapure water, which accounts for 50% of the total solution volume, to obtain the excipient solution. The specific components of the excipients and their mass fraction in the total solution volume are as follows: glycerin 5%, fragrance 0.5%, xylitol 5%, sodium lauryl sulfate 1%.

[0066] (2) Add honeysuckle extract, a herbal antibacterial ingredient, at a mass fraction of 15% of the total solution volume to the above solution, and stir thoroughly until the raw material is completely dissolved. The honeysuckle extract is from Mufan Biotechnology, product number MF-00323.

[0067] (3) Add ultrapure water to the above solution until the total volume of the solution is 80%, stir and add cetylpyridinium chloride, and stir thoroughly until the raw materials are completely dissolved.

[0068] (4) Add 3HB to the above solution to make its concentration reach 10mM, and stir thoroughly until completely dissolved.

[0069] (5) Add edible potassium nitrate to the above solution to make its mass fraction reach 3%, and stir thoroughly until completely dissolved.

[0070] (6) Add edible sodium bicarbonate to the above solution to make its mass fraction reach 5%, and stir thoroughly until completely dissolved.

[0071] (7) Measure and adjust the pH value of the solution to 6 at 25℃, add ultrapure water to the total volume of the solution, and the antibacterial and anti-inflammatory oral cleaning solution is obtained.

[0072] Example 2: Safety evaluation of oral cleaning solution

[0073] In this embodiment, the oral cleaning solution prepared in Example 1 was subjected to an in vivo safety evaluation experiment involving 7 consecutive days of oral administration to C57BL / 6 mice. The experimental steps are as follows:

[0074] Seven healthy female C57BL / 6 mice (8 weeks old) were selected for in vivo experiments. All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0075] Seven mice were randomly divided into two groups: a control group of three mice and a cleaning solution group of four mice.

[0076] The control group was fed normally each day, while the oral cleaning solution prepared in Example 1 was added to the drinking water of the cleaning solution group. C57BL / 6 mice weighing approximately 20g drank 10-15mL of water daily. The oral cleaning solution was added to the mice's drinking water, with the solution comprising 0.1% of the total volume, equivalent to each mouse ingesting at least 5μL / 10g / day of cleaning solution. Based on body weight conversion, this is equivalent to a 60kg adult user accidentally ingesting 30mL of oral cleaning solution daily.

[0077] Mice were cultured for 7 consecutive days, and their weight was measured daily. The results are shown in Figure 1. There was no statistically significant difference in weight between the two groups of mice from day 1 to day 7. No abnormalities were observed in the behavior of the mice.

[0078] Example 3: Evaluation of the antibacterial effect of oral cleaning solution

[0079] In this embodiment, an antibacterial experiment was conducted on the oral cleaning solution prepared in Example 1 and its diluted solutions, targeting *Porphyromonas gingivalis* (W83). The antibacterial effect of different dilutions of the oral cleaning solution was evaluated by detecting the growth curves of *Porphyromonas gingivalis* under the action of oral cleaning solutions at different dilutions. The experimental steps are as follows:

[0080] 1) Material pretreatment: Transfer the oral cleaning solution to a sterile centrifuge tube for later use.

[0081] 2) Streak the test bacteria on a blood agar plate and incubate at 37°C overnight until visible bacterial plaques are visible.

[0082] 3) Pick a single colony, streak it on a blood agar plate, and incubate at 37°C overnight until visible plaques are visible.

[0083] 4) Select an appropriate amount of mature colonies and incubate them in liquid culture medium at 37°C until the OD600 is approximately 0.6 before use. Divide them into the following groups:

[0084] A. Blank control group: Take 200 μL of bacterial culture from each sample, centrifuge and discard the supernatant, add 200 μL of bacterial culture medium to the precipitate, resuspend and add to a 96-well plate;

[0085] B. Oral cleaning solution group: Take 200 μL of bacterial culture for each sample, centrifuge and discard the supernatant, add 200 μL of the original cleaning solution to the precipitate, resuspend and add to a 96-well plate;

[0086] C. Oral cleaning solution diluted 10 times: Take 200 μL of bacterial culture, centrifuge and discard the supernatant, add 200 μL of cleaning solution diluted 10 times to the precipitate, resuspend and add to 96-well plate;

[0087] D. Oral cleaning solution diluted 100 times: Take 200 μL of bacterial culture, centrifuge and discard the supernatant, add 200 μL of cleaning solution diluted 100 times to the precipitate, resuspend and add to 96-well plate;

[0088] E. Oral cleaning solution diluted 1000 times: Take 200 μL of bacterial culture, centrifuge and discard the supernatant. Add 200 μL of cleaning solution diluted 1000 times to the precipitate, resuspend and add to a 96-well plate.

[0089] 5) Add each bacterial culture to a 96-well plate for anaerobic culture, 200 μL per well, 10 replicates per group.

[0090] 6) Collect OD600 values ​​every 4-8 hours during the 32-68 hours of incubation in the 96-well plate, and stop shaking 3 minutes before each OD measurement.

[0091] As shown in Figure 2, both the undiluted oral cleaning solution and its diluted solution (up to 1000 times) exhibit significant antibacterial properties, preventing the growth of Porphyromonas gingivalis.

[0092] Example 4: Evaluation of the rapid bactericidal effect of oral cleaning solution

[0093] In this embodiment, an antibacterial experiment was conducted on the oral cleaning solution prepared in Example 1 using *Porphyromonas gingivalis* (W83) as the target. The rapid bactericidal effect of the oral cleaning solution was evaluated by detecting the growth of *Porphyromonas gingivalis* after short-term contact with the oral cleaning solution, thus simulating its use as a mouthwash and rinse. The experimental steps are as follows:

[0094] 1) Material pretreatment: Transfer the oral cleaning solution to a sterile centrifuge tube for later use.

[0095] 2) Streak the test bacteria on a blood agar plate and incubate at 37°C overnight until visible bacterial plaques are visible.

[0096] 3) Pick a single colony, streak it on a blood agar plate, and incubate at 37°C overnight until visible plaques are visible.

[0097] 4) Select an appropriate amount of mature colonies and incubate them in liquid culture medium at 37°C until the OD600 reaches approximately 0.6 before use. Divide them into the following groups:

[0098] A. Blank control group: Take 1 mL of bacterial culture from each sample, centrifuge and discard the supernatant, add 1 mL of bacterial culture medium to the precipitate and resuspend for 30 seconds;

[0099] B. Oral cleaning solution 30s group: Take 1mL of bacterial culture from each sample, centrifuge and discard the supernatant, add 1mL of oral cleaning solution to the precipitate and resuspend for 30 seconds;

[0100] C. Oral cleaning solution 60s group: Take 1mL of bacterial culture from each sample, centrifuge and discard the supernatant, add 1mL of oral cleaning solution to the precipitate and resuspend for 60 seconds;

[0101] D. Oral cleaning solution 120s group: Take 1mL of bacterial culture from each sample, centrifuge and discard the supernatant, add 1mL of oral cleaning solution to the precipitate and resuspend for 120 seconds;

[0102] E. Oral cleaning solution 300s group: Take 1mL of bacterial culture from each sample, centrifuge and discard the supernatant, add 1mL of oral cleaning solution to the precipitate and resuspend for 300 seconds;

[0103] 5) After the reaction time is reached, quickly add each group of mixed liquids to a sufficient amount of liquid culture medium, centrifuge quickly, and resuspend the precipitate in liquid culture medium as bacterial solution.

[0104] 6) Spread the bacterial culture of each group onto a plate and incubate in an anaerobic incubator at 37°C for 1-3 days until the bacteria in the blank control group grow well. Remove the culture plates of each group, observe and photograph them, and count the colonies.

[0105] 7) Because the bacterial concentration in the blank control group was high, in order to ensure that individual colonies were clear and not mixed in the plates, the bacterial solution was diluted 10,000 times before actual plating (other groups were not diluted).

[0106] As shown in Figure 3, the undiluted oral cleaning solution achieves 100% sterilization within 30 seconds of contact with *Porphyromonas gingivalis*, preventing its growth. This indicates that the oral cleaning solution has a rapid bactericidal effect and provides ideal short-term sterilization when used as mouthwash or as cleaning water for oral rinsing / medication devices such as water flossers.

[0107] Example 5 evaluates the protective effect of 3HB on oral soft tissue cells in a simulated periodontitis environment.

[0108] In this embodiment, 3HB, the main functional component of oral cleaning solution, was used as a variable to conduct a therapeutic intervention experiment on gingival fibroblasts stimulated by *Porphyromonas gingivalis* (W83). RNAmics analysis was used to analyze the RNA expression of gingival fibroblasts with and without 3HB treatment 24 hours after *Porphyromonas gingivalis* stimulation, thereby evaluating the protective effect of 3HB on oral soft tissue cells under simulated periodontitis conditions. The experimental steps are as follows:

[0109] 1) Culture gingival fibroblasts to a suitable density and state.

[0110] 2) Divide the gingival fibroblasts into the following groups and treat them accordingly:

[0111] A. Simulated periodontitis group: Gingival fibroblasts were stimulated by Porphyromonas gingivalis for 24 hours.

[0112] B. Simulated 3HB treatment for periodontitis group: Gingival fibroblasts were stimulated by Porphyromonas gingivalis for 24 hours, and 3HB (10mM) was added at the same time.

[0113] 3) After stimulation, Trizol was used to collect tissue RNA, which was then extracted and subjected to RNA omics analysis.

[0114] 4) After the samples pass quality inspection, the omics results are analyzed using Gene Ontology (biological process) to compare the differences between the two groups.

[0115] GO_BP (Gene Ontology Biological Processes) refers to the use of Gene Ontology's classification of biological processes to perform functional annotation and enrichment analysis on differentially expressed genes, used to describe biological processes involving multiple genes, such as cell growth, differentiation, and maintenance.

[0116] Figure 4 shows the statistical data of GO_BP downregulation in the simulated 3HB-treated periodontitis group (B) compared to the simulated periodontitis group (A), and Figure 5 shows the statistical data of GO_BP upregulation in the simulated 3HB-treated periodontitis group (B) compared to the simulated periodontitis group (A). Figures 4 and 5 illustrate that 3HB can alter the inflammatory response process of gingival fibroblasts stimulated by Porphyromonas gingivalis, the main pathogen of periodontitis, suggesting that 3HB has a potential ameliorative effect on bacterial periodontitis. Some typical biological processes related to inflammatory factors and immune cell activation are highlighted in yellow, including but not limited to: the secretion of inflammatory factors such as granulocyte colony-stimulating factor, IL-1, IL-3, IFN-α, and TNF; immune response-dependent regulation of immune tolerance; defense response against pathogens; and activation of myeloid dendritic cells.

[0117] Example 6 evaluates the protective effect of 3HB on oral soft tissue cells under simulated smoking conditions.

[0118] In this embodiment, 3HB, the main functional component of oral cleaning solution, was used as a variable to conduct a therapeutic intervention experiment on gingival fibroblasts stimulated with nicotine (1.62 μg / mL). RNAmics analysis was used to analyze the RNA expression of gingival fibroblasts treated with and without 3HB 24 hours after nicotine stimulation, thereby evaluating the protective effect of 3HB on oral soft tissue cells under simulated smoking conditions. The experimental steps are as follows:

[0119] 1) Culture gingival fibroblasts to a suitable density and state.

[0120] 2) Divide the gingival fibroblasts into the following groups and treat them accordingly:

[0121] A. Simulated smoking group: Gingival fibroblasts were stimulated with nicotine (1.62 μg / mL) for 24 hours.

[0122] B. Simulated 3HB treatment for smoking group: Gingival fibroblasts were stimulated with nicotine (1.62 μg / mL) for 24 hours, and 3HB (10 mM) was added at the same time.

[0123] 3) After stimulation, Trizol was used to collect tissue RNA, which was then extracted and subjected to RNA omics analysis.

[0124] 4) After the samples pass quality inspection, the omics results are analyzed using Gene Ontology (biological process) to compare the differences between the two groups.

[0125] Figure 6 shows the statistical data of GO_BP downregulated in the simulated 3HB treatment smoking group (B) compared to the simulated smoking group (A), and Figure 7 shows the statistical data of GO_BP upregulated in the simulated 3HB treatment smoking group (B) compared to the simulated smoking group (A). Figures 6 and 7 illustrate that 3HB can alter some cellular metabolic biological processes related to nicotine stimulation of gingival fibroblasts, suggesting that 3HB has a potential ameliorative effect on discomfort symptoms such as abnormal metabolism of oral soft tissue cells caused by smoking. Its mechanism of action is based on the following biological processes, among which downregulated processes (Figure 6) include, but are not limited to: L-serine biosynthesis, norepinephrine transporter, hydrogen sulfide biosynthesis, G1 to G0 transition involved in cell differentiation, and cysteine ​​biosynthesis; upregulated processes (Figure 7) include, but are not limited to: positive regulation of cytoplasmic calcium ion concentration involved in the phospholipase CG protein-coupled signaling pathway, negative regulation of IL-1β production, amine metabolism, negative regulation of the retinoic acid receptor signaling pathway, and positive regulation of growth hormone secretion.

[0126] Example 7 evaluates the protective effect of 3HB on oral soft tissue cells under simulated areca nut chewing conditions.

[0127] In this embodiment, 3HB, the main functional component of the oral cleaning solution, was used as a variable to conduct a therapeutic intervention experiment on gingival fibroblasts stimulated with arecoline (15 μg / mL). RNAmics analysis was used to analyze the RNA expression of gingival fibroblasts treated with and without 3HB 24 hours after arecoline stimulation, thereby evaluating the protective effect of 3HB on oral soft tissue cells under simulated arecoline chewing conditions. The experimental steps are as follows:

[0128] 1) Culture gingival fibroblasts to a suitable density and state.

[0129] 2) Divide the gingival fibroblasts into the following groups and treat them accordingly:

[0130] A. Simulated chewing of areca nut group: Gingival fibroblasts were stimulated with arecoline (15 μg / mL) for 24 hours.

[0131] B. Simulated 3HB treatment of areca nut chewing group: Gingival fibroblasts were stimulated with arecoline (15 μg / mL) for 24 hours, and 3HB (10 mM) was added at the same time.

[0132] 3) After stimulation, Trizol was used to collect tissue RNA, which was then extracted and subjected to RNA omics analysis.

[0133] 4) After the samples pass quality inspection, the omics results are analyzed using Gene Ontology (biological process) to compare the differences between the two groups.

[0134] As shown in Figures 8 and 9, the GO_BP statistics of the simulated 3HB treatment group (B) downregulating GO_BP compared to the simulated areca nut chewing group (A) are shown in Figure 8, and the GO_BP statistics of the simulated 3HB treatment group (B) upregulating GO_BP compared to the simulated areca nut chewing group (A) are shown in Figure 9. Figures 8 and 9 illustrate that 3HB can alter some cellular metabolic-related biological processes in gingival fibroblasts under arecoline stimulation, suggesting that 3HB has a potential ameliorative effect on the abnormal metabolic processes in oral soft tissue cells caused by areca nut chewing. Its mechanism of action is based on the following biological processes, among which downregulated processes (Figure 8) include, but are not limited to: negative regulation of CREB transcription factor activity, apoptosis signaling pathway in endoplasmic reticulum stress response, response to reactive oxygen species, negative regulation of interleukin-2 production, PERK-mediated unfolded protein response, positive regulation of histone acetylation, and positive regulation of interleukin-4 production; upregulated processes (Figure 9) include, but are not limited to: cellular response to hypoxia, cellular response to salt stress, negative regulation of membrane potential, cellular response to retinoic acid, inositol triphosphate biosynthesis, and necrotic cell death.

[0135] The animal experiment implementation plan involved in this embodiment has been approved by the Biomedical Ethics Committee of Peking University (ethics approval number: LA2023480).

[0136] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oral cleaning solution comprising the following ingredients: 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, cetylpyridinium chloride, potassium nitrate, sodium bicarbonate, and a herbal antibacterial ingredient.

2. The oral cleaning solution of claim 1, wherein, The herbal antibacterial ingredient is an extract of a herbal plant with antibacterial efficacy; Preferably, the herbal plant comprises one or more of honeysuckle, mint, citrus, rosemary, spiny amaranth, tea tree oil, and resveratrol; Preferably, the herbal antibacterial ingredient is an extract of honeysuckle.

3. The oral cleaning solution according to claim 1 or 2, having one or more of the following characteristics: (1) the concentration of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof is 1 mM-100 mM; (2) the mass fraction of cetylpyridinium chloride in the oral cleaning solution is 0.5%-10%; (3) the mass fraction of potassium nitrate in the oral cleaning solution is 0.5%-10%; (4) the mass fraction of sodium bicarbonate in the oral cleaning solution is 1%-10%; (5) the mass fraction of the herbal antibacterial ingredient in the oral cleaning solution is 5%-35%.

4. The oral cleaning solution according to any one of claims 1-3, having a pH value of 6.0-7.

0.

5. The oral cleaning solution of any one of claims 1-4, wherein, The pharmaceutically acceptable salt is a sodium salt or a potassium salt of 3-hydroxybutyric acid.

6. The oral cleaning solution according to any one of claims 1-5, which is a mouthwash. Preferably, the oral cleaning solution further comprises an auxiliary material for a mouthwash, including one or more of a solvent (e.g., water), a solubilizer, a lubricant, a humectant, a flavoring, and a sweetener.

7. The oral cleaning solution according to any one of claims 1-6, which is a general product or a concentrated product.

8. The oral cleaning solution according to any one of claims 1-7, which is a water for cleaning an oral irrigation / dosing device. Preferably, the oral cleaning solution is a concentrated water for cleaning an oral irrigation / dosing device.

9. A method of preparing an oral cleaning solution according to any one of claims 1 to 8, said method comprising: 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, cetyl pyridinium chloride, potassium nitrate, sodium bicarbonate, and the herbal antibacterial ingredient are dissolved using a solvent (e.g., water), and the pH is adjusted.

10. The preparation method according to claim 9, comprising the following steps: (1) dissolving the auxiliary material for a mouthwash using a solvent to obtain an auxiliary material solution; (2) sequentially adding the herbal antibacterial ingredient, cetylpyridinium chloride, 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof, potassium nitrate, and sodium bicarbonate to the auxiliary material solution, and thoroughly stirring after each addition until the raw materials are completely dissolved; (3) measuring and adjusting the pH of the solution at room temperature, and adding a solvent to the total solution volume to obtain the oral cleaning solution.

11. Use of the oral cleaning solution according to any one of claims 1-8, selected from the group consisting of inhibiting the growth of pathogenic bacteria in the oral cavity, improving plaque removal efficiency, reducing the harm of smoking or chewing betel nuts to the oral soft tissue, reducing inflammation of the oral soft tissue, increasing the activity of cells in the oral soft tissue, accelerating the healing of oral wounds, and preventing / reducing tooth sensitivity.

12. Use of the oral cavity cleaning solution according to any one of claims 1 to 8 for the manufacture of a medicament for the prevention and / or treatment of oral cavity diseases in a patient, including but not limited to mild / severe periodontitis, dental caries, fungal infectious diseases (e.g. oral candidiasis); Preferably, the patient is a patient in the healing period of a post-intraoral surgery wound, a person with difficulty in brushing teeth, a long-term smoker, a long-term areca chewer.

13. Use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and / or treatment of oral cavity diseases, including but not limited to mild / severe periodontitis, oral submucous fibrosis, oral mucosa diseases; Preferably, the oral cavity diseases are associated with smoking and / or chewing areca.

14. Use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt for the improvement of the discomfort of the oral cavity soft tissue caused by smoking and / or chewing areca.

15. Use of 3-hydroxybutyric acid or a pharmaceutically acceptable salt of thereof for the manufacture of a preparation for down-regulating the RNA expression of gingival fibroblasts.

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