A composition for synergistically inhibiting porphyromonas gingivalis and application thereof

Abstract

The present application relates to a kind of composition of synergistic inhibition porphyromonas gingivalis, belong to oral care technical field.The present application provides a kind of composition of synergistic inhibition porphyromonas gingivalis, the composition is composed of peach geniposide and hinokitiol.The present application selects peach geniposide and hinokitiol from numerous existing components, finds that the composition formed by the combination of the two has synergistic effect, can improve the bacteriostatic efficacy of the composition, especially for the bacteriostatic effect of porphyromonas gingivalis, its inhibitory effect is stronger.

Description

Technical Field

[0001] This invention relates to the field of oral care technology, and in particular to a composition and its application that synergistically inhibits Porphyromonas gingivalis. Background Technology

[0002] Porphyromonas gingivalis ( Porphyromonas gingivalis,P. gingivalis It is a Gram-negative anaerobic bacillus, belonging to the phylum Bacteroidetes ( Bacteroidetes *Porphyromonas gingivalis* is one of the main pathogens of periodontal diseases (such as gingivitis and chronic periodontitis). It directly damages periodontal tissues through multiple mechanisms. Its secreted matrix metalloproteinases (MMPs) degrade collagen fibers in the periodontal ligament, leading to gingival separation from the tooth root and the formation of progressively deepening periodontal pockets. Simultaneously, this bacterium can invade gingival epithelial cells and fibroblasts, multiplying intracellularly and evading clearance by the host's immune system. Regarding alveolar bone, *Porphyromonas gingivalis* causes progressive alveolar bone resorption by stimulating osteoclast activation and inhibiting osteoblast function. Studies have also found that this bacterium can interfere with the host's coagulation system, promoting gingival bleeding and creating favorable conditions for the colonization of other bacteria. These pathological changes ultimately lead to irreversible destruction of the tooth-supporting tissues, a major cause of tooth loss in adults.

[0003] In addition, Porphyromonas gingivalis can manipulate the host's immune response. The bacterium's LPS activates immune cells such as macrophages through Toll-like receptors, leading to the release of large amounts of pro-inflammatory factors (such as IL-1β, TNF-α, and IL-6).

[0004] Recent studies have found that the pathogenic effects of *Porphyromonas gingivalis* are not limited to the oral cavity; the bacteria may enter the bloodstream through gingival ulcers, causing lesions in distant sites. In the cardiovascular system, gingival protease secreted by *Porphyromonas gingivalis* can promote the formation of atherosclerotic plaques; in diabetic patients, periodontal infection can exacerbate insulin resistance, creating a vicious cycle; most notably, *Porphyromonas gingivalis* and its toxic products have been detected in the brain tissue of Alzheimer's disease patients, suggesting that it may cross the blood-brain barrier to promote neuroinflammation and β-amyloid deposition.

[0005] Therefore, developing a drug or composition that can effectively inhibit Porphyromonas gingivalis is of great significance for the prevention and treatment of oral and other related diseases.

[0006] Current antibacterial methods for Porphyromonas gingivalis mainly include mechanical debridement, chemical antibacterial agents, and antibiotics. However, the following key issues still exist in practical applications and urgently need to be addressed or improved through technological innovation.

[0007] Mechanical debridement is insufficient to completely remove biofilms in complex areas. While mechanical debridement (such as subgingival scaling and root planing) is a core clinical treatment for periodontitis, its effectiveness in removing biofilms from anatomically complex areas like furcation zones and cementum depressions is limited. *Porphyromonas gingivalis* can resist mechanical forces by forming biofilms and secrete gingival proteases to destroy host tissue. Furthermore, debridement of deep periodontal pockets (>5mm) is even more challenging and requires auxiliary antibacterial measures.

[0008] Chemical antibacterial agents pose risks of side effects and drug resistance. While commonly used chemical antibacterial agents in oral care products (such as chlorhexidine gluconate and cetylpyridinium chloride) offer broad-spectrum antibacterial activity, they have significant drawbacks: ① Disruption of the oral microecology: Long-term use of chlorhexidine can lead to oral flora imbalance, causing Candida infections or tooth discoloration; ② Mucosal irritation: High concentrations of chemical antibacterial agents may damage the oral mucosa, reducing patient compliance; ③ Development of drug resistance: Resistant strains of Porphyromonas gingivalis to chlorhexidine gluconate have been reported, exhibiting enhanced resistance through upregulation of efflux pumps or biofilm matrix.

[0009] Antibiotics (such as metronidazole and tetracycline) are the main drugs for treating Porphyromonas gingivalis infections, but they have the following problems: ① Widespread drug resistance: The resistance rate of Porphyromonas gingivalis to metronidazole has increased from <5% in 2000 to 15%-20% currently. There are also reports of Porphyromonas gingivalis developing resistance to tetracyclines, β-lactams, macrolides (such as clindamycin), and other antibiotics; ② Short-term effectiveness and easy relapse after discontinuation: Antibiotics can only inhibit planktonic bacteria and have poor permeability to biofilms, making relapse easy after discontinuation; ③ Systemic side effects: Long-term use may lead to gastrointestinal reactions or hepatotoxicity, limiting their long-term use.

[0010] Although natural ingredients have shown some potential in inhibiting Porphyromonas gingivalis, the following problems still exist: ① The antibacterial effect of a single ingredient is limited, and high concentrations may lead to cytotoxicity or unpleasant taste; ② The synergistic effect has not been fully explored: the synergistic antibacterial effect between different natural ingredients has not been fully studied, and there is still much room for optimization of the combined formulation.

[0011] Therefore, there is an urgent need to develop a composition formulated from natural active ingredients that can synergistically enhance the antibacterial effect against Porphyromonas gingivalis while reducing the required concentration. This composition can be applied to oral care products such as mouthwash, toothpaste, and oral sprays, providing a safe and effective new option for the prevention and treatment of periodontal diseases. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition and its application for synergistic inhibition of Porphyromonas gingivalis.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a composition for synergistically inhibiting Porphyromonas gingivalis, the composition comprising tau tannin and cypressin.

[0014] Totarol is a natural active ingredient derived from Podocarpus macrophyllus. It is renowned for its excellent antibacterial, antioxidant, and preservative properties. In skincare, totarol effectively inhibits Propionibacterium acnes and Staphylococcus aureus, which cause acne, by disrupting their cell membranes and causing their death, thus reducing acne. Simultaneously, it possesses strong antioxidant properties, effectively inhibiting lipid peroxidation and delaying skin aging. Furthermore, totarol has a strong bactericidal effect against Gram-positive bacteria and can be used as a natural preservative.

[0015] Hinokitiol (also known as juniper alcohol) is a natural monoterpene compound derived from the juniper plant. Its chemical structure is a unique seven-membered ring phenol-ketone, classifying it as an environmentally friendly bioactive ingredient. Its most prominent characteristic is its high safety. As a plant-derived natural molecule, it exhibits excellent biocompatibility, is gentle and non-irritating to skin and mucous membranes, and is unlikely to induce drug resistance with long-term use. It is widely used in the daily chemical, pharmaceutical, and food industries. In terms of applications, hinokitiol possesses anti-dandruff and anti-itch properties, regulates scalp microecology, and controls sebum secretion, commonly used in hair care products. Furthermore, it can also be used as a food preservative, fruit and vegetable preservative, and wood antifungal agent, demonstrating its safety advantages across multiple applications.

[0016] This invention screened tau phenol and cypress phenol from a large number of existing components and found that the combination of the two has a synergistic effect, which can improve the antibacterial efficacy of the composition, especially against Porphyromonas gingivalis, where the inhibitory effect is stronger.

[0017] In a preferred embodiment of the first aspect, the mass ratio of taurine to cylindricaol is (1-2):(1-10). Exemplarily, the mass ratio of taurine to cylindricaol can be any value or any range of values ​​from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 2:9, or 2:10.

[0018] In a preferred embodiment of the first aspect, the mass ratio of taurine to cypressin is 1:(2-10).

[0019] In a preferred embodiment of the first aspect, the mass ratio of tau phenol to cypressin is 1:10.

[0020] This invention investigated the ratio of tau phenol to cypressin in the composition using the Chou-Talalay model.CI If the value is less than 1, it indicates that there is a synergistic effect between the two. CI A lower value indicates a better synergistic effect; CI =1 indicates that there is an additive effect; CI A value greater than 1 indicates the presence of an antagonistic effect.

[0021] This invention prepares taurine and pine phenol into an antibacterial solution of a certain mass concentration. It was found that when the mass ratio of taurine to pine phenol is in the range of (1-2):(1-10) (for example, the total mass concentration of taurine and pine phenol in the antibacterial solution is 10 μg / mL, and the mass concentrations of taurine and pine phenol are 0.09 μg / mL and 0.91 μg / mL, respectively, and their mass ratio is 1:10), CI A value <1 indicates a synergistic effect between the two within this range. When the mass ratio of tau phenol to cypressin is in the range of 1:(1-10), CI A value in the range of 0.239 to 0.670 indicates that this ratio range can enhance the synergistic effect of the two. When the mass ratio of taurine to cypressin is 1:10, CI A value of 0.239 indicates that the synergistic effect between the two is strongest at this mass ratio.

[0022] In a second aspect, the present invention provides the use of the composition described in the first aspect in the preparation of oral care products.

[0023] Porphyromonas gingivalis causes a series of oral lesions through its unique pathogenic mechanism. The composition of the present invention can effectively inhibit Porphyromonas gingivalis. Therefore, using the composition of the present invention in oral care products can effectively prevent and improve oral lesions caused by Porphyromonas gingivalis.

[0024] As a preferred embodiment of the second aspect, the composition for inhibiting Porphyromonas gingivalis is present in the oral care product at a content of 0.03-0.1% by mass.

[0025] As a preferred embodiment of the second aspect, the product includes toothpaste and mouthwash.

[0026] Thirdly, the present invention provides the use of the composition described in the first aspect in the preparation of products for the prevention or improvement of oral diseases.

[0027] Since the composition of the present invention can effectively inhibit Porphyromonas gingivalis, using the composition of the present invention in products for the prevention or improvement of oral diseases can play a role in preventing or improving oral diseases.

[0028] As a preferred embodiment of the third aspect, the oral disease is any one of halitosis, acute dental caries, dental caries, periodontitis, gingivitis, periodontitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcer, or thrush.

[0029] Halitosis, acute dental caries, periodontitis, gingivitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcers, and thrush are all oral diseases caused by oral pathogens. Inhibiting these oral pathogens can treat these oral diseases. In the oral environment, *Porphyromonas gingivalis* is a major pathogen causing oral lesions; therefore, the composition of this invention has the effect of preventing or improving oral diseases.

[0030] Fourthly, the present invention provides the use of the composition described in the first aspect in antibacterial products.

[0031] As a preferred embodiment of the fourth aspect, the bacteria is Porphyromonas gingivalis.

[0032] The composition of the present invention has a certain antibacterial effect, especially against Porphyromonas gingivalis, with a MIC of 10 μg / mL, while the MICs of tau phenol or cylindrica phenol alone are 80 μg / mL and 40 μg / mL, respectively. Obviously, the composition of the present invention has a stronger antibacterial ability, so it can be used as an antibacterial active ingredient to prepare antibacterial products.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through systematic screening of the compound ratio of tau tannin and physalis phenol, found that when the mass ratio of tau tannin to physalis phenol is (1-2):(1-10), CI <1, the antibacterial effect against Porphyromonas gingivalis is synergistic; among them, when the mass ratio of tau phenol to cypressin is in the range of 1:(1-10). CI The synergistic effect is enhanced, with a concentration of approximately 0.239–0.670. This compound system not only reduces the dosage of single ingredients and improves safety, but also provides a scientific basis for the development of oral care products such as mouthwash, toothpaste, and oral sprays. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the inhibition zone in Example 1; Figure 2 This is a schematic diagram of the inhibition zone in Example 2; Figure 3 This is a schematic diagram of the inhibition zone in Example 3; Figure 4 This is a schematic diagram of the antibacterial ring of the mouthwash containing the composition of Example 1. Detailed Implementation

[0035] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0036] The tau phenol used in this invention is from Shaanxi Huatai Bio-Fine Chemical Co., Ltd., and its product name is tau phenol. It is a yellow-orange or brownish-yellow powder with a special odor; the loss on drying is ≤5.0%, and the residue on ignition is ≤2.0%; the heavy metal indicators include lead ≤10 ppm, arsenic ≤2 ppm, mercury ≤1 ppm, and cadmium ≤5 ppm, all of which meet the standards; the microbiological indicators include a total bacterial count ≤100 CFU / g, molds and yeasts ≤10 CFU / g, and no pathogenic bacteria should be detected; the content (HPLC method) is not less than 80%.

[0037] The physalis phenol used in this invention comes from Guangzhou Hengguang Composite Materials Co., Ltd., and its product name is Higmild®-HK99. Its quality specifications are as follows: appearance is white to light yellow crystalline powder, active content is not less than 98%, drying loss is not more than 2%, and ignition residue is less than 1%.

[0038] The capsaicin used in this invention comes from Xi'an Feida Biotechnology Co., Ltd., and its quality specifications are: white to yellow powder with a characteristic odor, wherein the content of the main component capsaicin is 95~98%.

[0039] The cinnamon extract used in this invention comes from Luyuan Natural Fragrance Oil Refinery in Qingyuan District, Ji'an City. Its quality specifications are: light yellow to brownish-yellow liquid with a characteristic odor, the content of the main component cinnamaldehyde is 95~99%, the refractive index is 1.619~1.625, and the relative density is 1.046~1.053 g / mL.

[0040] The components and formulations of the compositions in Examples 1-5 and Comparative Examples 1-3 are shown in Table 1: Table 1. Components and their mass ratios of the compositions in Examples 1-3 and Comparative Examples 1-4 Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that capsaicin was used instead of physalisol.

[0041] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that cinnamon extract was used instead of tau phenol.

[0042] Test Example 1: Minimum Inhibitory Concentration (MIC) Test 1. Experimental materials Porphyromonas gingivalis ( Porphyromonas gingivalis ,Pg,ATCC 33277), purchased from Guangdong Provincial Microbial Culture Collection Center GDMCC.

[0043] Blood agar plates (TSA + 5% defibrinated sheep blood), brain and heart extract broth (BHI), vitamin K1 (0.1 mg / mL), heme chloride (0.5 mg / mL), Beijing Luqiao Technology Co., Ltd.; dimethyl sulfoxide, Sinopharm Group Pharmaceutical Co., Ltd.; tau phenol, Shaanxi Huatai Bio-Fine Chemical Co., Ltd.; cypressin, Guangzhou Hengguang Composite Materials Co., Ltd.

[0044] 2. Experimental apparatus Biosafety cabinet AC2-4S1, Singapore ESCO Technology Co., Ltd.; Biochemical incubator BD240, Binder Environmental Testing Equipment (Shanghai) Co., Ltd.; 0.01% electronic analytical balance PX84ZH, Ohaus Instruments (Shanghai) Co., Ltd.; Autoclave SX-700, Tomy Digital Biology, Japan; 20-200µL pipettes, 100-1000µL pipettes, Eppendorf, Germany.

[0045] 3. Experimental Methods 3.1 Preparation of culture medium Prepare the culture medium by weighing 24.5g of brain and heart extract broth culture base into 1L of distilled water, heating to boiling until completely dissolved, and autoclaving at 121℃ for 15 minutes. Before use, place the broth in an anaerobic environment to remove oxygen. When using it for culturing microorganisms, add 100μL each of vitamin K1 and heme chloride to every 10mL of broth culture medium.

[0046] 3.2 Activation of bacterial strains and preparation of bacterial suspension Dissolve the lyophilized bacterial culture in pre-deoxygenated sterile water, inoculate onto blood agar plates, and anaerobically incubate at 37°C for 7-10 days, then subculture 1-2 times. When the colonies on the blood agar plates turn from white to black, pick colonies in good growth condition and transfer them to brain and heart extract broth medium. Grind the culture finely and incubate anaerobically at 37°C for 48 hours. Centrifuge 10 mL of the bacterial suspension in a centrifuge tube for 2 minutes, remove the supernatant, collect the lower layer of cells, and then inoculate 10 μL into brain and heart extract broth medium. After incubating as described above, adjust the bacterial suspension concentration to 1.0 × 10⁻⁶. 9 CFU / mL, diluted 50 times with culture medium, for later use.

[0047] 3.3 Preparation for Drug Trial Preparation of mother liquors of tau phenol and cypress phenol: 320 mg of tau phenol and cypress phenol were weighed and dissolved in 2 mL of anhydrous ethanol. The solutions were then added to brain heart extract broth BHI medium and sonicated to prepare 100 mL of tau phenol and cypress phenol mother liquor with a concentration of 3200 μg / mL.

[0048] According to the ratio of tau phenol and cypress phenol in Table 1, brain heart extract broth BHI medium was used as the diluent, and 6% dimethyl sulfoxide (volume concentration) was added to prepare the corresponding concentration of the stock solution. The concentration of the stock solution of the composition was 1000 μg / mL.

[0049] 3.4 Measurement of MIC values ​​of samples using the 96-well plate method 3.4.1 Preparation and addition of working solution According to the paper "Study on the in vitro antibacterial function of quercetin extracted from Osmanthus fragrans against Porphyromonas gingivalis and Fusobacterium nucleatum" published in the journal *Occupational Health* in December 2024, the MIC determination of quercetin and cylindrical phenol as single herbs was performed using the two-fold dilution method. The stock solutions of quercetin and cylindrical phenol were diluted to 160 μg / mL using brain heart extract broth BHI medium. 100 μL of each solution was added to well 1 of a 96-well plate and then diluted two-fold with BHI medium to the following concentrations: 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.63, and 0.31 μg / mL. Well 11 was a positive control well, and well 12 was a negative control well. 100 and 200 μL of brain heart extract broth BHI medium were added to each well.

[0050] For the determination of MIC in Examples 1-6 and Comparative Examples 1-6, the stock solutions of the compositions were diluted to the following concentrations using brain heart extract broth BHI medium: 160, 140, 120, 100, 80, 60, 40, 20, 10, and 5 μg / mL. In a 96-well plate, 100 μL of the corresponding concentration of working solution was added to wells 1-10. Well 11 was a positive control well, and well 12 was a negative control well; 100 and 200 μL of brain heart extract broth BHI medium were added to these wells, respectively.

[0051] 3.4.2 Bacterial suspension loading The diluted bacterial suspension is approximately 2.0 × 10⁻⁶. 7 CFU / mL. Add 100 μL of diluted bacterial solution to each well from well 1 to well 11 of a 96-well plate. The final bacterial concentration in each well should then be 1.0 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0052] 3.4.3 Cultivation and Result Interpretation After anaerobic incubation at 37°C for 48 hours, the 96-well plate was removed and observed against a dark background. If the wells were clear and bright without any turbidity, it indicated that no bacteria were growing in the wells. The lowest concentration of the sample solution at this point was the MIC for Porphyromonas gingivalis.

[0053] 3.3.4 Calculation of the synergistic index for inhibiting Porphyromonas gingivalis The Chou-Talalay model is an effective method for quantifying synergistic effects, using a combination index.CI The synergistic effect of each composition is evaluated using the Chou-Talalay model. The calculation formula is as follows: Where Ca-b are the MICs of components A and B acting alone, respectively; C A-B C represents the concentration of each component at which the composition reaches the MIC. A-B The result = MIC experimental data of the composition × the proportion of components A and B in the composition. CI If the value is less than 1, it indicates the existence of a synergistic effect. CI =1 indicates that there is an additive effect; CI A value greater than 1 indicates the presence of an antagonistic effect.

[0054] 4 Experimental Results 4.1 Results of single-drug antibacterial activity The MIC test results of tau phenol and cylindrica phenol against *Porphyromonas gingivalis* are detailed in Table 2. Tau phenol is a natural diterpenoid phenolic compound with hydrophobic properties. It can disrupt the bacterial cell membrane structure, leading to leakage of cell contents, thereby exerting an antibacterial effect; the MIC of tau phenol alone is 80 μg / mL. Cylindrica phenol is a seven-membered ring phenol-ketone compound with strong metal ion chelating ability and membrane permeability. It can interfere with bacterial iron metabolism and energy metabolism, thus exhibiting broad-spectrum antibacterial activity; its MIC of cylindrica phenol alone is 40 μg / mL. Both tau phenol and cylindrica phenol have good antibacterial effects against *Porphyromonas gingivalis*, but the high concentrations required for their individual use limit their application in practical products. Therefore, further investigation is needed to explore the synergistic effect of tau phenol and cylindrica phenol in inhibiting *Porphyromonas gingivalis*. By using them in combination, it is possible to enhance the antibacterial effect through different mechanisms, thereby reducing the dosage of each.

[0055] Table 2. Antibacterial Results of Single Drugs 4.2 Antibacterial test results of the composition Tables 3-12 show the MIC test results of the combination of tau tannin and cylindrica phenol against *Porphyromonas gingivalis*. Examples 1-6 show the results when the mass ratio of tau tannin to cylindrica phenol is (1-2):(1-10). CI <1, the antibacterial effect against *Porphyromonas gingivalis* was synergistic, enhancing the inhibitory ability against *Porphyromonas gingivalis*. Among them, in Example 1, the combination of tsaourol and cylindricaol = 1:10 showed the best effect. CIThe lowest value (0.239) indicates the strongest synergistic effect; the MIC of the composition in Example 1 was reduced to 10 μg / mL, only 1.34% of the MIC of tsaool alone and 22.72% of the MIC of pine phenol alone, significantly reducing the amount of each component used. In Comparative Examples 1-3, the mass ratio of tsaool to pine phenol was between (4:1) and (8:1), and its... CI >1; Comparative Example 4: The mass ratio of pine phenol to cypress phenol was 1:15. CI Above 0.9 ， The ratio is close to 1. Therefore, when the mass ratio of taurine to cylindricaol exceeds the range of (1-2):(1-10), the inhibition of *Porphyromonas gingivalis* exhibits an antagonistic effect or a near-additive effect. Comparative Example 5 uses capsaicin instead of cylindricaol in Example 1. CI >1, the inhibition of *Porphyromonas gingivalis* showed an antagonistic effect; Comparative Example 6 used cinnamon extract instead of tau phenol in Example 1. CI >1. The inhibition of Porphyromonas gingivalis showed an antagonistic effect, as detailed in Tables 13 and 14.

[0056] Table 3. Synergistic test results of Example 1 (μg / mL) Table 4. Synergistic test results of Example 2 (μg / mL) Table 5. Synergistic test results of Example 3 (μg / mL) Table 6. Synergistic test results (μg / mL) of Example 4 Table 7. Synergistic test results of Example 5 (μg / mL) Table 8. Synergistic test results (μg / mL) of Example 6 Table 9. Results of synergistic testing in Comparative Example 1 (μg / mL) Table 10. Results of synergistic testing in Comparative Example 2 (μg / mL) Table 11. Results of synergistic testing in Comparative Example 3 (μg / mL) Table 12. Results of synergistic testing in Comparative Example 4 (μg / mL) Table 13. Results of synergistic testing in Comparative Example 5 (μg / mL) Table 14. Results of the synergistic test for Comparative Example 6 (μg / mL) Test Example 2: Antibacterial Zone Test 1. Experimental materials Porphyromonas gingivalis ( Porphyromonas gingivalis ,Pg,ATCC 33277), purchased from Guangdong Provincial Microbial Culture Collection Center GDMCC.

[0057] Blood agar plates (TSA + 5% defibrinated sheep blood), brain and heart extract broth (BHI), vitamin K1 (0.1 mg / mL), heme chloride (0.5 mg / mL), Beijing Luqiao Technology Co., Ltd.; dimethyl sulfoxide, Sinopharm Group Pharmaceutical Co., Ltd.; sterile water, Perfect (Guangdong) Daily Necessities Co., Ltd.; tau phenol, Shaanxi Huatai Bio-Fine Chemical Co., Ltd.; cypressin, Guangzhou Hengguang Composite Materials Co., Ltd.

[0058] 2. Experimental apparatus Biosafety cabinet AC2-4S1, Singapore Yisi Technology Co., Ltd.; Biochemical incubator BD240, Binder Environmental Testing Equipment (Shanghai) Co., Ltd.; 0.01% electronic analytical balance PX84ZH, Ohaus Instruments (Shanghai) Co., Ltd.; Autoclave SX-700, Tomy Digital Biology, Japan; 20-200µL adjustable pipette, 100-1000µL adjustable pipette, Eppendorf, Germany.

[0059] 3. Experimental Methods 3.1 Preparation of culture medium Prepare the culture medium by weighing 24.5g of brain and heart extract broth culture base into 1L of distilled water, heating to boiling until completely dissolved, and autoclaving at 121℃ for 15 minutes. Before use, place the broth in an anaerobic environment to remove oxygen. When using it for culturing microorganisms, add 100μL each of vitamin K1 and heme chloride to every 10mL of broth culture medium.

[0060] 3.2 Activation of bacterial strains and preparation of bacterial suspension Dissolve the lyophilized bacterial culture in pre-deoxygenated sterile water, inoculate onto blood agar plates, and anaerobically incubate at 37°C for 7-10 days, then subculture 1-2 times. When the colonies on the blood agar plates turn from white to black, pick colonies in good growth condition and transfer them to brain and heart extract broth medium. Grind the culture finely and incubate anaerobically at 37°C for 48 hours. Centrifuge 10 mL of the bacterial suspension in a centrifuge tube for 2 minutes, remove the supernatant, collect the lower layer of cells, and then inoculate 10 μL into brain and heart extract broth medium. After incubating as described above, adjust the bacterial suspension concentration to 1.0 × 10⁻⁶. 9 CFU / mL, diluted 50 times with culture medium, for later use.

[0061] 3.3 Preparation for Drug Trial Sterile water was used as a diluent for tau phenol and cypress phenol, and 6% dimethyl sulfoxide was added to prepare stock solutions of corresponding concentrations, so that the stock solution concentrations of tau phenol and cypress phenol were 1200 μg / mL, respectively.

[0062] According to the ratio of tau phenol and cypress phenol in Table 1, sterile water was used as the diluent, and 6% dimethyl sulfoxide was added to prepare the corresponding concentration of the stock solution, so that the concentration of the stock solution of each composition was 1200 μg / mL.

[0063] 3.4 Preparation of blood agar plates Prepare blood agar plates (TSA + 5% defibrinated sheep blood) in advance, add 100-200 μL of prepared bacterial solution, spread evenly on the entire surface of the blood agar plate, and let it dry.

[0064] 3.5 Sample addition Place three sterile Oxford cups alternately on the same dried blood agar plate. Add 200 μL of each sample solution to each Oxford cup. Add 200 μL of sterile distilled water to the Oxford cup of the blank control group.

[0065] 3.6 Measurement of Culture and Inhibition Zone Results The bacterial plates containing the added samples were placed in anaerobic refrigeration at 4°C for 4 hours or overnight for pre-diffusion. After pre-diffusion, they were removed and placed upright in anaerobic incubation at 36°C ± 1°C for 36-72 hours. The diameter of the inhibition zone formed in the test sample and the blank control group was measured using calipers. The sensitivity of *Porphyromonas gingivalis* to the drug was determined based on the size of the inhibition zone. The results were interpreted according to the National Committee for Standardization of Clinical Trials (NCCLS) standards: inhibition zone diameter <8 mm was considered insensitive, 8–13 mm was considered lowly sensitive, 13–19 mm was considered moderately sensitive, and >19 mm was considered highly sensitive.

[0066] 3.7 Data Processing Methods The analysis was performed using GraphPad Prism 10.0 software. In the table, identical letters indicate that there is no significant difference between two data points.P >0.05), different letters in the table indicate a significant difference between two data points. P <0.05).

[0067] 4. Results of the inhibition zone experiment Table 15 shows the inhibition zone test results of tau phenol, cypress phenol and their combination against Porphyromonas gingivalis. Figures 1 to 3 The images show the inhibition zone tests of *Porphyromonas gingivalis* in Examples 1-3. In Examples 1-3, when the mass ratio of tsulphol to cylindricaol was (1:10) to (1:6), the inhibition zones were significantly higher than those of cylindricaol alone, and *Porphyromonas gingivalis* showed high sensitivity to all examples. In Example 1, when the mass ratio of cylindricaol to cylindricaol was 1:10, the inhibition zone diameter was 25.24 ± 0.18 mm, which was 1.63 times that of tsulphol alone and 1.10 times that of cylindricaol alone, demonstrating the most significant synergistic effect. In Comparative Example 1, when the mass ratio of tsulphol to cylindricaol was 4:1, the inhibition zone was only 15.02 mm, smaller than that of tsulphol alone, suggesting antagonism or ineffective superposition. Furthermore, the inhibition zone of sterile water was 0 mm.

[0068] Table 15 Results of the Antibacterial Zone Test Application example: Oral spray test of Example 1 1. Experimental formula and process The oral spray formulation containing Example 1 is detailed in Table 16: Table 16 contains oral spray formulations from Example 1. The oral spray formulation containing Example 1 is prepared as follows: (1) Add water of phase A to the mixing pot, then add disodium EDTA and xylitol, turn on the stirring, heat the mixture to 75-80°C, and keep it warm for 25-30 minutes; (2) Turn on the cooling, cool it down to 55°C, and add sodium benzoate of phase B to the mixing pot; (3) Premix the raw materials of phase C, heat them appropriately, stir and dissolve them, and set them aside; (4) When the mixing pot cools down to 45°C, add the premixed phase C to the mixing pot; (5) Add the premixed phase D to the mixing pot; (6) If necessary, use phase E to adjust the pH to 5.5-6.0; (6) If the test is qualified, the product can be discharged.

[0069] 2. Oral spray antibacterial zone test The undiluted oral spray from Example 1 was used to test the inhibition zone against *Porphyromonas gingivalis*. The results of the inhibition zone test for the oral spray from Example 1 are detailed in Table 17. Figure 4Example 1: The inhibition zone size of the oral spray against *Porphyromonas gingivalis* was 16.24 mm. According to the NCCLS standard, it showed moderate sensitivity to *Porphyromonas gingivalis*, indicating that the oral spray containing Example 1 has a strong inhibitory effect on *Porphyromonas gingivalis*.

[0070] Table 17 shows the antibacterial zone test results for the oral spray from Example 1. 3. Sensory testing and detection of pH value, harmful substances and microorganisms. Referring to QB / T 2945-2012 "Oral Cleaning and Care Solution" and "Cosmetic Safety Technical Specifications" (2015 edition), sensory, pH, harmful substance, and microbial tests were conducted on the oral spray containing Example 1. Sensory tests included fragrance and clarity. Harmful substances included lead, arsenic, mercury, cadmium, and methanol. Microbial tests included total bacterial count, total mold and yeast count, thermotolerant coliforms, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0071] Fragrance testing was conducted directly by manual smelling. Clarity testing was performed by pouring 25ml of sample into a colorimetric tube under room temperature and natural light conditions, and visually observing from a distance of 30cm. pH value was tested according to QB / T 2945-2012. Methanol content was tested according to QB / T5703. Lead, arsenic, mercury, cadmium, total bacterial count, total mold and yeast count, thermotolerant coliforms, Staphylococcus aureus, and Pseudomonas aeruginosa were tested according to the "Cosmetic Safety Technical Specifications" (2015 edition). The quality standards and test results for the oral spray containing Example 1 are detailed in Table 18.

[0072] Table 18 contains the quality standards and test results of the oral spray from Example 1. As shown in Table 18, the oral spray containing Example 1 meets the quality standards in terms of fragrance, clarity, pH value, lead, arsenic, mercury, cadmium, methanol, total bacterial count, total mold and yeast count, thermotolerant coliforms, Staphylococcus aureus and Pseudomonas aeruginosa.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composition for synergistically inhibiting *Porphyromonas gingivalis*, characterized in that, The composition consists of tau phenol and cypress phenol; the mass ratio of tau phenol to cypress phenol is (1-2):(1-10).

2. The composition according to claim 1, characterized in that, The mass ratio of taurine and cypressin is 1:(2-10).

3. The use of the composition according to claim 1 in the preparation of oral care products.

4. The application as described in claim 3, characterized in that, The composition that inhibits Porphyromonas gingivalis is present in the oral care product at a content of 0.03-0.1% by weight.

5. The application as described in claim 4, characterized in that, The products include toothpaste and mouthwash.

6. The use of the composition of claim 1 in the preparation of products for the prevention or improvement of oral diseases.

7. The application as described in claim 6, characterized in that, The oral diseases mentioned are any one of halitosis, dental caries, periodontitis, pulpitis, oral mucositis, oral mucosal ulcers, and thrush.

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