Composition for synergistically inhibiting porphyromonas gingivalis and application

Abstract

The invention relates to a composition for synergistically inhibiting porphyromonas gingivalis, and belongs to the technical field of oral care. The invention provides a composition for synergistically inhibiting porphyromonas gingivalis. The composition is prepared from totarol and hinokiol. The totarol and the hinokiol are screened from numerous existing components, it is found that the composition formed by combining and compounding the totarol and the hinokiol has the synergistic interaction effect, the antibacterial effect of the composition can be improved, especially the antibacterial effect on porphyromonas gingivalis can be improved, and the inhibition effect of the composition is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral care, in particular to a composition for synergistically inhibiting Porphyromonas gingivalis and application thereof. BACKGROUND

[0002] Porphyromonas gingivalis (P. gingivalis) is a gram-negative anaerobic bacillus belonging to Bacteroidetes, which is one of the main pathogenic bacteria of periodontal diseases (such as gingivitis and chronic periodontitis). Porphyromonas gingivalis, P. gingivalis Bacteroidetes P. gingivalis directly damages periodontal tissues through various mechanisms. The matrix metalloproteinases (MMPs) secreted by P. gingivalis can degrade collagen fibers in the periodontal membrane, leading to the separation of the gums from the roots and the formation of deepening periodontal pockets. At the same time, P. gingivalis can invade gingival epithelial cells and fibroblasts, reproduce in the cells, and evade the clearance of the host immune system. In the alveolar bone, P. gingivalis causes progressive alveolar bone resorption by stimulating the activation of osteoclasts and inhibiting the function of osteoblasts. Studies have also found that P. gingivalis can interfere with the host's coagulation system, promote gum bleeding, and create favorable conditions for the colonization of other bacteria. These pathological changes eventually lead to irreversible destruction of the tooth support tissue and are the main cause of tooth loss in adults.

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

[0004] Recent studies have found that the pathogenic effects of P. gingivalis are not limited to the oral cavity; the bacteria can enter the blood circulation through gum ulcers and cause lesions at distant sites. In the cardiovascular system, the gingipains secreted by P. gingivalis can promote the formation of atherosclerotic plaques; for diabetic patients, periodontal infection can exacerbate insulin resistance, forming a vicious cycle; most notably, P. gingivalis and its toxic products have been detected in the brain tissue of patients with Alzheimer's disease, suggesting that they can promote neuroinflammation and β-amyloid deposition by crossing the blood-brain barrier.

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

[0006] Current methods for inhibiting P. gingivalis include mechanical debridement, chemical bacteriostatic agents, and antibiotics, but there are still key problems in practical application that need to be solved or improved through technological innovation.

[0007] ​Mechanical debridement is difficult to completely remove biofilm in complex areas. The core means of clinical treatment of periodontitis is mechanical debridement (such as subgingival scaling and root planing), but its effect on biofilm removal in anatomically complex areas such as furcation area and bone concave is limited. Porphyromonas gingivalis can resist mechanical force by forming biofilm and secrete gingipains to destroy host tissues. In addition, the debridement of deep periodontal pocket (>5mm) is more difficult and requires the assistance of antibacterial means.

[0008] Chemical antibacterial agents have side effects and resistance risks. Although the commonly used chemical antibacterial agents (such as chlorhexidine gluconate and cetylpyridinium chloride) in oral care products can inhibit bacteria in a broad spectrum, they have significant defects: ①oral microecological destruction: long-term application of chlorhexidine can lead to oral flora imbalance, causing candida infection or tooth staining; ②mucous membrane irritation: high concentration of chemical antibacterial agents may damage the oral mucosa, reducing patient compliance; ③resistance: drug-resistant strains of Porphyromonas gingivalis to chlorhexidine gluconate have been reported, which can enhance drug resistance through up-regulation of efflux pumps or biofilm matrix.

[0009] Antibiotics (such as metronidazole and tetracycline) are the main drugs for treating Porphyromonas gingivalis infection, but there are the following problems: ①resistance: the resistance rate of Porphyromonas gingivalis to metronidazole has increased from <5% in 2000 to 15%-20% at present, and there have also been reports of resistance of Porphyromonas gingivalis to antibiotics such as tetracyclines, β-lactams, and macrolides (such as clindamycin); ②short-term effectiveness and easy recurrence after drug withdrawal: antibiotics can only inhibit planktonic bacteria and have poor penetration of biofilm, and are prone to recurrence after drug withdrawal; ③systemic side effects: long-term use may cause gastrointestinal reactions or liver toxicity, limiting its long-term application.

[0010] Although natural ingredients show certain potential in inhibiting Porphyromonas gingivalis, there are still the following problems: ①the antibacterial effect of single ingredient is limited, and high concentration may cause cytotoxicity or unpleasant taste; ②the synergistic effect has not been fully explored: the synergistic antibacterial effect between different natural ingredients has not been fully studied, and there is a large optimization space for combination formula.

[0011] Therefore, it is urgent to develop a composition composed of natural active ingredients, which can improve the antibacterial effect on Porphyromonas gingivalis through synergistic effect and reduce the use concentration. Such a composition can be applied to oral care products such as mouthwash, toothpaste, and oral spray, providing a safe and efficient new choice for the prevention and treatment of periodontal diseases. SUMMARY

[0012] The purpose of the present application is to overcome the shortcomings of the prior art and provide a composition for synergistically inhibiting Porphyromonas gingivalis and applications thereof.

[0013] To achieve the above-mentioned purpose, the technical solution adopted by the present application is: In a first aspect, the present application provides a composition for synergistically inhibiting Porphyromonas gingivalis, the composition consisting of totarol and hinokitiol.

[0014] Totarol is a natural active ingredient derived from totara. Totarol is known for its excellent antibacterial, antioxidant and preservative effects. In the field of skin care, totarol can effectively inhibit Propionibacterium acnes and Staphylococcus aureus, which are the main bacteria causing acne, by destroying the bacterial cell membrane and leading to death, thereby playing a role in acne removal. At the same time, it has strong antioxidant properties and can effectively inhibit lipid peroxidation, thereby delaying skin aging. In addition, totarol has strong killing effect on gram-positive bacteria and can be used as a natural preservative.

[0015] Hinokitiol is a natural monoterpenoid compound derived from Chamaecyparis plants. Its chemical structure is a unique seven-membered ring phenolic ketone, and it is an environmentally friendly bioactive ingredient. The most prominent feature of this ingredient is its high safety. As a plant-derived natural molecule, it has good biocompatibility and is mild and non-irritating to the skin and mucous membranes, and long-term use is less likely to induce drug resistance. It is widely used in daily chemical, pharmaceutical and food industries. In terms of application, hinokitiol has the functions of removing dandruff, soothing itching, regulating scalp microecology and controlling oil secretion, and is often used in hair care products. In addition, it can also be used as a food preservative, fruit and vegetable preservative and wood mildew inhibitor, which reflects its safety advantage in multiple fields of application.

[0016] The present application screens totarol and hinokitiol from a large number of existing components, and finds that the combination of the two has a synergistic effect, which can improve the antibacterial effect of the composition, especially the antibacterial effect on Porphyromonas gingivalis.

[0017] As a preferred embodiment of the first aspect, the mass ratio of the totarol and the hinokitiol is (1-2):(1-10). Exemplarily, the mass ratio of the totarol and the hinokitiol can be any value or any value range in 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] As a preferred embodiment of the first aspect, the mass ratio of the totarol and the hinokitiol is 1:(2-10).

[0019] As a preferred embodiment of the first aspect, the mass ratio of the totarol and the hinokitiol is 1:10.

[0020] The present application studies the ratio of totarol and hinokitiol in the composition by Chou-Talalay model. When the mass ratio of totarol to hinokitiol is 1:10, the synergistic effect of the composition is the best.CI <1, indicating a synergistic effect, CI The lower the value, the better the synergistic effect; CI =1, indicating an additive effect; CI >1, indicating an antagonistic effect.

[0021] The present application configures the amentoflavone and the hinokitiol into an antibacterial solution with a certain mass concentration. It is found that when the mass ratio of the amentoflavone to the hinokitiol is within the range of (1-2):(1-10) (for example, the total mass concentration of the amentoflavone and the hinokitiol in the antibacterial solution is 10 μg / mL, the mass concentrations of the amentoflavone and the hinokitiol are 0.09 μg / mL and 0.91 μg / mL respectively, and the mass ratio is 1:10), CI <1, indicating that the two have a synergistic effect within the range. When the mass ratio of the amentoflavone to the hinokitiol is within the range of 1:(1-10), CI the value is within the range of 0.239-0.670, indicating that the synergistic effect of the two can be improved within the ratio range. When the mass ratio of the amentoflavone to the hinokitiol is 1:10, CI the value is 0.239, indicating that the synergistic effect of the two is the strongest when the mass ratio is the value.

[0022] In a second aspect, the present application provides the use of the composition of the first aspect in the preparation of an oral care product.

[0023] Porphyromonas gingivalis causes a series of oral diseases through its special pathogenic mechanism. The composition of the present application can effectively inhibit Porphyromonas gingivalis. Therefore, the composition of the present application can be used in an oral care product to effectively prevent and improve oral diseases caused by Porphyromonas gingivalis.

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

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

[0026] In a third aspect, the present application provides the use of the composition of the first aspect in the preparation of a product for preventing or improving oral diseases.

[0027] Since the composition of the present application can effectively inhibit Porphyromonas gingivalis, the composition of the present application can be used in a product for preventing or improving oral diseases to prevent or improve oral diseases.

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

[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 purslane phenol and physalis phenol, found that when the mass ratio of purslane phenol 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 peach geniposide used in the application is from Shaanxi Huatai Biological Fine Chemical Co., Ltd., and the product name is peach geniposide. The appearance is yellow orange or brown yellow powder, and has a special smell. The loss on drying is less than or equal to 5.0%, and the ignition residue is less than or equal to 2.0%. The heavy metal indexes including lead are less than or equal to 10 ppm, arsenic is less than or equal to 2 ppm, mercury is less than or equal to 1 ppm, and cadmium is less than or equal to 5 ppm, all of which meet the standards. The total number of bacteria in the microbial index is less than or equal to 100 CFU / g, the number of molds and yeasts is less than or equal to 10 CFU / g, and no pathogenic bacteria are detected. The content (HPLC method) is not less than 80%.

[0037] The hinokitiol used in the application is from Guangzhou Hengguang Composite Material Co., Ltd., and the product name is Higmild®-HK99. The quality specification is as follows: the appearance is white to light yellow crystalline powder, the active matter content is not less than 98%, the loss on drying is not more than 2%, and the ignition residue is less than 1%.

[0038] The capsaicin used in the application is from Xi'an Feida Biological Technology Co., Ltd., and the quality specification is as follows: white to yellow powder, with characteristic smell, and the content of main component capsaicin is 95-98%.

[0039] The cinnamon extract used in the application is from Jian City Qingyuan District Lv Yuan Natural Spice Oil Refining Factory, and the quality specification is as follows: light yellow to brown yellow liquid, with characteristic smell, and the content of 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 their proportions of the compositions of examples 1-5 and comparative examples 1-3 are shown in table 1: Table 1 Components and mass ratios of the compositions of examples 1-3 and comparative examples 1-4 Comparative example 5 The difference between comparative example 5 and example 1 is that capsaicin is used to replace hinokitiol.

[0041] Comparative example 6 The difference between comparative example 6 and example 1 is that cinnamon extract is used to replace peach geniposide.

[0042] Test example 1 Minimum inhibitory concentration (MIC) test 1. Experimental materials Porphyromonas gingivalis (Pg, ATCC 33277) was purchased from Guangdong Microbial Culture Collection Center (GDMCC). Porphyromonas gingivalis

[0043] ​Blood agar plates (TSA + 5% defibrinated sheep blood), brain heart infusion broth BHI, vitamin K1 (0.1 mg / mL), hematin chloride (0.5 mg / mL), Beijing Lüqiao Technology Co., Ltd.; dimethyl sulfoxide, Sinopharm Group Co., Ltd.; methyl 3, 5-dimethoxy-4-hydroxybenzoate, Shaanxi Huatai Biological Fine Chemical Co., Ltd.; hinokitiol, Guangzhou Hengguang Composite Material Co., Ltd.

[0044] 2. Experimental instruments Bio-safety cabinet AC2-4S1, Singapore Yisigao Technology Co., Ltd.; biochemical incubator BD240, BINDER Environmental Test Equipment (Shanghai) Co., Ltd.; electronic analytical balance of 1 / 10,000, Ohaus Instruments (Shanghai) Co., Ltd.; high-pressure steam sterilization pot SX-700, Japan Tomy Digital Biology Co., Ltd.; 20-200 μL pipette, 100-1000 μL pipette, Eppendorf, Germany.

[0045] 3. Experimental methods 3.1 Preparation of culture medium Prepare the culture medium, weigh 24.5 g of brain heart infusion broth culture medium in 1 L of distilled water, heat to boiling until completely dissolved, 121 ℃ high-pressure sterilization for 15 min, and reserve for use. Use within 24 h to remove oxygen in an anaerobic environment, add 100 μL of vitamin K1 and hematin chloride to each 10 mL of broth culture medium for culturing bacteria.

[0046] 3.2 Activation of bacteria and preparation of bacterial suspension Dissolve the freeze-dried bacteria in pre-deoxygenated sterile water, inoculate into blood agar plates, and incubate at 37 ℃ in an anaerobic environment for 7-10 days, and continue to subculture 1-2 times. When the colonies on the blood plate turn from white to black, select well-grown colonies in brain heart infusion broth medium, grind carefully, and incubate at 37 ℃ in an anaerobic environment for 48 h. Centrifuge 10 mL of bacterial solution in a centrifuge tube for 2 min, remove the supernatant, and take the lower bacterial body. Then, select 10 μL to inoculate into brain heart infusion broth medium, and incubate according to the above method. Adjust the concentration of the bacterial suspension to 1.0 × 10 9 CFU / mL, dilute 50 times with culture medium, and reserve for use.

[0047] 3.3 Preparation of reagents Preparation of methyl 3, 5-dimethoxy-4-hydroxybenzoate and hinokitiol stock solutions: weigh 320 mg of methyl 3, 5-dimethoxy-4-hydroxybenzoate and hinokitiol, respectively, dissolve in 2 mL of anhydrous ethanol, and then add to brain heart infusion broth BHI medium. After ultrasonic dissolution, prepare 100 mL of methyl 3, 5-dimethoxy-4-hydroxybenzoate and hinokitiol stock solutions with a concentration of 3200 μg / mL.

[0048] According to the ratio of pinoresinol and hinokitiol in Table 1, using brain heart infusion broth BHI medium as diluent, adding 6% dimethyl sulfoxide (volume concentration) to prepare the corresponding concentration of stock solution, the composition stock solution concentration is 1000 μg / mL.

[0049] 3.4 MIC value of sample measured by 96-well plate method 3.4.1 Preparation of working solution and sample addition According to the paper published in December 2024 in the journal of Occupation and Health, "Study on the antibacterial function of quercetin extracted from osmanthus in vitro against porphyromonas gingivalis and fusobacterium nucleatum", the MIC of pinoresinol and hinokitiol single herb was measured by double dilution method. The brain heart infusion broth BHI medium was used to dilute the pinoresinol and hinokitiol stock solution to 160 μg / mL, 100 μL was added to the first hole of the 96-well plate, and the BHI medium was double diluted to the following concentrations: 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 μg / mL; hole 11 is the positive control hole, hole 12 is the negative control hole, 100, 200 μL of brain heart infusion broth BHI medium is added respectively.

[0050] The MIC of examples 1-6 and comparative examples 1-6 was measured by using brain heart infusion broth BHI medium to dilute the composition stock solution to the following concentrations: 160, 140, 120, 100, 80, 60, 40, 20, 10, 5 μg / mL. In the 96-well plate, 100 μL of the corresponding concentration of working solution was added to hole 1 to hole 10 respectively. Hole 11 is the positive control hole, hole 12 is the negative control hole, 100, 200 μL of brain heart infusion broth BHI medium is added respectively.

[0051] 3.4.2 Bacterial solution addition The diluted bacterial suspension was about 2.0 × 10 7 CFU / mL. 100 μL of the diluted bacterial solution was added to each of the first to eleventh holes in the 96-well plate, at this time the final content of the bacterial solution concentration in each hole should be 1.0 × 10 7 CFU / mL.

[0052] 3.4.3 Culture and result interpretation After 48 h of anaerobic culture at 37℃, the 96-well plate was taken out and observed under dark background, clear and bright without turbidity appeared in the hole, indicating that there was no bacterial growth in the hole, at this time the minimum concentration of the sample was the MIC against porphyromonas gingivalis.

[0053] 3.3.4 Calculation of synergistic index for inhibiting porphyromonas gingivalis The "Chou-Talalay" model is an effective method for quantifying synergistic effect, through the combination index (CI),CI ) to evaluate the combined effect of each composition. Therefore, the synergistic effect of each composition was evaluated by the Chou-Talalay model, and the calculation formula is as follows: Wherein, Ca-b is the MIC of component A, B alone; C A-B represents the concentration of each component when the composition reaches the MIC, C A-B The results = composition MIC experimental data x A-B component in the composition. CI <1, indicating that there is a synergistic effect; CI = 1, indicating that there is an additive effect; CI > 1, indicating that there is an antagonistic effect.

[0054] 4 Experimental results 4.1 Single drug bacteriostatic results The MIC test results of amentoflavone and hinokitiol on P. gingivalis are shown in Table 2. Amentoflavone is a natural diterpene phenolic compound with hydrophobicity, which can destroy the structure of bacterial cell membrane and cause cell content leakage, thereby playing a bacteriostatic effect; the MIC of amentoflavone alone is 80 μg / mL. Hinokitiol is a seven-membered ring phenolic ketone compound with strong metal ion chelating ability and membrane penetration, which can interfere with the iron metabolism and energy metabolism of bacteria, thereby having a broad-spectrum bacteriostatic effect; the MIC of hinokitiol alone is 40 μg / mL. Amentoflavone and hinokitiol have good bacteriostatic effect on P. gingivalis, but the required concentration is high when used alone, which limits its application in actual products. Therefore, the synergistic effect of amentoflavone and hinokitiol on P. gingivalis was further explored, and through combination use, the bacteriostatic effect may be synergistically enhanced through different mechanisms, and the dosage of each component may be reduced.

[0055] Table 2 Single drug bacteriostatic results 4.2 Composition bacteriostatic test results Tables 3-12 are the MIC test results of the composition of amentoflavone and hinokitiol on P. gingivalis. Examples 1-6, i.e. the mass ratio of amentoflavone to hinokitiol is (1-2): (1-10), CI <1, the bacteriostatic effect on P. gingivalis shows synergistic effect, which improves the inhibition ability on P. gingivalis. Among them, example 1, the combination effect of amentoflavone: hinokitiol = 1:10 is optimal, CIThe value is the lowest (0.239), and the synergistic effect is the strongest; the MIC of the composition of Example 1 is reduced to 10 μg / mL, which is only 1.34% of the MIC of methylellagic acid and 22.72% of the MIC of hinokitiol, significantly reducing the amount of each component. The mass ratio of methylellagic acid to hinokitiol in Comparative Example 1-3 is (4:1) to (8:1), and the MIC of the composition is CI >1; the mass ratio of methylellagic acid to hinokitiol in Comparative Example 4 is 1:15, and the MIC of the composition is CI >1 ， >1. It can be seen that when the mass ratio of methylellagic acid to hinokitiol exceeds the range of (1-2):(1-10), the inhibition of P. gingivalis shows an antagonistic effect or is close to an additive effect. In Comparative Example 5, capsaicin is used to replace hinokitiol in Example 1, CI >1, showing an antagonistic effect on P. gingivalis; in Comparative Example 6, cinnamon extract is used to replace methylellagic acid in Example 1, CI >1, showing an antagonistic effect on P. gingivalis. See Tables 13 and 14 for details.

[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 of Example 4 (μg / mL) Table 7: Synergistic test results of Example 5 (μg / mL) Table 8: Synergistic test results of Example 6 (μg / mL) Table 9: Synergistic test results of Comparative Example 1 (μg / mL) Table 10: Synergistic test results of Comparative Example 2 (μg / mL) Table 11: Synergistic test results of Comparative Example 3 (μg / mL) Table 12: Synergistic test results of Comparative Example 4 (μg / mL) Table 13 Synergy test results of Comparative Example 5 (μg / mL) Table 14 Synergy test results of Comparative Example 6 (μg / mL) Test Example 2 Bacteriostatic circle test 1. Experimental materials Porphyromonas gingivalis (P. gingivalis, Pg, ATCC 33277) was purchased from Guangdong Microbial Culture Collection Center (GDMCC). Porphyromonas gingivalis , Pg, ATCC 33277) was purchased from Guangdong Microbial Culture Collection Center (GDMCC).

[0057] TSA+5% defibrinated sheep blood, brain heart infusion broth BHI, vitamin K1 (0.1 mg / mL), hemin chloride (0.5 mg / mL), Beijing Lq Technology Co., Ltd.; dimethyl sulfoxide, Sinopharm Group Co., Ltd.; sterile water, Perfect (Guangdong) Daily Products Co., Ltd.; methyl 3, 5-dihydroxybenzoate, Shaanxi Huatai Biological Fine Chemical Co., Ltd.; hinokitiol, Guangzhou Hengguang Composite Material Co., Ltd.

[0058] 2. Experimental instruments Bio-safety cabinet AC2-4S1, Singapore Yisigao Technology Co., Ltd.; biochemical incubator BD240, BINDER Environmental Test Equipment (Shanghai) Co., Ltd.; one-hundredth electronic analytical balance PX84ZH, Ohaus Instruments (Shanghai) Co., Ltd.; high-pressure steam sterilization pot SX-700, Japan Tomy Digital Biology Co., Ltd.; 20-200µL adjustable pipette, 100-1000µL adjustable pipette, Eppendorf, Germany.

[0059] 3. Experimental method 3.1 Preparation of culture medium Prepare the culture medium, weigh 24.5g brain heart infusion broth culture medium in 1L distilled water, heat to boiling until completely dissolved, 121℃ high pressure sterilization for 15min, and reserve for use. Within 24h before use, place in anaerobic environment to remove oxygen, when used for culture of bacterial strains, add vitamin K1 and hemin chloride 100μL respectively in each 10mL broth medium.

[0060] 3.2 Activation of bacterial strains and preparation of bacterial suspension The freeze-dried bacterial strain was dissolved in pre-deoxidized sterile water and inoculated into blood agar plates for anaerobic culture at 37°C for 7-10 days, and then subcultured for 1-2 times. When the colonies on the blood plate turned from white to black, well-grown colonies were selected and ground into a fine paste in brain heart infusion broth medium, and then cultured anaerobically at 37°C for 48 hours. Then, 10 mL of the bacterial solution was centrifuged in a centrifuge tube for 2 minutes, and the supernatant was removed. The lower bacterial body was then selected and inoculated into brain heart infusion broth medium at 10 μL. After culture according to the above method, the concentration of the bacterial suspension was adjusted to 1.0 × 10 9 CFU / mL, and then diluted 50 times with the culture medium for standby.

[0061] 3.3 Preparation of reagents Taoju phenol and cypress phenol were each diluted with sterile water as a diluent, and 6% dimethyl sulfoxide was added to prepare a corresponding concentration of a stock solution, so that the stock solution concentrations of Taoju phenol and cypress phenol were 1200 μg / mL, respectively.

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

[0063] 3.4 Preparation of bacterial-containing blood plates Blood plates (TSA + 5% defibrillated sheep blood) were prepared in advance, and 100-200 μL of the standby bacterial solution was added dropwise to evenly coat the entire surface of the blood plate, and then dried.

[0064] 3.5 Sample addition Three sterile Oxford cups were arranged at intervals on the same dry bacterial-containing blood plate, and 200 μL of each sample solution was added to each Oxford cup. The blank control group was added with 200 μL of sterile distilled water.

[0065] 3.6 Culture and measurement of inhibition zone results The bacterial-containing plate with added samples was placed in an anaerobic refrigerator at 4°C for 4 hours or overnight, then removed after pre-diffusion, and placed vertically in an anaerobic culture at 36°C ± 1°C for 36-72 hours. Then, the diameters of the inhibition zones formed by the test samples and the blank control group were measured with a vernier caliper. According to the size of the inhibition zone, the sensitivity of Porphyromonas gingivalis to the drug was determined, and the results were determined according to the standard of the National Clinical Laboratory Standardization Committee (NCCLS): inhibition zone diameter <8 mm is not sensitive, 8-13 mm is low sensitivity, 13-19 mm is moderate sensitivity, and >19 mm is high sensitivity.

[0066] 3.7 Data processing method GraphPad Prism 10.0 software was used for analysis. In the table, the same letter indicates that there is no significant difference between the two data.P >0.05), different letters in the table indicate that there is a significant difference between the two data P <0.05).

[0067] 4 Bacteriostatic circle experiment results Table 15 is the bacteriostatic circle test results of pinoresinol and hinokitiol and their combinations on Porphyromonas gingivalis. Figures 1 to 3 The following are the photos of the bacteriostatic circle test of Porphyromonas gingivalis in Examples 1-3. In Examples 1-3, i.e. when the mass ratio of pinoresinol to hinokitiol is (1:10) to (1:6), the bacteriostatic circle is significantly higher than that of hinokitiol, and Porphyromonas gingivalis is highly sensitive to the above examples. In Example 1, the mass ratio of pinoresinol to hinokitiol is 1:10, and the diameter of the bacteriostatic circle is 25.24±0.18 mm, which is 1.63 times that of pinoresinol alone and 1.10 times that of hinokitiol alone, and the synergistic effect is the most prominent. In Comparative Example 1, i.e. when the mass ratio of pinoresinol to hinokitiol is 4:1, the bacteriostatic circle is only 15.02 mm, which is smaller than the bacteriostatic circle of pinoresinol alone, indicating that it is antagonistic or ineffective. In addition, the bacteriostatic circle of sterile water is 0 mm.

[0068] Table 15 Bacteriostatic circle test results Application Example contains oral spray test of Example 1 1. Experimental formula and process The oral spray formula containing Example 1 is shown in Table 16: Table 16 Oral spray formula containing Example 1 The preparation process of the oral spray formula containing Example 1 is as follows: (1) add water in phase A to the stirring pot, then add disodium EDTA and xylitol, start stirring, and heat to 75-80°C, and keep for 25-30 minutes; (2) start cooling, cool to 55°C, and add sodium benzoate in phase B to the stirring pot; (3) pre-mix the raw materials in phase C, heat and stir to dissolve, and reserve; (4) when the stirring pot cools to 45°C, add the pre-mixed phase C to the stirring pot; (5) add the pre-mixed phase D to the stirring pot; (6) adjust the pH to 5.5-6.0 if necessary; (6) check and pass, and then discharge.

[0069] 2. Bacteriostatic circle test of oral spray The bacteriostatic circle test was performed using the stock solution of the oral spray containing Example 1. The bacteriostatic circle test results of the oral spray containing Example 1 on Porphyromonas gingivalis are shown in Table 17 and Figure 4The bacteriostatic circle size of the oral spray of Example 1 on Porphyromonas gingivalis is 16.24 mm, and according to the NCCLS standard, it shows moderate sensitivity to Porphyromonas gingivalis, indicating that the oral spray containing Example 1 has strong inhibitory ability on Porphyromonas gingivalis.

[0070] Table 17 contains the results of the bacteriostatic circle test of the oral spray of Example 1 3. Sensory test and PH value, harmful substance and microbial detection According to QB / T 2945-2012 "Oral cleaning and nursing liquid" and "Cosmetic safety technical specification" (2015 edition), the sensory, pH value, harmful substance and microbial detection of the oral spray containing Example 1 are carried out, wherein the sensory test includes fragrance type, clarity, harmful substance includes lead, arsenic, mercury, cadmium, methanol, and microorganism includes total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus and Pseudomonas aeruginosa.

[0071] The test method of fragrance type is directly artificial smelling. The clarity test method is: under the conditions of room temperature and natural light, 25 ml of sample is poured into a colorimetric tube, and observed by eyes at a distance of 30 cm. The pH value is tested according to QB / T 2945-2012. The methanol content is tested according to QB / T 5703. Lead, arsenic, mercury, cadmium, total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus and Pseudomonas aeruginosa are tested according to "Cosmetic safety technical specification" (2015 edition). The quality standards and test results of the oral spray containing Example 1 are shown in Table 18.

[0072] Table 18 contains the quality standards and test results of the oral spray containing Example 1 As shown in Table 18, the fragrance type, clarity, pH value, lead, arsenic, mercury, cadmium, methanol, total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus and Pseudomonas aeruginosa of the oral spray containing Example 1 meet the quality standards.

[0073] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A composition for synergistically inhibiting Porphyromonas gingivalis, characterized by comprising, The composition consists of peperin and hinokitiol.

2. The composition of claim 1, wherein The mass ratio of peperin and hinokitiol is (1-2):(1-10).

3. The composition of claim 2, wherein The mass ratio of peperin and hinokitiol is 1:(2-10).

4. Use of the composition according to any one of claims 1-3 in the preparation of an oral care product.

5. The use according to claim 4, wherein the compound is ###0002### The content of the composition for inhibiting Porphyromonas gingivalis in the oral care product is 0.03-0.1% by mass.

6. The use according to claim 4, wherein the compound is ###0002### The product includes toothpaste and mouthwash.

7. Use of the composition according to any one of claims 1-3 in the preparation of a product for preventing or improving oral diseases.

8. Use according to claim 7, wherein the compound is ###0002### The oral disease is any one of halitosis, acute tooth, dental caries, periodontal disease, gingivitis, periodontitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcer, and aphthous ulcer.

9. Use of the composition according to any one of claims 1-3 in a bacteriostatic product.

10. Use according to claim 9, wherein The bacterium is Porphyromonas gingivalis.

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