Porphyromonas gulae-derived cytokine production inhibitory composition, oral care composition for dog or cat, periodontal disease-preventing agent, intraoral biofilm formation inhibitor, Anti-inflammatory agent, and halitosis inhibitor

Abstract

[Problem] Provided are: a Porphyromonas gulae-derived cytokine production inhibitory composition in which active ingredients themselves match with the palatability of a pet; an oral care composition for a dog or a cat; a periodontal disease-preventing agent; an intraoral biofilm formation inhibitor; an anti-inflammatory agent; and a halitosis inhibitor. [Solution] The present invention pertains to a Porphyromonas gulae-derived cytokine production inhibitory composition containing at least one compound selected from among cyclodextran and derivatives thereof. The inhibitory composition can be applied to an oral care composition for a dog or a cat, a periodontal disease-preventing agent, an intraoral biofilm formation inhibitor, an anti-inflammatory agent, and a halitosis inhibitor.

Description

Composition for inhibiting cytokine production derived from Porphyromonas gulae, oral care composition for dogs or cats, agent for preventing periodontal disease, agent for inhibiting oral biofilm formation, anti-inflammatory agent, and agent for suppressing bad breath

[0001] The present invention relates to a composition for suppressing the production of cytokines derived from Porphyromonas gulae, an oral care composition for dogs or cats, a preventive agent for periodontal disease, an inhibitor of oral biofilm formation, an anti-inflammatory agent, and an agent for suppressing bad breath.

[0002] Like humans, dogs and cats suffer from various oral problems such as periodontal disease. Because the oral environments of humans and pets are completely different, the bacterial flora present in the oral cavity is also different. Specifically, the main causative bacterium of periodontal disease in humans is Porphyromonas gingivalis, whereas the main causative bacterium of periodontal disease in dogs and cats is Porphyromonas gulae, which is different from the causative bacterium in humans.

[0003] As a measure to address oral problems such as periodontal disease in pets, a periodontal disease preventive agent against Porphyromonas gulae containing Angelica keiskei extract as an active ingredient has been proposed (see Patent Document 1). According to the preventive agent described in Patent Document 1, the Angelica keiskei extract exerts an antibacterial effect (growth inhibitory effect) against Porphyromonas gulae, which is expected to prevent periodontal disease in dogs and cats.

[0004] JP 2015-067539 A

[0005] However, since Angelica keiskei extract liquid is not palatable to pets, the preventive agent described in Patent Document 1 must be used in combination with one or more selected from carrageenan, pectin, gellan gum, alginic acid, and salts thereof. Pets will not tolerate products with poor palatability, so palatability is extremely important when using oral compositions for pets. Furthermore, since pets swallow products without gargling, high safety is required, and the method of use differs from that for humans. For this reason, technologies for human oral compositions often cannot be directly applied to pets. Therefore, there is a need for a composition in which the active ingredient that contributes to the prevention of periodontal disease itself is palatable to pets without being used in combination with other ingredients, thereby allowing for greater freedom in development.

[0006] The present invention has been made in view of the above problems, and aims to provide a composition for inhibiting cytokine production derived from Porphyromonas gulae, an oral care composition for dogs or cats, a periodontal disease preventive agent, an oral biofilm formation inhibitor, an anti-inflammatory agent, and an agent for suppressing bad breath, the active ingredients of which themselves match the preferences of pets.

[0007] As a result of extensive research, the present inventors have discovered that cyclodextran, a cyclic oligosaccharide, itself exhibits the function of suppressing the production of cytokines derived from Porphyromonas gulae, and have thus completed the present invention. Specifically, the present invention provides the following.

[0008] A first aspect of the present invention provides a composition for inhibiting the production of cytokines derived from Porphyromonas gulae, which contains at least one compound selected from cyclodextran and its derivatives.

[0009] A second aspect of the invention provides an oral care composition for dogs or cats containing the inhibitory composition according to the first aspect. A third aspect of the invention provides a periodontal disease preventive agent for dogs or cats containing the inhibitory composition according to the first aspect. A fourth aspect of the invention provides an oral biofilm formation inhibitor for dogs or cats containing the inhibitory composition according to the first aspect. A fifth aspect of the invention provides an anti-inflammatory agent for dogs or cats containing the inhibitory composition according to the first aspect. A sixth aspect of the invention provides a bad breath suppressant for dogs or cats containing the inhibitory composition according to the first aspect.

[0010] FIG. 1 shows an example of the structural formula of cyclodextran. Cyclodextran is a cyclic isomaltooligosaccharide in which 4 to 33 glucose units are cyclically linked via α-1,6 glucoside bonds. It has no sweetness, is tasteless, and is odorless. Therefore, it does not affect the taste or aroma of food, making it highly palatable to dogs and cats. Furthermore, cyclodextran is highly water-soluble, dissolving in an equal or less amount of water, making it safe enough for dogs and cats to swallow without gargling. Furthermore, cyclodextran is stable against heat, acid, and alkali, allowing for a high degree of development flexibility in providing oral care compositions, periodontal disease preventatives, oral biofilm formation inhibitors, anti-inflammatory agents, and halitosis suppressants.

[0011] According to the present invention, it is possible to provide a composition for inhibiting cytokine production derived from Porphyromonas gulae, an oral care composition for dogs or cats, a periodontal disease preventive agent, an oral biofilm formation inhibitor, an anti-inflammatory agent, and an agent for suppressing bad breath, the active ingredients of which themselves match the preferences of pets.

[0012] Figure 1 shows an example of the structural formula of cyclodextran (CI). Figure 2 shows an example of the structural formula of cyclodextrin (CD). Figure 3 shows the results of [Test 1]: the bactericidal effect of CI-Dextran mix on P. gulae. Figure 4 shows the results of [Test 2]: the inhibitory effect of CI-Dextran mix on P. gulae biofilm formation (relationship between CI-Dextran mix concentration and OD). Figure 5 shows the results of [Test 3]: electron microscopy observation of P. gulae and its biofilm on a hydroxyapatite disk (control). Figure 6 shows the results of [Test 3]: electron microscopy observation of P. gulae and its biofilm on a hydroxyapatite disk (CI-Dextran mix concentration 5%). Figure 7 shows the results of [Test 4]: the inhibitory effect of CI-Dextran mix on P. gulae biofilm formation (control). Figure 8 shows the effect of CI-Dextran mix on inflammatory cytokine production in mouse macrophage cells caused by P. gulae. Figure 8 shows the results of [Test 6]: the deodorizing effect of CI-Dextran mix on substances that cause bad breath (methyl mercaptan, etc.). Figure 9 shows the results of [Test 7]: the inhibitory effect of CI-Dextran mix on insoluble glucan production by P. gulae.

[0013] Specific embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0014] <Composition for Inhibiting Cytokine Production Derived from Porphyromonas gulae> The composition for inhibiting cytokine production according to the present invention contains at least one compound selected from cyclodextran and its derivatives.

[0015] [Cyclodextran and its derivatives] Figure 1 shows an example of the structural formula of cyclodextran (CI). Cyclodextran is a cyclic isomaltooligosaccharide in which 4 to 33 glucose units are linked in a ring via α-1,6 glycosidic bonds.

[0016] Cyclodextrin is known as a compound similar to cyclodextran. Figure 2 shows an example of the structural formula of cyclodextrin (CD). CI and CD are similar in that they are cyclic oligosaccharides in which multiple glucose units are linked in a ring, but they differ in the following ways. First, CI has 4 to 33 glucose units, while CD has 6 to 8 glucose units. Second, CI has glucose units linked via α-1,6 glycosidic bonds, while CD has glucose units linked via α-1,4 glycosidic bonds. Third, the molecular structure of CI is large and shallow, while the molecular structure of CD is small and deep. Fourth, CI has high solubility in water, while CD has low solubility in water.

[0017] For example, CI can be obtained from the culture medium of a microorganism of the genus Bacillus or from the reaction mixture of cyclic isomaltose synthase (see Japanese Patent Nos. 3075873 and 3117328). When CI is obtained using a microorganism or an enzyme, it is obtained as a mixture of unbranched CI and branched CI. Cyclodextran derivatives include the branched CI from this mixture.

[0018] [Optional Components (Additives)] In addition to at least one selected from CI and its derivatives, the cytokine production inhibitory composition of the present invention may contain optional components (additives) in normal amounts, either alone or in appropriate combinations of two or more, as needed, within the scope that does not impair the effects of the present invention.

[0019] Optional components include surfactants, abrasives, binders, wetting agents, foaming agents, retention agents, flavorings, acidulants, colorants, pH adjusters, preservatives, sweeteners, active ingredients, thickeners, etc. Specific examples of optional components are shown below, but the components that can be blended in the composition of the present invention are not limited to these.

[0020] [Surfactant] Any surfactant can be used without particular limitation as long as it is suitable for incorporation into a composition to be administered to the oral cavity of a dog or cat. Examples of the surfactant include nonionic surfactants, anionic surfactants, and amphoteric surfactants.

[0021] Examples of nonionic surfactants include fatty acid esters of sugars or sugar alcohols, such as sucrose fatty acid esters, maltose fatty acid esters, maltitol fatty acid esters, maltotriitol fatty acid esters, maltotetraitol fatty acid esters, maltopentaitol fatty acid esters, maltohexaitol fatty acid esters, maltoheptaitol, fatty acid esters, sorbitan fatty acid esters, lactose fatty acid esters, lactitol fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oil, polyoxyethylene-polyoxypropylene block copolymers, polyoxyethylene ethers of glycerin esters, and alkylolamides. The fatty acids preferably have 12 to 18 carbon atoms.

[0022] Examples of anionic surfactants include N-acylamino acid salts, α-olefin sulfonates, alkyl sulfates, etc. Specific examples include sodium α-olefin sulfonate having an alkyl chain with 10 to 16 carbon atoms, sodium lauryl sulfate, sodium N-acyl glutamates such as sodium N-lauroyl glutamate and sodium N-palmitoyl glutamate, sodium N-acyl sarcosinates such as sodium N-lauroyl sarcosinate and sodium N-myristoyl sarcosinate, and sodium N-methyl-N-acyltaurates such as sodium N-lauroyl methyl taurate and sodium N-myristoyl methyl taurate.

[0023] Examples of amphoteric surfactants include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, alkyldiaminoethylglycine hydrochloride, and laurylmethylaminoacetic acid betaine.

[0024] As the surfactant, nonionic surfactants and anionic surfactants are preferred, and from the viewpoint of the feeling upon use such as irritation to the oral mucosa, nonionic surfactants are preferred, and fatty acid esters of sugars or sugar alcohols are particularly preferred.

[0025] [Abrasives] Examples of abrasives include silica-based abrasives such as crystalline silica, amorphous silica, silica gel, and aluminosilicate, zeolite, calcium hydrogen phosphate anhydrate, calcium hydrogen phosphate dihydrate, calcium pyrophosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, tribasic magnesium phosphate, zirconium silicate, tribasic calcium phosphate, hydroxyapatite, quaternary calcium phosphate, and synthetic bark-based abrasives.

[0026] [Binder] Examples of binders include gelatin, methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, xanthan gum, gum arabic, guar gum, locust bean gum, polyvinyl alcohol, and polyvinylpyrrolidone.

[0027] [Humectant] As the humectant, one or more polyhydric alcohols such as glycerin, concentrated glycerin, diglycerin, sorbitol, maltitol, dipropylene glycol, propylene glycol, 1,3-butylene glycol, xylitol, and polyethylene glycol can be used.

[0028] [Foaming Agent] Examples of the foaming agent include sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium alkyl sulfosuccinate, sodium coconut oil fatty acid monoglycerin sulfonate, sodium α-olefin sulfonate, N-acylamino acid salts such as N-acyl glutamate, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, maltitol fatty acid esters, sucrose fatty acid esters, polyglycerin fatty acid esters, fatty acid diethanolamides, polyoxyethylene sorbitan monostearate, polyoxyethylene hydrogenated castor oil, and polyoxyethylene fatty acid esters, and these can be used alone or in combination of two or more.

[0029] [Retention Agent] As a retention agent for retaining (sustaining) the active ingredient of the cytokine production-suppressing composition, liquid paraffin, gelling hydrocarbons which are mixtures of liquid paraffin and polyethylene, vegetable oils, beeswax, etc. can be used, and these can be used alone or in combination of two or more. Note that the gelling hydrocarbons also function as a gelling agent.

[0030] [Fragrance] The fragrance is preferably one or more types of oil-based fragrances, such as natural fragrances and synthetic fragrances, or powdered fragrances, but is not particularly limited to these. The fragrance is preferably one or more types of oil-based fragrances, such as natural fragrances and synthetic fragrances, or powdered fragrances, but is not particularly limited to these. Examples of natural fragrances include mastic oil, parsley oil, anise oil, eucalyptus oil, wintergreen oil, cassia oil, menthol oil, spearmint oil, peppermint oil, lemon oil, coriander oil, orange oil, mandarin oil, lime oil, lavender oil, laurel oil, chamomile oil, cardamom oil, caraway oil, bay oil, lemongrass oil, pine needle oil, neroli oil, rose oil, jasmine oil, iris concrete, peppermint absolute, rose absolute, orange flower, citrus oil, mixed fruit oil, strawberry oil, cinnamon oil, clove oil, and grape oil.

[0031] Examples of single fragrances include leaf alcohol, carvone, anethole, methyl salicylate, cinnamic aldehyde, linalool, linalyl acetate, limonene, menthone, menthyl acetate, pinene, octyl aldehyde, citral, pulegone, carbyl acetate, anisaldehyde, ethyl acetate, ethyl butyrate, allyl cyclohexane propionate, methyl anthranilate, ethyl methyl anthranilate, vanillin, undecalactone, hexanal, ethynone alcohol, propyl alcohol, butanol, isoamyl alcohol, hexenol, dimethyl sulfide, cyclotene, furfural, trimethylpyrazine, ethyl lactate, and ethyl lioacetate. Examples of blended flavors including single flavors and / or natural flavors include strawberry flavor, apple flavor, banana flavor, pineapple flavor, grape flavor, mango flavor, melon flavor, tropical fruit flavor, watermelon flavor, butter flavor, milk flavor, yogurt flavor, fruit mix flavor, herb mint flavor, etc. The flavor may be in the form of an essential oil, extract, solid, or a spray-dried powder thereof.

[0032] [Acidulant] Examples of the acidulant include organic acids such as citric acid, malic acid, succinic acid, and tartaric acid.

[0033] [Coloring Agent] Examples of coloring agents include natural pigments such as safflower red pigment, gardenia yellow pigment, gardenia blue pigment, perilla pigment, red koji pigment, red cabbage pigment, carrot pigment, hibiscus pigment, cacao pigment, spirulina blue pigment, and coumarind pigment; legally designated pigments such as Red No. 3, Red No. 104, Red No. 105, Red No. 106, Yellow No. 4, Yellow No. 5, Green No. 3, and Blue No. 1; riboflavin, copper chlorophyll sodium, and titanium dioxide.

[0034] [pH Adjuster] Examples of pH adjusters include organic acids such as citric acid, malic acid, tartaric acid, and succinic acid, and their sodium salts and potassium salts; phosphates such as sodium phosphate and sodium hydrogen phosphate; and carbonates such as sodium carbonate and sodium hydrogen carbonate.

[0035] [Preservatives / Antimicrobial Agents] Examples of preservatives / antimicrobial agents include benzoic acid and its salts, parabens (parahydroxybenzoic acid esters) such as methylparaben, ethylparaben, propylparaben, and butylparaben, sorbic acid and its salts, ethylenediaminetetraacetate, and benzalkonium chloride.

[0036] [Other Sweeteners] Other sweeteners include saccharin sodium, aspartame, trehalose, stevioside, stevia extract, p-methoxycinnamaldehyde, neohesperidyl dihydrochalcone, perillartine, and xylitol.

[0037] [Active Ingredient] The cytokine production-suppressing composition of the present invention may further contain active ingredients in addition to those described above. Such active ingredients include lysozyme chloride, sodium fluoride, potassium fluoride, sodium monofluorophosphate, potassium nitrate, sodium polyphosphate, polyethylene glycol, polyvinylpyrrolidone, hinokitiol, ascorbic acid (vitamin C), ascorbate salts, chlorhexidine salts, cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, bisabolol, triclosan, isopropylmethylphenol, tocopherol, tocopherol acetate, ε-aminocaproic acid, tranexamic acid, aluminum hydroxyl allantoin, aluminum lactate, dihydrocholesterol, glycyrrhetinic acid, glycyrrhizinate salts, copper chlorophyllin salt, sodium chloride, guaiazulene sulfonate, dextranase, pyridoxine hydrochloride, and medicinal hydroxyapatite, and one or more of these may be incorporated.

[0038] [Dosage Form] The dosage form of the cytokine production-inhibiting composition of the present invention is not particularly limited. When the cytokine production-inhibiting composition is a liquid preparation, water or a lower alcohol having 3 or less carbon atoms, such as ethanol or propanol, may be blended as a solvent. The dosage form of the cytokine production-inhibiting composition may be selected from toothpaste, gel, liquid toothpaste, powder toothpaste, mouthwash, film, chewing gum, pet food additive, pasta, etc.

[0039] The amount of water to be added is not particularly limited as long as the CI content is appropriate, but is preferably 20 to 98% by mass per 100 parts by mass of the cytokine production inhibitory composition, and if a lower alcohol is added, the content is preferably 1 to 20% by mass.

[0040] The cytokine production-suppressing composition can be obtained by mixing various components, for example, by mixing the various components in a solvent such as water and stirring the mixture.

[0041] The pH of the cytokine production-inhibitory composition at 25° C. is preferably 4.0 to 9.0, and more preferably 5.0 to 8.0. When used as a paste-like composition, the pH at 25° C. is preferably 5.5 to 9.0, when used as a spray composition, the pH is preferably 4.5 to 8.5, and when used as an impregnation solution for a sheet or the like, the pH is preferably 4.5 to 8.5.

[0042] Furthermore, from the viewpoint of usability, the viscosity of the cytokine production-suppressing composition at 25°C, as measured using a Brookfield viscometer, is preferably 1 to 50,000 mPa·s, and more preferably 5 to 20,000 mPa·s. When used as a gel or paste composition, the viscosity at 25°C is preferably 10 to 50,000 mPa·s, when used as a spray composition it is preferably 1 to 1,000 mPa·s, and when used as an impregnation liquid for sheets or the like it is preferably 1 to 500 mPa·s.

[0043] [Method of Use] The cytokine production-suppressing composition of the present invention is applied to the oral cavity of pets, particularly dogs or cats. The oral composition is first applied to the tooth surfaces, gums, spaces between teeth, and spaces between the teeth and gums. Application methods include application with the owner's finger, application with tools such as a toothbrush, cotton swab, appropriately shaped gauze, sheet, toothbrushing toy, or rope, or application directly from a container equipped with a nozzle or spray bottle. After application, the owner's finger or the tool may be used to scrub and clean the tooth surfaces, spaces between teeth, and spaces between the teeth and gums. The tool may be a set in which the cytokine production-suppressing composition has been pre-applied by application or impregnation, or the cytokine production-suppressing composition may be applied to the tool immediately before application. The liquid composition may also be administered as a stock solution or by mixing with drinking water.

[0044] The cytokine production-inhibiting composition may also be incorporated into dog food, cat food, snacks, or supplements, and provided to pets in the form of eating the dog food or the like.

[0045] The cytokine production-inhibiting composition of the present invention contains at least one compound selected from cyclodextran and its derivatives. Cyclodextran is a cyclic isomaltooligosaccharide in which 4 to 33 glucose units are cyclically linked via α-1,6 glucoside bonds, and is tasteless and odorless. Therefore, it does not affect the taste or aroma of food, making it highly palatable to dogs and cats. Furthermore, cyclodextran is highly water-soluble, dissolving in an equal or less amount of water, making it safe enough for dogs and cats to swallow without gargling. Thus, the present invention provides a cytokine production-inhibiting composition derived from Porphyromonas gulae, the active ingredient of which is itself palatable to pets.

[0046] <Applications of the cytokine production-inhibiting composition> Cyclodextran is stable against heat, acid, and alkali, and therefore can be used in oral care compositions for dogs or cats, periodontal disease preventive agents, oral biofilm formation inhibitors, anti-inflammatory agents, and bad breath suppressants under the same formulation conditions as the cytokine production-inhibiting composition of the present invention.

[0047] <Composition for Inhibiting Glucan Production Derived from Porphyromonas gulae> The present invention also provides a composition for inhibiting glucan production derived from Porphyromonas gulae, comprising at least one compound selected from cyclodextran and its derivatives. While the present invention is not bound by any theory, compositions containing at least one compound selected from cyclodextran and its derivatives have been shown to inhibit the production of water-soluble and water-insoluble glucans derived from Porphyromonas gulae. Therefore, administration of such compositions inhibits biofilm formation in the oral cavity of dogs and cats. Furthermore, the inhibition of biofilm formation inhibits the adhesion of bacteria such as Porphyromonas gulae in the oral cavity of dogs and cats, thereby suppressing the occurrence of inflammation and maintaining a healthy oral cavity.

[0048] In addition, with regard to the various optional components that can be incorporated into the glucan production-inhibiting composition of this embodiment, as well as the dosage form and method of use of the glucan production-inhibiting composition, the contents explained in relation to the cytokine production-inhibiting composition are directly incorporated.

[0049] <Applications of the glucan production-inhibiting composition> Because cyclodextran is stable against heat, acid, and alkali, it can be used as an oral biofilm formation inhibitor or anti-inflammatory agent for dogs or cats under the same formulation conditions as the glucan production-inhibiting composition of the present invention. In other words, the present invention also provides an oral biofilm formation inhibitor or anti-inflammatory agent for dogs or cats that contains the glucan production-inhibiting composition.

[0050] <Composition for Inhibiting the Production of Methyl Mercaptan and / or Hydrogen Sulfide Derived from Porphyromonas gulae> The present invention also provides a composition for inhibiting the production of methyl mercaptan derived from Porphyromonas gulae, which contains at least one compound selected from cyclodextran and its derivatives, and a composition for inhibiting the production of hydrogen sulfide derived from Porphyromonas gulae, which contains at least one compound selected from cyclodextran and its derivatives. While the present invention is not bound by any theory, compositions containing at least one compound selected from cyclodextran and its derivatives have been shown to have the effect of inhibiting the production of methyl mercaptan and hydrogen sulfide derived from Porphyromonas gulae. Therefore, by administering such a composition, the generation of odorous substances that cause bad breath in the oral cavity of dogs and cats is suppressed, resulting in the suppression of bad breath and the maintenance of favorable oral health.

[0051] The contents explained for the cytokine production inhibitory composition are incorporated herein by reference as they relate to the various optional components that can be incorporated into the methyl mercaptan and / or hydrogen sulfide production inhibitory composition of this embodiment, as well as the dosage form and method of use of the methyl mercaptan and / or hydrogen sulfide production inhibitory composition.

[0052] <Application of the Composition That Inhibits Methyl Mercaptan and / or Hydrogen Sulfide Production> Because cyclodextran is stable against heat, acid, and alkali, it can be used in halitosis suppressants for dogs or cats under the same formulation conditions as the composition that inhibits methyl mercaptan and / or hydrogen sulfide production of the present invention. That is, the present invention also provides a halitosis suppressant for dogs or cats containing the composition that inhibits methyl mercaptan and / or hydrogen sulfide production.

[0053] In this study, we confirmed the bactericidal effect of CI against Porphyromonas gulae (P. gulae), a typical periodontal pathogen in dogs and cats, its effect in reducing bad breath, and its anti-inflammatory effect against inflammation induced by periodontal pathogens. As a control, we also conducted similar tests on Porphyromonas gingivalis (P. gingivalis), a typical periodontal pathogen in humans.

[0054] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0055] <Test Method>

[0056] [Test 1] Bactericidal effect of CI on P. gulae Focusing on the periodontal pathogenic bacteria P. gulae and P. gulae, the growth inhibitory effect of CI-Dextran mix (containing at least CI and dextran, with a CI content of 13% or more, Nisshin Sugar Co., Ltd.) co-culture was investigated in vitro. P. gulae was inoculated onto sheep blood agar medium for anaerobic bacteria (Nippon Becton Dickinson Co., Ltd.) and cultured under anaerobic conditions for 3 days, after which the turbidity was adjusted to 1.0 with horse lysed blood-supplemented Brucella broth liquid medium (Kyokuto Pharmaceutical Industries Co., Ltd.). The suspension of S. glae was mixed with CI-Dextran mix at a ratio of 0.01 to 2.5%, and the mixture was cultured at 37°C under anaerobic conditions for 10 minutes, 1 hour, 4 hours, and 24 hours. The viability of the cultured S. glae was then measured using the BacTiter-Glo® Microbial Cell Viability Assay (Promega).

[0057] As a control, a test was also performed in which P. gulae was replaced with P. gingivalis.

[0058] [Test 2] Inhibitory effect of CI-Dextran mix on biofilm formation by P. gulae Periodontal disease bacteria, including P. gulae, adhere to teeth and form biofilms by congregating with other bacteria. Dental plaque, a mass of bacteria, is also a type of biofilm, and the formation of biofilms makes them resistant to external treatments, including antibiotics. In this test, the biofilm formation ability of CI-Dextran mix (0.039% to 5.000%) co-cultured with a Biofilm Formation Assay Kit (DOJINDO) and a P. gulae suspension was investigated.

[0059] 180 μL of the CI-Dextran mix mixed culture solution was added to 20 μL of the P. gulae culture solution prepared in [Test 1], and mixed to a CI-Dextran mix of 0.039% to 5.000%. 200 μL of each concentration was added to 8 wells of a 96-well plate, covered with a 96-peg lid, and cultured for 24 hours under anaerobic conditions in a 37 ° C incubator. After 24 hours, 200 μL of the bacteria-free CI-Dextran mix mixed culture solution was seeded on another 96-well plate, and the 96-peg lid was transferred from the plate after 24 hours of culture and covered, and the plate was cultured for an additional 72 hours under anaerobic conditions in a 37 ° C incubator under anaerobic conditions in the dark. After 72 hours, the 96-peg lid was washed twice with saline, stained with crystal violet for 30 minutes, washed again with saline, and the stained solution was extracted with ethanol. The absorbance of the extracted solution at 590 nm was measured using a plate reader. The same test was repeated twice to confirm reproducibility.

[0060] As a control, a test was also performed in which P. gulae was replaced with P. gingivalis.

[0061] [Test 3] Electron Microscopic Observation of P. gulae and Its Biofilm on Hydroxyapatite Disks The biofilm formation inhibitory effect of CI-Dextran mix confirmed in [Test 2] was confirmed by scanning electron microscopy using hydroxyapatite disks with the same components as actual teeth. This suggests that the excretion and activity of toxins from periodontal disease bacteria may be inhibited by binding to CI-Dextran mix. In this test, the binding ability of P. gulae to each CI-Dextran mix was investigated using a scanning electron microscope. P. gulae cultured in the same manner as [Test 2] was examined. Gulae bacteria were mixed with CI-Dextran mix (1 mL) suspended in culture medium to a final concentration of 5%, and co-cultured with hydroxyapatite disks in a 12-well plate for 24 hours. After 24 hours of co-culture, the culture medium was replaced with a CI-Dextran mix containing no bacteria. After further culture for 72 hours, the periodontal disease bacteria and biofilm on the hydroxyapatite were observed using a scanning electron microscope.

[0062] [Test 4] Effect of CI-Dextran mix on inflammatory cytokine production in mouse macrophage cells (RAW264.7) caused by P. gulae The amount of inflammatory cytokine (IL-6) produced by mouse macrophage cells (RAW264.7) caused by P. gulae treated with low and high concentrations of CI-Dextran mix was measured by ELISA.

[0063] RAW264.7 cells were subcultured in DMEM medium supplemented with 10% fetal bovine serum, and 1 × 10 ４ The cells were seeded at 1000 cells / well into a 96-well culture plate and incubated at 37°C, 5% CO until the cells reached confluence. ２ The bacteria were cultured under the following conditions. Immediately before inoculation with cells, P. gulae was adjusted to a turbidity of 1.0 in each medium containing 10% fetal bovine serum and antibiotics, and purified water or CI-Dextran mix was added at a ratio of 0.0045 to 2.5%. The mixture was then allowed to stand for 5 minutes. The bacterial suspension was mixed with an equal volume of DMEM medium containing 10% fetal bovine serum and antibiotics, and IL-6 in the culture medium 24 hours after inoculation with cells was measured using ELISA.

[0064] As a control, a test was also performed in which P. gulae was replaced with P. gingivalis.

[0065] [Test 5] Statistical Tests For each test item from Test 1 to Test 4, the presence or absence of statistically significant differences between the control group and each bentonite-administered group was analyzed at the 5% and 1% risk levels. Data between the vehicle control group and the test substance group were first analyzed using Bartlett's test for equality of variance. If this test determined that the variances across all dose groups were uniform (p>0.05), a one-way analysis of variance was used to examine the presence or absence of significant differences between the groups. If a significant difference was observed between the groups (p≦0.05), Dunnett's multiple comparison test was performed to determine the presence or absence of significant differences between the control group and each administration group. If Bartlett's test for equality of variance determined that the variances of each group were not equal (p≦0.05), a Kruskal-Wallis test was used to examine the presence or absence of significant differences between the groups. As a result, when a significant difference was observed between the groups (p≦0.05), the presence or absence of a significant difference between the control group and each treatment group was determined using Dunnett's non-parametric multiple comparison method.

[0066] [Test 6] Deodorizing effect of CI-Dextran mix on substances that cause bad breath (e.g., methyl mercaptan) First, we focused on methyl mercaptan, the main substance that causes bad breath in periodontal disease, and investigated the direct deodorizing effect of CI-Dextran mix (containing at least CI and dextran, with a CI-7 to CI-12 content of approximately 20.4%, manufactured by Nisshin Sugar Co., Ltd.; the same applies to Test 6) on methyl mercaptan. A methyl mercaptan standard (Fujifilm Wako Pure Chemical Industries, Ltd.) and the CI-Dextran mix were mixed at a ratio of 0.625 to 2.5% and allowed to stand in a gas collection bottle. The methyl mercaptan concentration after 30 minutes was evaluated using OralChroma (simple gas chromatography, Nissha FIS Inc.). 1 mL of gas was collected using a 1 mL syringe and added to the OralChroma for measurement. Next, we focused on the periodontal pathogen P. gulae, which is closely related to the progression of periodontal disease in dogs, and investigated the inhibitory effect of co-culturing with CI-Dextran mix on the production of halitosis substances in vitro. P. gulae was inoculated onto sheep blood agar medium for anaerobic bacteria (Nippon Becton Dickinson Co., Ltd.) and cultured under anaerobic conditions for 3 days, after which the turbidity was adjusted to 1.0 in horse lysed blood-supplemented Brucella broth liquid medium (Kyokuto Pharmaceutical Industries Co., Ltd.). CI-Dextran mix was mixed with the P. gulae suspension at a ratio of 0.625 to 2.5%, and after 24 hours of culture at 37°C under anaerobic conditions, the production of methyl mercaptan and hydrogen sulfide was measured using OralChroma.

[0067] [Test 7] Inhibitory effect of CI-Dextran mix on insoluble glucan production by P. gulae Periodontal disease bacteria, including P. gulae, adhere to teeth and form biofilms by congregating multiple bacteria. Previous studies have shown that CI-Dextran mix inhibits biofilm formation by P. gulae in a concentration-dependent manner. Biofilms are formed by insoluble glucan (mutan) produced from sugars by glucosyltransferase-I (GTF) and other enzymes. In this experiment, we examined the effect of CI-Dextran mix on the production of insoluble glucan, a major component of biofilms. P. gulae was co-cultured with CI-Dextran mix (0.625-2.5%). A suspension of S. glulae and glucose (2 g / L) was cultured in a 37°C incubator under anaerobic conditions in the dark for 72 hours, followed by repeated centrifugation (10,000 g, 4°C, 10 minutes) and washing with sterile distilled water or cold 70% ethanol. The insoluble glucan obtained by culturing in 1 M NaOH for 2 hours was quantified by the phenol-sulfuric acid method. Glucose was used as the standard sugar.

[0068] [Test 8] Statistical Testing For each test item in Tests 6 and 7, the presence or absence of statistically significant differences between the control group and the CI-Dextran mix-administered group was analyzed at the 5% and 1% risk levels. A one-way analysis of variance was used to examine the presence or absence of significant differences between the groups. If a significant difference was observed between the groups (p≦0.05), Dunnett's multiple comparison test was performed to determine the presence or absence of significant differences between the control group and each administration group.

[0069] <Results> [Test 1] Bactericidal effect of CI-Dextran mix against P. gulae The results of Test 1 are shown in Figure 3. No bactericidal effect on P. gulae was observed when mixing with CI-Dextran mix. Similarly, no bactericidal effect was observed on P. gingivalis.

[0070] A slight decrease in the number of bacteria was observed one hour after culturing P. gingivalis, but there was no effect at other times, and the decrease was very slight, so it was determined that there was no bactericidal effect. Although there were occasional points at which statistically significant increases were observed at other time points, there was wide variation in these results, suggesting that there was no effect on the number of live bacteria.

[0071] [Test 2] Inhibitory effect of CI-Dextran mix on biofilm formation by P. gulae The results of Test 2 are shown in Figure 4. Significant inhibition of biofilm formation was observed for both P. gulae and P. gingivalis (control), periodontal disease bacteria, at concentrations of 2.5% or higher compared to the control, but the effect was more pronounced for P. gulae. In both experiments, biofilm formation was significantly suppressed at a 5% concentration compared to the control (containing no CI-Dextran mix).

[0072] [Test 3] Electron microscopic observation of P. gulae and its biofilm on hydroxyapatite discs. P. gulae was co-cultured with 5% CI-Dextran mix on hydroxyapatite discs according to the same schedule as in the biofilm formation evaluation. Figure 5 shows the observation results for the control, and Figure 6 shows the observation results for the 5% CI-Dextran mix. Figures 5 and 6 confirm that CI-Dextran inhibits biofilm formation by P. gulae.

[0073] [Test 4] Effect of CI-Dextran mix on inflammatory cytokine production in mouse macrophage cells (RAW264.7) induced by P. gulae The results of Test 4 are shown in Figure 7. The effect of CI-Dextran mix on anti-inflammatory effects was investigated by measuring the amount of inflammatory cytokine (IL-6) produced by macrophage cells induced by P. gulae that had been previously treated with CI-Dextran mix. Compared to the untreated group (Untreated), the group to which only P. gulae was added to the culture medium showed a significant increase in IL-6 production, while the increase in production was significantly reduced by CI-Dextran mix treatment in a concentration-dependent manner.

[0074] The amount of IL-6 produced also increased significantly in the group in which only P. gingivalis was added to the culture medium, and treatment with the CI-Dextran mix did not have any inhibitory effect on cytokine production.

[0075] This suggests that cyclodextran and its derivatives can specifically suppress the production of cytokines derived from P. gulae. It is speculated that the excretion and activity of toxins from the periodontal disease bacterium P. gulae are specifically inhibited by binding to cyclodextran and its derivatives.

[0076] [Test 6] Deodorizing effect of CI-Dextran mix on substances that cause bad breath (e.g., methyl mercaptan) Figure 8 shows the results of [Test 6]: Deodorizing effect of CI-Dextran mix on substances that cause bad breath (e.g., methyl mercaptan). No direct deodorizing effect on methyl mercaptan was observed when CI-Dextran mix was added. However, co-culturing P. glae with CI-Dextran mix resulted in a concentration-dependent decrease in the production of methyl mercaptan and hydrogen sulfide. Methyl mercaptan production was significantly inhibited by 1.25% and 2.5% CI-Dextran mix, and hydrogen sulfide production was significantly inhibited by 2.5% CI-Dextran mix.

[0077] [Test 7] Investigation of the inhibitory effect of CI-Dextran mix on insoluble glucan production by P. gulae The results of evaluating the insoluble glucan formation ability when CI-Dextran mix was co-cultured with P. gulae at concentrations of 0.625% to 2.5% are shown in Figure 9. The amount of insoluble glucan (mutan) produced decreased in a concentration-dependent manner with CI-Dextran mix concentrations of 1.25% and 2.5% showing a significant decrease compared to the control.

[0078] <Discussion> In this study, we investigated the bactericidal effect of CI-Dextran mix against P. gulae, a typical periodontal pathogen in dogs and cats, its biofilm-forming ability, and its anti-inflammatory effect against the inflammatory response induced by P. gulae.

[0079] When the periodontal bacterial solution was mixed with the CI-Dextran mix, no bactericidal effect was observed between 10 minutes and 24 hours after exposure.

[0080] Next, we investigated the effect of CI-Dextran mix on biofilm formation by periodontal disease bacteria. Results showed that P. gulae concentrations of 2.5% or higher significantly inhibited biofilm formation compared to the control, while biofilm formation was virtually nonexistent at 5% concentrations. Furthermore, the inhibitory effect on P. gulae was greater than that on P. gingivalis.

[0081] Furthermore, the inhibitory effects of 5% CI-Dextran mix on the growth of P. gulae and on biofilm formation were also clearly observed through electron microscopy using hydroxyapatite discs.

[0082] We also investigated whether the CI-Dextran mix affected the levels of inflammatory cytokines produced by macrophages, which are primarily involved in oral inflammation. The results showed that exposure to the CI-Dextran mix significantly reduced IL-6 production induced by P. glae, which is associated with gingivitis, periodontitis, and tooth decay. However, no effect was observed on cytokine production induced by P. gingivalis.

[0083] These results suggest that cyclodextran significantly inhibits glucan formation by P. gulae and P. gingivalis, thereby inhibiting biofilm formation and subsequent plaque formation. Furthermore, by preventing the colonization of periodontal disease bacteria, cyclodextran is expected to have the effect of suppressing bad breath and the progression of inflammation, making it useful for periodontal disease care in humans and small animals.

Claims

1. A composition for inhibiting the production of cytokines derived from Porphyromonas gulae, which contains at least one compound selected from cyclodextran and its derivatives.

2. An oral care composition for dogs or cats containing the inhibitory composition of claim 1.

3. A periodontal disease preventive agent for dogs or cats containing the inhibitory composition according to claim 1.

4. An oral biofilm formation inhibitor for dogs or cats, comprising the inhibitory composition described in claim 1.

5. An anti-inflammatory agent for dogs or cats containing the inhibitory composition according to claim 1.

6. A breath odor suppressant for dogs or cats, comprising the suppressant composition according to claim 1.

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