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

To provide: a Porphyromonas gulae-derived cytokine production inhibitory composition whose active ingredients themselves are compatible with pets' palatability; an oral care composition for a dog or 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 or more compounds selected from among cyclodextran and derivatives thereof. The inhibitory composition can be applied to an oral care composition for a dog or cat, a periodontal disease-preventing agent, an intraoral biofilm formation inhibitor, an anti-inflammatory agent, and a halitosis inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] Dogs and cats, like humans, suffer from various oral problems such as periodontal disease. The oral environments of humans and pets are completely different, so the bacterial flora present in the oral cavity is also different. Specifically, the main bacterium responsible for periodontal disease in humans is Porphyromonas gingivalis, while the main bacterium responsible for periodontal disease in dogs and cats is Porphyromonas gulae, which is different from the bacterium responsible for human periodontal disease.

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

[0004] [Patent Document 1] JP 2015-067539 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, since the Angelica extract liquid is not palatable to pets, in the case of the preventive agent described in Patent Document 1, it is required to use it in combination with one or more selected from carrageenan, pectin, gellan gum, alginic acid and its salts. Pets do not accept things that are not palatable, so palatability is extremely important when using oral compositions for pets. In addition, since pets swallow without gargling, high safety is required, and the method of use is different from that of humans. For this reason, there are many cases where the technology of oral compositions for humans cannot be applied directly to pets. Therefore, there is a demand for a composition in which the active ingredient that contributes to the prevention of periodontal disease itself matches the palatability of pets without being used in combination with other ingredients, and which has even greater freedom of development.

[0006] The present invention has been made in consideration of such 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 inhibitor of oral biofilm formation, an anti-inflammatory agent, and an agent for suppressing bad breath, the active ingredients of which themselves match the preferences of pets. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have found that cyclodextran, a cyclic oligosaccharide, itself exhibits a 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] The second aspect of the invention provides an oral care composition for dogs or cats, which contains the inhibitory composition according to the first aspect. The third aspect of the invention provides a periodontal disease preventive agent for dogs or cats, which contains the inhibitory composition according to the first aspect. The fourth aspect of the invention provides an oral biofilm formation inhibitor for dogs or cats, which contains the inhibitory composition according to the first aspect. The fifth aspect of the invention provides an anti-inflammatory agent for dogs or cats, which contains the inhibitory composition according to the first aspect. The sixth aspect of the invention provides a breath odor suppressant for dogs or cats, which contains the inhibitory composition according to the first aspect.

[0010] FIG. 1 is an example of the structural formula of cyclodextran. Cyclodextran is a cyclic isomaltooligosaccharide in which 4 to 33 glucose units are cyclically linked by α-1,6 glucoside bonds, and is tasteless and odorless. Therefore, it does not affect the taste or aroma of food, and is highly popular with dogs and cats. In addition, cyclodextran is extremely water-soluble and dissolves in an equal or less amount of water, so it is safe enough that dogs and cats can swallow it without gargling. In addition, cyclodextran is stable against heat, acid, and alkali, so there is a high degree of freedom in development when providing oral care compositions, periodontal disease preventive agents, oral biofilm formation inhibitors, anti-inflammatory agents, and oral odor suppressants. Effect of the Invention

[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 inhibitor of oral biofilm formation, an anti-inflammatory agent, and an agent for suppressing bad breath, the active ingredients of which themselves match the preferences of pets. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is an example of the structural formula of cyclodextran (CI). [Diagram 2] FIG. 2 is an example of the structural formula of cyclodextrin (CD). [Diagram 3]Figure 3 shows the results of the bactericidal effect of [Test 1] CI-Dextran mix against P. gulae. [Figure 4] Figure 4 shows the results of the inhibitory effect of [Test 2] CI-Dextran mix on P. gulae biofilm formation (relationship between CI-Dextran mix concentration and OD). [Diagram 5] Figure 5 shows the results of electron microscopic observation of P. gulae and its biofilm on a hydroxyapatite disk [Test 3] (control). [Figure 6] Figure 6 shows the results of electron microscopic observation of P. gulae and its biofilm on a hydroxyapatite disk [Test 3] (CI-Dextran mix concentration 5%). [Figure 7] Figure 7 is a diagram showing the effect of CI-Dextran mix on the production of inflammatory cytokines by mouse macrophage cells by P. gulae [Test 4]. [Figure 8] Figure 8 shows the results of the deodorizing effect of [Test 6] CI-Dextran mix on malodor-causing substances (such as methyl mercaptan). [Figure 9] Figure 9 shows the results of the investigation of the inhibitory effect of [Test 7] CI-Dextran mix on the production of insoluble glucan by P. gulae bacteria.

Mode for Carrying Out the Invention

[0013] Hereinafter, specific embodiments of the present invention will be described in detail. However, 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] <Cytokine Production Inhibiting Composition Derived from Porphyromonas gulae> The cytokine production inhibiting composition according to the present invention contains at least one or more compounds selected from cyclodextrin 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 glucoside 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 points. First, CI has 4 to 33 glucose units linked, while CD has 6 to 8 glucose units linked. Second, CI has glucose units linked by α-1,6 glucoside bonds, while CD has glucose units linked by α-1,4 glucoside bonds. Third, the molecular structure of CI has a large and shallow aperture, while the molecular structure of CD has a small and deep aperture. 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 solution of cyclic isomaltose synthase (see 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 the mixture.

[0018] [Optional ingredients (additives)] In addition to at least one type selected from CI and its derivatives, the cytokine production inhibitory composition of the present invention can contain optional components (additives) alone or in appropriate combinations of two or more types in normal amounts 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] [Surfactants] The surfactant can be any surfactant that can be used without particular limitation as long as it is blended 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 sucrose fatty acid esters, which are fatty acid esters of sugars or sugar alcohols, 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, alkylolamides, etc. It is preferable to use fatty acids having 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 carbon number of 10 to 16, sodium lauryl sulfate, sodium N-acyl glutamate such as sodium N-lauroyl glutamate and sodium N-palmitoyl glutamate, sodium N-acyl sarcosine such as sodium N-lauroyl sarcosine and sodium N-myristoyl sarcosine, sodium N-methyl-N-acyltaurate such as sodium N-lauroyl methyl taurine and sodium N-myristoyl methyl taurine, etc.

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

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

[0025] [Abrasive] 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, magnesium triphosphate, zirconium silicate, calcium triphosphate, hydroxyapatite, calcium quaternary phosphate, and synthetic bark-based abrasives.

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

[0027] [Wetting agent] As the humectant, one or more of 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 foaming agents include sodium lauryl sulfate, sodium lauroyl sarcosine, 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 diethanolamide, polyoxyethylene sorbitan monostearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid esters, and the like, and one or more of these can be used in combination.

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

[0030] [Fragrance] As the flavoring, it is preferable to use one or more types of oil and fat flavorings such as natural flavorings and synthetic flavorings, and powder flavorings, but there is no particular limitation. As the flavoring, it is preferable to use one or more types of oil and fat flavorings such as natural flavorings and synthetic flavorings, and powder flavorings, but there is no particular limitation. For example, natural flavorings 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, absolute peppermint, absolute rose, orange flower, citrus oil, mixed fruit oil, strawberry oil, cinnamon oil, clove oil, grape oil, etc.

[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, an extract, a solid, or a powder obtained by spray drying these.

[0032] [Acidulant] Acidulants include organic acids such as citric acid, malic acid, succinic acid, and tartaric acid.

[0033] [Coloring agent] Examples of coloring agents include natural dyes such as safflower red dye, gardenia yellow dye, gardenia blue dye, perilla dye, red koji dye, red cabbage dye, carrot dye, hibiscus dye, cacao dye, spirulina blue dye, and coumarind dye; legal dyes 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, sodium copper chlorophyll, and titanium dioxide.

[0034] [pH adjuster] Examples of pH adjusters include organic acids such as citric acid, malic acid, tartaric acid, succinic acid, and their sodium salts or 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] Preservatives and antiseptics include benzoic acid and its salts, parabens (paraoxybenzoic acid esters) such as methylparaben, ethylparaben, propylparaben, and butylparaben, sorbic acid and its salts, ethylenediaminetetraacetate, and benzalkonium chloride.

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

[0037] [Active ingredients] The cytokine production suppression composition of the present invention may contain other active ingredients in addition to the 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, medicinal hydroxyapatite, etc., and one or more of these may be contained.

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

[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 mass% per 100 mass parts of the cytokine production inhibitory composition, and if a lower alcohol is added, the content is preferably 1 to 20 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-inhibiting composition at 25° C. is preferably 4.0 to 9.0, and more preferably 5.0 to 8.0. When the composition is in the form of a paste, the pH at 25° C. is preferably 5.5 to 9.0, when the composition is for spraying, the pH is preferably 4.5 to 8.5, and when the composition is for impregnation of a sheet or the like, the pH is preferably 4.5 to 8.5.

[0042] 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 a sheet or the like it is preferably 1 to 500 mPa·s.

[0043] [How to use] The cytokine production suppression composition of the present invention is applied to the oral cavity of pets, particularly dogs or cats. The method of use is to first apply the oral composition to the tooth surface, gums, between teeth, between teeth, etc. Examples of application methods include application with the owner's finger, application with tools such as toothbrushes, cotton swabs, appropriately shaped gauze, sheets, or toys or ropes for brushing teeth, and application directly by filling the composition into a container with a nozzle or a spray, etc. After application, the owner's finger or the above tool may be used to scrub and clean the surface of the teeth, between teeth, and between teeth and gums. The above tool may be a set in which the cytokine production suppression composition is attached to the tool in advance by application or impregnation, or the cytokine production suppression composition may be attached 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-suppressing composition may also be incorporated into dog food, cat food, snacks, or supplements, and provided to pets in the form of dog food or the like.

[0045] The cytokine production inhibitory composition of the present invention contains at least one compound selected from cyclodextrin and its derivatives. Cyclodextrin is a cyclic isomaltooligosaccharide in which 4 to 33 glucoses are linked cyclically by α-1,6 glucoside bonds, has no sweetness, and is tasteless and odorless. Therefore, since it does not affect the taste and aroma of food, it has high palatability for dogs and cats. In addition, cyclodextrin has extremely high water solubility and dissolves in water equal to or less than its own weight, so it has sufficient safety that there is no problem even if dogs and cats gargle and swallow it. Therefore, according to the present invention, a cytokine production inhibitory composition derived from Porphyromonas gulae in which the active ingredient itself matches the palatability of pets can be provided.

[0046] <Application of the cytokine production inhibitory composition> Since cyclodextrin is stable against heat, acids, and alkalis, it can be applied to oral care compositions, periodontal disease preventives, oral biofilm formation inhibitors, anti-inflammatory agents, and bad breath suppressants for dogs or cats under the same formulation conditions as the cytokine production inhibitory composition of the present invention.

[0047] <Glucan production inhibitory composition derived from Porphyromonas gulae> The present invention further provides a glucan production inhibitory composition derived from Porphyromonas gulae containing at least one compound selected from cyclodextrin and its derivatives. Although the present invention is not bound by any theory, compositions containing at least one compound selected from cyclodextrin and its derivatives are also recognized to have the effect of suppressing the production of water-soluble glucan and insoluble glucan derived from Porphyromonas gulae. Therefore, by taking such a composition, the formation of biofilm is inhibited in the oral cavity of dogs and cats. And by inhibiting the formation of biofilm, the adhesion of bacterial cells such as Porphyromonas gulae in the oral cavity of dogs and cats is suppressed, so as a result, the occurrence of inflammation is suppressed and the oral health state is preferably maintained.

[0048] Note that regarding various optional components that can be incorporated into the glucan production inhibitory composition of the present embodiment, and the dosage form and method of use of the glucan production inhibitory composition, the content described in the cytokine production inhibitory composition is incorporated as it is.

[0049] <Application of Glucan Production Inhibitory Composition> Since cyclodextrin is stable against heat, acids, and alkalis, it can be applied to an inhibitor for inhibiting the formation of oral biofilms and an anti-inflammatory agent for dogs or cats under the same blending conditions as the glucan production inhibitory composition of the present invention. That is, the present invention also provides an inhibitor for inhibiting the formation of oral biofilms and an anti-inflammatory agent for dogs or cats containing the glucan production inhibitory composition.

[0050] <Composition for Inhibiting the Production of Methyl Mercaptan and / or Hydrogen Sulfide Derived from Porphyromonas gulae> The present invention further provides a composition for inhibiting the production of methyl mercaptan derived from Porphyromonas gulae containing at least one compound selected from cyclodextrin and its derivatives, and a composition for inhibiting the production of hydrogen sulfide derived from Porphyromonas gulae containing at least one compound selected from cyclodextrin and its derivatives. Although the present invention is not bound by any theory, in a composition containing at least one compound selected from cyclodextrin and its derivatives, an action of inhibiting the production of methyl mercaptan and hydrogen sulfide derived from Porphyromonas gulae is also recognized. Therefore, by taking such a composition, the generation of odor-causing substances in the oral cavity of dogs and cats is suppressed, and as a result, the generation of bad breath is suppressed, and the oral health state is suitably maintained.

[0051] Note that regarding various optional components that can be incorporated into the composition for inhibiting the production of methyl mercaptan and / or hydrogen sulfide of the present embodiment, and the dosage form and method of use of the composition for inhibiting the production of methyl mercaptan and / or hydrogen sulfide, the content described in the cytokine production inhibitory composition is incorporated as it is.

[0052] <Application of methyl mercaptan and / or hydrogen sulfide production inhibitor composition> Cyclodextran is stable against heat, acid, and alkali, and therefore can be used in a dog or cat bad breath suppressant under the same formulation conditions as the methyl mercaptan and / or hydrogen sulfide production inhibitor composition of the present invention. That is, the present invention also provides a dog or cat bad breath suppressant containing methyl mercaptan and / or hydrogen sulfide production inhibitor composition. EXAMPLES

[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 of reducing bad breath, and its anti-inflammatory effect against inflammation induced by periodontal bacteria. As a control, we also conducted similar confirmations for Porphyromonas gingivalis (P.gingivalis), a typical periodontal pathogen in humans.

[0054] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. There is none.

[0055] <Test Method>

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

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

[0058] [Test 2] Inhibitory effect of CI-Dextran mix on biofilm formation by P. gulae Periodontal disease bacteria, including P. gulae, form biofilms by adhering to teeth and gathering together. Dental plaque, a mass of bacteria, is also a type of biofilm, and the formation of a biofilm makes it resistant to external treatments, including antibiotics. In this study, we investigated the biofilm formation ability of P. gulae in co-culture with a CI-Dextran mix (0.039%-5.000%) using the Biofilm Formation Assay Kit (DOJINDO) and a suspension of P. gulae bacteria.

[0059] 180μL of CI-Dextran mix mixed bacteria solution was added to 20μL of P.gulae bacteria solution prepared in [Test 1], and mixed to CI-Dextran mix 0.039% to 5.000%. 200μL was added to a 96-well plate so that each concentration was 8 wells, and the plate was covered with a 96-peg lid and cultured for 24 hours in a 37℃ incubator under light-shielded anaerobic conditions. After 24 hours, 200μL of CI-Dextran mix mixed culture solution without bacteria was seeded on another 96-well plate, and the plate was transferred from the plate after 24 hours of culture and covered with a 96-peg lid, and cultured for another 72 hours in a 37℃ incubator under light-shielded anaerobic conditions. After 72 hours, the 96-peg lid was washed twice with saline, stained with crystal violet solution for 30 minutes, washed again with saline solution, and the stained solution was extracted with ethanol, and the 590nm absorbance of the extracted solution was measured with a plate reader. The test was repeated twice under the same conditions to confirm reproducibility.

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

[0061] [Test 3] Electron microscopic observation of P. gulae and its biofilm on hydroxyapatite disks The inhibitory effect of CI-Dextran mix on biofilm formation confirmed in [Test 2] was confirmed by scanning electron microscopy using hydroxyapatite disks similar to the components of actual teeth. It is suggested 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 bacteria to each CI-Dextran mix was investigated by scanning electron microscopy. P.gulae bacteria cultured in the same manner as in [Test 2] 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 which the culture medium was replaced with CI-Dextran mix containing no bacteria, and the periodontal disease bacteria and biofilm on the hydroxyapatite after further culture for 72 hours were observed by scanning electron microscopy.

[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) infected with 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 100 cells / well in a 96-well culture plate and cultured at 37°C under 5% CO2 conditions until the cells reached confluence. Just before inoculation of the cells, P. gulae was adjusted to a turbidity of 1.0 in each medium without 10% fetal bovine serum or antibiotics, and purified water or CI-Dextran mix was mixed at a ratio of 0.0045-2.5% and left to stand for 5 minutes. The bacterial solution was mixed with the same amount of DMEM medium without 10% fetal bovine serum or antibiotics, and IL-6 in the culture medium 24 hours after inoculation was measured using ELISA.

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

[0065] [Test 5] Statistical Test 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% levels of risk. For data between the vehicle control group and the test substance group, Bartlett's test for equal variances was first performed. If this test determined that the variances in all dose groups were uniform (p>0.05), the presence or absence of significant differences between the groups was examined using one-way analysis of variance. 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 administered group. If the Bartlett's test for equal variances determined that the variances of each group were not equal (p≦0.05), the Kruskal-Wallis test was used to determine the presence or absence of significant differences between the groups. If a significant difference was observed between the groups (p≦0.05), the presence or absence of significant differences between the control group and each administered group was examined using a Dunnett-type nonparametric multiple comparison test.

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

[0067] [Test 7] Investigation of the inhibitory effect of CI-Dextran mix on insoluble glucan production by P. gulae Periodontal disease bacteria, including P. gulae, form biofilms by adhering to teeth and gathering together. 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 solution and glucose (2g / L) were co-cultured with CI-Dextran mix (0.625-2.5%) in a 37°C incubator under anaerobic conditions and dark conditions for 72 hours, and then centrifuged (10,000g, 4°C, 10 minutes) and washed with sterile distilled water or cold 70% ethanol repeatedly. Insoluble glucan obtained by culturing in 1M NaOH for 2 hours was quantified by the phenol-sulfuric acid method. Glucose was used as the standard sugar.

[0068] [Exam 8] Statistical Test For each test item in Study 6 and Study 7, the presence or absence of statistically significant differences between the control group and the CI-Dextran mix group was analyzed at the risk level of 5% and 1%. The presence or absence of significant differences between the groups was examined using one-way analysis of variance. If a significant difference was found 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 treatment group.

[0069] <Result> [Test 1] Bactericidal effect of CI-Dextran mix against P.gulae The results of Test 1 are shown in Figure 3. No bactericidal effect was observed against P.gulae by the addition of CI-Dextran mix. Similarly, no bactericidal effect was observed against 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. There were also points where a statistically significant increase was observed on the other time axes, but all of these were widely scattered, 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. Both periodontal disease bacteria, P. gulae and P. gingivalis (control), showed significant inhibition of biofilm formation at concentrations of 2.5% or more compared to the control, but the effect was more pronounced for P. gulae. In both experiments, biofilm formation was significantly suppressed at a concentration of 5% compared to the control (not containing CI-Dextran mix).

[0072] [Test 3] Electron microscopic observation of P. gulae and its biofilm on hydroxyapatite disks CI-Dextran mix 5% and P.gulae were co-cultured on hydroxyapatite disks using 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 CI-Dextran mix 5%. 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) caused by P.gulae The results of Test 4 are shown in Figure 7. The effect of CI-Dextran mix on anti-inflammation was investigated by measuring the amount of inflammatory cytokines (IL-6) produced by macrophage cells caused by P.gulae that had been treated in advance with CI-Dextran mix. Compared to the untreated group, the group in which only P.gulae was added to the culture medium showed a significant increase in the amount of IL-6 produced, while the increase in the amount of production was significantly reduced in a concentration-dependent manner by treatment with CI-Dextran mix.

[0074] The amount of IL-6 produced was also significantly increased in the group in which only P. gingivalis was added to the culture medium, and the CI-Dextran mix treatment did not have an 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 of toxins and activity of 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 against substances that cause bad breath (methyl mercaptan, etc.) Figure 8 shows the results of [Test 6] on the deodorizing effect of CI-Dextran mix on substances that cause bad breath (methyl mercaptan, etc.). No direct deodorizing effect on methyl mercaptan was observed when CI-Dextran mix was mixed. On the other hand, by co-culturing P. gulae with CI-Dextran mix, the production of methyl mercaptan and hydrogen sulfide decreased in a concentration-dependent manner, and the production of methyl mercaptan was significantly inhibited by CI-Dextran mix at concentrations of 1.25% and 2.5%, and hydrogen sulfide was significantly inhibited by CI-Dextran mix at a concentration of 2.5%.

[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 depending on the concentration of CI-Dextran mix, and a significant decrease was observed at concentrations of 1.25% and 2.5% compared to the control.

[0078] <Consideration> 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 10 minutes to 24 hours after exposure.

[0080] Next, we investigated the effect of CI-Dextran mix on biofilm formation from periodontal disease bacteria. As a result, significant inhibition of biofilm formation was observed at P.gulae concentrations of 2.5% or more compared to the control, and biofilm formation was almost not observed at a concentration of 5%. Furthermore, the inhibitory effect of P.gulae was greater than that of P.gingivalis.

[0081] Furthermore, the inhibitory effect of 5% CI-Dextran mix on the proliferation and biofilm formation of P. gulae was also clearly observed by electron microscopy using hydroxyapatite disks.

[0082] We also examined whether the CI-Dextran mix affected the amount of inflammatory cytokines produced by macrophage cells, which are primarily involved in inflammation in the oral cavity. As a result, IL-6 production induced by P.gulae, which is related to gingivitis, periodontitis, and tooth dissolution, was significantly reduced by exposure to the CI-Dextran mix. However, no effect was observed on cytokine production induced by P.gingivalis.

[0083] Cyclodextran significantly inhibited glucan formation by P.gulae and P.gingivalis, suggesting the possibility that it may inhibit biofilm formation and subsequent plaque formation. In addition, since it is expected to have the effect of suppressing bad breath and the progression of inflammation by preventing the establishment of periodontal disease bacteria, cyclodextran is considered to be useful for periodontal disease care in humans and small animals.

Claims

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

2. 10. An oral care composition for dogs or cats comprising the inhibitor composition of claim 1.

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

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

5. An anti-inflammatory agent for dogs or cats, comprising 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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