Inhibitor of cysteine ​​protease activity produced by periodontal disease-causing bacteria

Abstract

To provide a composition used for inhibiting proteolytic activity of periodontal pathogen-producing cysteine protease, a pathogenic factor of periodontal pathogen.SOLUTION: A composition for inhibiting activity of periodontal pathogen-producing cysteine protease, particularly a composition for inhibiting activity of gingipain, is prepared by using at least one selected from the group consisting of processed products of hinokitiol and Paeonia lactiflora as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition used for suppressing the activity of cysteine protease produced by periodontal disease-causing bacteria.

Background Art

[0002] Periodontal disease is a chronic infectious disease caused by the infection of periodontal disease-causing bacteria. When inflammation progresses due to the infection of the causative bacteria, not only does the gum swell and bleed or pus, but the alveolar bone that supports the teeth is destroyed and eventually the teeth are lost. Furthermore, periodontal disease is known to be a risk factor for systemic diseases such as diabetes, heart disease, and low birth weight in pregnant women. Therefore, the prevention or treatment of periodontal disease is very important not only for maintaining and restoring healthy oral function but also for reducing the risk of systemic diseases.

[0003] In recent years, among periodontal disease-causing bacteria, Porphyromonas gingivalis, a Gram-negative anaerobic rod (hereinafter referred to as "P. gingivalis"), is the most important causative bacterium, and it is known that the proteases produced by it decompose periodontal tissue components such as collagen and serum proteins involved in the body's defense system, and it has been clarified that it is deeply related to the pathogenicity of periodontal disease. In particular, basic amino acid-gingipain such as arginine-gingipain is a cysteine protease having trypsin-like protease activity produced by P. gingivalis, and is considered to be the most potent pathogenic factor for periodontal disease. Specifically, basic amino acid-gingipain has an important function in obtaining nutrients for the bacteria themselves, and further decomposes the extracellular matrix constituting the periodontal tissue, such as collagen and fibronectin, decomposes antibodies and complements, which are important elements of the biological reaction to eliminate the bacteria, and further inhibits the function of neutrophils, thereby sustaining the infection and chronicizing the inflammation. Therefore, in order to treat or prevent periodontal disease, the development of a preparation for suppressing the activity of basic amino acid-gingipain is expected.

[0004] Among the basic amino acid gingipaines, cysteine ​​protease inhibitors such as antipain, leupeptin, TLCK (tosyl-lysine-chloromethyl ketone hydrochloride), or E-64 are known to inhibit the activity of arginine-gingipain. Furthermore, focusing on the substrate characteristic of arginine-gingipain that selectively degrades the arginine terminus, peptide derivatives have been proposed as antagonistic inhibitors (see Patent Documents 1 or 2). However, the safety of any of these has not been confirmed, and they have not been put into practical use. In addition, since gabexate mesylate, which is used as a treatment for pancreatitis and disseminated intravascular coagulation, exhibits a high specific activity inhibitory effect on arginine-gingipain, various arginine-gingipain activity inhibitors using it have been proposed (see Patent Documents 3 to 6).

[0005] On the other hand, hinokitiol has long been known for its antibacterial properties and, due to its broad antibacterial spectrum, is widely used as a food preservative, hair growth agent, and cosmetic additive. It is also known to have matrix metalloproteinase activity inhibitory properties, but it is not known to inhibit the activity of cysteine ​​proteases, particularly the basic amino acid gingipain, which have trypsin-like protease activity produced by periodontal disease-causing bacteria. Furthermore, peony is known to have muscle relaxation, vasodilation, anti-inflammatory, sedative, and analgesic effects, and its roots are used as a raw material for crude drugs (Japanese Pharmacopoeia). Peony extract is also listed in the standards for quasi-drug raw materials and has been used as a raw material for quasi-drugs for a long time. However, it is not known that peony or its extract has the effect of inhibiting the activity of cysteine ​​proteases, particularly the basic amino acid gingipain, produced by periodontal disease-causing bacteria. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 11-228526 [Patent Document 2] Japanese Patent Publication No. 2001-89436 [Patent Document 3] Japanese Patent Publication No. 2008-150325 [Patent Document 4] Japanese Patent Publication No. 2009-137895 [Patent Document 5] Japanese Patent Publication No. 2010-1228 [Patent Document 6] Japanese Patent Publication No. 2012-131726 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a composition for use in suppressing the proteolytic activity of cysteine ​​proteases produced by periodontal disease-causing bacteria, particularly the basic amino acid gingipain, which are pathogenic factors in periodontal disease. Furthermore, the present invention aims to provide new applications based on novel effects discovered regarding hinokitiol and peony. [Means for solving the problem]

[0008] The inventors of the present invention have been diligently studying to solve the aforementioned problems, and as shown in the examples described below, they have discovered that hinokitiol and peony extract have the effect of suppressing the protein degradation activity of cysteine ​​protease produced by periodontal disease-causing bacteria. Based on this finding, they have further studied the subject and completed the present invention. The present invention has the following embodiments.

[0009] (I) Composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria (I-1) A composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria, comprising at least one selected from the group consisting of hinokitiol and peony extract as an active ingredient. (I-2) A composition for inhibiting gingipain activity, comprising at least one selected from the group consisting of hinokitiol and peony extract as an active ingredient. (I-3) A composition for inhibiting basic amino acid-gingipain activity, comprising at least one selected from the group consisting of hinokitiol and peony extract as an active ingredient. (I-4) A basic amino acid-gingipain activity inhibitory composition as described in (I-3), wherein the basic amino acid is arginine or lysine. (I-5) An oral composition for inhibiting activity as described in any of (I-1) to (I-4). (I-6) An activity-inhibiting composition according to any one of (I-1) to (I-5), wherein the proportion of hinokitiol in the activity-inhibiting composition is 0.001 to 0.5% by mass, preferably 0.05 to 0.2% by mass, and more preferably 0.01 to 0.2% by mass. (I-7) An activity-inhibiting composition according to any one of (I-1) to (I-6), wherein the proportion of peony processed product in the activity-inhibiting composition is 0.00001 to 1% by mass on a dry basis, preferably 0.00005 to 0.1% by mass, and more preferably 0.0001 to 0.01% by mass.

[0010] (II) A method for imparting an inhibitory effect on the activity of cysteine ​​protease produced by periodontal disease-causing bacteria. (II-1) A method for imparting an inhibitory effect on the activity of cysteine ​​protease produced by periodontal disease-causing bacteria to an oral composition, comprising the step of blending the oral composition with at least one selected from the group consisting of hinokitiol and peony extract. This method can be rephrased as "a method for producing an oral composition having an inhibitory effect on the activity of cysteine ​​protease produced by periodontal disease-causing bacteria, comprising the step of blending the oral composition with at least one selected from the group consisting of hinokitiol and peony extract." (II-2) The method described in (II-1), wherein the cysteine ​​protease produced by the periodontal disease causative bacteria is gingipain, preferably a basic amino acid-gingipain. (II-3) The method described in (II-2), wherein the basic amino acid is arginine or lysine. (II-4) The method according to any one of (II-1) to (II-3), comprising the step of blending an oral composition so that the content of hinokitiol is 0.001 to 0.5% by mass, preferably 0.05 to 0.2% by mass, and more preferably 0.01 to 0.2% by mass. (II-5) The method according to any one of (II-1) to (II-4), comprising the step of blending the oral composition so that the content of peony extract is 0.00001 to 1% by mass, preferably 0.00005 to 0.1% by mass, and more preferably 0.0001 to 0.01% by mass, on a dry content basis. [Effects of the Invention]

[0011] The present invention provides a composition for inhibiting the activity of cysteine ​​protease, gingipain, or basic amino acid-gingipain produced by periodontal disease-causing bacteria, which can suppress the activity of these proteolytic enzymes produced by P. gingivalis, a bacterium that causes periodontal disease. Furthermore, the method of the present invention can impart to an oral composition the ability to inhibit the activity of cysteine ​​protease, gingipain, or basic amino acid-gingipain produced by P. gingivalis, a bacterium that causes periodontal disease. [Brief explanation of the drawing]

[0012] [Figure 1] In Experimental Example 1, we evaluated whether the decomposition of Bz-L-Arg-pNA·HCl (substrate) in P. gingivalis-produced protease solution (crude enzyme solution) was inhibited by the presence of the following test samples: a test sample containing neither hinokitiol nor peony extract (Comparative Example 1), test samples containing 0.01%, 0.02%, or 0.04% hinokitiol (Examples 1, 2, and 3), test samples containing 0.0001%, 0.001%, or 0.01% peony extract (Examples 4, 5, and 6), and test samples containing both hinokitiol and peony extract (Examples 7-8). [Figure 2]In Experimental Example 1, the results of evaluating whether the degradation of Ac-Lys-pNA·HCl (substrate) in the protease solution produced by P. gingivalis (crude enzyme solution) is inhibited by the test samples that contain neither hinokitiol nor processed peony (Comparative Example 2), the test samples that contain 0.01%, 0.02% or 0.04% hinokitiol (Examples 9, 10 and 11), and the test samples that contain 0.0001%, 0.001% or 0.01% peony extract (Examples 12, 13 and 14) are shown.

Mode for Carrying Out the Invention

[0013] (I) Composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria The composition targeted by the present invention is a composition containing at least one selected from the group consisting of hinokitiol and processed peony as an active ingredient, and is characterized by being used for suppressing the activity of cysteine protease produced by periodontal disease-causing bacteria. Here, the periodontal disease-causing bacteria targeted by the present invention is Porphyromonas gingivalis (P. gingivalis). Further, examples of the cysteine protease produced by P. gingivalis include preferably gingipain, particularly basic amino acid-gingipains such as arginine-gingipain having an action of cleaving the C-terminal of an arginine residue and lysine-gingipain having an action of cleaving the C-terminal of a lysine residue. Therefore, the composition of the present invention is preferably a composition for suppressing the activity of gingipain, more preferably a composition for suppressing the activity of basic amino acid-gingipain, and particularly preferably arginine-gingipain or lysine-gingipain. Note that the basic amino acid refers to an amino acid having an amino group or imino group that can receive a proton (H + ) and examples thereof include arginine, lysine, histidine, tryptophan, and ornithine. Preferably arginine and lysine. In the present specification, hereinafter, the term "Cys protease" is used as a general term for cysteine proteases including gingipain and basic amino acid-gingipain.

[0014] In the present invention, the suppression of Cys protease activity means attenuating or eliminating the proteolytic activity (trypsin-like protease activity) of the Cys protease produced by P. zingiberis. As shown in Experimental Example 1 described later, the inhibitory effect on the Cys protease activity is evaluated by reacting the Cys protease with Bz-L-Arg-pNA·HCl (Benzoyl-L-arginine p-nitroanilide monohydrochloride) (substrate for arginine-dingipine) or Ac-Lys-pNA·HCl (N-α-Acetyl-L-lysine p-nitroanilide monohydrochloride) (substrate for lysine-dingipine), which are used as its substrates, in the presence or absence of a test sample, and measuring the absorbance of the luminescence derived from p-nitroaniline (pNA) generated. When the absorbance of the reaction solution when reacted in the presence of the test sample is lower than the absorbance of the reaction solution when reacted in the absence, or when the difference in absorbance before and after the reaction when reacted in the presence of the test sample is lower than the difference in absorbance before and after the reaction when reacted in the absence, it can be determined that the test sample has an inhibitory effect on Cys protease activity.

[0015] Thiocolchicoside, which is contained as an active ingredient in the composition for suppressing Cys protease activity of the present invention, is an unsaturated seven-membered ring compound contained in Taiwan cypress and Japanese cypress, and is also called β-tuyapricin and 4-isopropyltropolone. It has been conventionally known to have an antibacterial effect and is a well-known compound widely used as an additive for food preservatives, hair tonics, cosmetics, etc. It forms a salt when contacting metal ions, and in the present invention, these salts are also collectively referred to as thiocolchicoside. Thiocolchicoside can be isolated from essential oils extracted from Taiwan cypress and Japanese cypress, but it can be easily obtained commercially.

[0016] The hinokitiol content in the Cys protease activity inhibitory composition of the present invention is not particularly limited as long as it exerts a Cys protease activity inhibitory effect, and can be appropriately set depending on the purpose and use. For example, if the Cys protease activity inhibitory composition of the present invention is an oral composition that is applied directly to the oral cavity, the content can be appropriately set from, for example, 0.001 to 0.5% by mass. Preferably, it is 0.005 to 0.2% by mass, and more preferably 0.01 to 0.2% by mass. If the Cys protease activity inhibitory composition of the present invention is an oral composition that is diluted with water at the time of use and applied to the oral cavity, it is sufficient that it contains hinokitiol so that the concentration after dilution is within the above range. For example, although not limited, one example is that when used after diluting 10 times with water at the time of use, the oral composition can be prepared to contain hinokitiol at a concentration of 0.01 to 5% by mass. Furthermore, when the Cys protease activity inhibitory composition of the present invention is used as an additive to impart Cys protease activity inhibitory properties to the oral composition described above (including those used as is and those diluted before use), the content of hinokitiol is not particularly limited, as long as the oral composition can be prepared, and can be appropriately set from, for example, 0.1 to 100% by mass.

[0017] The peony product contained as an active ingredient in the Cys protease activity inhibitory composition of the present invention is a processed root of Paeonia lactiflora Pallas of the Paeoniaceae family or other closely related plants, and may be a crushed root, a finely chopped root, a dried root thereof, or a solvent extract (peony extract). Peony extract is preferred. Examples of extraction solvents used for extracting peony extract include water, organic solvents (lower alcohols such as ethanol, methanol, and isopropanol; polyhydric alcohols such as propylene glycol and 1,3-butylene glycol; ethers, hexane, benzene, chloroform, acetone, pentane, ethyl acetate, etc.) or mixtures thereof. Preferably, it is water, ethanol, 1,3-butylene glycol, or a mixture thereof (e.g., aqueous ethanol).

[0018] The extraction method used in the production of peony extract is not particularly limited, and any general extraction method used in the production of plant extracts is acceptable. For example, methods include immersing peony roots (preferably dried and pulverized) in an extraction solvent by cold immersion, hot immersion, etc., and stirring as necessary; percolation method; steam distillation method, etc. The obtained extract can be recovered by removing solid matter from the extract by filtration or centrifugation as necessary. In the Cys protease activity inhibitory composition of the present invention, the liquid peony extract obtained by the above extraction process may be used as is, but if necessary, some or all of the solvent may be removed and used as a concentrated liquid or dried product (extract powder, dried extract). Furthermore, these concentrates or dried products may be subjected to further purification, and they may also be dissolved or suspended in a suitable solvent before use.

[0019] Since peony products (such as crushed peony roots or peony extract) are commercially available, commercially obtained products may be used in this invention.

[0020] The content of peony extract in the Cys protease activity inhibitory composition of the present invention is not particularly limited as long as it exerts a Cys protease activity inhibitory effect, and can be appropriately set depending on the purpose and use. For example, if the Cys protease activity inhibitory composition of the present invention is an oral composition that is applied directly to the oral cavity, the content can be appropriately set from, for example, 0.00001 to 1% by mass when converted to the dried peony extract. Preferably it is 0.00005 to 0.1% by mass, and more preferably 0.0001 to 0.01% by mass. If the Cys protease activity inhibitory composition of the present invention is an oral composition that is diluted with water at the time of use and applied to the oral cavity, it is sufficient that it contains peony extract so that the concentration after dilution is within the above range. For example, although not limited, one example is that when used after diluting 10 times with water at the time of use, the oral composition can be prepared to contain peony extract at a concentration of 0.000001 to 0.1% by mass. Furthermore, when the Cys protease activity inhibitory composition of the present invention is used as an additive to impart a Cys protease activity inhibitory effect to the oral composition described above (including those used as is and those diluted at the time of use), the content of the peony extract is not particularly limited, as long as the oral composition can be prepared, and can be appropriately set from, for example, 0.1 to 100% by mass.

[0021] As described above, the Cys protease activity inhibitory composition of the present invention may be used as an oral composition applied to the oral cavity either as is or diluted with water, or it may be used as an additive to prepare an oral composition having a Cys protease activity inhibitory effect by being added to such oral compositions.

[0022] Since hinokitiol is a crystalline substance that is slightly soluble in water, when using the Cys protease activity inhibitory composition of the present invention as an additive to an oral composition (hereinafter, the composition of the present invention in this case will be referred to as "the additive of the present invention"), it is preferable to prepare it in the form of a liquid preparation in which hinokitiol is dissolved in a soluble solvent such as ethanol or propylene glycol.

[0023] When the Cys protease activity inhibitory composition of the present invention is used as an oral composition, either as is or diluted with water (hereinafter, the composition of the present invention in this case will be referred to as "the oral composition of the present invention"), examples of its dosage form include, without limitation, toothpaste (paste, powder toothpaste, gel toothpaste, liquid toothpaste), mouthwash (mouth rinse, mouthwash, dental rinse), oral ointment, and oral freshener (liquid preparations such as mouth spray, tablets (including candy tablets), oral hygiene agents, capsules, granules, gummies, gum, etc.). Liquid dosage forms such as mouthwash may be of the type used as is, or they may be of the type that is concentrated and diluted before use. These may be used not only for the purpose of inhibiting the activity of Cys protease, but also for the purpose of cleaning and washing the inside of the mouth, such as brushing teeth, for the purpose of providing a refreshing sensation in the mouth, for the purpose of sterilizing the inside of the mouth, and / or for the purpose of removing or preventing bad breath.

[0024] The oral compositions of the present invention can be prepared in a desired dosage form according to conventionally known methods by adding additives such as abrasives, foaming agents, thickeners, fragrances, sweeteners, wetting agents, antioxidants, pH adjusters, colorants, and preservatives, as well as pharmacologically active ingredients and solvents, to the extent that they do not impair the effects of the present invention, depending on the type of oral composition, dosage form, and purpose.

[0025] As abrasives, various abrasives commonly used in oral compositions can be incorporated. While not particularly limited, examples include monocalcium phosphate, discalcium phosphate dihydrate and anhydrous, tricalcium phosphate, calcium carbonate, calcium pyrophosphate, aluminum hydroxide, alumina, silica gel, anhydrous silicic acid, aluminum silicate, precipitated silica, insoluble sodium metaphosphate, trimagnesium phosphate, magnesium carbonate, calcium sulfate, polymethyl methacrylate, bentonite, zirconium silicate, and titanium-bonded silicates.

[0026] As a foaming agent, various foaming agents (foaming surfactants) commonly used in oral compositions can be incorporated. While not particularly limited, anionic surfactants, nonionic surfactants, cationic surfactants, and / or amphoteric surfactants can be incorporated. More specifically, anionic surfactants include, for example, amino acid-based surfactants such as lauroyl methyl taurine or its salts, lauroyl glutamic acid or its salts, lauroyl sarcosine or its salts; sodium dodecylbenzenesulfonate, hydrogenated coconut fatty acid monoglyceride monosulfate sodium, α-olefin sulfonate sodium, etc. Nonionic surfactants include, for example, sucrose fatty acid esters, maltose fatty acid esters, maltitol fatty acid esters, lactol fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan monostearate, polyoxyethylene higher alcohol ethers, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene copolymer, polyoxyethylene polyoxypropylene fatty acid esters, polyglycerin fatty acid esters, etc. Examples of cationic surfactants include lauryltrimethylammonium chloride, stearyltrimethylammonium chloride, benzethonium chloride, cetylpyridinium chloride, benzalkonium chloride, and stearyldimethylbenzylammonium chloride. Examples of amphoteric surfactants include coconut oil fatty acid amidopropyl betaine, lauryldimethylaminoacetic acid betaine, lauryldimethylamine oxide, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolium betaine, N-lauryldiaminoethylglycine, N-myristyldiaminoethylglycine, and sodium N-alkyl-1-hydroxyethylimidazoline betaine.

[0027] Various thickeners commonly used in oral compositions can be incorporated as thickeners. While not particularly limited, examples include xanthan gum, sodium alginate, polyvinyl alcohol, hydroxyethylcellulose, sodium polyacrylate, carrageenan, sodium carboxymethylcellulose, and polyvinylpyrrolidone.

[0028] As for the fragrance, various fragrances commonly used in oral compositions can be incorporated. While not particularly limited, these include l-menthol, l-carbone, cinnamic aldehyde, orange oil, anethole, 1,8-cineole, methyl salicylate, eugenol, thymol, linalool, limonene, menthone, menthyl acetate, citral, camphor, borneol, pinene, spiranthol, ethyl acetate, ethyl butyrate, isoamyl acetate, hexanal, hexenal, methyl anthranilate, ethyl methylphenyl glycidate, benzaldehyde, vanillin, ethyl vanillin, furaneol, maltol, ethyl maltol, and gamma / deltadecalac. Fragrance ingredients include ton, gamma / deltaundecalactone, N-ethyl-p-menthane-3-carboxamide, menthyl lactate, and ethylene glycol-l-menthyl carbonate; natural essential oils include peppermint oil, spearmint oil, eucalyptus oil, clove oil, thyme oil, sage oil, cardamom oil, rosemary oil, marjoram oil, lemon oil, nutmeg oil, lavender oil, and paracreth oil; and blended flavors include apple, banana, strawberry, blueberry, melon, peach, pineapple, grape, muscat, wine, cherry, squash, coffee, brandy, and yogurt.

[0029] As sweeteners, various sweeteners commonly used in oral compositions can be incorporated. While not particularly limited, examples include sodium saccharin, acesulfamyl potassium, stevioside, neohespolydyl dihydrochalcone, glycyrrhizin, perillartin, thaumatin, aspartylphenylalanine methyl ester, p-methoxycinnamic aldehyde, aspartame, xylitol, and erythritol.

[0030] Various humectants commonly used in oral compositions can be incorporated as humectants. While not particularly limited, examples include sorbitol, glycerin, concentrated glycerin, ethylene glycol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, and polypropylene glycol.

[0031] As antioxidants, various antioxidants commonly used in oral compositions can be incorporated. While not particularly limited, examples include rosemary extract, stevia extract, sunflower seed extract, propyl gallate, dibutylhydroxytoluene, butylhydroxyanisole, L-cysteine ​​hydrochloride, phytic acid, hydroquinone and its glycosides, nordihydroguaiaretinic acid, guaiac butter, polyphenols, tocopherol acetate, pine extract, and ascorbic acid.

[0032] Various pH adjusters commonly used in oral compositions can be incorporated as pH adjusters. While not particularly limited, examples include phthalic acid, phosphoric acid, citric acid, succinic acid, acetic acid, fumaric acid, malic acid, carbonate and their potassium, sodium, or ammonium salts, and sodium hydroxide. The pH of the oral composition of the present invention is not particularly limited as long as it is within a range that poses no safety concerns for the oral cavity and the human body. For example, when 30 g of the oral composition is dissolved in 70 mL of water at 25°C, the pH range is 3 to 9. The pH of the oral composition of the present invention can be adjusted to this range using the aforementioned pH adjusters as needed.

[0033] Various colorants commonly used in oral compositions can be incorporated as colorants. While not particularly limited, examples include Red No. 2, Red No. 3, Red No. 225, Red No. 226, Yellow No. 4, Yellow No. 5, Yellow No. 205, Blue No. 1, Blue No. 2, Blue No. 201, Blue No. 204, Green No. 3, Titanium Mica, Titanium Oxide, and the like.

[0034] As preservatives, various preservatives commonly used in oral compositions can be incorporated. While not particularly limited, examples include sodium benzoate, parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, cetylpyridinium chloride, alkyldiaminoethylglycine hydrochloride, and potassium sorbate.

[0035] As pharmacologically active ingredients, various pharmacologically active ingredients commonly used in oral compositions can be incorporated. While not particularly limited, examples include bactericidal or antibacterial agents such as chlorohexidine, triclosan, isopropylmethylphenol, cetylpyridinium chloride, zinc gluconate, and zinc citrate; anti-inflammatory agents such as tranexamic acid and dipotassium glycyrrhizate; astringents such as allantoin, allantoin chlorohydroxyaluminum, ascorbic acid, lysozyme chloride, glycyrrhizic acid and its salts, sodium chloride, and aluminum lactate; enzymes such as dextranase, amylase, protease, mutanase, and lysozyme; and hypersensitivity inhibitors such as strontium chloride and potassium nitrate. These can be used within pharmaceutically acceptable limits.

[0036] Ethanol, isopropyl alcohol, and other solvents can be used.

[0037] The content of optional components in the oral composition of the present invention can be appropriately set depending on the amount of each component, the carrier, the type of additive, etc., but the total amount is typically 1 to 80% by weight.

[0038] Furthermore, the oral composition of the present invention may contain conventionally known pharmacologically acceptable carriers. Examples of such carriers include aqueous carriers such as water (ion-exchanged water, distilled water, purified water, etc.) and physiological saline; and oily carriers such as higher fatty acids, vegetable oils, animal oils, silicone oils, and metal soaps. In the present invention, the content of these carriers is adjusted to correspond to the remainder of the oral composition, excluding hinokitiol, which is the active ingredient, and other optional components.

[0039] The amount of the oral composition of the present invention used per application is not particularly limited and can be adjusted as appropriate depending on the application. For example, in the case of toothpaste, it is 0.2 to 1 g, and in the case of liquid toothpaste or mouthwash, it is 5 to 20 mL. Furthermore, the users of the oral composition of the present invention are preferably adults, and more preferably people suffering from periodontal disease or people who are susceptible to periodontal disease.

[0040] (II) Method for conferring inhibitory effect on Cys protease activity As shown in Experimental Example 1 described later, hinokitiol and peony extract have the effect of suppressing the activity of Cys protease produced by P. gingivalis, a bacterium that causes periodontal disease. Therefore, by incorporating at least one selected from hinokitiol and peony extract into an oral composition, it is possible to impart to the oral composition the effect of suppressing the activity of Cys protease produced by P. gingivalis.

[0041] Accordingly, the present invention provides a method for imparting an inhibitory effect on the activity of Cys protease produced by periodontal disease-causing bacteria to an oral composition, comprising the step of blending the oral composition with at least one selected from hinokitiol and peony extract. The oral composition may be blended with hinokitiol alone, with peony extract alone or in combination of two or more peony extracts with different forms, or further blended with hinokitiol and peony extract. A suitable peony extract to be blended here is peony extract.

[0042] Here, the oral compositions to which hinokitiol and / or peony extract are to be added are the oral compositions described above, except that they do not contain hinokitiol and peony extract as their dosage form and constituent components. The amount of hinokitiol added to the oral composition will vary depending on the type, dosage form and purpose of the oral composition being prepared, but for example, if the oral composition being prepared is an oral composition that is applied directly to the oral cavity, it can be appropriately set from, for example, 0.001 to 0.5% by mass. Preferably, it is 0.005 to 0.2% by mass, and more preferably 0.01 to 0.2% by mass. If the oral composition being prepared is an oral composition that is diluted with water at the time of use and applied to the oral cavity, the amount of hinokitiol added should be such that the concentration of hinokitiol in the diluted oral composition falls within the above range. For example, although not limited, one example is an oral composition that is diluted 10 times with water before use. In this case, the oral composition can be prepared by incorporating hinokitiol in a proportion of 0.01 to 5% by mass.

[0043] The amount of peony extract to be added to an oral composition varies depending on the type, dosage form, and purpose of the oral composition being prepared. For example, if the oral composition to be prepared is to be applied directly to the oral cavity, the amount can be appropriately set from, for example, 0.00001 to 1% by mass on a dry weight basis. Preferably, it is 0.00005 to 0.1% by mass, and more preferably 0.0001 to 0.01% by mass. If the oral composition to be prepared is to be diluted with water before application to the oral cavity, the peony extract should be added so that the concentration of the peony extract in the diluted oral composition falls within the aforementioned range. For example, although not limited, one example is an oral composition that is diluted 10 times with water before use. In this case, the oral composition can be prepared by adding peony extract at a ratio of 0.000001 to 10% by mass.

[0044] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.” [Examples]

[0045] The present invention will be described below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. The following experiments were conducted at room temperature (25±5℃) and atmospheric pressure unless otherwise specified. Also, unless otherwise specified, "%" means mass%.

[0046] Experimental Example 1: Evaluation of the inhibitory effect on gingipain activity. The inhibitory effects of hinokitiol and peony extract on the protease (gingipain) activity (gingipain activity) produced by Porphyromonas gingivalis (P. gingivalis) were evaluated using the following tests.

[0047] (1) Test method (1-1) Purification of P. gingivalis-produced protease P. gingivalis (JCM 12257) was cultured anaerobically at 37°C for 2 days in GAM liquid medium containing 50 μg / mL hemin and 1 μg / mL menadione. After culturing, the culture medium was centrifuged (10000 rpm, 4°C, 10 minutes), and ammonium sulfate was added to the collected supernatant to 80% saturation. The mixture was stirred with a stirrer for 2 hours under 4°C conditions. This was then centrifuged (10000 rpm, 4°C, 30 minutes), and the resulting precipitate was dissolved in Buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM CaCl2), and this was placed on a dialysis membrane and dialyzed for 1 day in Buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM CaCl2, 0.05% Tween20) (4°C). After dialysis, the dialysate collected from the dialysis membrane was centrifuged (10,000 rpm, 4°C, 30 minutes), and glycerin was added to the resulting supernatant to a final concentration of 25% to obtain P. gingivalis-produced protease solution (crude enzyme solution).

[0048] (1-2) Measurement of gingipain activity In 0.1 M Tris-HCl (pH 7.4), a final concentration of 0.05 mM cysteine, test samples (for examples and comparative examples) with the compositions shown in Table 1, a 100-fold dilution of the P. gingivalis-produced protease solution (crude enzyme solution) prepared by the above method, and 1 mM Bz-L-Arg-pNA·HCl[L-BAPA] (Benzoyl-L-arginine p-nitroanilide monohydrochloride) (substrate for arginine-gingipain) or 1 mM Ac-Lys-pNA·HCl (N-α-Acetyl-L-lysine p-nitroanilide monohydrochloride) (substrate for lysine-gingipain) were mixed and incubated at 37°C for 5 hours. The concentrations of each component listed here are the final concentrations after addition and mixing. Before and after incubation, 0.5 mL of the reaction solution was transferred to a 48-well microplate and the absorbance at a wavelength of 430 nm was measured. The amount of p-nitroaniline (pNA) produced by the reaction, i.e., the substrate degradation activity (gingipain activity), was evaluated by the difference between the absorbance before incubation (initial value) and the absorbance after incubation.

[0049] [Table 1]

[0050] (2) Test results The results when using the arginine-gingipain substrate are shown in Table 2 and Figure 1, and the results when using the lysine-gingipain substrate are shown in Table 3 and Figure 2, respectively.

[0051] [Table 2]

[0052] [Table 3]

[0053] As shown in Table 2 and Figure 1, in Comparative Example 1, which contained neither hinokitiol nor peony extract, luminescence derived from pNA was confirmed, indicating arginine-gingipain activity. In contrast, in Examples 1 (containing 0.01%), 2 (containing 0.02%), and 3 (containing 0.04%), the activity was confirmed to be suppressed in a dose-dependent manner. From these results, it was found that hinokitiol has the effect of suppressing arginine-gingipain activity. Furthermore, in Examples 4 (containing 0.0001%), 5 (containing 0.001%), and 6 (containing 0.01%), the activity was also confirmed to be suppressed in a dose-dependent manner. From these results, it was found that peony extract and other peony-derived products have the effect of suppressing arginine-gingipain activity. Furthermore, the results from Examples 7 and 8 confirmed that when hinokitiol is used in combination with peony extract, the arginine-gingipain activity inhibitory effect of hinokitiol is maintained or enhanced without being impaired.

[0054] As shown in Table 3 and Figure 2, in Comparative Example 2, which contained neither hinokitiol nor peony extract, luminescence derived from pNA was confirmed, indicating lysine-gingipain activity. In contrast, in Example 9, Example 102, and Example 11, which contained 0.01% hinokitiol, the activity was confirmed to be suppressed in a dose-dependent manner. From these results, it was found that hinokitiol has the effect of suppressing not only arginine-gingipain activity but also lysine-gingipain activity. Furthermore, in Example 12, Example 13, and Example 14, which contained 0.0001% peony extract, the activity was also confirmed to be suppressed in a dose-dependent manner. From these results, it was found that peony extract also has the effect of suppressing not only arginine-gingipain activity but also lysine-gingipain activity. These experimental results revealed that hinokitiol and peony extract have an inhibitory effect on the activity of the basic amino acid gingipain.

[0055] Table 4 illustrates the present invention's composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria, which is in the form of toothpaste.

[0056] [Table 4]

[0057] Table 5 illustrates the present invention's composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria, which is in the form of a mouthwash.

[0058] [Table 5]

[0059] Table 6 illustrates the present invention's composition for inhibiting the activity of cysteine ​​protease produced by periodontal disease-causing bacteria, which is in the form of a gel-like ointment (semi-solid preparation).

[0060] [Table 6]

Claims

1. A composition for inhibiting gingipain activity, containing hinokitiol and peony extract in proportions that exert an effective inhibitory effect on gingipain activity: However, this excludes the following compositions: (1) Toothpaste containing glycerin, sorbitol, hydrated silica, water, alcohol, calcium pyrophosphate, silica, polyoxyethylene hydrogenated castor oil, titanium dioxide, sodium edetate, allantoin, sodium chloride, angelica extract, peony extract, aluminum lactate, sodium N-lauroyl-L-glutamate, carrageenan, fragrance, aminocaproic acid, dipotassium glycyrrhizate, hinokitiol, parabens, soapberry extract, and pigments; (2) Mouthwash containing water, alcohol, glycerin, polyoxyethylene hydrogenated castor oil, N-coconut oil fatty acid acyl-N-carboxymethoxyethyl-N-carboxymethylethylenediamine disodium, sodium edetate, stearyl glycyrrhetinate, fragrance, dipotassium glycyrrhizate, hinokitiol, angelica extract, peony extract, soapberry extract, parabens, and l-menthol; (3) An oral composition containing at least one herbal extract and hinokitiol, and at least one selected from allantoin, allantoin chlorohydroxyaluminum and allantoin dihydroxyaluminum, wherein the herbal extract is at least one of rosemary extract, loquat leaf extract, licorice extract, tea extract, peony extract, angelica extract, chamomile extract, bamboo grass extract, soapberry extract, balloon flower extract, citrus extract, ginkgo extract, hydrangea extract, and clove extract; (4) An oral composition containing hinokitiol, peony extract, and angelica extract.

2. A composition for inhibiting basic amino acid-gingipain activity, containing hinokitiol and peony extract in proportions that exert an effective inhibitory effect on basic amino acid-gingipain activity: However, this excludes the following compositions: (1) Toothpaste containing glycerin, sorbitol, hydrated silica, water, alcohol, calcium pyrophosphate, silica, polyoxyethylene hydrogenated castor oil, titanium dioxide, sodium edetate, allantoin, sodium chloride, angelica extract, peony extract, aluminum lactate, sodium N-lauroyl-L-glutamate, carrageenan, fragrance, aminocaproic acid, dipotassium glycyrrhizate, hinokitiol, parabens, soapberry extract, and pigments; (2) Mouthwash containing water, alcohol, glycerin, polyoxyethylene hydrogenated castor oil, N-coconut oil fatty acid acyl-N-carboxymethoxyethyl-N-carboxymethylethylenediamine disodium, sodium edetate, stearyl glycyrrhetinate, fragrance, dipotassium glycyrrhizate, hinokitiol, angelica extract, peony extract, soapberry extract, parabens, and l-menthol; (3) An oral composition containing at least one herbal extract and hinokitiol, and at least one selected from allantoin, allantoin chlorohydroxyaluminum and allantoin dihydroxyaluminum, wherein the herbal extract is at least one of rosemary extract, loquat leaf extract, licorice extract, tea extract, peony extract, angelica extract, chamomile extract, bamboo grass extract, soapberry extract, balloon flower extract, citrus extract, ginkgo extract, hydrangea extract, and clove extract; (4) An oral composition containing hinokitiol, peony extract, and angelica extract.

3. An activity-inhibiting composition according to claim 1 or 2, which is an oral composition.

4. A method for imparting gingipain activity inhibitory effects to an oral composition, comprising the step of blending hinokitiol and peony extract in an effective ratio that exhibits gingipain activity inhibitory effects: However, the following compositions shall be excluded from the oral composition obtained through the compounding process; (1) Toothpaste containing glycerin, sorbitol, hydrated silica, water, alcohol, calcium pyrophosphate, silica, polyoxyethylene hydrogenated castor oil, titanium dioxide, sodium edetate, allantoin, sodium chloride, angelica extract, peony extract, aluminum lactate, sodium N-lauroyl-L-glutamate, carrageenan, fragrance, aminocaproic acid, dipotassium glycyrrhizate, hinokitiol, parabens, soapberry extract, and pigments; (2) Mouthwash containing water, alcohol, glycerin, polyoxyethylene hydrogenated castor oil, N-coconut oil fatty acid acyl-N-carboxymethoxyethyl-N-carboxymethylethylenediamine disodium, sodium edetate, stearyl glycyrrhetinate, fragrance, dipotassium glycyrrhizate, hinokitiol, angelica extract, peony extract, soapberry extract, parabens, and l-menthol; (3) An oral composition containing at least one herbal extract and hinokitiol, and at least one selected from allantoin, allantoin chlorohydroxyaluminum and allantoin dihydroxyaluminum, wherein the herbal extract is at least one of rosemary extract, loquat leaf extract, licorice extract, tea extract, peony extract, angelica extract, chamomile extract, bamboo grass extract, soapberry extract, balloon flower extract, citrus extract, ginkgo extract, hydrangea extract, and clove extract; (4) An oral composition containing hinokitiol, peony extract, and angelica extract.

Images