Oral cavity composition for Anti-inflammation in aged tissue

Abstract

The main purpose set by the present inventors is to provide a technology for suppressing inflammation particularly in aged tissue in the oral cavity.　The present inventors have discovered that it is possible to suppress inflammation particularly in aged tissue in the oral cavity by an oral cavity composition that contains β-glycyrrhetinic acid and at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, methylhesperidin, and chemically possible salts thereof.

Description

Oral composition for anti - inflammation in aging tissues

[0001] The present disclosure relates to an oral composition for anti - inflammation in aging tissues and the like.

[0002] Periodontal disease is an inflammatory disease including gingivitis and periodontitis. Since progression of the pathological condition of periodontal disease may lead to tooth loss, prevention and / or improvement of inflammation in oral tissues can contribute to an extension of healthy life span.

[0003] Japanese Patent Application Laid - Open No. 2019 - 196329

[0004] It is known that the risk of periodontal disease increases with aging, but the essential mechanism has not yet been elucidated. Therefore, it cannot always be said that the prior art can effectively suppress inflammation in aging tissues. In recent years, there has been an increasing demand for health management according to an individual's age, gender, lifestyle, health condition, genetic tendency, etc., such as personalized medicine and personalized supplements. In view of such circumstances, the present inventors aimed mainly at providing a technique for suppressing inflammation particularly in aging tissues in the oral cavity.

[0005] The present inventors have found that at least one selected from the group consisting of β - glycyrrhetinic acid, glycyrrhizic acid, ascorbic acid, nicotinic acid tocopherol, tocopherol acetate, pyridoxine, retinol acetate, riboflavin, phylloquinone, calciferol, anthraquinone, menaquinone - 4, and L - aspartic acid, and chemically possible salts thereof, and quercetin, genistein, fisetin, nobioretin, kenferol, apigenin, and naringenin, and glycosides thereof can exhibit an anti - inflammatory effect particularly in aging tissues in the oral cavity. Further, they have found that at least one selected from the group consisting of retinol acetate, retinol, all - trans - retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L - histidine, and methyl hesperidin, and chemically possible salts thereof can further enhance the above - mentioned anti - inflammatory effect of β - glycyrrhetinic acid. Then, through repeated improvements, the present disclosure has been completed.

[0006] This disclosure includes, for example, the subject matter described in the following section: Section 1. An oral composition for anti-inflammatory use in aging tissues, comprising β-glycyrrhetinic acid, glycyrrhizic acid, ascorbic acid, tocopherol nicotinate, tocopherol acetate, pyridoxine, retinol acetate, riboflavin, phylloquinone, calciferol, anthraquinone, menaquinone-4, and L-aspartic acid, and chemically possible salts thereof, and at least one selected from the group consisting of quercetin, genistein, fisetin, nobiletin, kaempferol, apigenin, and naringenin, and glycosides thereof. Item 2. An oral composition for anti-inflammatory effects in aging tissue, comprising β-glycyrrhetinic acid and at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methylhesperidin, and chemically possible salts thereof. Item 3. The oral composition according to item 1 or 2, wherein the aging tissue is the periodontal ligament. Item 4. The oral composition according to any one of items 1 to 3, for use against an inflammatory response induced by endotoxins. Item 5. The oral composition according to any one of items 1 to 4, wherein the anti-inflammatory effect is the suppression of the expression of at least one inflammation-related factor selected from the group consisting of inflammatory cytokines and chemokines. Item 6. The oral composition according to any one of items 1 to 5, wherein the anti-inflammatory effect is the suppression of the expression of at least one inflammation-related factor selected from the group consisting of IL-6, CCL2, IL-8, CXCL1, ICAM-1, and MMP-1. Item 7. The oral composition according to any one of items 1 to 6, wherein the aging tissue is the tissue of a subject aged 40 years or older.

[0007] According to this disclosure, a technology may be provided to suppress inflammation, particularly in aging tissues in the oral cavity.

[0008] Test 1: The left figure shows a fluorescence microscope image of SA-β-gal positive cells detected using the β-galactosidase detection fluorescent reagent SPiDER-βGal, and the right figure shows the fluorescence intensity (relative value with fluorescence intensity at p.4 set to 1). In the fluorescence microscope image, SA-β-gal is stained red and the nucleus is stained blue. In the figure, the top of the bar graph shows the mean value, and the error bars show the standard deviation (the same applies hereafter). ** p < 0.01, n = 4. Test 1: Gene expression levels of p16, p21, and p53 are shown. ** p < 0.01, * p < 0.05, n = 5. Test 1: Gene expression levels of CDK2 and CDK4 are shown. ** p < 0.01, * p < 0.05, n = 5. Test 1: Gene expression levels of IL-6, CCL2, and ICAM-1 are shown. ** p < 0.01, n = 5. Test 1: Results of analyzing the protein expression levels of p16 and ICAM-1 by Western blotting are shown. The upper part of the figure shows the quantitative results of band intensity performed using Image J. ** p < 0.01, n = 3. Test 2: Results of analyzing the expression levels of CCL2, IL-6, IL-8, and CXCL1 genes in p.4 and p.18 cells with or without the addition of Porphyromonas gingivalis (hereinafter, Pg) LPS (indicated as "Pg LPS" in the figure). The results represent relative values ​​with the gene expression level when p.4 cells were cultured without Pg LPS set to 1. ** p < 0.01, n = 5. Test 3: CCL2 expression levels when each test substance was added are shown. The results for p.4 are shown as relative values ​​with the CCL2 expression level in p.4 without the test substance added set to 1 before the addition of Pg LPS, and for p.18, the results are shown as relative values ​​with the CCL2 expression level in p.18 without the test substance added set to 1 before the addition of Pg LPS. (Continued from Figure 7) Test 3: Shows the CCL2 expression levels when each test substance is added. All of the test substances shown in this figure are flavonoids. Test 3: Shows the IL-6 expression levels when each test substance is added.The results for p.4 are shown as relative values ​​with the IL-6 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS, and for p.18, the relative values ​​with the IL-6 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS. (Continued from Figure 9) Test 3: Shows the IL-6 expression level when each test substance is added. Test 3: Shows the IL-8 expression level when each test substance is added. The results for p.4 are shown as relative values ​​with the IL-8 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS, and for p.18, the relative values ​​with the IL-8 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS. (Continued from Figure 11) Test 3: Shows the IL-8 expression level when each test substance is added. Test 3: Shows the CXCL1 expression level when each test substance is added. The results are shown as follows: for p.4, the relative value is set to 1 when the CXCL1 expression level in p.4 without the test substance was added before the addition of Pg LPS; and for p.18, the relative value is set to 1 when the CXCL1 expression level in p.18 without the test substance was added before the addition of Pg LPS. (Continued from Figure 13) Test 3: Shows the CXCL1 expression levels when each test substance was added.

[0009] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, oral compositions for anti-inflammatory purposes in aging tissues, and encompasses everything disclosed herein and recognizable to those skilled in the art.

[0010] I. Oral Compositions of the Disclosure The oral compositions included in the Disclosure contain β-glycyrrhetinic acid and at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methylhesperidin, and chemically possible salts thereof. Hereinafter, the oral compositions included in the Disclosure may be referred to as "Oral Compositions of the Disclosure." Furthermore, β-glycyrrhetinic acid, retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methylhesperidin, and chemically possible salts thereof, may be collectively referred to as "Active Ingredients of the Disclosure."

[0011] β-Glycyrrhetinic acid is an organic compound obtained by the hydrolysis of glycyrrhizic acid. The molar mass of β-glycyrrhetinic acid is 470.69 g / mol. The structure of β-glycyrrhetinic acid is shown below.

[0012] Ascorbic acid is a compound commonly known as vitamin C. The molar mass of ascorbic acid is 176.12 g / mol. While not particularly limited, the sodium salt of ascorbic acid (sodium ascorbate) is preferred in the techniques of this disclosure. The structure of ascorbic acid is shown below.

[0013] Cobalamin has a choline ring structure with cobalt as the central metal, and is generally considered a B vitamin. 12 Cobalamins are a general term for compounds known as cobalamins. Cobalamins include cyanocobalamin, hydroxocobalamin, methylcobalamin, and adenosylcobalamin. Although not particularly limited, hydroxocobalamin or a salt thereof is preferred in the art of this disclosure, and hydroxocobalamin acetate (hydroxocobalamin acetate) is more preferred. The molar mass of hydroxocobalamin is 1346.37 g / mol.

[0014] Retinol is a type of vitamin A. Its molar mass is 286.45 g / mol. The structure of retinol is shown below.

[0015] Retinyl acetate is a compound having a structure in which retinol and acetic acid are esterified. The molar mass of retinyl acetate is 328.49 g / mol. The structure of retinyl acetate is shown below.

[0016] All-trans-retinoic acid is an oxidized form of retinol, a compound having all-trans conjugated double bond chains and terminal carboxylic acid groups. The molar mass of all-trans-retinoic acid is 300.44 g / mol. The structure of all-trans-retinoic acid is shown below.

[0017] In this disclosure, calciferol is used as a general term for ergocalciferol and cholecalciferol. Ergocalciferol is generally known as vitamin D2, and cholecalciferol is generally known as vitamin D3. The molar mass of ergocalciferol is 396.65 g / mol, and the molar mass of cholecalciferol is 384.65 g / mol. In the art of this disclosure, either ergocalciferol or cholecalciferol can be preferably used, and the use of ergocalciferol is more preferable. The structure of ergocalciferol is shown below. The structure of cholecalciferol is shown below.

[0018] Anthraquinone is a type of aromatic organic compound. Its molar mass is 208.22 g / mol. The structure of anthraquinone is shown below.

[0019] L-histidine is a compound classified as an amino acid having an amino group at the α-position and an imidazole ring as a side chain. The molar mass of L-histidine is 155.15 g / mol. Although not particularly limited, in the art of this disclosure, it is preferable to use the hydrochloride salt of L-histidine (L-histidine hydrochloride). The structure of L-histidine is shown below.

[0020] Methylhesperidin is a compound that has a structure in which a methoxy group is introduced into the aglycone portion of hesperidin, a type of flavonoid. The molar mass of methylhesperidin is 624.59 g / mol. The structure of methylhesperidin is shown below.

[0021] Each of the above components may optionally form chemically possible salts. Specific examples of salts include, for example, salts with inorganic bases such as aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, potassium salts, sodium salts, and zinc salts. Also, for example, salts with organic bases include salts of primary, secondary, or tertiary amines. Specific examples of amines include, for example, arginine, betaine, caffeine, choline-N-N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine-N-ethylmorpholine-N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine. Examples of salts produced by inorganic acids include hydrochlorides, hydrobroms, sulfates, phosphates, and nitrates. Examples of salts produced by organic acids include acetates, benzoates, tartrates, maleates, fumarates, succinates, citrates, oxalates, p-toluenesulfonates, benzenesulfonates, methanesulfonates, and trifluoroacetates.

[0022] The content of the active ingredients in this disclosure is not particularly limited and may be, for example, 0.000001 to 1% by mass relative to the total oral composition. The upper or lower limits of the above range are 0.000001, 0.000005, 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.008, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024, 0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044, 0.046, 0.048, 0. It may be 0.5, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1% by mass. The content of the active ingredient of this disclosure is preferably 0.000001 to 0.8% by mass, more preferably 0.00005 to 0.5% by mass, and even more preferably 0.0001 to 0.3% by mass, relative to the total oral composition.

[0023] In addition to the active ingredients of this disclosure, the oral compositions may further contain optional ingredients that can be incorporated into the oral compositions, such as surfactants, flavoring agents, sweeteners, humectants, binders, preservatives, colorants, pH adjusters, abrasives, and pharmaceutically active ingredients other than the active ingredients of this disclosure, either individually or in combination of two or more.

[0024] For example, at least one surfactant selected from the group consisting of nonionic surfactants, anionic surfactants, and amphoteric surfactants can be incorporated.

[0025] Specifically, examples of nonionic surfactants include polyoxyalkylene alkyl ethers, polyoxyalkylene glycols, polyoxyalkylene fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyalkylene glycerin fatty acid esters, polyoxyalkylene fatty acid amides, polyoxyalkylene glycol fatty acid esters, polyoxyalkylene castor oil derivatives, polyoxyalkylene hydrogenated castor oil, polyoxyalkylene hydrogenated castor oil derivatives, polyglycerin fatty acid esters, monoglycerin fatty acid esters, sorbitan fatty acid esters, sorbitan fatty acid esters, alkyl glycol fatty acid esters, alkyl polyglycosides, sugar fatty acid esters (sucrose fatty acid esters, maltose fatty acid esters, lactose fatty acid esters, etc.), fatty acid alkanolamides, and diethyl sebacate. When a nonionic surfactant has a polyoxyalkylene moiety, it is preferable that the polyoxyalkylene moiety is a polyoxyethylene moiety.

[0026] Examples of anionic surfactants include sulfate ester salts such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinate salts such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; acyl amino acid salts such as sodium cocoyl sarcosinate and sodium lauroyl methylalanine; and acyl methyl taurate salts such as sodium cocoyl methyl taurate.

[0027] Examples of amphoteric surfactants include betaine-type surfactants such as lauryldimethylaminoacetic acid betaine and coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine; and imidazoline-type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine sodium. Surfactants can be formulated individually or in combination of two or more types.

[0028] As flavoring agents, for example, menthol, carvone, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronellol, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineole, linalool, ethyl linalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d-borneol, fennel oil, cinnamon oil, cinnamaldehyde, peppermint oil, vanillin, and other fragrances can be used. Flavoring agents can be used individually or in combination of two or more.

[0029] In addition, sweeteners such as sodium saccharin, potassium acesulfamethamate, stevia, stevioside, neohesperidyl dihydrochalcone, perillartin, thaumatin, aspartylphenylalanyl methyl ester, and p-methoxycinnamic aldehyde can be used. The sweeteners can be used individually or in combination of two or more.

[0030] As a wetting agent, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, xylitol, maltitol, lactitol, polyoxyethylene glycol, etc., can be used individually or in combination of two or more.

[0031] Examples of binders include cellulose derivatives such as sodium carboxymethylcellulose, carboxymethyl ethylcellulose salt, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, crystalline cellulose, and crystalline cellulose / carmellose sodium; microbially produced polymers such as xanthan gum; natural polymers or natural rubbers such as tragacanth gum, karaya gum, arabic gum, carrageenan, dextrin, agar, pectin, pullulan, gellan gum, locust bean gum, and sodium alginate; synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyvinyl methyl ether, and sodium polyacrylate; inorganic binders such as thickening silica and bee gum; and cationic binders such as O-[2-hydroxy-3-(trimethylammonio)propyl]hydroxyethylcellulose chloride. Binders can be used individually or in combination of two or more.

[0032] As preservatives, parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride may be included. Preservatives can be used individually or in combination of two or more.

[0033] As colorants, legally approved pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral pigments such as ultramarine, enhanced ultramarine, and navy blue, and titanium dioxide may be included. The colorants may be used individually or in combination of two or more.

[0034] As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphate, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, or chemically possible salts thereof, or sodium hydroxide may be included. The pH adjuster can be added individually or in combination of two or more to ensure that the pH of the oral composition is in the range of 4 to 8, preferably 5 to 7. The amount of pH adjuster added may be, for example, 0.01 to 2% by mass of the total oral composition.

[0035] Stabilizers such as POE hydrogenated castor oil, titanium dioxide, EDTA-2 sodium, EDTA-3 sodium, and sodium citrate may be included. Stabilizers can be used individually or in combination of two or more.

[0036] Examples of abrasives include calcium carbonate, magnesium carbonate, dicalcium phosphate, tricalcium phosphate, magnesium phosphate, silica, zeolite, sodium metaphosphate, aluminum hydroxide, magnesium hydroxide, calcium pyrophosphate, iron oxide, calcium sulfate, and anhydrous silicic acid. Abrasives can be used individually or in combination of two or more.

[0037] The oral composition disclosed herein further contains, as pharmaceutically active ingredients, amphoteric bactericides such as dodecyldiaminoethylglycine, nonionic bactericides such as isopropylmethylphenol and triclosan, anionic bactericides such as sodium lauroyl sarcosinate, cationic bactericides such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, enzymes such as dextranase, amylase, protease, mutanase, lysozyme, and lytic enzymes (Litec enzyme), and alkali gold such as sodium monofluorophosphate and potassium monofluorophosphate. Fluorides such as monofluorophosphates, sodium fluoride, and stannous fluoride, alkali metal salts of dl-α-tocopherol 2-L-ascorbic acid phosphate, tranexamic acid, epsilon-aminocaproic acid, aluminum chlorohydroxyl allantoin, allantoin, dihydrocholesterol, hinokitiol, copper chlorophyllin sodium, glycerophosphate, chlorophyll, sodium chloride, caropeptide, carbazochrome, potassium nitrate, and palatinite can be formulated individually or in combination of two or more.

[0038] Furthermore, water, alcohols, silicones, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, Plastibase, etc., can be added as bases. These can be formulated individually or in combination of two or more.

[0039] The method for preparing the oral composition of the present disclosure is not particularly limited as long as the effects of the present disclosure are achieved. For example, it can be prepared according to methods known in the art. More specifically, for example, it can be prepared by appropriately mixing the active ingredient of the present disclosure and other components, etc.

[0040] The dosage form of the oral composition of the present disclosure is not particularly limited and can be a solid composition, a semi-solid composition, a liquid composition, etc. More specifically, for example, dosage forms such as ointments, pastes, pastas, gels, liquids, sprays, mouthwashes, liquid dentifrices, toothpastes, gums, tablets, drops, etc. may be used. Among them, ointments, pastes, gels, liquids, mouthwashes, liquid dentifrices, and toothpastes are preferred.

[0041] II. Uses The inventors have found that at least one selected from the group consisting of β-glycyrrhetinic acid, glycyrrhizic acid, ascorbic acid, nicotinic acid tocopherol, tocopherol acetate, pyridoxine, retinol acetate, riboflavin, phylloquinone, calciferol, anthraquinone, menaquinone-4, and L-aspartic acid, and chemically possible salts thereof, and quercetin, genistein, fisetin, nobiletin, kenferol, apigenin, and naringenin, and glycosides thereof, can exhibit an anti-inflammatory effect, particularly in senescent cells. It has also been found that at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methyl hesperidin, and chemically possible salts thereof, can further enhance the anti-inflammatory effect of β-glycyrrhetinic acid. More specifically, it has been found that at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methyl hesperidin, and chemically possible salts thereof, can further enhance the effect of β-glycyrrhetinic acid in suppressing the expression of inflammation-related factors, particularly in senescent cells.

[0042] In addition, examples of inflammation-related factors include inflammatory cytokines, chemokines, and other factors. Inflammatory cytokines are a general term for proteins involved in the inflammatory response. Inflammatory cytokines are mainly produced by immune cells, endothelial cells, epithelial cells, etc., and function as signal transduction molecules. Specific examples of inflammatory cytokines include interleukins such as IL-1 (Interleukin-1) and IL-6, and tumor necrosis factor (Tumor Necrosis Factor, TNF-α).

[0043] Chemokines are low molecular weight signal transduction proteins that play a role in inducing immune cells such as leukocytes to the site of inflammation or infection. Specific examples of chemokines include IL-8, CCL2 (C-C motif chemokine ligand 2), and CXCL1 (C-X-C motif chemokine ligand 1).

[0044] Other factors included in the inflammation-related factors include, for example, ICAM-1 (Intercellular Adhesion Molecule-1), MMP-1 (Matrix Metalloproteinase-1), and the like.

[0045] Further, the inventors have confirmed that senescent cells have an increased sensitivity to endotoxin compared to normal cells (i.e., non-senescent cells), and that the expression of inflammation-related factors is significantly increased when endotoxin is added to senescent cells. And it has also been found that the active ingredient of the present disclosure can suppress the expression of inflammation-related factors induced by endotoxin in senescent cells. Incidentally, endotoxin is a pathogenic factor also called lipopolysaccharide (LPS), and is a component constituting the outer membrane of the cell wall of gram-negative bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Escherichia coli.

[0046] The oral compositions of this disclosure can be used for anti-inflammatory applications in aging tissue (preferably aged periodontal ligament tissue), and for suppressing the expression of inflammation-related factors (preferably at least one selected from the group consisting of IL-6, CCL2, IL-8, CXCL1, ICAM-1, and MMP-1) in aging tissue (preferably aged periodontal ligament tissue).

[0047] The periodontal ligament is connective tissue that surrounds the tooth root, located between the alveolar bone and cementum, and plays a role in fixing the tooth within the jawbone. Furthermore, in the progression of periodontal disease, the periodontal ligament fibers rupture, forming a periodontal pocket, suggesting that the periodontal ligament plays a very important role in the transition from gingivitis to periodontitis. Therefore, the oral compositions disclosed herein can be preferably used for anti-periodontal disease (prevention and / or improvement of periodontal disease) applications.

[0048] In one preferred embodiment, though not particularly limited, the oral compositions of the present disclosure are used to suppress endotoxin-induced inflammatory responses. In another preferred embodiment, the oral compositions of the present disclosure are used to suppress the expression of endotoxin-induced inflammation-related factors (preferably at least one selected from the group consisting of IL-6, CCL2, IL-8, CXCL1, ICAM-1, and MMP-1).

[0049] In one preferred embodiment, though not particularly limited, the aging tissue is the tissue of a subject aged 40 years or older. More preferably, the aging tissue is the tissue of a subject aged 45 years or older, even more preferably, the tissue is the tissue of a subject aged 50 years or older or 55 years or older, and particularly preferably, the tissue is the tissue of a subject aged 60 years or older. The lower limit of the range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0050] In this disclosure, senescent tissue refers to tissue containing senescent cells. Senescent cells are known to exhibit several characteristics that differ from normal cells. One such characteristic of senescent cells is that they are SA-β-gal (senescence-associated beta galactosidase) positive.

[0051] The anti-inflammatory effect of the oral composition of this disclosure can be confirmed, for example, by adding the oral composition of this disclosure to an evaluation system (preferably an evaluation system including LPS) containing senescent cells (preferably senescence-induced periodontal ligament cells), and observing a decrease in the expression level of at least one inflammation-related factor compared to when the composition is not added. In this disclosure, the term "factor" is used to include both genes and proteins.

[0052] Examples of animals to which the oral compositions of this disclosure may be applied include mammals, including humans (e.g., dogs, cats, mice, rats, sheep, horses, cattle, monkeys, etc.), with humans being particularly preferred. Although not particularly limited, the oral compositions of this disclosure are preferably applied to humans aged 40 years or older, more preferably to humans aged 45 years or older, even more preferably to humans aged 50 years or older or 55 years or older, and particularly preferably to humans aged 60 years or older. The lower limit of the above range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0053] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0054] Furthermore, the various characteristics (properties, structure, numerical values, functions, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinatable characteristics described herein.

[0055] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. The density of the culture medium used for cell culture was approximately 1.0 g / mL in all cases.

[0056] Experiment 1. Induction of senescence and confirmation of senescence characteristics in human periodontal ligament cells 1-1. Method 1-1-1. Evaluation of SA-β-gal-positive cells (Staining - Microscope) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Then, p.3 and p.17 were seeded into 12-well plates, respectively, to become p.4 and p.18, and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest) and 1% Antibiotics (Gibco) (hereafter simply referred to as "medium") until approximately 80% confluence was reached. After washing once with PBS(-), Bafilomycin A1 (Focus Biomolecules) was added to a concentration of 100 nM and incubated for 1 hour. After 1 hour, the cells were washed with PBS(-) and fixed for 3 minutes using 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako Pure Chemical). After washing with PBS(-), SPiDER-βGal (Dojindo) was added and incubated for 30 minutes. Then, Hoechst 33342 (Thermo Scientific), diluted in PBS(-) containing 0.3% Triton X-100 (Sigma-Aldrich), was added and incubated for 15 minutes. After washing with PBS(-), fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo Scientific). Fluorescence intensity was quantified by processing the fluorescence images using Celleste 5 Image Analysis Software (Thermo Scientific).

[0057] 1-1-2. Evaluation of the expression of inflammation-related genes and cell cycle regulatory genes (qPCR) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Subsequently, p.3 and p.17 were seeded into 12-well plates, respectively, to become p.4 and p.18, and cultured in medium until approximately 80% confluence was reached.

[0058] Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (p16, p21, p53, CDK2, CDK4, IL-6, CCL2, ICAM-1, and 18S rRNA) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected to the expression levels of 18S rRNA. The primer sets used to quantify each gene expression in this example are described in "6. Appendix".

[0059] 1-1-3. Expression Evaluation of p16, ICAM-1 Protein (Western Blotting) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Then, p.3 and p.17 were seeded into 6-well plates, respectively, to become p.4 and p.18, and cultured until confluence was reached. After that, the cells were washed with PBS and harvested. Protein was extracted from the harvested cells by a conventional method, and the total protein concentration of the obtained protein extract was measured by BCA assay (Thermo Scientific). After standardizing the protein concentration, Laemmli Sample Buffer (BIO-RAD) was added, and the proteins were denatured by boiling. The denatured proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes with the transferred proteins were blocked with PBS containing 5% skim milk and 0.05% Tween-20. After blocking, the film was sequentially reacted with primary and secondary antibodies, followed by the addition of substrates to induce chemiluminescence. The emission intensity was detected using an Amersham Imager 680 RGB (Cytiva) system. Band intensity quantification was performed using ImageJ.

[0060] 1-2. Results: Senescent cells are known to be positive for SA-β-gal (senescence-associated beta galactosidase). Figure 1 shows fluorescence microscope images (left) and fluorescence intensity (relative value with fluorescence intensity at p.4 set to 1, right) when SA-β-gal positive cells were detected using the β-galactosidase detection fluorescent reagent SPiDER-βGal. In the fluorescence microscope image on the left, SA-β-gal is stained red and the nucleus is stained blue. As shown in Figure 1, the expression of SA-β-gal, a characteristic of aging, is increased at p.18.

[0061] The expression levels of each gene are shown in Figures 2-4. Note that all the genes evaluated are those reported to be associated with aging (aging-related genes). The genes shown in Figure 2 are known to have a function in arranging the cell cycle, and all showed significantly increased expression at p.18. This result is consistent with previous findings that the expression of these genes is induced in senescent cells. The genes shown in Figure 3 are known to have a function in accelerating the cell cycle, and all showed significantly decreased expression. This result is consistent with previous findings that the expression of these genes is decreased in senescent cells. The genes shown in Figure 4 are inflammation-related genes, and all showed significantly increased expression at p.18. This result is consistent with previous findings that the expression of these genes is induced in senescent cells.

[0062] Figure 5 shows the results of analyzing the protein expression levels of p16 and ICAM-1 by Western blotting. Consistent with the gene expression levels shown in Figures 2 and 3, the protein expression levels of p16 and ICAM-1 were significantly increased in p.18.

[0063] These results confirm that aging is induced in human periodontal ligament fibroblasts that have undergone repeated subculturing.

[0064] Experiment 2. Evaluation of the response of inflammation-related factor gene expression to P. gingivalis LPS 2-1. Method Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Then, p.3 and p.17 were seeded in 12-well plates to become p.4 and p.18, respectively, and cultured until approximately 80% confluence was reached. Subsequently, 100 ng / mL of Porphyromonas gingivalis-derived lipopolysaccharides (Sigma-aldrich, hereafter Pg LPS) was added, or not added, and incubated for 6 hours.

[0065] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (CCL2, IL-6, IL-8, CXCL1, and 18S rRNA) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected to the expression level of 18S rRNA. Relative values ​​were calculated with the gene expression level when p.4 was cultured in a medium without Pg LPS added set to 1.

[0066] 2-2. Results The results are shown in Figure 6. As shown in Figure 6, the expression levels of CCL2, IL-6, IL-8, and CXCL1 were significantly higher in p.18 than in p.4 when Pg LPS was added. These results suggest that senescent cells have increased sensitivity to endotoxins and an increased inflammatory response to endotoxins.

[0067] Test 3. Evaluation of the anti-inflammatory effect of the test substance 13-1. Method Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 4, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Then, p.3 and p.17 were seeded in 12-well plates, respectively, to become p.4 and p.18, and cultured in medium until approximately 80% confluence was reached.

[0068] For each of the p.4 and p.18 cultured as described above, β-glycyrrhetinic acid, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, allantoin, sodium ascorbate, tocopherol nicotinate, tocopherol acetate, hinokitiol, pyridoxine hydrochloride, retinol acetate, riboflavin, nicotinic acid, nicotinamide, phylloquinone, menaquinone-4, ergocalciferol (sometimes simply referred to as "calciferol" in this example), sodium D-pantothenate, biotin, anthraquinone, L-glutamic acid, and L-aspartic acid were added at a concentration of 200 μM or not, and incubated for 1 hour. Corylin, genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin were added at a concentration of 20 μM or not, and incubated for 24 hours. Furthermore, coriolin, genistein, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin are flavonoids.

[0069] After 1 or 24 hours, the cells were washed with PBS and incubated with 100 ng / mL of Pg LPS for 6 hours. Total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (CCL2, IL-6, IL-8, CXCL1, and 18S rRNA) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected for the expression level of 18S rRNA.

[0070] 3-2. Results 3-2-1. CCL2 The CCL2 expression levels when each test substance was added are shown in the following tables and Figures 7 and 8. For p.4, the relative value is shown with the CCL2 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS. For p.18, the relative value is shown with the CCL2 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS.

[0071]

[0072]

[0073] As shown in the table above and in Figures 7 and 8, β-glycyrrhetinic acid, sodium ascorbate, tocopherol nicotinate, tocopherol acetate, pyridoxine hydrochloride, retinol acetate, riboflavin, phylloquinone, calciferol, anthraquinone, L-aspartic acid, quercetin, fisetin, nobiletin, kaempferol, apigenin, and naringenin were able to suppress CCL2 expression by more than 30% on p.18, and this suppressive effect on CCL2 expression was greater than that on p.4.

[0074] Furthermore, while monoammonium glycyrrhizinate, menaquinone-4, genistein, and fisetin resulted in CCL2 expression levels exceeding 1 (i.e., enhanced expression) on p.4, CCL2 expression levels were less than 1 on p.18. These results suggest that monoammonium glycyrrhizinate, menaquinone-4, genistein, and fisetin have a CCL2 expression inhibitory effect specifically on aging tissue.

[0075] 3-2-2. IL-6 The IL-6 expression levels when each test substance is added are shown in the following tables and Figures 9 and 10. For p.4, the relative value is shown with the IL-6 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS. For p.18, the relative value is shown with the IL-6 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS.

[0076]

[0077]

[0078] As shown in the table above and in Figures 9 and 10, sodium ascorbate, tocopherol nicotinate, tocopherol acetate, retinol acetate, nobiletin, and apigenin were able to suppress IL-6 expression by more than 30% on p.18, and this IL-6 expression suppression effect was greater than that on p.4.

[0079] Furthermore, dipotassium glycyrrhizinate, pyridoxine hydrochloride, quercetin, fisetin, and naringenin resulted in IL-6 expression levels exceeding 1 (i.e., enhanced) on p.4, but IL-6 expression levels were less than 1 on p.18. These results suggest that dipotassium glycyrrhizinate, pyridoxine hydrochloride, quercetin, fisetin, and naringenin have IL-6 expression inhibitory effects specifically on aging tissues.

[0080] 3-2-3. IL-8 The IL-8 expression levels when each test substance is added are shown in the following tables and Figures 11 and 12. For p.4, the relative value is shown with the IL-8 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS. For p.18, the relative value is shown with the IL-8 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS.

[0081]

[0082]

[0083] As shown in the table above and in Figures 11 and 12, sodium ascorbate, tocopherol nicotinate, retinol acetate, quercetin, nobiletin, and apigenin were able to suppress IL-8 expression by more than 30% on p.18, and this IL-8 expression suppression effect was greater than that on p.4.

[0084] Furthermore, dipotassium glycyrrhizinate, sodium ascorbate, and fisetin resulted in IL-8 expression levels exceeding 1 (i.e., enhanced expression) on p.4, while IL-8 expression levels were less than 1 on p.18. These results suggest that dipotassium glycyrrhizinate, sodium ascorbate, and fisetin have IL-8 expression inhibitory effects specifically on aging tissues.

[0085] 3-2-4. CXCL1 The CXCL1 expression levels when each test substance was added are shown in the following tables and Figures 13 and 14. For p.4, the relative value is shown with the CXCL1 expression level at p.4 without the test substance added set to 1 before the addition of Pg LPS. For p.18, the relative value is shown with the CXCL1 expression level at p.18 without the test substance added set to 1 before the addition of Pg LPS.

[0086]

[0087]

[0088] As shown in the table above and Figures 13 and 14, sodium ascorbate, tocopherol nicotinate, tocopherol acetate, retinol acetate, riboflavin, nobiletin, apigenin, and naringenin were able to suppress CXCL1 expression by more than 30% on p.18, and this CXCL1 expression suppression effect was greater than that on p.4.

[0089] Furthermore, while β-glycyrrhetinic acid resulted in CXCL1 expression levels exceeding 1 (i.e., enhanced expression) in p.4, CXCL1 expression levels were less than 1 in p.18. These results suggest that β-glycyrrhetinic acid has a CXCL1 expression inhibitory effect specifically on aging tissues.

[0090] 3-3. Discussion The above results suggest that β-glycyrrhetinic acid, glycyrrhizic acid, ascorbic acid, tocopherol nicotinate, tocopherol acetate, pyridoxine, retinol acetate, riboflavin, phylloquinone, calciferol, anthraquinone, menaquinone-4, and L-aspartic acid, as well as chemically possible salts thereof, and quercetin, genistein, fisetin, nobiletin, kaempferol, apigenin, and naringenin, as well as their glycosides, may exert anti-inflammatory effects, particularly in aging tissues in the oral cavity.

[0091] Test 4. Evaluation of the Inflammatory Effect of the Test Substance 24-1. Method Human periodontal ligament fibroblasts from p.17 were seeded in a 12-well plate to form p.18, and cultured until approximately 80% confluence was reached. The test substance was prepared in 0.1% DMSO-supplemented medium to a final concentration of 200 μM and added to p.18. After incubation for 1 hour, washing with PBS(-) was performed, and then Pg LPS was added to a final concentration of 100 ng / mL, followed by treatment for 6 hours. After treatment, total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the obtained cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene and the intercalator method using TB Green Premix Ex Taq II (FAST) qPCR (Takara Bio). The expression levels of each gene were corrected using 18S rRNA, and the relative expression level (%) was calculated with the expression level under Pg LPS treatment without the test substance added set to 100%.

[0092] 4-2. Results The results when each test substance was added are shown in the table below. Items that were not measured are indicated with "-".

[0093] Test 5. Evaluation of the anti-inflammatory effect of the test substance 35-1. Method Human periodontal ligament fibroblasts from p.17 were seeded in a 12-well plate to form p.18, and cultured until approximately 80% confluence was reached. β-glycyrrhetinic acid and each test substance were prepared in 0.1% DMSO-supplemented medium to a final concentration of 100 μM and added to p.18. After incubation for 1 hour, the cells were washed with PBS(-), and then Pg LPS was added to a final concentration of 100 ng / mL, followed by treatment for 6 hours. After treatment, total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted RNA using the PrimeScript RT reagent Kit (Takara Bio). The obtained cDNA was used as a template to quantify gene expression using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with specific primers for each gene and an intercalator method using TB Green Premix Ex Taq II (FAST) qPCR (Takara Bio). The expression levels of each gene were corrected with 18S rRNA, and the relative expression level (%) was calculated with the expression level when β-glycyrrhetinic acid was added alone set to 100%.

[0094] 5-2. Results The results when each test substance was added in combination with 100 μM β-glycyrrhetinic acid are shown in the table below. Items that were not measured are indicated with "-".

[0095] 5-3. Discussion As shown in Experiment 3, β-glycyrrhetinic acid can exert anti-inflammatory effects, particularly in aging tissues in the oral cavity. As shown in the table above, retinol acetate, retinol, all-trans-retinoic acid, sodium ascorbate, cobalamin, ergocalciferol, anthraquinone, methylhesperidin, and L-histidine were suggested to further enhance the anti-inflammatory effects of β-glycyrrhetinic acid. Among these compounds, some, such as all-trans-retinoic acid on IL-6 expression and methylhesperidin on IL-6 and IL-8, did not show inhibitory effects on expression on their own, but when combined with β-glycyrrhetinic acid, further enhanced its anti-inflammatory effects. Furthermore, although dipotassium glycyrrhizinate, tocopherol nicotinate, tocopherol acetate, allantoin, phylloquinone, L-aspartic acid, and riboflavin can exert anti-inflammatory effects individually, as shown in Tests 3 and 4, they did not further enhance the anti-inflammatory effect of β-glycyrrhetinic acid when combined with it.

[0096] 6. Appendix The primer sets used to quantify each gene expression in the examples of this disclosure are shown in the table below.

[0097]

Claims

1. An oral composition for anti-inflammatory purposes in aging tissue, comprising β-glycyrrhetinic acid and at least one selected from the group consisting of retinol acetate, retinol, all-trans-retinoic acid, ascorbic acid, cobalamin, calciferol, anthraquinone, L-histidine, and methylhesperidin, and chemically possible salts thereof.

2. The oral composition according to claim 1, wherein the aging tissue is the periodontal ligament.

3. The oral composition according to claim 1 or 2, for use against inflammatory responses induced by endotoxins.

4. The oral composition according to claim 1 or 2, wherein the anti-inflammatory effect is the suppression of the expression of at least one inflammation-related factor selected from the group consisting of inflammatory cytokines and chemokines.

5. The oral composition according to claim 1 or 2, wherein the anti-inflammatory effect is the suppression of the expression of at least one inflammation-related factor selected from the group consisting of IL-6, CCL2, IL-8, CXCL1, ICAM-1, and MMP-1.

6. The oral composition according to claim 1 or 2, wherein the aging tissue is the tissue of a subject aged 40 years or older.

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