JP2023148032A - Composition for oral use - Google Patents

Abstract

To provide a composition for oral use, the composition containing a dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt.SOLUTION: The present invention provides compositions for oral use, containing a dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt (where, the compositions for oral use do not include ones with at least one selected from the group consisting of polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl ether, and polyoxyethylene fatty acid ester).SELECTED DRAWING: None

Description

The present disclosure relates to oral compositions, and more particularly, to oral compositions containing dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt.

dl-α-tocopherol 2-L-ascorbic acid phosphodiester (herein sometimes referred to as “EPC”) is a compound in which ascorbic acid (vitamin C) and tocopherol (vitamin E) are combined via phosphoric acid. It is a compound with an ester bond, has antioxidant effect and moisturizing effect, and is used in cosmetics such as hair growth agents (Patent Document 1).

Although EPC is expected to be applied in the oral cavity field, there have been no reports yet on what effects it actually exerts in the oral cavity field.

International Publication No. 2020/196852

Periodontol 2000. 2015 Oct;69(1):7-17. Molecular aspects of the pathogenesis of periodontitis J Periodontal Res. 2016 Dec;51(6):748-757. Influence of retinoic acid on human gingival epithelial barriers Int J Dent. 2012; 2012: 821383.Published online 2012 Jul 26. Host-Bacteria Crosstalk at the Dentogingival Junction Arch Oral Biol. 2016 Jun;66:30-7. The traditional Japanese medicine hangeshashinto alleviates oral ulcer-induced pain in a rat model. Aust Dent J. 2009 Dec;54(4):347-54. Localization of matrix metalloproteinases (MMPs-2, 8, 9 and 20) in normal and carious dentine.

An object of the present disclosure is to provide an oral composition comprising a dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt.

The present inventors demonstrated that dl-α-tocopherol 2-L-ascorbic acid phosphodiester alkali metal salt suppresses destruction of oral mucosal epithelial barrier function and inhibits matrix metalloproteinase (MMP) activity. After discovering that it inhibits the use of anti-oxidants, they made further improvements.

The present disclosure encompasses, for example, the subject matter described in the following sections.
Item 1.
Oral composition containing dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt (provided that from the group consisting of polyoxyethylene alkylphenyl ether, polyoxyethylene alkyl ether, and polyoxyethylene fatty acid ester) (excluding oral compositions containing at least one selected type).
Item 2.
Item 2. The composition according to Item 1, containing 0.001 to 1% by mass of dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt.
Item 3.
Item 3. The composition according to item 1 or 2, wherein the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt is dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester potassium salt.
Item 4.
Item 4. The composition according to any one of items 1 to 3, which is used for anti-periodontal disease, anti-oral mucositis, or anti-caries.
Item 5.
Item 5. The composition according to any one of Items 1 to 4, which is used to enhance the barrier function of oral mucosal epithelium.
Item 6.
Item 6. The composition according to any one of Items 1 to 5, which is used to inhibit matrix metalloprotease activity in the oral cavity.

The composition of the present disclosure contains dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt, and can suppress destruction of oral mucosal epithelial barrier function and/or inhibit MMP activity.

The transmittance (%) when EPC-K (dl-α-tocopherol 2-L-ascorbic acid phosphate diester potassium salt) is added is shown. The transmittance (%) when EPC-K is added is shown. The transmittance (%) is shown when EPC-K, APM (Mg ascorbic acid phosphate), VC (ascorbic acid), VEA (tocopherol acetate), and VEN (tocopherol nicotinate) are added. The results of measuring MMP-8 activity are shown. The results of measuring MMP-1 activity are shown. Shows the cell survival rate (%) when sorbeth-60 tetraoleate, diethyl sebacate, polyoxyethylene hydrogenated castor oil 10, polyoxyethylene (9) lauryl ether, and polyoxyethylene (20) stearyl ether are added. . When polyoxyethylene (9) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (10) octylphenyl ether, and polyoxyethylene glycol monolaurate are added. Cell viability (%) is shown.

Each embodiment included in the present disclosure will be described in further detail below.
Oral compositions encompassed by the present disclosure include dl-α-tocopherol 2-L-ascorbic acid phosphate diester alkali metal salt. In this specification, the composition may be referred to as "the oral composition of the present disclosure."

dl-α-tocopherol 2-L-ascorbic acid phosphodiester (EPC) is a compound in which ascorbic acid (vitamin C) and tocopherol (vitamin E) are ester-bonded via phosphoric acid, and is called (ascorbyl/tocopheryl). Also called phosphoric acid. The chemical formula is shown below.

Examples of the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt include dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester potassium salt, dl-α-tocopherol 2-L-ascorbic acid Examples include phosphoric acid diester sodium salt, dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester lithium salt, and the like. Among these, dl-α-tocopherol 2-L-ascorbic acid phosphate diester potassium salt is preferred.
Further, the alkali metal salt of dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester may be a monosalt or a di-salt.

The content of the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt contained in the oral composition of the present disclosure can be, for example, about 0.001 to 1% by mass. The upper limit or lower limit of the range is, for example, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0. It may be about .09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9% by mass. More specifically, for example, it may be about 0.002 to 0.5% by mass, or about 0.005 to 0.3% by mass.

The oral composition of the present disclosure has the effect of suppressing destruction of the oral mucosal epithelial barrier function by containing the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt. Therefore, the oral cavity composition of the present disclosure can be suitably used for enhancing the barrier function of the oral mucosal epithelium. Oral mucosal epithelium is not particularly limited, and includes epithelium that constitutes masticatory mucosa (e.g., gingiva, etc.) (e.g., gingival epithelium such as adherent epithelium, gingival sulcus epithelium, etc.), and covering mucosa (e.g., buccal mucosa, etc.). Examples include epithelium constituting special mucous membranes (for example, tongue mucosa, etc.).

Furthermore, the oral composition of the present disclosure exhibits the effect of inhibiting MMP activity by containing the alkali metal salt of dl-α-tocopherol 2-L-ascorbic acid phosphate diester. Therefore, the oral composition of the present disclosure can be suitably used for inhibiting matrix metalloproteinase (MMP) activity in the oral cavity. In addition, since the oral composition of the present disclosure exhibits an MMP activity inhibiting effect, it can inhibit the activity of already produced neutrophil collagenase.
Examples of matrix metalloproteinases (MMPs) in the oral cavity include MMP-1, 2, 3, 8, 9, and 13. Among these, MMP-1 and 8 are preferred. Note that MMP-1 (EC 3.4.24.7) is also called interstitial collagenase. MMP-8 (EC 3.4.24.34) is also called neutrophil collagenase.

Furthermore, it has been reported that the gingival epithelium forms a barrier against invading microorganisms and protects the periodontal tissue from infection (Non-Patent Document 1). Furthermore, periodontal disease bacteria such as Porphyromonas gingivalis are known to secrete bacterial protease and destroy the gingival epithelium, thereby achieving invasion into the periodontal tissue (Non-patent Document 2). . It is known that as the invasion of periodontal disease bacteria into tissues progresses, immune cells such as neutrophils accumulate in order to eliminate them, and the amount of MMPs produced increases. MMPs are proteolytic enzymes that degrade extracellular matrices (eg, collagen such as type I collagen, type II collagen, and type III collagen), which destroys periodontal tissue and progresses periodontal disease. In fact, increases in MMP-1, 2, 3, 8, 9, and 13 have been observed in the gingival crevicular fluid (GCF) of patients with periodontal disease (Non-Patent Document 3).
The oral composition of the present disclosure contains the alkali metal salt of dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester, thereby suppressing the destruction of the oral mucosal epithelial barrier function and/or suppressing the activity of MMP. Since it has the effect of inhibiting periodontal disease, it can be suitably used as an anti-periodontal disease agent. That is, the oral composition of the present disclosure can prevent periodontal disease bacteria from progressing to the periodontal tissue by enhancing the barrier function of the oral mucosal epithelium. Furthermore, the oral cavity composition of the present disclosure can prevent periodontal tissue destruction by inhibiting matrix metalloprotease activity in the oral cavity. Therefore, it can be expected to be applied to all periodontal disease situations, from early stages to advanced stages. In addition, in this specification, "anti-periodontal disease" means prevention of onset of periodontal disease and/or suppression of progression of periodontal disease.

Additionally, it has been reported that when ulcers are formed on the oral mucosa (oral mucositis) due to bites, burns, side effects of chemotherapy, radiation therapy, etc., the barrier function of the epithelium is lost, leading to oral bacterial infection. (Non-patent Document 4). The oral composition of the present disclosure has the effect of suppressing destruction of the oral mucosal epithelial barrier function by containing the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt. It can be suitably used for flames. In addition, in this specification, "anti-oral mucositis" means prevention of the onset of oral mucositis and/or suppression of the progression of oral mucositis.

Further, dentin contains approximately 20% collagen, which is an organic substance, and the progression of dentin caries is accompanied by demineralization and collagen decomposition. It is thought that collagen is degraded by MMPs in saliva penetrating into dentin and decomposing it (Non-Patent Document 5). The oral composition of the present disclosure exhibits the effect of inhibiting MMP activity by containing the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt, and is therefore suitably used for anti-caries purposes. be able to. In addition, in this specification, "anti-caries" means the prevention of the onset of caries and/or the suppression of the progression of caries.

Examples of subjects to which the oral composition of the present disclosure can be applied include mammals including humans (eg, dogs, cats, mice, rats, sheep, horses, cows, monkeys, etc.). Among these, humans are preferred.
Humans to whom the oral composition of the present disclosure is applied include, for example, periodontal disease patients or suspected patients; oral mucositis patients or suspected patients; caries patients or suspected patients; Not limited to people with a weakened barrier function of the oral mucosal epithelium; people with increased intraoral matrix metalloproteinase activity; healthy people (e.g., people who want to prevent the onset of periodontal disease, people with periodontal disease) people who want to suppress the progression of oral mucositis, people who want to prevent the onset of oral mucositis, people who want to suppress the progression of oral mucositis, people who want to prevent the onset of caries, people who want to suppress the progression of caries, etc.) Good too.

The oral composition of the present disclosure can be, for example, a solid composition, a liquid composition, or the like. In addition, the oral composition of the present disclosure can be prepared in a conventional manner, for example, as an ointment, a paste, a paste, a gel, a liquid, a spray, a mouthwash, a liquid dentifrice, a toothpaste, a gum, a tablet, or a drop. It can be made into the following forms (dosage forms). Among these, mouth rinses, liquid dentifrices, toothpastes, ointments, pastes, liquids, and gels are preferred.

The oral composition of the present disclosure further contains, in addition to dl-α-tocopherol 2-L-ascorbic acid phosphate diester alkali metal salt, one or more optional components that can be incorporated into the oral composition, for example. It's okay.

For example, a nonionic surfactant, an anionic surfactant, or an amphoteric surfactant can be blended as the surfactant. Specifically, for example, nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid ester, maltose fatty acid ester, and lactose fatty acid ester; fatty acid alkanolamides; glycerin fatty acid ester; sorbitan fatty acid ester; fatty acid monoglyceride; diethyl sebacate. ; polyoxyethylene hydrogenated castor oil; fatty acid polyoxyethylene sorbitan, and the like. Examples of anionic surfactants include sulfuric ester salts such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; sodium cocoyl sarcosine and lauroyl methylalanine. Acyl amino acid salts such as sodium; sodium cocoyl methyl taurate and the like. Examples of zwitterionic surfactants include betaine acetate type surfactants such as betaine lauryldimethylaminoacetate and betaine coconut oil fatty acid amidopropyldimethylaminoacetate; imidazoline type surfactants such as sodium N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine; Examples include activators and the like. These surfactants may be used alone or in combination of two or more. The amount incorporated is usually 0.1 to 5% by mass based on the total amount of the composition.
In addition, from the oral composition of the present disclosure, for example, an oral composition containing at least one selected from the group consisting of polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl ether, and polyoxyethylene fatty acid ester. is preferably excluded.
Examples of the polyoxyethylene alkylphenyl ether include polyoxyethylene alkylphenyl ether in which the average number of added moles of ethylene oxide is 1 to 40 and the number of carbon atoms in the alkyl group is 7 to 30. Examples of polyoxyethylene alkyl ethers include polyoxyethylene alkyl ethers in which the average number of added moles of ethylene oxide is 1 to 40 and the number of carbon atoms in the alkyl group is 1 to 18. Examples of the polyoxyethylene fatty acid ester include polyoxyethylene fatty acid esters in which the average number of added moles of ethylene oxide is 1 to 40 and the number of carbon atoms in the alkyl group is 1 to 20.

Further, 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, cineol, linalool, Ethyl linalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamic oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d- Flavoring agents such as borneol, fennel oil, cinnamon oil, cinnamaldehyde, peppermint oil, and vanillin can be used. These can be blended alone or in combination of two or more in an amount of, for example, 0.001 to 1.5% by mass based on the total amount of the composition.

Further, as a sweetening agent, for example, saccharin sodium, acesulfame potassium, stevioside, neohesperidyl dihydrochalcone, perillartin, thaumatin, asparatylphenylalanyl methyl ester, p-methoxycinnamic aldehyde, etc. can be used. These can be blended alone or in combination of two or more in an amount of, for example, 0.01 to 1% by mass based on the total amount of the composition.

Furthermore, as a wetting agent, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylit, maltit, lactit, polyoxyethylene glycol, etc. may be blended alone or in combination of two or more. can.

As a binder, cellulose derivatives such as carboxymethylcellulose sodium, carboxymethylethylcellulose salt, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, crystalline cellulose, crystalline cellulose/carmellose sodium, and microorganism-produced substances such as xanthan gum. Molecules, natural polymers or natural gums such as gum tragacanth, gum karaya, gum arabic, carrageenan, dextrin, agar, pectin, pullulan, gellan gum, locust bean gum, sodium alginate, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyvinyl methyl ether, Examples include synthetic polymers such as sodium polyacrylate, thickening silica, inorganic binders such as Veegum, and cationic binders such as O-[2-hydroxy-3-(trimethylammonio)propyl]hydroxyethylcellulose chloride. It will be done. These binders can be used alone or in combination of two or more.

As preservatives, parabens such as methylparaben, ethylparaben, propylparaben, butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. can be blended. These can be used alone or in combination of two or more.

As a coloring agent, legal pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral pigments such as ultramarine, reinforced ultramarine, and deep blue, titanium oxide, etc. may be blended. These can be used alone or in combination of two or more.

As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or chemically possible salts thereof, sodium hydroxide, etc. may be blended. These can be used alone or in combination of two or more so that the pH of the composition is in the range of 4 to 8, preferably 5 to 7. The amount of the pH adjuster may be, for example, 0.01 to 2% by weight.

The oral composition of the present disclosure further contains vitamin E such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate, an amphoteric bactericide such as dodecyldiaminoethylglycine, triclosan, Nonionic disinfectants such as isopropylmethylphenol, anionic disinfectants such as sodium lauroylsarcosine, cationic disinfectants such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, benzethonium chloride, dextranase, amylase, protease, Enzymes such as mutanase, lysozyme, and lytic enzymes (Litech Enzyme), alkali metal monofluorophosphates such as sodium monofluorophosphate and potassium monofluorophosphate, fluorides such as sodium fluoride and stannous fluoride, tranexamic acid or epsilon aminocaproic acid, aluminum chlorohydroxyallantoin, dihydrocholesterol, glycyrrhetinic acid, glycyrrhizic acid, copper chlorophyllin sodium, glycerophosphate, chlorophyll, sodium chloride, calopeptide, allantoin, carbazochrome, potassium nitrate, palatinit, etc. alone or in combination of two or more. Can be blended in combination.

It is also possible to add alcohols, silicon, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastibase, etc. as a base. These can be used alone or in combination of two or more.

The oral composition of the present disclosure can be prepared by a known method or a method easily derived from a known method. For example, it can be prepared by appropriately mixing dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt and other components as necessary.

Note that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Additionally, the present disclosure encompasses any and all combinations of the features described herein.

Further, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure described above may be combined in any manner in order to specify the subject matter covered by the present disclosure. That is, the present disclosure encompasses all subject matter consisting of any and all combinations of the combinable features described herein.

The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited thereto. In the following, unless otherwise specified, experiments are conducted under atmospheric pressure and room temperature conditions. Further, unless otherwise specified, "%" means "% by mass". In addition, the blending amount of each component listed in each table is expressed in "% by mass" unless otherwise specified.

1. Epithelial barrier function evaluation test
Cell preparation Gingival epithelial cell line Epi4 cells were cultured on transwells until confluent. At this time, the Epi4 cells were cultured using a medium prepared by adding 1/100th the amount of Supplemental S7 (Thermo Fisher) to Epilife (Thermo Fisher) as a cell culture medium. The transwell is designed to be suspended so that the insert is placed in the middle of the well, and is a tool that enables cell permeability evaluation.

Bacterial adjustment P. Porphyromonas gingivalis W83 was cultured in a modified GAM medium (Nissui Pharmaceutical Co., Ltd.), recovered by centrifugation at 10,000 rpm, and cultured in a cell culture medium to achieve an OD ₆₀₀ of 1.0. g Bacteria concentration was adjusted.

Material Preparation Each evaluation material was prepared in a cell culture medium containing 2.5% DMSO to the respective evaluation concentration. The materials used were EPC-K (dl-α-tocopherol 2-L-ascorbic acid phosphate diester potassium salt), APM (Mg ascorbic acid phosphate), VC (ascorbic acid), VEA (tocopherol acetate), and VEN. (tocopherol nicotinate).

Barrier Function Evaluation The culture medium was removed from the Epi4 cells cultured on the transwells, 300 μl of the prepared medium containing each material was added, and the cells were incubated for 1 hour at 37° C. under a 5% CO ₂ concentration. At this time, a medium treatment containing no material was used as a control.
Next, the adjusted P. 100 μl of bacterial solution was added and incubated for 2 hours at 37° C. under a CO ₂ concentration of 5% by volume. At this time, for the control, P. (P.g.(+)), and P.g. A culture medium containing no Pg bacteria was added and incubated (Pg (-)).
After incubation, the medium was removed, washed with PBS, and FITC (fluoresceinisothiocyanate)-dextran (4 kDa) adjusted to 1 mg/ml was added, followed by incubation at 37° C. for 1 hour. FITC-dextran that had passed through the transwell was collected, and the fluorescence intensity (excitation light: 490 nm, emission light: 520 nm) was measured using a fluorescence plate reader (Gemini XPS). P. vs. control. The amount of FITC permeation when G.g. bacterial solution was added (without P.g. (+) material) was 100%, and P.g. The amount of FITC permeation when only a medium containing no P.g. bacteria was added (P.g.(-)) was converted to 0%, and the transmittance when materials of each concentration were treated was calculated. The results are shown in Figures 1-3. Note that the material concentration in the figure is the concentration at the time of preparation, and the final concentration (concentration after addition of P.g bacteria) is 3/4 of the concentration value. Furthermore, it can be said that the lower the permeability, the higher the epithelial barrier function.

As shown in Figures 1 to 3, EPC-K has P. It was confirmed that it suppresses the destruction of the oral mucosal epithelial barrier caused by G bacteria. Furthermore, similar effects were not confirmed for ascorbic acid and its derivative APM, and tocopherol derivatives VEA and VEN.

2. MMP activity measurement test
Material Preparation Each evaluation material was adjusted to the respective evaluation concentration with a solvent (buffer included in the kit described below + 5% DMSO) to prepare an evaluation material solution. The materials used were EPC-K (dl-α-tocopherol 2-L-ascorbic acid phosphate diester potassium salt), VC (ascorbic acid), VEA (tocopherol acetate), and VEN (tocopherol nicotinate).

Enzyme activity measurement MMP-8 (neutrophil collagenase) activity was measured using MMP-8 fluorimetric drug discovery kit (Enzo Life Sciences, Inc., BML-AK415-0001), and MMP-1 (fibroblast collagenase) activity was measured using MMP-8 fluorimetric drug discovery kit (Enzo Life Sciences, Inc., BML-AK415-0001). - 1 The evaluation was performed using a fluorimetric drug discovery kit (Enzo Life Sciences, Inc., BML-AK405-0001).
Mix 0.1 μl of MMP-8 included in the kit and 69.9 μl of buffer included in the kit, or 0.2 μl of MMP-1 included in the kit and 69.8 μl of buffer included in the kit to 20 μl of each prepared material solution. , and reacted at 37°C for 60 minutes. At this time, as a positive control (Cont.), we used a sample in which no material was added and an equal amount of buffer was added, and to measure the background of only the substrate, MMP-8 or MMP-1 was not added and an equal amount of buffer was added. A sample (NC) was prepared.
The fluorescent substrate attached to the kit was diluted 10 times with a buffer, and 10 μl was added to the solution after 60 minutes of reaction. After the addition, MMP-8 was reacted at 37°C for 20 minutes and MMP-1 was reacted at 37°C for 60 minutes, and then the fluorescence intensity of excitation light: 328 nm and emission light: 420 nm was measured. The value obtained by subtracting the fluorescence intensity (NC) of only the substrate as a background from the fluorescence intensity of the positive control sample (Cont.) is set as 100%, and the fluorescence intensity (NC) of only the substrate is calculated as the background from the fluorescence intensity of each material solution added sample. The ratio of the value subtracted as the ground was calculated as the enzyme activity. In addition, MMP-8 was more reactive than MMP-1 with respect to the reaction substrates attached to both kits, so in this test, MMP-8 and MMP-1 Tests were conducted by changing the dilution rate and reaction time. The results are shown in Figures 4 and 5. Note that the material concentration in the figure is the concentration at the time of material preparation, and the final concentration (concentration at the time of fluorescence intensity measurement) is 1/5 of the concentration value.

As shown in FIGS. 4 and 5, EPC-K was confirmed to inhibit MMP activity (MMP-8 and MMP-1) in a concentration-dependent manner. Furthermore, similar effects were not confirmed for ascorbic acid and tocopherol derivatives VEA and VEN.

3. Cytotoxicity evaluation test
Cell Culture Oral epithelial cell line (Ca9-22 cells) was cultured in a 96-well plate until confluent.

The final concentration of each surfactant in the material preparation evaluation solution was adjusted to 1% using PBS containing 0.5% DMSO as a solvent. The surfactants used were polyoxyethylene (9) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (10) octylphenyl ether, and polyoxyethylene glycol monolaur. ester, sorbeth-60 tetraoleate, diethyl sebacate, and polyoxyethylene hydrogenated castor oil 10.

Cytotoxicity evaluation 100 μl of the prepared evaluation solution was added to cultured Ca9-22 cells, and the mixture was allowed to stand at room temperature for 10 minutes. After 10 minutes, the plate was washed three times with 200 μl of PBS and reacted with WST-1 reagent (Takara Bio) for 30 minutes. After the reaction, the absorbance at 450 nm and 600 nm was measured, and the value obtained by subtracting the absorbance at 600 nm from the absorbance at 450 nm was calculated. Among the calculated values, the cell survival rate was calculated by setting the value of the solvent-only treatment as 100%. The results are shown in Figures 6 and 7.

As shown in FIGS. 6 and 7, polyoxyethylene alkyl phenyl ether (polyoxyethylene (10) octylphenyl ether), polyoxyethylene alkyl ether (polyoxyethylene (9) lauryl ether, polyoxyethylene (20) cetyl ether, It was confirmed that polyoxyethylene (20) stearyl ether) and polyoxyethylene fatty acid ester (polyoxyethylene glycol monolaurate) were cytotoxic to oral cavity-derived cells. On the other hand, it was confirmed that other surfactants such as sorbeth-60 tetraoleate, diethyl sebacate, and polyoxyethylene hydrogenated castor oil 10 did not cause cytotoxicity in oral cavity-derived cells.

Claims (6)

Oral composition containing dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt (provided that from the group consisting of polyoxyethylene alkylphenyl ether, polyoxyethylene alkyl ether, and polyoxyethylene fatty acid ester) (excluding oral compositions containing at least one selected type). The composition according to claim 1, containing 0.001 to 1% by mass of dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt. The composition according to claim 1 or 2, wherein the dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester alkali metal salt is dl-α-tocopherol 2-L-ascorbic acid phosphoric acid diester potassium salt. The composition according to any one of claims 1 to 3, which is used for anti-periodontal disease, anti-oral mucositis, or anti-caries. The composition according to any one of claims 1 to 4, which is used to enhance the barrier function of oral mucosal epithelium. The composition according to any one of claims 1 to 5, which is used for inhibiting matrix metalloprotease activity in the oral cavity.

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