JP2024175353A - Collagenase-type matrix metalloprotease (mmp) activity inhibitor and oral composition - Google Patents

Abstract

The present invention provides a collagenase-type matrix metalloproteinase (MMP) activity inhibitor and an oral composition that exhibit excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity.
[Solution] The collagenase-type matrix metalloproteinase (MMP) activity inhibitor of the present invention contains 0.35% by mass or more of citric acids. The collagenase-type matrix metalloproteinase (MMP) activity inhibitor of the present invention contains 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid. The liquid oral composition of the present invention contains 0.35% by mass or more of citric acids. The oral composition of the present invention contains 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid.
[Selection diagram] None

Description

The present invention relates to a collagenase-type matrix metalloproteinase (MMP) activity inhibitor and an oral composition.

In general, the basic skeleton of a living body is formed by hard tissues such as bones and teeth, and soft tissues such as ligaments, tendons, synovial membranes, and periodontal ligaments that surround and connect these tissues and aid in movement. These hard and soft tissues are connected by attachment structures, helping the two tissues to function as a single composite tissue rather than separately. It is also known that matrix metalloproteinases (MMPs) are strongly involved in the modification and reconstruction of these tissues, as well as in inflammatory diseases such as rheumatism, arthritis, and periodontal disease, in these attachment structures.

MMP is a general term for extracellular matrix degrading enzymes that are characterized by having a zinc (II) ion at the active site. There are more than 20 known types of MMP enzyme molecules, including collagenase type (MMP-1, 8, 13), stromelysin type (MMP-3), and gelatinase type (MMP-2, 9), and these are produced by many types of cells.

Known collagenase-type MMPs include MMP-1, a tissue collagenase produced by fibroblasts and macrophages, MMP-8, a neutrophil collagenase, and MMP-13, produced by chondrocytes.

In periodontal diseases such as periodontitis, tissue destruction caused by a disruption in the metabolic balance of synthesis and decomposition of periodontal tissue due to abnormal expression of collagenase-type MMPs is largely involved. For example, in response to bacteria in the periodontal pocket, neutrophils infiltrate and accumulate in the gingival crevicular fluid, producing MMP-8. This destroys the connective tissue attachment of the periodontal pocket, leading to increased pocket depth and gingival recession, and as the condition progresses, the teeth become loose and fall out. By inhibiting the action of these collagenase-type MMPs, the breakdown of collagen that constitutes periodontal tissue can be suppressed.

MMP inhibitors have been known for some time, for example those disclosed in Patent Document 1. Such MMP inhibitors disclose a component consisting of gold nanoparticles having on their surface a protective agent that contains a functional group capable of forming a coordinate bond with the zinc ion at the enzyme active site.

JP 2016-138091 A

It is believed that inhibiting the activity of collagenase-type MMPs leads to the prevention of periodontal disease and the progression of periodontal disease. There is a demand for collagenase-type matrix metalloproteinase (MMP) activity inhibitors and oral compositions that exhibit excellent collagenase-type MMP activity inhibitory effects.

As a result of research conducted by the inventors to solve the above problems, they have newly discovered that citric acid at a specific concentration or higher exerts an excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity.

Various aspects for solving the above problems will be described below.
The collagenase-type matrix metalloproteinase (MMP) activity inhibitor of aspect 1 is characterized by containing 0.35% by mass or more of citric acids.

The collagenase-type matrix metalloproteinase (MMP) activity inhibitor of aspect 2 is characterized by containing 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid.

Aspect 3 is a collagenase-type matrix metalloproteinase (MMP) activity inhibitor according to aspect 1 or 2, which is an agent for inhibiting destruction of periodontal tissue and promoting regeneration, an agent for inhibiting decomposition and promoting regeneration of periodontal tissue collagen, an agent for inhibiting decomposition and promoting regeneration of gingival collagen, or an agent for inhibiting deepening of periodontal pockets and promoting repair.

The liquid oral composition of aspect 4 is characterized by containing 0.35% by weight or more of citric acids.
The liquid oral composition of aspect 5 is characterized in that it contains 0.35% by mass or more of citric acids and is for inhibiting collagenase-type matrix metalloproteinase (MMP) activity.

The oral composition of aspect 6 is characterized by containing 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid.
The oral composition of aspect 7 is characterized in that it contains 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid, and is used for inhibiting collagenase-type matrix metalloproteinase (MMP) activity.

The present invention can exert an excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity.

First Embodiment
A first embodiment of the collagenase-type matrix metalloproteinase (MMP) activity inhibitor (hereinafter referred to as "MMP activity inhibitor") and liquid oral composition of the present invention will be described.

The component contained in the MMP activity inhibitor of this embodiment is a citric acid.
The liquid oral composition of this embodiment contains the same components as the MMP activity inhibitor. The MMP activity inhibitor and the liquid oral composition may contain only one of the above components, or may contain two of the above components in combination. The liquid oral composition may be for inhibiting collagenase-type matrix metalloproteinase (MMP) activity.

The above-mentioned components contained in the MMP activity inhibitor and the liquid oral composition are explained below. Since the MMP activity inhibitor and the liquid oral composition contain the same components as those mentioned above, the liquid oral composition is explained below. Regarding the MMP activity inhibitor, the differences from the liquid oral composition are explained. In the present invention, the liquid oral composition means one that is intended to be used primarily in the oral cavity. It includes not only oral preparations that are discharged from the oral cavity after use, but also ingestible foods and beverages. The MMP activity inhibitor is not limited to one that is intended to be used in the oral cavity.

<Citric acid>
The citric acid salts contained in the liquid oral composition are at least one selected from citric acid and citrates. Examples of citrates include alkali metal salts of citric acid and alkaline earth metal salts of citric acid. Examples of citrates include sodium citrate, magnesium citrate, potassium citrate, calcium citrate, etc. These may contain only one type alone or may contain two or more types in combination.

The lower limit of the citric acid content in the liquid oral composition is 0.35% by mass, and preferably 0.4% by mass. When the lower limit of the content is 0.35% by mass, an excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity can be exhibited.

The upper limit of the citric acid content in the liquid oral composition is set as appropriate, but is preferably 5% by mass, and more preferably 4% by mass. When the upper limit of the content is 5% by mass, the collagenase-type matrix metalloproteinase (MMP) activity inhibitory effect can be efficiently exerted.

The upper or lower limit of the citric acid content may be, for example, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0% by mass. The citric acid content is expressed as the content converted to citric acid.

<Other ingredients>
The liquid oral composition may contain other components other than the above-mentioned components depending on the application purpose, form, use, etc. Examples of other components include surfactants, flavoring agents, sweeteners, humectants, binders, preservatives, colorants, pH adjusters, chelating agents, medicinal ingredients, bases, abrasives, stabilizers, etc. The other components may be any known components that are blended into liquid oral compositions. The liquid oral composition may contain only one of the above other components alone, or may contain two or more of them in combination.

Examples of the surfactant include nonionic surfactants, anionic surfactants, amphoteric surfactants, and cationic surfactants.
Specific examples of nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid esters, maltose fatty acid esters, and lactose fatty acid esters; fatty acid alkanolamides; glycerin fatty acid esters; sorbitan fatty acid esters; fatty acid monoglycerides; polyoxyethylene alkyl ethers having a polyoxyethylene addition coefficient of 8 to 10 and an alkyl group having 13 to 15 carbon atoms; polyoxyethylene alkylphenyl ethers having a polyoxyethylene addition coefficient of 10 to 18 and an alkyl group having 9 carbon atoms; diethyl sebacate; polyoxyethylene hydrogenated castor oil; and polyoxyethylene sorbitan fatty acid.

Specific 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 lauroyl methyl alanine; and sodium cocoyl methyl taurate.

Specific examples of amphoteric surfactants include acetate betaine type surfactants such as lauryl dimethylamino acetate betaine and coconut oil fatty acid amidopropyl dimethylamino acetate betaine; imidazoline type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethyl ethylenediamine sodium; and amino acid type surfactants such as N-lauryl diaminoethyl glycine.

Specific examples of cationic surfactants include distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, N-coconut oil fatty acid acyl-L-arginine ethyl-DL-pyrrolidone carboxylate, etc.

Specific examples of flavoring agents include menthol, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronellol, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineole, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d-borneol, fennel oil, cinnamon oil, anise oil, cinnamaldehyde, peppermint oil, and vanillin.

Specific examples of sweeteners include saccharin sodium, acesulfame potassium, stevioside, neohesperidyl dihydrochalcone, perillartine, thaumatin, aspartyl phenylalanyl methyl ester, and p-methoxycinnamic aldehyde.

Specific examples of humectants include sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, polyoxyethylene glycol, etc.

Specific examples of binders include cellulose derivatives such as sodium carboxymethylcellulose, carboxymethylethylcellulose salts, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, crystalline cellulose, and crystalline cellulose-carmellose sodium; microbial polymers such as xanthan gum; natural polymers or natural rubbers such as tragacanth gum, karaya gum, gum arabic, guar 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; thickening silica, inorganic binders such as veegum, and cationic binders such as O-[2-hydroxy-3-(trimethylammonio)propyl]hydroxyethylcellulose chloride.

Specific examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride.

Specific examples of colorants include legal dyes such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3; mineral dyes such as ultramarine, enhanced ultramarine, and Prussian blue; and titanium oxide.
Specific examples of pH adjusters include citric acid, phosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or chemically acceptable salts thereof, sodium hydroxide, and the like.

Specific examples of the chelating agent include edetic acid, sodium edetate, potassium edetate, and phytic acid.
Specific examples of medicinal ingredients include vitamin E such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate; vitamin C such as ascorbic acid, sodium ascorbate, or magnesium ascorbyl phosphate; vitamin B6 such as pyridoxine hydrochloride; amphoteric bactericides such as glycyrrhizinate and its derivatives, glycyrrhetinic acid, and dodecyldiaminoethylglycine; nonionic bactericides such as triclosan, isopropylmethylphenol, and polyoxyethylene lauryl ether; anionic bactericides such as sodium cocoyl sarcosine and sorbic acid; cetylpyridinium chloride, chlorhexidine hydrochloride, and chlorhexidine gluconate. cationic bactericides such as methacrylate crosslinker, benzalkonium chloride, benzethonium chloride, etc.; enzymes such as dextranase, amylase, protease, mutanase, lysozyme, bacteriolytic enzyme (Retech Enzyme), etc.; alkali metal monofluorophosphates such as sodium monofluorophosphate, potassium monofluorophosphate, etc.; fluorides such as sodium fluoride, stannous fluoride, etc.; tranexamic acid, epsilon aminocaproic acid, aluminum chlorohydroxyl allantoin, dihydrocholesterol, hinokitiol, copper chlorophyllin sodium, chlorophyll, sodium chloride, callopeptide, allantoin, carbazochrome, potassium nitrate, palatinit, etc.

Specific examples of bases include alcohols, silicone, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastibase, etc.

Specific examples of the alcohols include ethyl alcohol, lauryl alcohol, and myristyl alcohol.
Specific examples of abrasives include calcium carbonate, magnesium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, magnesium phosphate, silica, zeolite, sodium metaphosphate, aluminum hydroxide, magnesium hydroxide, calcium pyrophosphate, red iron oxide, calcium sulfate, and silicic anhydride.

Specific examples of the stabilizer include sodium thiosulfate, sodium sulfite, calcium lactate, lanolin, triacetin, castor oil, magnesium sulfate, and the like.
<Application form, use, and dosage form>
The application form of the MMP activity inhibitor and the liquid oral composition is not particularly limited, and can be used as, for example, medicines, quasi-drugs, cosmetics, etc. The use of the MMP activity inhibitor and the liquid oral composition can be appropriately adopted from known uses used as liquid dosage forms. Specifically, for example, chewing agents, oral dissolving agents, oral disintegrating agents, tongue care agents, mouth fresheners, mouthwashes, gargles, liquid dentifrice, biofilm dispersants, oral odor prevention agents, gum massage agents, oral moisturizing agents, tongue coating removers, oral coating agents, oral germicides, throat germicides, oral throat agents, periodontal disease treatment agents, denture coating agents, denture stabilizers, denture preservatives, denture cleaners, implant care agents, etc. The MMP activity inhibitor may also be used as a toothpaste or denture attachment agent.

The MMP activity inhibitor and the liquid oral composition contain a solvent, such as water or alcohol. The dosage form of the MMP activity inhibitor is not particularly limited, and can be applied in the form (dosage form) of, for example, ointment, paste, spray, gel, liquid, suspension, gum, tablet, drop, etc.

<Action>
The MMP activity inhibitor and the liquid oral composition exhibit excellent inhibitory effects against collagenase-type matrix metalloproteinase (MMP) activity due to the above-mentioned components. Specific examples of collagenase-type MMPs include MMP-1, MMP-8, and MMP-13. Among these, the MMP activity inhibitor exhibits a more excellent inhibitory effect against MMP-8.

In periodontal diseases such as periodontitis, tissue destruction caused by a disruption in the metabolic balance between synthesis and decomposition of periodontal tissue due to abnormal expression of collagenase-type MMPs is closely related. By inhibiting the action of collagenase-type MMPs, the degradation of collagen that constitutes periodontal tissues can be suppressed.

The MMP activity inhibitor exerts an effect of inhibiting the destruction and promoting the regeneration of periodontal tissues such as junctional epithelium, gingival sulcus epithelium, and periodontal ligament, by inhibiting MMP activity. In other words, the MMP activity inhibitor can be preferably used as an agent for inhibiting the destruction and promoting the regeneration of periodontal tissues. Furthermore, the MMP activity inhibitor exerts an effect of inhibiting the decomposition and promoting the regeneration of periodontal tissue collagen by inhibiting MMP activity. In other words, the MMP activity inhibitor can be preferably used as an agent for inhibiting the decomposition and promoting the regeneration of periodontal tissue collagen. Furthermore, the MMP activity inhibitor exerts an effect of inhibiting the decomposition and promoting the regeneration of gingival collagen by inhibiting MMP activity. In other words, the MMP activity inhibitor can be preferably used as an agent for inhibiting the decomposition and promoting the regeneration of gingival collagen.

In inflammatory diseases such as periodontal disease, for example, neutrophils infiltrate and accumulate in the gingival crevicular fluid in response to bacteria in the periodontal pocket, producing MMP-8. This destroys the connective tissue attachment of the periodontal pocket, leading to an increase in pocket depth and gingival recession, and as the condition progresses, the teeth become loose and fall out. MMP activity inhibitors inhibit the activity of neutrophil collagenase that has already been produced by inhibiting MMP activity, thereby exerting an effect of suppressing the deepening of the periodontal pocket and promoting repair. In other words, MMP activity inhibitors can be preferably used as agents for suppressing the deepening of periodontal pockets and promoting repair.

Due to the above-mentioned effects, the MMP activity inhibitor and liquid oral composition of this embodiment can be used to inhibit gingival recession, inhibit gingival destruction, inhibit periodontal tissue destruction, inhibit gum recession, inhibit gingival collagen decomposition, inhibit attachment loss, protect periodontal tissue collagen, inhibit periodontal tissue destruction associated with chronic inflammation, and/or protect dentin collagen, etc.

In this specification, "inhibiting attachment loss" means inhibiting the gingival epithelium from peeling off from the tooth surface and the migration of the attachment position between the gingiva and the tooth toward the tooth root. "Inhibiting gingival recession" means inhibiting the entire gingiva from migrating toward the tooth root. "Inhibiting gingival recession" means inhibiting exposure of the tooth root that accompanies the migration of the entire gingiva toward the tooth root.

The effects of this embodiment will be described.
(1-1) The MMP activity inhibitor and the liquid oral composition of the present embodiment contain 0.35% by mass or more of citric acids, and therefore can exhibit excellent collagenase-type matrix metalloproteinase (MMP) activity inhibitory effect.

(1-2) By inhibiting collagenase-type matrix metalloproteinase (MMP) activity, it is expected that the effects of suppressing gingival recession, suppressing gingival destruction, suppressing periodontal tissue destruction, suppressing gum recession, suppressing gingival collagen degradation, suppressing attachment loss, protecting periodontal tissue collagen, suppressing periodontal tissue destruction associated with chronic inflammation, and/or protecting dentin collagen can be expected.

Second Embodiment
A second embodiment of the MMP activity inhibitor and oral composition of the present invention will be described below, focusing on the differences from the first embodiment described above.

The components contained in the MMP activity inhibitor of this embodiment are citric acids and β-glycyrrhetinic acid.
The oral composition of this embodiment contains the same components as the MMP activity inhibitor. The MMP activity inhibitor and the oral composition may contain only one of the above components, or may contain two of the above components in combination. The oral composition may be for inhibiting collagenase-type matrix metalloproteinase (MMP) activity.

<Citric acid>
Specific examples of the citric acids are the same as those described in the first embodiment.
The lower limit of the content of the citric acid in the oral composition of this embodiment is 0.05% by mass, preferably 0.1% by mass. When the lower limit of the content is 0.05% by mass, excellent inhibitory effect against collagenase-type matrix metalloproteinase (MMP) activity can be exhibited by combining with β-glycyrrhetinic acid.

The upper limit of the content of citric acids in the oral composition is set as appropriate, but is preferably 5% by mass, and more preferably 4% by mass. When the upper limit of the content is 5% by mass, the collagenase-type matrix metalloproteinase (MMP) activity inhibitory effect can be efficiently exerted.

The upper or lower limit of the citric acid content may be, for example, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0% by mass. The citric acid content is expressed as the content converted to citric acid.

<β-Glycyrrhetinic Acid>
β-Glycyrrhetinic acid is a component contained in plants of the genus Glycyrrhiza in the family Fabaceae. β-Glycyrrhetinic acid is the aglycone of glycyrrhizinic acid.

The lower limit of the content of β-glycyrrhetinic acid in the oral composition is 0.025% by mass, and preferably 0.03% by mass. When the lower limit of the content is 0.025% by mass, an excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity can be achieved by using it in combination with citric acids.

The upper limit of the content of β-glycyrrhetinic acid in the oral composition is set appropriately, but is preferably 2% by mass, and more preferably 1% by mass. When the upper limit of the content is 2% by mass, the collagenase-type matrix metalloproteinase (MMP) activity inhibitory effect can be efficiently exerted.

The upper or lower limit of the β-glycyrrhetinic acid content may be, for example, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or 2% by mass.

<Other ingredients>
The oral composition may contain other components other than the above-mentioned components depending on the application purpose, form, use, etc. Examples of other components include surfactants, flavoring agents, sweeteners, humectants, binders, preservatives, colorants, pH adjusters, chelating agents, medicinal ingredients, bases, abrasives, stabilizers, etc. The other components may be any known components that are blended into oral compositions. The oral composition may contain only one of the above other components alone, or may contain two or more of them in combination. Specific examples of other components are the same as those described in the first embodiment.

<Application form, use, and dosage form>
The application form of the MMP activity inhibitor and the oral composition is not particularly limited, and can be used as, for example, a medicine, a quasi-drug, a cosmetic, etc. The use of the MMP activity inhibitor and the oral composition can be appropriately adopted from known uses. Specific examples include chewing agents, oral dissolving agents, oral disintegrating agents, tongue care agents, oral fresheners, toothpastes, mouthwashes, gargles, liquid dentifrices, biofilm dispersants, oral fouling agents, gum massaging agents, oral moisturizing agents, tongue coating removers, oral application agents, oral germicides, throat germicides, oral throat agents, periodontal disease treatment agents, denture mounting agents, denture coating agents, denture stabilizers, denture preservatives, denture cleaners, implant care agents, etc.

The dosage form of the MMP activity inhibitor and the oral composition is not particularly limited, and can be applied in the form (dosage form) of an ointment, paste, spray, gel, liquid, suspension, gum, tablet, drop, etc. by including a solvent such as water or alcohol.

<Action>
This is the same as the description of the first embodiment.
The effects of this embodiment will be described.

(2-1) The MMP activity inhibitor and oral composition of this embodiment contain 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid. Therefore, they can exert an excellent inhibitory effect on collagenase-type matrix metalloproteinase (MMP) activity.

(2-2) By inhibiting collagenase-type matrix metalloproteinase (MMP) activity, it is expected that the effects of suppressing gingival recession, suppressing gingival destruction, suppressing periodontal tissue destruction, suppressing gum recession, suppressing gingival collagen degradation, suppressing attachment loss, protecting periodontal tissue collagen, suppressing periodontal tissue destruction associated with chronic inflammation, and/or protecting dentin collagen can be expected.

The above embodiment can be modified as follows: This embodiment and the following modifications can be combined with each other to the extent that no technical contradiction occurs.
- It may also be applied to pets, livestock and other captive animals other than humans.

The MMP activity inhibitor and oral composition of this embodiment will be described in more detail based on the following examples. Note that the MMP activity inhibitor and oral composition are not limited to the configurations described in the Examples section.

<Test Example 1: MMP-8 activity inhibition test by citric acids>
(Preparation)
As MMP-8 activity inhibitors, sample solutions of Examples 1 and 2 and Comparative Examples 1 to 3 were prepared. The types of materials contained in the sample solutions of Examples 1 and 2 and Comparative Examples 1 to 3 are as shown in Table 1 below. The sample solutions of Examples 1 and 2 and Comparative Examples 1 to 3 were diluted with a buffer + 5% DMSO solution included in the kit described below so that the concentrations of each material were as shown in Table 1.

(Measurement of MMP-8 activity inhibition)
MMP-8 (neutrophil collagenase) activity was measured using an MMP-8 fluorimetric drug discovery kit (Enzo Life Sciences, Inc.), essentially following the instructions in the manual.

That is, 20 μL of 200-fold diluted MMP-8 was added to 70 μL of each solution prepared as described above, and the mixture was allowed to react at 37°C for 60 minutes. At this time, a sample was prepared as a positive control in which no material was added but an equal amount of buffer was added, and a sample was prepared as a positive control in which no MMP-8 was added but an equal amount of buffer was added to measure the background of the substrate alone.

A fluorescent substrate (methylcoumarinamide fluorescent substrate: Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ ) was dissolved and diluted 10-fold with buffer (concentration 40 μM). 10 μL of the diluted fluorescent substrate was added, and after reacting at 37° C. for 20 minutes, the fluorescence intensity was measured at excitation light: 328 nm and emission light: 420 nm. The value (NC) obtained by subtracting the fluorescence intensity of the substrate alone as background from the fluorescence intensity of the positive control sample was taken as 100%, and the ratio of the value obtained by subtracting the fluorescence intensity of the substrate alone as background from the fluorescence intensity of each sample was calculated as the enzyme activity. The lower the ratio value, the stronger the inhibition of MMP-8 activity. Measurements were performed with N=3, and the calculated average values and standard deviations (SD) are shown in Table 1.

As shown in Table 1, it was confirmed that the citric acid solutions of the predetermined concentrations in Examples 1 and 2 exhibited excellent MMP-8 activity inhibitory effects. On the other hand, the results of Comparative Examples 2 and 3, which had low citric acid concentrations, both showed low MMP-8 activity inhibitory effects.

<Test Example 2: MMP-8 activity inhibition test by citric acids and β-glycyrrhetinic acid>
(Preparation)
As MMP-8 activity inhibitors, sample solutions of Examples 3 and 4 and Comparative Examples 4 and 5 were prepared. The types of materials contained in the sample solutions of Examples 3 and 4 and Comparative Examples 4 and 5 are as shown in Table 2 below. The sample solutions of Examples 3 and 4 and Comparative Examples 4 and 5 were diluted with a buffer + 5% DMSO solution included in the kit described below so that the concentrations of each material were as shown in Table 2.

(Measurement of MMP-8 activity inhibition)
The measurement was performed in the same manner as in Test Example 1. Measurement was performed with N=3, and the calculated average value and standard deviation (SD) are shown in Table 2.

As shown in Table 2, Comparative Example 5, in which β-glycyrrhetinic acid was used alone, showed a low MMP-8 activity inhibitory effect. On the other hand, Example 3 or Example 4, in which β-glycyrrhetinic acid was used in combination with citric acid at the same concentrations as Comparative Example 2 or Comparative Example 3 of Test Example 1, was confirmed to exhibit an excellent MMP-8 activity inhibitory effect.

Claims (7)

A collagenase-type matrix metalloproteinase (MMP) activity inhibitor characterized by containing 0.35% by mass or more of citric acid. A collagenase-type matrix metalloproteinase (MMP) activity inhibitor characterized by containing 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid. The collagenase-type matrix metalloproteinase (MMP) activity inhibitor according to claim 1 or 2, which is an agent for inhibiting destruction of periodontal tissue and promoting regeneration, an agent for inhibiting decomposition and promoting regeneration of periodontal tissue collagen, an agent for inhibiting decomposition and promoting regeneration of gingival collagen, or an agent for inhibiting deepening of periodontal pockets and promoting repair. A liquid oral composition containing 0.35% by mass or more of citric acids. A liquid oral composition containing 0.35% by mass or more of citric acids and used to inhibit collagenase-type matrix metalloproteinase (MMP) activity. An oral composition containing 0.05% by mass or more of citric acids and 0.025% by mass or more of β-glycyrrhetinic acid. An oral composition comprising 0.05% by mass or more of a citric acid and 0.025% by mass or more of β-glycyrrhetinic acid, and used for inhibiting collagenase-type matrix metalloproteinase (MMP) activity.