JP7553620B2 - Anti-inflammatory Composition - Google Patents

Description

The present invention relates to an anti-inflammatory composition containing glycyrrhizinic acid or a salt thereof, and more particularly to an anti-inflammatory composition containing glycyrrhizinic acid or a salt thereof and a specific plant extract.

Glycyrrhizic acid or its salts are ingredients contained in the herbal medicine liquorice.
It is known to have an anti-inflammatory effect. Glycyrrhizic acid or its salts are widely used in mouthwashes for suppressing or preventing the symptoms of inflammatory diseases such as periodontal disease, topical skin preparations for suppressing skin inflammation, hair growth agents, soaps, etc., due to their anti-inflammatory effect.

For example, Patent Document 1 discloses a liquid oral composition containing glycyrrhizinate as an anti-inflammatory agent, and states that it can prevent oral diseases such as gingivitis.
Furthermore, Patent Document 2 discloses an external skin preparation containing glycyrrhizinic acid, and states that it is effective against inflammation such as allergic inflammation and atopic dermatitis.

JP 2001-261576 A JP 2006-327965 A

As the number of elderly people suffering from advanced inflammation is increasing in the super-aging society, there is a demand for anti-inflammatory compositions with higher anti-inflammatory effects. Therefore, the object of the present invention is to provide an anti-inflammatory composition containing glycyrrhizinic acid or a salt thereof with enhanced anti-inflammatory effects.

As a result of extensive research aimed at solving the above-mentioned problems, the inventors discovered that the anti-inflammatory effect of glycyrrhizinic acid or a salt thereof is enhanced by adding a certain plant extract together with glycyrrhizinic acid or a salt thereof, and thus completed the present invention.

That is, the present invention has been able to solve the above problems by the means described below.
1. An anti-inflammatory composition comprising glycyrrhizic acid or a salt thereof and at least one extract selected from the group consisting of extracts of horsetail, hawthorn, peony root, witch hazel, white birch and sage.
2. The anti-inflammatory composition according to 1 above, which is an oral composition.

The anti-inflammatory composition of the present invention contains glycyrrhizinic acid or a salt thereof and a specific plant extract, thereby making it possible to enhance the anti-inflammatory effect of glycyrrhizinic acid or a salt thereof.

FIG. 1 is a diagram showing the relative expression level of IL-6 mRNA in Example 1-1. FIG. 2 is a diagram showing the relative expression level of IL-6 mRNA in Example 1-2.

The present invention will be explained in more detail below.

Anti-inflammatory Composition
The anti-inflammatory composition of the present invention is characterized by containing glycyrrhizic acid or a salt thereof and at least one extract selected from the group consisting of extracts of horsetail, hawthorn, peony, witch hazel, white birch and sage.

Glycyrrhizic acid or its salts are components contained in the roots and stems of licorice, a type of herbal medicine. Glycyrrhizic acid or its salts are known to have the effect of suppressing inflammation of the mouth and throat, that is, to have an anti-inflammatory effect.
Examples of glycyrrhizinic acid or salts thereof in the present invention include glycyrrhizinic acid, dipotassium glycyrrhizinate ( _GK2 ), trisodium glycyrrhizinate, monoammonium glycyrrhizinate, diammonium glycyrrhizinate, disodium glycyrrhizinate, etc., and these can be used alone or in combination of two or more. Among these, dipotassium glycyrrhizinate is more suitable from the viewpoint of obtaining the effects of the present invention to a high degree.
Glycyrrhizic acid or a salt thereof can be obtained by extraction from licorice using a conventionally known method, or a commercially available product such as dipotassium glycyrrhizinate or monoammonium glycyrrhizinate manufactured by Alps Pharmaceutical Co., Ltd. or Maruzen Pharmaceutical Co., Ltd. can be used.

The amount of glycyrrhizinic acid or a salt thereof may be, for example, 0.
0001 to 1.0 mass%, preferably 0.0005 to 0.1 mass%, more preferably 0.
The content can be 0.001 to 0.015% by mass. By making the content 0.0001% by mass or more, the anti-inflammatory effect of glycyrrhizinic acid or a salt thereof is expected to be enhanced, and by making the content 1.0% by mass or less, formulation becomes easier and a good feeling of use can be obtained, particularly in the case of a liquid oral composition.

The anti-inflammatory composition of the present invention contains at least one extract selected from the group consisting of extracts of horsetail, hawthorn, peony, witch hazel, birch and sage.
The anti-inflammatory composition of the present invention is a combination of glycyrrhizinic acid or a salt thereof and the above-mentioned specific plant extract, i.e., by combining glycyrrhizinic acid or a salt thereof with the above-mentioned specific plant extract from among many plant extracts, the anti-inflammatory effect is enhanced.

In particular, from the viewpoint of the effect of the present invention of enhancing the anti-inflammatory effect, it is preferable to contain at least one extract selected from the group consisting of extracts of horsetail, hawthorn, peony root, and witch hazel, and it is more preferable to contain an extract of at least one of horsetail and hawthorn.
When three or more of the above extracts are contained, it is preferable that, in addition to the extracts of horsetail and hawthorn, at least an extract of peony, hamamelis, white birch or sage is contained.

The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is preferably glycyrrhizinic acid or a salt thereof: horsetail extract, hawthorn extract, peony extract, witch hazel extract, white birch extract, and sage extract=600:1 to 5:4, more preferably 450:1 to 3:2, and even more preferably 300:1.
The ratio is preferably 200:1 to 2:1, and most preferably 200:1 to 10:3. In the above content ratio, "horsetail extract, hawthorn extract, peony extract, witch hazel extract, birch extract, and sage extract" means the total content of each plant extract.

In the case where three or more of the above extracts are contained, the mass ratio is glycyrrhizinic acid or a salt thereof:
The ratio of horsetail extract:hawthorn extract:peony extract, hamamelis extract, birch extract, or sage extract is preferably 600:1:1:1 to 1.25:1:1:1, more preferably 450:1:1:1 to 1.5:1:1:1, even more preferably 300:1:1:1 to 2:1:1:1, and most preferably 200:1:1:1 to 3:1:
The ratio is 1:1. In addition, in the above content ratio, "peony extract, witch hazel extract, birch extract, or sage extract" means the content of any one of the plant extracts.

The content ratio of the plant extracts (in terms of solid matter) contained in the anti-inflammatory composition of the present invention is preferably, in terms of mass ratio, glycyrrhizinic acid or a salt thereof:Equisetum arvense extract=600:1 to 5:4.
It is more preferable that the ratio is from 450:1 to 3:2, and even more preferable that the ratio is from 300:1 to 2:1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, preferably glycyrrhizinic acid or a salt thereof: hawthorn extract = 600:1 to 5:4, more preferably 450:1 to 3:2, and even more preferably 300:1 to 2:1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, preferably glycyrrhizinic acid or a salt thereof: peony extract, witch hazel extract, white birch extract, or sage extract = 600:1 to 5:4, more preferably 450:1 to 3:2, and even more preferably 300:1 to 2:1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, preferably horsetail extract: hawthorn extract=1:10,000 to 10,000:1, more preferably 1:1,000 to 1,000:1, and more preferably 1:500 to 500:
It is even more preferred that it is 1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, preferably horsetail extract:peony extract, witch hazel extract, white birch extract, or sage extract=1:10,000 to 10,000:1, and more preferably 1:1,000 to 1,000:1.
More preferably, the ratio is 1:500 to 500:1, and even more preferably.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is preferably hawthorn extract:peony extract, hamamelis extract, white birch extract, or sage extract=1:10,000 to 10,000:1, more preferably 1:1,000 to 1:1,000 to 1
It is more preferably 000:1, and even more preferably 1:500 to 500:1.
In addition, in the above content ratio, "peony extract, witch hazel extract, birch extract, or sage extract" means the content of any one of the respective plant extracts.

Furthermore, when the anti-inflammatory composition of the present invention is a liquid composition, it preferably contains at least one of horsetail extract, hawthorn extract, and witch hazel extract, from the viewpoint of being less likely to form aggregates even during long-term storage.

The above plant extract may be a commercially available one as described later in the Examples, or may be one extracted from a plant by a method described later. The method for extracting the plant extract is not particularly limited, and may be any conventionally known method. For example, an extract may be obtained by squeezing, solvent extraction, water distillation, steam distillation, or the like from any part of the above plant as is or after cutting, crushing, or the like. As the method for solvent extraction, any method known in the art may be adopted, and conventionally known extraction methods such as water (including warm water and hot water) extraction, alcohol extraction, supercritical extraction, microwave extraction, and squeeze extraction may be used.

When solvent extraction is performed, examples of the solvent include water; alcohols (whether anhydrous or hydrous) such as methanol, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, and 1,3-butylene glycol; ketones such as acetone; ethers such as diethyl ether and dioxane; nitriles such as acetonitrile; esters such as ethyl acetate; hexane, xylene, benzene, and chloroform.
The solvent for the solvent extraction is preferably water, alcohols, ketones, hexane, etc., more preferably water, alcohols, and ketones. These solvents may be used alone or in combination of two or more.

The obtained extract may be used as it is, or may be dried before use. If necessary, the obtained extract may be purified, concentrated, or the like. Examples of the purification process include filtration, or adsorption, decolorization, and separation using an ion exchange resin or an activated carbon column. Examples of the concentration process include conventional methods such as an evaporator. These may be further dried, such as freeze-dried, or may be powdered according to a conventional method. If necessary, the extract thus obtained may be dissolved in water, ethanol, or the like before use.

When the anti-inflammatory composition of the present invention is a composition for oral cavity, the total amount of the above-mentioned plant extract (in terms of solid matter) is, for example, 0.001 ppm by mass to 3 mass %, preferably 0.01 ppm by mass to 0.1
By making the content 0.001 ppm by mass or more, the extract (converted into solid matter) can exhibit its anti-inflammatory effect, and by making the content 3% by mass or less, the extract is less likely to discolor and has little effect on the flavor.

The content ratio of the plant extract (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, preferably glycyrrhizinic acid or a salt thereof:(plant extract)=450:1 to 3:2, more preferably 300:1 to 2:1, and most preferably 200:1 to 10:3. Here, the "plant extract" in the above content ratio means the total amount of the plant extract contained in the composition.

The use of the anti-inflammatory composition of the present invention is not particularly limited as long as it is an application that can enjoy the anti-inflammatory effect of glycyrrhizinic acid or a salt thereof, and it can be used for various applications. For example,
Examples include oral compositions, compositions for topical use on the skin, cosmetics, and cleansing agents.

When the anti-inflammatory composition of the present invention is a composition for oral cavity, it is effective in treating various inflammatory diseases occurring in the oral cavity,
For example, it can be applied to periodontal diseases such as gingivitis and periodontitis, inflammation caused by stomatitis or fungal infections such as Candida, mucositis caused by contact with prosthetic appliances such as dentures, and swelling of wounds associated with dental treatment.
The oral composition may be in the form of, for example, mouthwash, toothpaste, mouth freshener, gargle (mouthwash), toothpaste such as liquid toothpaste and toothpaste, lozenge, chewing gum, and the like.
In particular, in order to ensure that the entire composition is sufficiently distributed throughout the oral cavity, the composition is preferably used as a mouthwash, which is administered in an appropriate amount into the oral cavity. To prepare a mouthwash, for example, the composition may be prepared by a conventional method using water, ethanol, or the like as a solvent.
Furthermore, for example, liquid dentifrices may be used that contain abrasives such as calcium hydrogen phosphate, aluminum hydroxide, anhydrous silicic acid, and calcium carbonate, as necessary.

Furthermore, the oral composition may contain other optional components as long as they do not impair the effects of the present invention.
For example, fluorides such as sodium fluoride and sodium monofluorophosphate; azulene;
Azulene sulfonate, β-glycyrrhetinic acid, dihydrocholesterol, epidihydrocholesterol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate,
Anti-inflammatory agents such as ε-aminocaproic acid, tranexamic acid, allantoin, ascorbic acid, etc.;
Anti-tartar agents such as phosphates, polyphosphates, methoxyethylene maleic anhydride copolymers, zinc chloride, and organic zinc salts; astringents such as hinokitiol, aluminum allantoin chlorhydroxyl, aluminum allantoin dihydroxyl, and sodium chloride; humectants such as glycerin, sorbitol, and polyethylene glycol; foaming agents such as sodium lauryl sulfate; pinene, peppermint oil, cinnamon oil, clove oil, eugenol, lemon oil,
Fragrances such as vanillin, cineole, and eucalyptus oil; saccharin, sodium saccharin,
Sweeteners such as sucralose, xylitol, erythritol, sorbitol, maltitol, and stevia; Blue No. 1, Yellow No. 5, Yellow No. 4, Yellow No. 203, Green No. 3, and Green No. 201;
Coloring agents such as Red No. 102; preservatives such as parabens and sodium benzoate; pH adjusters such as monosodium phosphate, disodium phosphate, citric acid and sodium citrate; POE hydrogenated castor oil, POE-POP block polymer, POE-POP alkyl ether, POE
nonionic surfactants such as alkyl ethers, POE alkyl phenyl ethers, POE fatty acid esters, POE higher alcohol ethers, POE-POP fatty acid esters, POE sorbitan fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, and propylene glycol fatty acid esters; sodium lauryl sulfate, sodium myristyl sulfate, POE alkyl ether sulfates, sodium lauroyl sarcosine, sodium myristoyl sarcosine, alkyl ether carboxylates,
Anionic surfactants such as alkyl phosphates, POE alkyl ether phosphates, N-acyltaurine salts, POE alkyl ether phosphoric acid/phosphate salts, and sulfonates; cationic surfactants such as alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, and POE alkylamine/fatty acid amide; 2-alkyl-N-carboxymethyl-N
-Hydroxyethylimidazolinium betaine, coconut oil fatty acid amidopropyl betaine,
Examples of the surfactant include amphoteric surfactants such as sodium lauryldiaminoethylglycine.

Furthermore, when the anti-inflammatory composition of the present invention is a composition for external use on the skin, it can be applied to various inflammatory diseases that occur on the skin, such as allergic dermatitis caused by contact with allergens or insect bites, endogenous skin diseases such as atopic dermatitis, and dermatitis associated with a decrease in barrier function, such as xerosis.
The composition for external use on the skin can be designed as desired without any particular limitation, for example, in the form of a lotion, an emulsion such as milky lotion or cream, or an oil.
The skin external composition can contain any component that is generally used in skin external preparations such as cosmetics, and can be appropriately selected and used according to the above-mentioned desired formulation.Such optional components include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japan wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, Japanese laurel wax, lanolin, reduced lanolin, hard lanolin, jojoba wax, etc.; hydrocarbons such as liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, vaseline, microcrystalline wax, etc.;
higher fatty acids such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, and undecylenic acid; higher alcohols such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol; synthetic ester oils such as cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, diisopropyl adipate, 2-ethylhexyl sebacate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glycerin di-2-heptylundecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, and pentane erythritol tetra-2-ethylhexanoate;
Chain polysiloxanes such as dimethylpolysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane, etc.; cyclic polysiloxanes such as octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexanesiloxane, etc.; oils such as silicone oils including modified polysiloxanes such as amino-modified polysiloxane, polyether-modified polysiloxane, alkyl-modified polysiloxane, fluorine-modified polysiloxane, etc.; fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate,
Anionic surfactants such as alkyl triethanolamine ether sulfate; cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, laurylamine oxide; amphoteric surfactants such as imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaine, amido betaine, sulfobetaine, etc.), and acyl methyl taurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (
POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.),
POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE
E alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonyl phenyl ether, etc.), Pluronic types, POE/PO
Nonionic surfactants such as P alkyl ethers (POE/POP 2-decyl tetradecyl ether, etc.), tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexanediol, 1,2-hexanediol, and 1,2-octanediol; Moisturizing ingredients such as sodium lolidonecarboxylate, lactic acid, sodium lactate, etc.; powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, barium sulfate, etc., which may be surface-treated; inorganic pigments such as red iron oxide, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine, Prussian blue, titanium oxide, and zinc oxide, which may be surface-treated; pearling agents such as titanium mica, fish phosphorus foil, and bismuth oxychloride, which may be surface-treated; Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 2, which may be laked, etc.
No. 23, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 2
Organic dyes such as No. 01, Purple No. 201, and Red No. 204; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomers; para-aminobenzoic acid-based ultraviolet absorbers; anthranilic acid-based ultraviolet absorbers; salicylic acid-based ultraviolet absorbers; cinnamic acid-based ultraviolet absorbers; benzophenone-based ultraviolet absorbers; sugar-based ultraviolet absorbers; 2-(
2'-hydroxy-5'-t-octylphenyl)benzotriazole, 4-methoxy-
Ultraviolet absorbers such as 4'-t-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; vitamin A or its derivatives, vitamin B6 hydrochloride, vitamin B
vitamin B such as vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, vitamin B15 or a derivative thereof; α-tocopherol,
Examples include vitamins such as β-tocopherol, γ-tocopherol, vitamin E acetate, and the like, vitamins D, vitamin H, pantothenic acid, pantethine, pyrroloquinoline quinone, and the like; and antibacterial agents such as phenoxyethanol, and the like.

The anti-inflammatory effect of the anti-inflammatory composition of the present invention can be evaluated, for example, by measuring the expression levels of mRNA and protein of inflammatory cytokines in tissues and cells collected from various organs experiencing inflammation, or in cultured cells stimulated with LPS or the like, as described below in the Examples.
Inflammatory cytokines such as IL-6 and TNF-α can be used as inflammation markers.

For example, when evaluating the anti-inflammatory effect based on the expression level of IL-6 mRNA, the evaluation can be performed by determining the relative expression level of IL-6 mRNA based on various measurements of the expression levels of IL-6 mRNA under the conditions described in the Examples below.
Details will be described later in the Examples.

Alternatively, when evaluating the anti-inflammatory effect based on the protein expression level of TNF-α, the inflammation inhibition rate can be calculated from the protein expression level of TNF-α measured in various ways under the conditions described in the Examples described below, using the following formula, and the inflammation inhibition rate in each sample can be calculated by setting the inflammation inhibition rate in the case where no plant extract is used ( _GK2 treatment: application of only _GK2 and LPS stimulation) to 1.
Inflammation inhibition rate of " _GK2 treatment" = (TNF-α amount of "LPS treatment" - TNF-α amount of " _GK2 treatment"
(TNF-α amount in "LPS treatment" - TNF-α amount in "untreated")
Inflammation inhibition rate in each sample = {(Amount of TNF-α in "LPS treatment" - Amount of TNF-α in each sample) / (Amount of TNF-α in "LPS treatment" - Amount of TNF-α in "untreated")} / Inflammation inhibition rate of _GK2 treatment. Details will be described later in the Examples.

The inflammation inhibition rate is preferably 1.0 or more, and more preferably 2.0 or more.
The relative expression level of IL-6 mRNA is preferably 1.0 or less, and more preferably 0.
It is more preferable that the ratio is 0.5 or less.

The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples.

Example 1: Evaluation of anti-inflammatory effect using the mRNA expression level of inflammatory cytokines as an index (Example 1-1)
In this test, the anti-inflammatory effect of the anti-inflammatory composition of the present invention was evaluated by measuring the mRNA expression level of IL-6, a type of inflammatory cytokine, in cells in which inflammation was induced with LPS derived from Porphyromonas gingivalis (hereinafter referred to as P.g.).
Specifically, first, a GK ₂ solution containing dipotassium glycyrrhizinate (GK ₂ ), a plant extract solution containing one type of plant extract, and a LPS solution containing P. g.-derived LPS that stimulates cells were prepared.
Then, cells were cultured in the presence of the prepared solutions, and the mRNA expression level of IL-6, a type of inflammatory cytokine, was measured to determine whether the expression level of IL-6 was increased or decreased.
The anti-inflammatory effect was evaluated.
In this test, the control sample was “ _GK2 ” which was stimulated with LPS.
Tests were also performed with " _GK2- treated" (without plant extract), "LPS-treated" (without _GK2 and plant extract), in which only LPS stimulation was performed, and "untreated" (without _GK2 , plant extract, and LPS stimulation), in which neither _GK2 nor plant extract was applied and neither LPS stimulation was performed.
The cell culture medium used was E-MEM medium supplemented with 1% by mass FBS.

[method]
1. Preparation of dipotassium glycyrrhizinate (GK ₂ ) solution GK ₂ (Maruzen Pharmaceutical Co., Ltd.), polyoxyethylene hydrogenated castor oil (Nikko Chemicals Co., Ltd., HCO-100), and purified water were dissolved in propylene glycol to a concentration of 1% by mass to obtain a GK ₂ stock solution. This GK ₂ stock solution was diluted with 1% FBS-supplemented E-MEM medium for 25 min.
A _GK2 solution was prepared by diluting the solution 100-fold.

2. Preparation of plant extract solutions Plant extract was weighed out so that the concentration was 0.1% by mass, and _GK2 , polyoxyethylene hydrogenated castor oil, and purified water were added so that each was 1% by mass, and dissolved in propylene glycol to obtain a plant extract stock solution. Various plant extract solutions were prepared by diluting this plant extract stock solution 2500 times with E-MEM medium supplemented with 1% by mass FBS.

3. Preparation of LPS solution LPS derived from P. g. (Invivogen) was diluted 500-fold with E-MEM medium containing 1% by mass FBS to prepare an LPS solution.

4. Preparation of test cells In a 12-well plate, culture medium and human oral squamous cell carcinoma cells: Ca9-22 at 10 ⁶ cells/w
The cells were seeded in 100 ml of each medium and cultured in a CO ₂ incubator (CO ₂ : 5%, 37° C.) for 24 hours to prepare test cells.

5. Induction of inflammation by LPS, exposure to samples, and RNA extraction (1) After removing the medium from the test cells, _GK2 solution, plant extract solution, and LPS solution were added to the test cells in a total volume of 2 mL as described below in (i) to (iv), and the cells were cultured in a _CO2 incubator ( _CO2 : 5%, 37°C) for 24 hours.
(i) 1 mL each of various plant extract solutions and LPS solutions was added to each sample (sample containing plant extract) to give final concentrations of 2.0 ppm by mass for _GK2 and 0.2 ppm by mass for the plant extract.
It was set to m.
(ii) " _GK2 treatment" (control sample) contained 1 mL each of _GK2 solution and LPS solution.
was added one by one.
(iii) "LPS treatment" (control sample) was performed using LPS solution and 1% FBS-added E-M
1 mL of EM medium was added to each well.
(iv) To the "untreated" (control sample), 2 mL of E-MEM medium containing 1% by mass FBS was added.
(2) Total RNA was extracted and purified from the cells using RNeasy Mini Kit (QIAGEN).

6. Measurement of mRNA expression level (RT-PCR)
RNA extracted from cells was purified using Transcriptor Universal cDNA
cDNA was synthesized using Master (Roche).

(Real-time PCR)
FastStart Essential DNA Green Master (Ro
Real-time PCR was carried out using a PCR kit (manufactured by Che). Real-time PCR was carried out for 600 seconds at 95°C for one cycle, and 50 cycles in total, each cycle consisting of 10 seconds at 95°C and 30 seconds at 60°C. The primers used are shown below.
GAPDH
Forward Primer: 5'-GCACCGTCAAGGCTGAGAAC-3
'
Reverse Primer: 5'-TGGTGAAGACGCCAGTGGA-3'
IL-6
Forward Primer: 5'-GCCAGAGCTGTGCAGATGAG-3
'
Reverse Primer: 5'-TCAGCAGGCTGGCATTTG-3'

[result]
The values (relative expression levels) of each sample, where the relative expression level of IL-6 in "GK ₂ treatment" corrected by the expression level of GAPDH was set to 1, are shown in Figure 1 (only each sample and "GK ₂ treatment" are shown). Note that the relative expression levels of IL-6 in Figure 1 show the average of a total of two tests.
The various plant extracts shown in FIG. 1 were as follows:
・Horsetail: Maruzen Pharmaceutical Co., Ltd. (product name) Horsetail Extract BG
Hawthorn: Manufactured by Ichimaru Falcos, (product name) Falcolex Hawthorn B
Peony: Maruzen Pharmaceutical Co., Ltd. (product name) Peony Extract BG-JC
・Witch hazel: Maruzen Pharmaceutical Co., Ltd. (product name) Witch hazel extract BG-J
・Birch: Maruzen Pharmaceutical Co., Ltd. (product name) Birch Extract BG
Sage: Falcos (product name) Falcolex Sage B, manufactured by Ichimaru Falcos
・Houttuynia cordata: Manufactured by Ichimaru Falcos Co., Ltd. (product name) Falcolex Houttuynia cordata B

As can be seen from the results in FIG. 1, when extracts of horsetail, hawthorn, peony, witch hazel, white birch, or sage were added in addition to dipotassium glycyrrhizinate, the expression level of IL-6 mRNA was reduced compared to the "GK ₂ treatment" in which no plant extract was added.
On the other hand, when the extract of Houttuynia cordata was added, the relative expression level of IL-6 increased compared to the " _GK2 treatment" in which no plant extract was added.
Therefore, it was confirmed that the anti-inflammatory effect was improved by selecting an extract of horsetail, hawthorn, peony, witch hazel, white birch, or sage from among plant extracts and adding it to dipotassium glycyrrhizinate.

(Example 1-2)
The anti-inflammatory effect was evaluated in the same manner as in Example 1-1, except that the plant extract was a plant extract of horsetail and/or hawthorn, and the final concentration of the plant extract was set to the concentrations shown in Figure 2.

[result]
The values (relative expression levels) of each sample, where the relative expression level of IL-6 in " _GK2 treatment" corrected by the expression level of GAPDH was set to 1, are shown in FIG. 2 (only each sample and " _GK2 treatment" are shown).
The relative expression level of IL-6 in FIG. 2 shows the average value of two tests performed in total.

As can be seen from the results in Figure 2, when both horsetail and hawthorn extracts were added in addition to dipotassium glycyrrhizinate, the expression level of IL-6 mRNA was reduced compared to "GK ₂ treatment" in which no plant extracts were added. In particular, the expression level of IL-6 mRNA was most reduced when both 0.2 ppm by mass of horsetail and 0.2 ppm by mass of hawthorn were added. In addition, for example, the expression level of IL-6 mRNA was more reduced when 0.04 ppm by mass of horsetail and 0.0 ppm by mass of hawthorn were added than when 0.2 ppm by mass of horsetail alone or 0.2 ppm by mass of hawthorn alone was added.
The expression level of IL-6 mRNA was decreased when the extracts of horsetail and hawthorn were added in combination at 4 ppm by mass, and the expression level of IL-6 mRNA was decreased when the extracts of horsetail and hawthorn were added in combination, compared to when the extracts of horsetail or hawthorn were added alone.
It was found that the expression level of A was decreased.

(Examples 1 to 3)
In Example 1-1, the plant extracts used were the three plant extracts shown in Table 1.
The anti-inflammatory effects were evaluated in the same manner as in Example 1-1, except that the final concentrations of _{No. 2} and each plant extract were set to the concentrations shown in Table 1.

[result]
The values (relative expression levels) of each sample are shown in Table 1 below, assuming that the relative expression level of IL-6 in " _GK2 treatment" corrected for the expression level of GAPDH is 1. The relative expression levels of IL-6 in Table 1 show the average of a total of two tests.

As can be seen from the results in Table 1, even when extracts of peony, hamamelis, birch, or sage were added in addition to the extracts of horsetail and hawthorn as plant extracts,
Compared to " _GK2 treatment" without the addition of the plant extract, the expression level of IL-6 mRNA was decreased.

(Examples 1 to 4)
In this study, Aggregatibacter Actinomycetemcomi
In the case of cells in which inflammation was induced with LPS derived from A.a.
The anti-inflammatory effect of the anti-inflammatory composition of the present invention was evaluated by measuring the mRNA expression levels of various inflammatory cytokines (IL-6, TNF-α).
Specifically, first, a GK ₂ solution containing dipotassium glycyrrhizinate (GK ₂ ), a plant extract solution containing a plant extract, an LPS solution containing A. a.-derived LPS that stimulates cells,
Then, cells were cultured in the presence of the above-prepared solutions, and the anti-inflammatory effect was evaluated by measuring the mRNA expression levels of IL-6 and TNF-α, which are types of inflammatory cytokines.
In this test, the control sample was “ _GK2 ” which was stimulated with LPS.
Tests were also performed with " _GK2- treated" (without plant extract), "LPS-treated" (without _GK2 and plant extract), in which only LPS stimulation was performed, and "untreated" (without _GK2 , plant extract, and LPS stimulation), in which neither _GK2 nor plant extract was applied and neither LPS stimulation was performed.
The cell culture medium was Keratinocyte-SFM (1x) [+] L-Gl
Bovine Pituitary Extract (final concentration: 25 μg
/mL), epidermal growth factor (final concentration: 0.05ng/mL), epidermal growth factor (final concentration: 0.05ng/
mL), penicillin (final concentration: 100 U/mL), streptomycin
(final concentration: 100 mg/mL) was added, and the mixture was sterilized by filtration (hereinafter referred to as K-SF
M medium) was used.

[method]
1. Preparation of dipotassium glycyrrhizinate (GK ₂ ) solution GK ₂ (Maruzen Pharmaceuticals), polyoxyethylene hydrogenated castor oil (H
GK2 _stock solution was obtained by dissolving 1% by mass each of GK2 extract (CO-100) and purified water in propylene glycol, and then diluting this GK2 _stock solution 2500 times with K-SFM medium to prepare a _GK2 solution.
2. Preparation of plant extract solution _GK2 (Maruzen Pharmaceuticals), polyoxyethylene hydrogenated castor oil (H
CO-100) and purified water were each 1% by mass, and the plant extract was 0.05% by mass or 0.
The extract was dissolved in propylene glycol to a concentration of 0.01% by mass to obtain a plant extract stock solution, which was then diluted 2500-fold with K-SFM medium to prepare various plant extract solutions.
3. Preparation of LPS solution A. a.-derived LPS was diluted with K-SFM medium to a concentration of 2 μg/mL to prepare an LPS solution.
4. Preparation of control solution Polyoxyethylene hydrogenated castor oil (HCO-100, manufactured by Nikko Chemicals Co., Ltd.) and purified water were each dissolved in propylene glycol to a concentration of 1% by mass, and the solution was further diluted 2500-fold with K-SFM medium to prepare a control solution.
5. Preparation of test cells In a 12-well plate, culture medium and immortalized oral mucosal epithelial cells: RT7 were added at 1.0 x 10 ⁵ cells.
The cells were seeded at 1 1/well and incubated in a _CO2 incubator ( _CO2 : 5%, 37°C) for 24 h.
The cells were cultured for 2 hours and used as test cells.

6. Induction of inflammation by LPS, exposure to samples, and extraction of RNA (1) After removing the medium from the test cells, 500 μL of fresh medium was added to each well and incubated at 37° C. for 4
After culturing for 8 hours, the medium was removed, and the prepared _GK2 solution, plant extract solution, LPS solution, and control solution were added to a total volume of 1 mL as described below in (i) to (iv), and the cells were left to stand for 3 hours in a _CO2 incubator ( _CO2 : 5%, 37°C), after which the cells were harvested.
(i) To each sample (sample containing plant extract), 500 μL each of various plant extract solutions and LPS solution was added. The final concentrations of _GK2 and plant extract were set to the concentrations shown in Table 2.
(ii) " _GK2 treatment" (control sample) was prepared by adding 500 mL of _GK2 solution and LPS solution.
Each μL was added.
(iii) To the "LPS treatment" (control sample), 500 μL each of the LPS solution and the control solution was added.
(iv) To the "untreated" (control) sample, 500 μL each of K-SFM medium and the control solution was added.
(2) Total RNA was extracted and purified from the cells using RNeasy Mini Kit (QIAGEN).

7. Measurement of mRNA expression level (RT-PCR)
RNA extracted from cells was purified using Transcriptor Universal cDNA
cDNA was synthesized by RT-PCR using Master (manufactured by Roche).

(Real-time PCR)
FastStart Essential DNA Green Master (Ro
Real-time PCR was carried out using a PCR kit (manufactured by Che). Real-time PCR was carried out for 1 cycle at 95°C for 60 seconds, followed by 45 cycles each consisting of 95°C for 10 seconds and 60°C for 30 seconds. The primers used are shown below.
GAPDH
Forward Primer: 5'-GCACCGTCAAGGCTGAGAAC-3
'
Reverse Primer: 5'-TGGTGAAGACGCCAGTGGA-3'
IL-6
Forward Primer: 5'-GCCAGAGCTGTGCAGATGAG-3
'
Reverse Primer: 5'-TCAGCAGGCTGGCATTTG-3'
・Human TNF-α
Forward Primer: 5'-ACAACCCTCAGACGCCACAT-3
'
Reverse Primer: 5'-GTGGAGCCGTGGGTCAGTAT-3
'

[result]
The values (relative expression levels) of each sample, where the relative expression level of IL-6 in " _GK2 treatment" corrected for the expression level of GAPDH was set to 1, are shown in Table 2. The relative expression levels of IL-6 in Table 2 show the average of a total of three tests.
Table 3 shows the numerical value (relative expression level) of each sample when the relative expression level of TNF-α in " _GK2 treatment" corrected by the expression level of GAPDH is set to 1. The relative expression level of TNF-α in Table 3 shows the average value of a total of five tests.

As can be seen from the results in Tables 2 and 3, when extracts of horsetail, hawthorn, peony, witch hazel, white birch, or sage were added in addition to dipotassium glycyrrhizinate, the mRNA expression levels of IL-6 and TNF-α were reduced compared to "GK ₂ treatment" in which no plant extract was added.
Therefore, it was confirmed that the anti-inflammatory effect was improved by selecting an extract of horsetail, hawthorn, peony, witch hazel, white birch, or sage and adding it to dipotassium glycyrrhizinate.

Example 2: Evaluation of anti-inflammatory effect using the protein expression level of TNF-α as an index (Example 2-1)
In this test, the anti-inflammatory effect of the anti-inflammatory composition of the present invention was evaluated by measuring the protein expression level of TNF-α, a type of inflammatory cytokine, in cells in which inflammation was induced with P. g.-derived LPS using the ELISA method described below.

[method]
1. 10% by mass FBS-supplemented RPMI 1640 medium was added with 10 nM PMA, and THP-1
The cells were seeded at 10 ⁵ cells/well onto a 24-well plate and cultured for 7 days to induce differentiation into macrophages.
2. After confirming induction of differentiation, the medium was replaced with RPMI 1640 medium supplemented with 1% by mass FBS, and cultured for 24 hours.
3. 1 mL of the diluted test sample solution, which was prepared by diluting the test sample described below 5000 times with RPMI 1640 medium, was added to the cells, and the diluted test sample solution was removed after 2 hours. After that, the cells were washed once with RPMI 1640 medium, and stimulated with P. g.-derived LPS (0.2 μg/mL, medium containing 1% FBS by mass).
4. Six hours after stimulation, the supernatant was collected and the amount of TNF-α protein was measured by ELISA.

The inflammation inhibition rate was calculated using the following formula, and the inflammation inhibition rate for each sample was determined assuming that the inflammation inhibition rate for the "GK ₂ treatment" described below was 1.0.
Inflammation inhibition rate of " _GK2 treatment" = (TNF-α amount of "LPS treatment" - TNF-α amount of " _GK2 treatment"
(TNF-α amount in "LPS treatment" - TNF-α amount in "untreated")
Inflammation inhibition rate for each sample = {(Amount of TNF-α in "LPS treatment" - Amount of TNF-α in each sample) / (Amount of TNF-α in "LPS treatment" - Amount of TNF-α in "untreated")} / Inflammation inhibition rate for " _GK2 treatment" In the formula, "Amount of TNF-α in LPS treatment" refers to the amount of TNF-α when the test is performed using liquid culture medium instead of the diluted sample liquid in the above method 3.
In addition, the "amount of TNF-α without treatment" refers to the amount of TNF-α without LPS stimulation and with 1% by mass FB instead.
The amount of TNF-α when the test was performed using the supernatant obtained after culturing in SRPMI medium for 6 hours is shown.

[Subject]
Sample containing plant extract: 1% by mass of GK ₂ + polyoxyethylene hydrogenated castor oil (HCO-100, manufactured by Nikko Chemicals) + plant extract (added so that the final concentration of GK ₂ was 2.0 ppm by mass and the final concentration of the plant extract was 0.05 ppm by mass or 0.01 ppm by mass)
GK ₂ treatment: 1% by mass of GK ₂ + polyoxyethylene hydrogenated castor oil (HCO-100, manufactured by Nikko Chemicals Co., Ltd.) (added so that the final concentration of GK ₂ becomes 2.0 ppm by mass)

[result]
The results are shown in Table 2. The plant extracts used are as follows:
・Horsetail: Maruzen Pharmaceutical Co., Ltd. (product name) Horsetail Extract BG
Hawthorn: Manufactured by Ichimaru Falcos, (product name) Falcolex Hawthorn B
Peony: Maruzen Pharmaceutical Co., Ltd. (product name) Peony Extract BG-JC
・Houttuynia cordata: Manufactured by Ichimaru Falcos Co., Ltd. (product name) Falcolex Houttuynia cordata B
Linden: Maruzen Pharmaceutical Co., Ltd. (product name) Linden Extract BG-J
・ Tea: Maruzen Pharmaceutical Co., Ltd. (product name) Green Tea Extract BG
Lily: Maruzen Pharmaceutical Co., Ltd. (product name) Lily Extract BG
・Seaweed: Ichimaru Falcos Co., Ltd. (product name) IPF-100K

As can be seen from the results in Table 4, when extracts of horsetail, hawthorn, or peony were added in addition to dipotassium glycyrrhizinate, the inflammation inhibition rate was higher than that of "GK ₂ treatment" in which no plant extract was added.
On the other hand, when extracts of plants such as Houttuynia cordata, Linden, tea, lily, or seaweed were added, the inflammation inhibition rate was lower than that of the " _GK2 treatment" in which no plant extract was added.

(Example 2-2)
In this test, the anti-inflammatory effect of the anti-inflammatory composition of the present invention was evaluated by measuring the protein expression level of TNF-α, a type of inflammatory cytokine, in cells in which inflammation was induced with A. a.-derived LPS using the ELISA method described below.
The dipotassium glycyrrhizinate (GK ₂ ) solution, plant extract solution, LPS solution, control solution and test cells were prepared in the same manner as in Examples 1-4 above.

[method]
1. After removing the medium from the test cells, _GK2 solution, plant extract solution, LPS solution,
Control solutions were added as follows (i) to (iv).
(i) To each sample (sample containing plant extract), 50 μL each of various plant extract solutions and LPS solution was added. The final concentrations of _GK2 and plant extract were set as shown in Table 5.
(ii) " _GK2 treatment" (control sample) contained 50 μl each of _GK2 solution and LPS solution.
L was added at a time.
(iii) To the "LPS treatment" (control sample), 50 μL each of the LPS solution and the control solution was added.
(iv) To the "untreated" (control) sample, 50 μL each of K-SFM medium and the control solution was added.
2. Each sample was placed in a CO ₂ incubator (CO ₂ : 5%, 37° C.), and the cells and supernatant were collected after 6 hours.
3. Human TNF-alpha DuoSet ELISA (R&D System
The test was performed using a FT-IR spectrometer (manufactured by ms) according to the manufacturer's instructions.
4. The inflammation inhibition rate was determined in the same manner as in Example 2-1 above.

[result]
The results are shown in Table 5. The plant extract used was the same as in Example 2-1.

As can be seen from the results in Table 5, when extracts of horsetail, hawthorn, peony, witch hazel, birch, or sage were added in addition to dipotassium glycyrrhizinate,
The inflammation suppression rate was higher than that of the " _GK2 treatment" in which no plant extract was added.

From the results of the above examples, it was found that a composition containing glycyrrhizinic acid or a salt thereof and at least one extract selected from the group consisting of extracts of horsetail, hawthorn, peony, witch hazel, white birch and sage has an anti-inflammatory effect.

Claims (2)

An anti-inflammatory composition for oral cavity, comprising glycyrrhizic acid or a salt thereof and at least one extract selected from the group consisting of extracts of hawthorn and witch hazel with alcohols. An anti-inflammatory composition for the oral cavity, which contains glycyrrhizinic acid or a salt thereof, and at least two extracts selected from the group consisting of extracts of hawthorn, horsetail, witch hazel, peony, sage, and birch with alcohols (however, excluding an anti-inflammatory composition for the oral cavity which contains glycyrrhizinic acid or a salt thereof, an extract of horsetail with alcohols, and an extract of birch with alcohols, and an anti-inflammatory composition for the oral cavity which contains glycyrrhizinic acid or a salt thereof, an extract of peony with alcohols, and an extract of sage with alcohols ).

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