JP7596059B2 - Anti-inflammatory Composition - Google Patents

Description

The present invention relates to an anti-inflammatory composition containing glycyrrhizinic acid or a salt thereof, and in particular 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, and are known to have anti-inflammatory properties. Due to their anti-inflammatory properties, glycyrrhizic acid or its salts are widely used in mouthwashes to suppress or prevent the symptoms of inflammatory diseases such as periodontal disease, topical skin preparations to suppress skin inflammation, hair growth agents, soaps, etc.

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 a skin preparation for external use that contains 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 we enter a super-aging society, the number of elderly people suffering from advanced inflammation is increasing, and there is a demand for anti-inflammatory compositions such as those described above that have a higher anti-inflammatory effect. Therefore, the object of the present invention is to provide an anti-inflammatory composition containing glycyrrhizinic acid or a salt thereof that has an enhanced anti-inflammatory effect.

As a result of intensive research conducted by the inventors to solve the above problems, they 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, 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 enhancing 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.

Glycyrrhizic acid or a salt thereof can be contained in an amount of, for example, 0.0001 to 1.0% by mass, preferably 0.0005 to 0.1% by mass, and more preferably 0.001 to 0.015% by mass, based on the total amount of the anti-inflammatory composition of the present invention. By making the amount 0.0001% by mass or more, the anti-inflammatory effect of glycyrrhizic acid or a salt thereof is expected to be enhanced, and by making the amount 1.0% by mass or less, formulation becomes easier and a good usability 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 its salt: horsetail extract, hawthorn extract, peony extract, witch hazel extract, birch extract, and sage extract = 600:1 to 5:4 by mass, more preferably 450:1 to 3:2, even more preferably 300:1 to 2:1, and most preferably 200:1 to 10:3. Note that "horsetail extract, hawthorn extract, peony extract, witch hazel extract, birch extract, and sage extract" in the above content ratio means the total content of each plant extract.

When three or more of the above extracts are contained, the mass ratio of glycyrrhizinic acid or its salt: horsetail extract: hawthorn extract: peony extract, witch hazel extract, white 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 3:1:1:1. In the above content ratio, "peony extract, witch hazel extract, white birch extract, or sage extract" means the content of any one of the plant extracts.

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:Equisetum arvense 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: 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 Equisetum arvense extract: Hawthorn extract = 1:10,000 to 10,000:1, more preferably 1:1,000 to 1,000:1, and even more preferably 1:500 to 500:1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, in mass ratio, preferably horsetail extract:peony extract, witch hazel extract, white birch extract, or sage extract=1:10,000 to 10,000:1, more preferably 1:1,000 to 1,000:1, and even more preferably 1:500 to 500:1.
The content ratio of the plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, in mass ratio, preferably hawthorn extract:peony extract, witch hazel extract, white birch extract, or sage extract=1:10,000 to 10,000:1, more preferably 1:1,000 to 1,000:1, and even more preferably 1:500 to 500:1.
In addition, in the above content ratio, "peony extract, witch hazel extract, white birch extract, or sage extract" means the content of any one of the respective plant extracts.

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 extracts may be commercially available as described later in the Examples, or may be extracted from plants by the methods 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, either as is or after cutting or crushing. As the solvent extraction method, any method known in the art may be used, and conventionally known extraction methods such as water (including warm water and hot water) extraction, alcohol extraction, supercritical extraction, microwave extraction, and compression 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 and concentrated. Examples of purification include filtration, or adsorption, decolorization, and separation using ion exchange resins or activated carbon columns. Examples of concentration include conventional methods such as evaporators. These may be further dried, such as freeze-dried, or powdered according to a conventional method. The extract thus obtained may be dissolved in water, ethanol, or the like, as necessary, before use.

When the anti-inflammatory composition of the present invention is an oral composition, it can contain, for example, 0.001 ppm to 3% by mass, preferably 0.01 ppm to 0.1% by mass of the above plant extract as a whole (solid matter equivalent). By making it 0.001 ppm by mass or more, the above extract (solid matter equivalent) can have an anti-inflammatory effect, and by making it 3% by mass or less, it 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 preferably glycyrrhizinic acid or a salt thereof:(plant extract)=450:1 to 3:2 by mass, more preferably 300:1 to 2:1, and most preferably 200:1 to 10:3. Here, "plant extract" in the above content ratio means the total amount of plant extract contained in the composition.

The anti-inflammatory composition of the present invention can be used for a variety of purposes without particular limitations, so long as the anti-inflammatory effect of glycyrrhizinic acid or its salts can be enjoyed. Examples include oral compositions, compositions for external use on the skin, cosmetics, and cleaning agents.

When the anti-inflammatory composition of the present invention is a composition for oral cavity, it can be applied to various inflammatory diseases that occur in the oral cavity, for example, periodontal diseases such as gingivitis and periodontitis, inflammation caused by stomatitis or fungal infections such as Candida fungi, 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 (gargle), toothpaste such as liquid toothpaste and toothpaste, lozenge, chewing gum, and the like.
In particular, the composition is preferably used as a mouthwash in which an appropriate amount is placed in the oral cavity so that the entire composition can be sufficiently distributed throughout 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; anti-inflammatory agents such as azulene, azulene sulfonate, β-glycyrrhetinic acid, dihydrocholesterol, epidihydrocholesterol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, ε-aminocaproic acid, tranexamic acid, allantoin, and ascorbic acid; tartar prevention agents such as phosphates, polyphosphates, methoxyethylene maleic anhydride copolymers, zinc chloride, and zinc organic acids; astringents such as hinokitiol, aluminum chlorhydroxyl allantoin, aluminum dihydroxyl allantoin, and sodium chloride. ; humectants such as glycerin, sorbitol, polyethylene glycol; foaming agents such as sodium lauryl sulfate; fragrances such as pinene, peppermint oil, cinnamon oil, clove oil, eugenol, lemon oil, vanillin, cineole, eucalyptus oil; sweeteners such as saccharin, saccharin sodium, sucralose, xylitol, erythritol, sorbitol, maltitol, stevia; colorants such as Blue No. 1, Yellow No. 5, Yellow No. 4, Yellow No. 203, Green No. 3, Green No. 201, Red No. 102; preservatives such as parabens and sodium benzoate; monosodium phosphate, disodium phosphate, citric acid, chrysocolla, etc. pH adjusters such as sodium enoate; nonionic surfactants such as POE hydrogenated castor oil, POE-POP block polymer, POE-POP alkyl ether, POE alkyl ether, POE alkyl phenyl ether, POE fatty acid ester, POE higher alcohol ether, POE-POP fatty acid ester, POE sorbitan fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, and propylene glycol fatty acid ester; sodium lauryl sulfate, sodium myristyl sulfate, POE alkyl ether sulfate, sodium lauroyl sarcosyl, Examples of the surfactants include anionic surfactants such as sodium myristoyl sarcosine, alkyl ether carboxylates, 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 amides; and amphoteric surfactants such as 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, coconut oil fatty acid amidopropyl betaine, and 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, for example, optional components that are usually used in skin external preparations such as cosmetics, and can be appropriately selected and used according to the 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, hardened coconut oil, hardened oil, Japan wax, hardened castor oil, beeswax, candelilla wax, carnauba wax, ivory 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, undecylenic acid, etc.; cetyl alcohol, stearyl ... Synthesis of higher alcohols such as allyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol; synthesis of 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, etc. Ester oils; 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.; anionic surfactants such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl triethanolamine ether sulfate; stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxychloride, etc. cationic surfactants such as amides; imidazoline-based amphoteric surfactants (e.g., 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt), betaine-based surfactants (e.g., alkyl betaines, amido betaines, sulfobetaines), amphoteric surfactants such as 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 monostearate, etc.), non-ionic interfaces such as POE sorbitan, 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 alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE/POP alkyl ethers (POE/POP 2-decyltetradecyl 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. Active agents: 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 pyrrolidone carboxylate, lactic acid, and sodium lactate; powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, and barium sulfate, which may be surface-treated; red iron oxide, yellow iron oxide, black iron oxide, and cobalt oxide, which may be surface-treated. , ultramarine, Prussian blue, titanium oxide, and zinc oxide; pearling agents such as titanium dioxide, fish phosphate foil, and bismuth oxychloride, which may be surface-treated; organic dyes such as 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. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, and Red No. 204, which may be laked; 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; Examples of such an ultraviolet absorbing agent include sugar-based ultraviolet absorbing agents; ultraviolet absorbing agents such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; vitamin A or a derivative thereof, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, vitamin B15 or a derivative thereof; vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate, vitamin D, vitamin H, pantothenic acid, pantethine, pyrroloquinoline quinone, and antibacterial agents such as phenoxyethanol.

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 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 of "LPS treatment" - TNF-α amount of "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.
Moreover, the relative expression level of the above-mentioned IL-6 mRNA is preferably 1.0 or less, and more preferably 0.5 or less.

The present invention will be further explained 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 an LPS solution containing P. g.-derived LPS that stimulates cells were prepared. Then, cells were cultured in the presence of the above-prepared solutions, and the anti-inflammatory effect was evaluated by measuring the mRNA expression level of IL-6, a type of inflammatory cytokine.
In addition, in this test, as control samples, "GK ₂ treatment" (no plant extract) in which only GK ₂ was applied and LPS stimulation was performed, "LPS treatment" (no GK ₂ and plant extract) in which only LPS stimulation was performed, and "untreated" (no GK ₂ , plant extract, or LPS stimulation) in which neither GK ₂ nor plant extract was applied and no LPS stimulation was performed were also tested.
The cell culture medium used was E-MEM medium supplemented with 1% by mass of 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 2500-fold with E-MEM medium supplemented with 1% by mass FBS to prepare a GK ₂ solution.

2. Preparation of plant extract solutions Plant extracts were weighed out to a concentration of 0.1% by mass, and GK ₂ , polyoxyethylene hydrogenated castor oil, and purified water were added to give 1% by mass each, 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 Human oral squamous cell carcinoma cells: Ca9-22 were seeded in a 12-well plate at 10 ⁶ cells/well 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) To each sample (sample containing plant extract), 1 mL each of various plant extract solutions and LPS solution was added, so that the final concentrations of _GK2 were 2.0 ppm by mass and the plant extracts were 0.2 ppm by mass.
(ii) " _GK2 treatment" (control sample) contained 1 mL each of _GK2 solution and LPS solution.
(iii) To the "LPS treatment" (control sample), 1 mL each of the LPS solution and E-MEM medium supplemented with 1% by mass FBS was added.
(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 the cells was used to synthesize cDNA using Transcriptor Universal cDNA Master (Roche).

(Real-time PCR)
Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). Real-time PCR was performed with 1 cycle of 600 seconds at 95°C, 10 seconds at 95°C and 30 seconds at 60°C for a total of 50 cycles. 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 is 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 relative expression level of IL-6 in " _GK2 treatment" corrected by the expression level of GAPDH was set to 1, and the numerical value (relative expression level) of each specimen is shown in Figure 2 (only each specimen and " _GK2 treatment" are shown). The relative expression level of IL-6 in Figure 2 shows the average of two tests in total.

As can be seen from the results in FIG. 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 the "GK ₂ treatment" in which no plant extract was added. In particular, the expression level of IL-6 mRNA was reduced most when both horsetail (0.2 mass ppm) and hawthorn (0.2 mass ppm) were added. In addition, it was found that the expression level of IL-6 mRNA was reduced more when both horsetail and hawthorn extracts were added in combination than when horsetail or hawthorn extracts were added alone, just as the expression level of IL-6 was reduced more when horsetail (0.04 mass ppm) and hawthorn (0.04 mass ppm) were added in combination than when horsetail was added alone at 0.2 mass ppm or hawthorn was added alone at 0.2 mass ppm.

(Examples 1 to 3)
The anti-inflammatory effect was evaluated in the same manner as in Example 1-1, except that the plant extracts used were the three types of plant extracts shown in Table 1, and the final concentrations of GK ₂ and each plant extract were 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, white birch, or sage were added in addition to extracts of horsetail and hawthorn as plant extracts, the expression level of IL-6 mRNA was reduced compared to the " _GK2 treatment" in which no plant extract was added.

(Examples 1 to 4)
In this test, 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) in cells in which inflammation was induced with LPS derived from Aggregatibacter Actinomycetemcomitance (hereinafter referred to as A.a.).
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 to stimulate cells, and a control solution were prepared. 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 addition, in this test, as control samples, "GK ₂ treatment" (no plant extract) in which only GK ₂ was applied and LPS stimulation was performed, "LPS treatment" (no GK ₂ and plant extract) in which only LPS stimulation was performed, and "untreated" (no GK ₂ , plant extract, or LPS stimulation) in which neither GK ₂ nor plant extract was applied and no LPS stimulation was performed were also tested.
The cell culture medium used was Keratinocyte-SFM (1x) [+] L-Glutamin to which Bovine Pituitary Extract (final concentration: 25 µg/mL), epidermal growth factor (final concentration: 0.05 ng/mL), penicillin (final concentration: 100 U/mL), and streptomycin (final concentration: 100 mg/mL) were added, followed by filter sterilization (hereinafter referred to as K-SFM medium).

[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 each 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 2500 times with K-SFM medium to prepare a GK ₂ solution.
2. Preparation of plant extract solutions _GK2 (Maruzen Pharmaceuticals), polyoxyethylene hydrogenated castor oil (Nikko Chemicals, HCO-100) and purified water were each dissolved at 1% by mass, and the plant extract was dissolved at 0.05% by mass or 0.01% by mass in propylene glycol to obtain a plant extract stock solution. This plant extract stock solution was 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 A control solution was prepared by dissolving polyoxyethylene hydrogenated castor oil (HCO-100, manufactured by Nikko Chemicals Co., Ltd.) and purified water in propylene glycol to a concentration of 1% by mass, and then diluting the solution 2,500 times with K-SFM medium.
5. Preparation of test cells Medium and immortalized oral mucosal epithelial cells: RT7 were seeded at 1.0 x 10 ⁵ cells/well into a 12-well plate and cultured in a CO ₂ incubator (CO ₂ : 5%, 37°C) for 24 hours to prepare test cells.

6. Induction of inflammation by LPS, exposure to samples, and RNA extraction (1) After removing the medium from the test cells, 500 μL of fresh medium was added to each well and cultured for 48 hours at 37° C. After removing the medium, 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.) and then 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) contained 500 μL each of _GK2 solution and LPS solution.
(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 the cells was subjected to RT-PCR using Transcriptor Universal cDNA Master (Roche).

(Real-time PCR)
Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). Real-time PCR was performed with 1 cycle of 95°C for 60 seconds, and 45 cycles 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, birch, or sage and adding it to dipotassium glycyrrhizinate.

Example 2: Evaluation of anti-inflammatory effect using TNF-α protein expression level 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 nM PMA was added to RPMI1640 medium supplemented with 10% by mass FBS, and THP-1 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 of "LPS treatment" - TNF-α amount of "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.
The "amount of untreated TNF-α" refers to the amount of TNF-α when the test was performed without LPS stimulation, but instead using the supernatant obtained by culturing in 1% by mass FBS RPMI medium for 6 hours.

[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, and control solution 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.
(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 Systems) was used 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 inhibition rate was higher than that of "GK ₂ treatment" in which no plant extract was added.

The results of the above examples show 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 use containing glycyrrhizinic acid or a salt thereof and an extract of horsetail with alcohols (excluding compositions containing extracts of Houttuynia cordata, Japanese linden, tea, lily or seaweed) . An anti-inflammatory composition for oral use (excluding compositions containing extracts of Houttuynia cordata, Japanese linden, tea, lily or seaweed) containing glycyrrhizinic acid or a salt thereof, an extract of horsetail with alcohols, and an extract of white birch with alcohols .

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