Anti-inflammatory composition

Abstract

To provide an anti-inflammatory composition having enhanced anti-inflammatory effect and containing glycyrrhizinic acid or a salt thereof.SOLUTION: An anti-inflammatory composition contains glycyrrhizinic acid or a salt thereof, and at least one extract selected from the group consisting of extracts of Equisetum arvense, Crataegus oxycantha, Paeonia lactiflora, Hamamelis virginiana, Betula platyphylla and Salvia officinalis.SELECTED DRAWING: None

Description

[Technical field]

[0001] 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. [Background technology]

[0002] Glycyrrhizic acid or its salts are components 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.

[0003] For example, Patent Document 1 discloses a liquid oral composition containing glycyrrhizinate as an anti-inflammatory agent, and describes that the composition 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. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2001-261576 A [Patent Document 2] JP 2006-327965 A Summary of the Invention [Problem to be solved by the invention]

[0005] 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. [Means for solving the problem]

[0006] As a result of intensive research conducted by the inventors in order to solve the above-mentioned 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.

[0007] That is, the present invention has been able to solve the above problems by the means described below. 1. An anti-inflammatory composition comprising 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. 2. The anti-inflammatory composition described in 1 above, which is an oral composition. Effect of the Invention

[0008] 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. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing the relative expression level of IL-6 mRNA in Example 1-1. [Diagram 2] FIG. 2 is a diagram showing the relative expression level of IL-6 mRNA in Example 1-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described in further detail.

[0011] <Anti-inflammatory composition> The anti-inflammatory composition of the present invention is characterized by 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.

[0012] 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 its salts 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 effect 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.

[0013] Glycyrrhizic acid or its salt may be contained in an amount of, for example, 0.0001 to 1.0% by mass, preferably 0.0005 to 0.1% by mass, 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 it 0.0001% by mass or more, the anti-inflammatory effect of glycyrrhizic acid or its salt is expected to be enhanced, and by making it 1.0% by mass or less, formulation becomes easy and a good feeling of use can be obtained, especially in the liquid oral composition.

[0014] 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 specific plant extract, which is selected from among many plant extracts and combined with glycyrrhizinic acid or a salt thereof, thereby enhancing the anti-inflammatory effect.

[0015] 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, the composition contains at least an extract of peony, hamamelis, white birch or sage.

[0016] 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, 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.

[0017] In addition, when three or more of the above extracts are contained, the mass ratio of glycyrrhizic acid or its salt: horsetail extract: hawthorn extract: peony extract, hamamelis 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, hamamelis extract, white birch extract, or sage extract" means the content of any one of the plant extracts.

[0018] 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 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 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 even more preferably 1:500 to 500:1. The content ratio of 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, more preferably 1:1,000 to 1,000:1, and even more preferably 1:500 to 500:1. The content ratio of plant extracts (converted into solid matter) contained in the anti-inflammatory composition of the present invention is, by mass, hawthorn extract:peony extract, witch hazel extract, white birch extract, or sage extract=1:10,000 to 10,000:1, preferably 1:1,000 to 1,000:1, 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] When the anti-inflammatory composition of the present invention is a composition for oral cavity, it can contain, for example, 0.001 ppm by mass to 3% by mass, preferably 0.01 ppm by mass to 0.1% by mass of the whole plant extract (converted into solid matter). By making it 0.001 ppm by mass or more, the extract (converted into solid matter) can have an anti-inflammatory effect, and by making it 3% by mass or less, it is less likely to be discolored and has little effect on the flavor.

[0024] 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, more preferably 300:1 to 2:1, and most preferably 200:1 to 10:3 by mass. Here, the "plant extract" in the above content ratio means the total amount of the plant extract contained in the composition.

[0025] 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 in various applications, such as oral compositions, external skin compositions, cosmetics, and cleansing agents.

[0026] 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, mouthwash (gargle), toothpastes such as liquid toothpaste and toothpaste, lozenges, 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.

[0027] 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 organic zinc acids; hinokitiol, allantoin chlorohydroxyaluminum, and allantoin di astringents such as hydroxyaluminum, sodium chloride, etc.; humectants such as glycerin, sorbitol, polyethylene glycol, etc.; foaming agents such as sodium lauryl sulfate, etc.; fragrances such as pinene, peppermint oil, cinnamon oil, clove oil, eugenol, lemon oil, vanillin, cineole, eucalyptus oil, etc.; sweeteners such as saccharin, saccharin sodium, sucralose, xylitol, erythritol, sorbitol, maltitol, stevia, etc.; colorants such as Blue No. 1, Yellow No. 5, Yellow No. 4, Yellow No. 203, Green No. 3, Green No. 201, Red No. 102, etc. ;preservatives such as parabens and sodium benzoate;pH adjusters such as monosodium phosphate, disodium phosphate, citric acid, and sodium citrate;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;anionic surfactants such as sodium lauryl sulfate, sodium myristyl sulfate, POE alkyl ether sulfate, sodium lauroyl sarcosinate, sodium myristoyl sarcosinate, alkyl ether carboxylate, alkyl phosphate, POE alkyl ether phosphate, N-acyltaurate, POE alkyl ether phosphoric acid / phosphate, and sulfonate;cationic surfactants such as alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, and POE alkylamine / fatty acid amide;Examples of amphoteric surfactants include 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, coconut oil fatty acid amidopropyl betaine, and sodium lauryl diaminoethyl glycine.

[0028] 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 usually 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, 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, ivory wax, lanolin, reduced lanolin, hard lanolin, jojoba wax, and other oils and waxes; liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, vaseline, microcrystals, Hydrocarbons such as talin wax; 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; cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, diisopropyl adipate, and 2-ethyl sebacate. synthetic ester oils such as ethylhexyl, 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; linear polysiloxanes such as dimethylpolysiloxanes, methylphenylpolysiloxanes, and diphenylpolysiloxanes; Polysiloxanes; cyclic polysiloxanes such as octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexanesiloxane, etc.; oils such as silicone oils, including modified polysiloxanes such as amino-modified polysiloxanes, polyether-modified polysiloxanes, alkyl-modified polysiloxanes, and fluorine-modified polysiloxanes; anionic surfactants such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl sulfate triethanolamine ether;Cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, laurylamine oxide, etc.; imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaines, amido betaines, sulfobetaines, etc.), acyl methyl taurine, etc.; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glyceryl monostearate, etc.), serine, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitan fatty acid esters (POE-sorbitan monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), ether, 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, alkyl glucosides and other nonionic surfactants; polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, etc. polyhydric alcohols such as butyl ether, 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 pyrrolidonecarboxylate, 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;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; pearlescent agents such as titanium mica, fish phosphorus 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 Examples of such substances include absorbents, benzophenone-based ultraviolet absorbers, sugar-based ultraviolet absorbers, ultraviolet absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane, lower alcohols such as ethanol and isopropanol, vitamin Bs such as vitamin A or a derivative thereof, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof, vitamins such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate, vitamins Ds, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone, and antibacterial agents such as phenoxyethanol.

[0029] 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.

[0030] For example, when evaluating the anti-inflammatory effect based on the expression level of IL-6 mRNA, the evaluation can be performed by calculating the relative expression level of IL-6 mRNA based on the expression levels of IL-6 mRNA measured in various ways under the conditions described in the Examples below. Details will be described later in the Examples.

[0031] Alternatively, when evaluating the anti-inflammatory effect based on the protein expression level of TNF-α, the inflammation inhibition rate can be calculated using the following formula from the protein expression levels of TNF-α measured in various ways under the conditions described in the Examples below, and the inflammation inhibition rate for 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 suppression 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 = {(TNF-α amount in "LPS treatment" - TNF-α amount in each sample) / (TNF-α amount in "LPS treatment" - TNF-α amount in "untreated")} / Inflammation inhibition rate for GK2 treatment Details will be described later in the Examples.

[0032] The above 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. EXAMPLES

[0033] The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples.

[0034] Example 1: Evaluation of anti-inflammatory effects using the mRNA expression level of inflammatory cytokines as an indicator (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 Pg). Specifically, we first prepared a GK2 solution containing dipotassium glycyrrhizinate (GK2), a plant extract solution containing one type of plant extract, and an LPS solution containing Pg-derived LPS to stimulate cells. We then cultured cells in the presence of the above-prepared solutions, and evaluated the anti-inflammatory effect by measuring the mRNA expression level of IL-6, a type of inflammatory cytokine. In addition, in this study, control samples were also tested: "GK2 treatment" (no plant extracts), in which only GK2 was applied and LPS stimulation was performed; "LPS treatment" (no GK2 and plant extracts), in which only LPS stimulation was performed; and "untreated" (no GK2, plant extracts, or LPS stimulation), in which neither GK2 nor plant extracts were applied and no LPS stimulation was performed. The cell culture medium used was E-MEM medium supplemented with 1% by mass of FBS.

[0035] [method] 1. Preparation of dipotassium glycyrrhizinate (GK2) solution GK2 (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 GK2 stock solution. This GK2 stock solution was diluted 2500 times with E-MEM medium supplemented with 1% by mass FBS to prepare a GK2 solution.

[0036] 2. Preparation of Plant Extract Solutions The 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 the mixture was dissolved in propylene glycol to obtain a plant extract stock solution. This plant extract stock solution was diluted 2500 times with E-MEM medium supplemented with 1% by mass FBS to prepare various plant extract solutions.

[0037] 3. Preparation of LPS Solution Pg-derived LPS (Invivogen) was diluted 500-fold with E-MEM medium supplemented with 1% by mass FBS to prepare an LPS solution.

[0038] 4. Preparation of Test Cells In a 12-well plate, add medium and human oral squamous cell carcinoma cells (Ca9-22) for 10 min. 6 The cells were seeded at 10 cells / well and cultured in a CO2 incubator (CO2: 5%, 37°C) for 24 hours to prepare test cells.

[0039] 5. Induction of inflammation with LPS, specimen exposure, and RNA extraction (1) After removing the medium from the test cells, GK2 solution, plant extract solution, and LPS solution were added to a total volume of 2 mL as described below in (i) to (iv), and the cells were cultured for 24 hours in a CO2 incubator (CO2: 5%, 37°C). (i) To each sample (sample containing plant extract), 1 mL each of various plant extract solutions and LPS solution was added, with the final concentrations of GK2 being 2.0 ppm by mass and the plant extracts being 0.2 ppm by mass. (ii) To the "GK2 treatment" (control sample), 1 mL each of GK2 solution and LPS solution was added. (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 supplemented with 1% by mass FBS was added. (2) Total RNA was extracted and purified from cells using the RNeasy Mini Kit (QIAGEN).

[0040] 6. Measurement of mRNA expression level (RT-PCR) RNA extracted from the cells was used to synthesize cDNA using Transcriptor Universal cDNA Master (Roche).

[0041] (Real-time PCR) Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). Real-time PCR was performed for 600 seconds at 95°C for 1 cycle, and 50 cycles in total 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'-TGGTGAAGACGCAGTGGA-3'IL-6 Forward Primer:5'-GCCAGAGCTGTGCAGATGAG-3' Reverse Primer:5'-TCAGCAGGCTGGCATTTG-3'

[0042] [result] The values ​​(relative expression levels) for each sample are shown in Figure 1, where the relative expression level of IL-6 in "GK2 treatment" corrected for the expression level of GAPDH is set to 1 (only each sample and "GK2 treatment" are shown). Note that the relative expression levels of IL-6 in Figure 1 show the average of a total of two tests. In addition, the various plant extracts used in Figure 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 Hamamelis: Manufactured by Maruzen Pharmaceuticals, (product name) Hamamelis Extract BG-J Birch: Maruzen Pharmaceutical Co., Ltd. (product name) Birch extract BG-JC Sage: Falcos (product name) Falcolex Sage B, manufactured by Ichimaru Falcos Houttuynia cordata: Manufactured by Ichimaru Falcos, (product name) Falcolex Houttuynia cordata B

[0043] As can be seen from the results in Figure 1, when extracts of horsetail, hawthorn, peony, witch hazel, birch, or sage were added in addition to dipotassium glycyrrhizinate, the mRNA expression level of IL-6 was reduced compared to the "GK2 treatment" in which no plant extract was added. On the other hand, when Houttuynia cordata extract 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.

[0044] (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.

[0045] [result] The relative expression level of IL-6 in "GK2 treatment" corrected by the expression level of GAPDH was set to 1, and the values ​​(relative expression level) of each specimen are shown in Figure 2 (only each specimen and "GK2 treatment" are shown). Note that the relative expression level of IL-6 in Figure 2 shows the average of a total of two tests.

[0046] 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 "GK2 treatment" in which no plant extract was added. In particular, the expression level of IL-6 mRNA was most reduced 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 0.04 mass ppm and hawthorn 0.04 mass ppm were added in combination than when horsetail 0.2 mass ppm or hawthorn 0.2 mass ppm was added alone.

[0047] (Examples 1-3) In Example 1-1, the plant extracts used were the three types of plant extracts shown in Table 1, and the final concentrations of GK2 and each plant extract were the concentrations shown in Table 1, but the anti-inflammatory effect was evaluated in the same manner as in Example 1-1.

[0048] [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.

[0049] [Table 1]

[0050] As can be seen from the results in Table 1, even when extracts of peony, witch hazel, 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 extracts were added.

[0051] (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 Aggregatibactor Actinomycetemcomitance (hereinafter referred to as Aa). Specifically, first, a GK2 solution containing dipotassium glycyrrhizinate (GK2), a plant extract solution containing a plant extract, an LPS solution containing Aa-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 effects were evaluated by measuring the mRNA expression levels of IL-6 and TNF-α, which are types of inflammatory cytokines. In addition, in this study, control samples were also tested: "GK2 treatment" (no plant extracts), in which only GK2 was applied and LPS stimulation was performed; "LPS treatment" (no GK2 and plant extracts), in which only LPS stimulation was performed; and "untreated" (no GK2, plant extracts, or LPS stimulation), in which neither GK2 nor plant extracts were applied and no LPS stimulation was performed. The cell culture medium used was Keratinocyte-SFM(1x)[+]L-Glutamin supplemented with 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), which was then sterilized by filtration (hereafter referred to as K-SFM medium).

[0052] [method] 1. Preparation of dipotassium glycyrrhizinate (GK2) solution GK2 (Maruzen Pharmaceuticals), polyoxyethylene hydrogenated castor oil (Nikko Chemicals, HCO-100), and purified water were each dissolved in propylene glycol to a concentration of 1% by mass to obtain a GK2 stock solution. This GK2 stock solution was diluted 2500 times with K-SFM medium to prepare a GK2 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 times with K-SFM medium to prepare various plant extract solutions. 3. Preparation of LPS Solution Aa-derived LPS was diluted with K-SFM medium to a concentration of 2 μg / mL to prepare an LPS solution. 4. Preparation of Control Solutions A control solution was prepared by dissolving polyoxyethylene hydrogenated castor oil (HCO-100, manufactured by Nikko Chemicals) and purified water in propylene glycol to a concentration of 1% by mass, and then diluting the solution 2,500-fold with K-SFM medium. 5. Preparation of Test Cells In a 12-well plate, culture medium and immortalized oral mucosal epithelial cells: RT7 (1.0 × 10 5 The cells were seeded at 100 cells / well and cultured in a CO2 incubator (CO2: 5%, 37°C) for 24 hours to prepare the test cells.

[0053] 6. Induction of inflammation with LPS, specimen exposure, and RNA extraction (1) After removing the medium from the test cells, 500 μL of fresh medium was added to each well and cultured at 37° C. for 48 hours. 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 in a CO2 incubator (CO2: 5%, 37° C.) for 3 hours, after which the cells were harvested. (i) 500 μL each of various plant extract solutions and LPS solutions were added to each sample (samples containing plant extracts). The final concentrations of GK2 and plant extracts were as shown in Table 2. (ii) To the "GK2 treatment" (control sample), 500 μL each of GK2 solution and LPS solution 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) specimen, 500 μL each of K-SFM medium and control solution was added. (2) Total RNA was extracted and purified from cells using the RNeasy Mini Kit (QIAGEN).

[0054] 7. Measurement of mRNA expression level (RT-PCR) RNA extracted from the cells was subjected to RT-PCR using Transcriptor Universal cDNA Master (Roche).

[0055] (Real-time PCR) Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). Real-time PCR was performed for 60 seconds at 95°C for 1 cycle, and 45 cycles in total 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'

[0056] [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 values ​​(relative expression levels) 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 levels of TNF-α in Table 3 show the average of a total of five tests.

[0057] [Table 2]

[0058] [Table 3]

[0059] As can be seen from the results in Tables 2 and 3, when extracts of horsetail, hawthorn, peony, witch hazel, birch, or sage were added in addition to dipotassium glycyrrhizinate, the mRNA expression levels of IL-6 and TNF-α were reduced compared to the "GK2 treatment" in which no plant extracts were 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.

[0060] Example 2: Evaluation of anti-inflammatory effect using TNF-α protein expression level as an indicator (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 Pg-derived LPS using the ELISA method described below.

[0061] [method] 1. 10% FBS-supplemented RPMI1640 medium was added with 10 nM PMA, and THP-1 cells were cultured at 10 5The cells were seeded at 1 cell / 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 RPMI1640 supplemented with 1% by mass FBS and cultured for 24 hours. 3. 1 mL of the diluted test sample, which was prepared by diluting the test sample described below 5000 times with RPMI1640 medium, was added to the cells, and the diluted test sample was removed after 2 hours. After that, the cells were washed once with RPMI1640 medium, and stimulated with Pg-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.

[0062] The inflammation inhibition rate was calculated using the following formula, and the inflammation inhibition rate for each sample was calculated assuming that the inflammation inhibition rate for the "GK2 treatment" described below was 1.0. Inflammation suppression 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 = {(TNF-α amount in "LPS treatment" - TNF-α amount in each sample) / (TNF-α amount in "LPS treatment" - TNF-α amount in "untreated")} / Inflammation inhibition rate for "GK2 treatment" In the formula, "amount of TNF-α after LPS treatment" refers to the amount of TNF-α when the test was performed using liquid medium instead of the diluted test sample solution in the above method 3. Moreover, 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.

[0063] [Subject] Sample containing plant extract: 1% by mass of GK2 + polyoxyethylene hydrogenated castor oil (Nikko Chemicals, HCO-100) + plant extract (added so that the final concentration of GK2 is 2.0 ppm by mass and the final concentration of plant extract is 0.05 ppm by mass or 0.01 ppm by mass) GK2 treatment: 1% by mass of GK2 + polyoxyethylene hydrogenated castor oil (Nikko Chemicals, HCO-100) (added so that the final concentration of GK2 is 2.0 ppm by mass)

[0064] [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, (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 Pharmaceuticals, (product name) Lily Extract BG Seaweed: Ichimaru Falcos Co., Ltd. (product name) IPF-100K

[0065] [Table 4]

[0066] 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 the "GK2 treatment" in which no plant extract was added. On the other hand, when plant extracts were added, such as extracts of Houttuynia cordata, linden, tea, lily, or seaweed, the inflammation inhibition rate was lower than that of the "GK2 treatment" in which no plant extract was added.

[0067] (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 Aa-derived LPS using the ELISA method described below. The dipotassium glycyrrhizinate (GK2) solution, plant extract solution, LPS solution, control solution and test cells were prepared in the same manner as in Examples 1-4 above.

[0068] [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) 50 μL each of various plant extract solutions and LPS solutions were added to each sample (samples containing plant extracts). The final concentrations of GK2 and plant extracts were as shown in Table 5. (ii) To the "GK2 treatment" (control sample), 50 μL each of GK2 solution and LPS solution was added. (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) specimen, 50 μL each of K-SFM medium and control solution was added. 2. Each sample was placed in a CO2 incubator (CO2: 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.

[0069] [result] The results are shown in Table 5. The plant extract used was the same as in Example 2-1.

[0070] [Table 5]

[0071] 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 the "GK2 treatment" in which no plant extract was added.

[0072] 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, birch and sage has an anti-inflammatory effect.

Claims

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 claim 1 , which is an oral composition.

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