Anti-inflammatory components

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 glycyrrhizic acid or a salt thereof, and particularly to an anti-inflammatory composition containing glycyrrhizic acid or a salt thereof and a specific plant extract.

Background Art

[0002] Glycyrrhizic acid or a salt thereof is a component contained in the crude drug licorice, and is known to exhibit an anti-inflammatory effect. Due to its anti-inflammatory effect, glycyrrhizic acid or a salt thereof is widely used in mouthwashes for suppressing or preventing symptoms of periodontal disease, which is a type of inflammatory disease, skin external preparations for suppressing skin inflammation, hair growth agents, soaps, and the like.

[0003] For example, Patent Document 1 discloses a liquid oral composition containing a glycyrrhizate as an anti-inflammatory agent, and describes that it can prevent oral diseases such as gingivitis. Further, Patent Document 2 discloses a skin external preparation containing glycyrrhizic acid, and describes that it is effective for inflammations such as allergic inflammation and atopic dermatitis.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Given the increasing number of elderly people suffering from advanced inflammation in our rapidly aging society, there is a growing demand for anti-inflammatory compositions with enhanced anti-inflammatory effects. Therefore, the object of the present invention is to provide an anti-inflammatory composition containing glycyrrhizic acid or a salt thereof that exhibits enhanced anti-inflammatory effects. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors of the present invention have discovered that the anti-inflammatory effect of glycyrrhizic acid or its salt is enhanced by including a certain plant extract together with glycyrrhizic acid or its salt, and have completed the present invention.

[0007] In other words, the present invention has been able to solve the above problems by the means described below. 1. An anti-inflammatory composition 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, birch, and sage. 2. An anti-inflammatory composition for oral use, as described in 1 above. [Effects of the Invention]

[0008] The anti-inflammatory composition of the present invention can enhance the anti-inflammatory effect of glycyrrhizic acid or its salt by containing glycyrrhizic acid or its salt and a specific plant extract. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the relative expression levels of IL-6 mRNA in Example 1-1. [Figure 2] Figure 2 shows the relative expression levels of IL-6 mRNA in Example 1-2. [Modes for carrying out the invention]

[0010] The present invention will be described in more detail below.

[0011] <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, birch, and sage.

[0012] Glycyrrhizic acid or its salts are components found in the roots and stems of licorice, a type of herbal medicine. Glycyrrhizic acid or its salts are known to have an anti-inflammatory effect, meaning they are effective in suppressing inflammation of the mouth, throat, and other areas. Examples of glycyrrhizic acid or its salts in the present invention include glycyrrhizic acid, dipotassium glycyrrhizate (GK2), trisodium glycyrrhizate, monoammonium glycyrrhizate, diammonium glycyrrhizate, and disodium glycyrrhizate. These can be used individually or in combination of two or more. Among these, dipotassium glycyrrhizate is more preferable from the viewpoint of obtaining the effects of the present invention to a high degree. Glycyrrhizic acid or its salts can be obtained by extraction from licorice using conventionally known methods, or commercially available products can be used. Examples of commercially available products include dipotassium glycyrrhizinate and monoammonium glycyrrhizinate manufactured by Alps Pharmaceutical Co., Ltd. and Maruzen Pharmaceutical Co., Ltd.

[0013] Glycyrrhizic acid or its salt can be contained in, 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 using 0.0001% by mass or more, the anti-inflammatory effect of glycyrrhizic acid or its salt is expected to be enhanced, while by using 1.0% by mass or less, formulation becomes easier, and a good user experience can be obtained, especially in liquid oral compositions.

[0014] The anti-inflammatory composition of the present invention contains at least one extract selected from the group consisting of horsetail, hawthorn, peony, witch hazel, birch, and sage. The anti-inflammatory composition of the present invention is a combination of glycyrrhizic acid or a salt thereof with the above-mentioned specific plant extract. In other words, the anti-inflammatory effect is enhanced by combining glycyrrhizic acid or a salt thereof with the above-mentioned specific plant extract, selected from among many plant extracts.

[0015] In particular, from the viewpoint of the present invention's effect of enhancing anti-inflammatory activity, it is preferable to contain at least one extract selected from the group consisting of horsetail, hawthorn, peony, and witch hazel extracts, and it is more preferable to contain at least one extract of horsetail and hawthorn. Furthermore, if the above extracts are contained in three or more types, it is preferable to contain at least one extract of peony, witch hazel, birch, or sage, in addition to the horsetail and hawthorn extracts.

[0016] Furthermore, the content ratio of plant extracts (on a solid basis) contained in the anti-inflammatory composition of the present invention is preferably, by mass ratio, glycyrrhizic acid or its salt: horsetail extract, hawthorn extract, peony extract, witch hazel extract, birch extract, and sage extract = 600:1 to 5:4, more preferably 450:1 to 3:2, even more preferably 300:1 to 2:1, and most preferably 200:1 to 10:3. In the above content ratio, "horsetail extract, hawthorn extract, peony extract, witch hazel extract, birch extract, and sage extract" refers to the total content of each plant extract.

[0017] When containing three or more of the above extracts, in terms of mass ratio, glycyrrhizic acid or its salt: extract of Stellaria media: extract of Crataegus pinnatifida: extract of Paeonia lactiflora, extract of Hamamelis virginiana, extract of Betula platyphylla, or extract of Salvia officinalis = 600:1:1:1 to 1.25:1:1:1 is preferable, more preferably 450:1:1:1 to 1.5:1:1:1, still more preferably 300:1:1:1 to 2:1:1:1, and most preferably 200:1:1:1 to 3:1:1:1. Here, the "extract of Paeonia lactiflora, extract of Hamamelis virginiana, extract of Betula platyphylla, or extract of Salvia officinalis" in the above content ratio means the content of any one of the plant extracts.

[0018] The content ratio of the plant extracts (in terms of solid content) contained in the anti-inflammatory composition of the present invention is preferably 600:1 to 5:4, more preferably 450:1 to 3:2, and still more preferably 300:1 to 2:1 in terms of mass ratio of glycyrrhizic acid or its salt: extract of Stellaria media. The content ratio of the plant extracts (in terms of solid content) contained in the anti-inflammatory composition of the present invention is preferably 600:1 to 5:4, more preferably 450:1 to 3:2, and still more preferably 300:1 to 2:1 in terms of mass ratio of glycyrrhizic acid or its salt: extract of Crataegus pinnatifida. The content ratio of the plant extracts (in terms of solid content) contained in the anti-inflammatory composition of the present invention is preferably 600:1 to 5:4, more preferably 450:1 to 3:2, and still more preferably 300:1 to 2:1 in terms of mass ratio of glycyrrhizic acid or its salt: extract of Paeonia lactiflora, extract of Hamamelis virginiana, extract of Betula platyphylla, or extract of Salvia officinalis. The content ratio of the plant extracts (in terms of solid content) contained in the anti-inflammatory composition of the present invention is preferably 1:10000 to 10000:1, more preferably 1:1000 to 1000:1, and still more preferably 1:500 to 500:1 in terms of mass ratio of extract of Stellaria media: extract of Crataegus pinnatifida. The content ratio of the plant extracts (in terms of solids) contained in the anti-inflammatory composition of the present invention is preferably a mass ratio of shepherd's purse extract: peony extract, witch hazel extract, birch extract, or sage extract = 1:10000 to 10000:1, more preferably 1:1000 to 1000:1, and even more preferably 1:500 to 500:1. The content ratio of the plant extracts (in terms of solids) contained in the anti-inflammatory composition of the present invention is preferably a mass ratio of Japanese quince extract: peony extract, witch hazel extract, birch extract, or sage extract = 1:10000 to 10000:1, more preferably 1:1000 to 1000:1, and even more preferably 1:500 to 500:1. In addition, the "peony extract, witch hazel extract, birch extract, or sage extract" in the above content ratio means the content of any one of the plant extracts.

[0019] When the anti-inflammatory composition of the present invention is a liquid composition, from the viewpoint of being less likely to form aggregates even during long-term storage, it is preferable to contain at least one of shepherd's purse extract, Japanese quince extract, and witch hazel extract.

[0020] As for the above plant extracts, commercially available ones can be used as described in the examples later, or those extracted from plants by the methods described later can also be used. The extraction method of the plant extracts is not particularly limited, and it may follow a conventionally known method. For example, any part of the above plants can be used as it is, or after cutting, grinding, etc., extracts can be obtained by squeezing, solvent extraction, water distillation, steam distillation, etc. As the solvent extraction method, a method known in the technical field may be adopted. For example, conventionally known extraction methods such as water (including warm water and hot water) extraction, alcohol extraction, supercritical extraction, microwave extraction, and pressing extraction can be used.

[0021] Examples of solvents used in solvent extraction include water; alcohols such as methanol, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, and 1,3-butylene glycol (whether anhydrous or hydrated); 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. Preferably, the solvent used in solvent extraction is water, alcohols, ketones, and hexane, and more preferably water, alcohols, and ketones. These solvents may be used individually or in combination of two or more.

[0022] The obtained extract may be used as is or after drying. Furthermore, the obtained extract may be purified and concentrated as needed. Examples of purification methods include filtration or adsorption, decolorization, and separation using ion exchange resins or activated carbon columns. Examples of concentration methods include conventional methods such as evaporators. Further drying treatments, such as freeze-drying, may be performed, or the extract may be powdered according to conventionally known methods. The extract thus obtained may also be dissolved in water, ethanol, or other solvents as needed before use.

[0023] When the anti-inflammatory composition of the present invention is an oral composition, it may contain, for example, 0.001 ppm to 3% by mass, preferably 0.01 ppm to 0.1% by mass, of the entire plant extract (on a solid basis). By setting the concentration to 0.001 ppm or more, the extract (on a solid basis) is at a concentration that can exert its anti-inflammatory effect, and by setting it to 3% by mass or less, it is less likely to cause discoloration and has little effect on flavor.

[0024] Furthermore, the content ratio of plant extracts (on a solid basis) contained in the anti-inflammatory composition of the present invention is preferably glycyrrhizic acid or its salt : (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 refers to the total amount of plant extract contained in the composition.

[0025] The anti-inflammatory composition of the present invention is not particularly limited in its use as long as it can benefit from the anti-inflammatory effects of glycyrrhizic acid or its salts, and can be used in a variety of applications. Examples include oral compositions, external skin compositions, cosmetics, and cleansing agents.

[0026] When the anti-inflammatory composition of the present invention is an oral composition, it can be applied to various inflammatory diseases occurring in the oral cavity, such as periodontal diseases like gingivitis and periodontitis, inflammation caused by stomatitis and fungal infections such as Candida, mucositis caused by contact with prosthetics such as dentures, and swelling of wounds associated with dental treatment. Oral compositions can take the form of, for example, mouthwash, toothpaste, oral fresheners, gargles (mouthwashes), toothpastes such as liquid toothpaste and paste toothpaste, lozenges, chewing gum, and the like. In particular, it is suitable for use as a mouthwash, where an appropriate amount is placed in the mouth for use, in order to ensure that the entire composition is thoroughly distributed throughout the oral cavity. To prepare it as a mouthwash, for example, water or ethanol can be used as a solvent and prepared by conventional methods. Furthermore, for example, liquid toothpaste can be used that contains abrasives such as calcium hydrogen phosphate, aluminum hydroxide, anhydrous silicic acid, and calcium carbonate as needed.

[0027] Furthermore, the oral composition may contain any other 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 preventive agents such as phosphates, polyphosphates, methoxyethylene maleic anhydride copolymer, zinc chloride, and zinc organic acid; hinokitiol, allantoin chlorohydroxyaluminum, allantoin di Astringents such as hydroxyaluminum and sodium chloride; humectants such as glycerin, sorbitol, and polyethylene glycol; foaming agents such as sodium lauryl sulfate; fragrances such as pinene, peppermint oil, cinnamon oil, clove oil, eugenol, lemon oil, vanillin, cineole, and eucalyptus oil; sweeteners such as saccharin, sodium saccharin, sucralose, xylitol, erythritol, sorbitol, maltitol, and stevia; colorants such as Blue No. 1, Yellow No. 5, Yellow No. 4, Yellow No. 203, Green No. 3, Green No. 201, and Red No. 102. ; 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 alkylphenyl 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-acyl taurine salt, POE alkyl ether phosphate, sulfonate; Cationic surfactants such as alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, and POE alkylamine fatty acid amide;Examples include amphoteric surfactants such as 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, coconut oil fatty acid amidopropyl betaine, and sodium lauryldiaminoethylglycinate.

[0028] Furthermore, when the anti-inflammatory composition of the present invention is a topical skin composition, it can be applied to various inflammatory diseases occurring 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 topical skin composition is not particularly limited and can be designed arbitrarily, for example, as a lotion, an emulsifier such as a milky lotion or cream, or an oil. A topical skin composition may contain any ingredients commonly used in topical skin preparations such as cosmetics, and can be appropriately selected and used according to the desired dosage form. Such optional ingredients include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japanese wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, privet wax, lanolin, reduced lanolin, hard lanolin, jojoba wax, liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, petrolatum, and microcristal; and liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, petrolatum, and microcristal. 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 hexyl, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glyceryl di-2-heptylundecanoate, glyceryl tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane erythritol tetra-2-ethylhexanoate, etc.; chain-like substances such as dimethylpolysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane, etc. Polysiloxanes; cyclic polysiloxanes such as octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, and dodecamethylcyclohexanesiloxane; modified polysiloxanes such as amino-modified polysiloxanes, polyether-modified polysiloxanes, alkyl-modified polysiloxanes, and fluorine-modified polysiloxanes; silicone oils and other oily agents; fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl sulfate triethanolamine ethers; anionic surfactants;Cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide; amphoteric surfactants such as imidazoline-based surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.), and amphoteric surfactants such as acylmethyl taurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), and glycerin fatty acids (glyceryl monostearate). Serine, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.) Nonionic surfactants such as POE alkyl ethers (POE2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE·POP alkyl ethers (POE·POP2-decyltetradecyl ether, etc.), Tetronic types, POE castor oil / hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, alkyl glucosides, etc.; polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol Polyhydric alcohols such as 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, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate, which may have surface treatments;Inorganic pigments such as red iron oxide, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine, Prussian blue, titanium dioxide, and zinc oxide, which may be surface-treated; pearlescent agents such as titanium mica, fish scale foil, and bismuth oxychloride, which may be surface-treated; organic dyes such as Red 202, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue 1, Green 201, Violet 201, and Red 204, which may be lake-formed; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomers; para-aminobenzoic acid-based UV absorbers; anthranilic acid-based UV absorbers; salicylic acid-based UV absorbers; cinnamic acid-based UV absorbers Examples of UV absorbers include: benzophenone-based UV absorbers; sugar-based UV absorbers; UV absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; vitamin B compounds such as vitamin A or its derivatives, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or its derivatives, vitamin B12, vitamin B15 or its derivatives; vitamin E compounds such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D compounds; 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 inflammatory cytokine mRNA and proteins in tissues and cells collected from various organs where inflammation is occurring, or in cultured cells stimulated with LPS or the like, as described later in the examples. Inflammatory cytokines such as IL-6 and TNF-α can be used as inflammatory markers.

[0030] For example, when evaluating the anti-inflammatory effect based on the IL-6 mRNA expression level, the evaluation can be performed by determining the relative expression level of IL-6 mRNA based on the IL-6 mRNA expression levels measured under the conditions described in the examples below. Further details will be provided in the examples below.

[0031] Alternatively, when evaluating the anti-inflammatory effect based on the TNF-α protein expression level, the inflammation suppression rate can be calculated using the following formula from the TNF-α protein expression levels measured under the conditions described in the examples below. This rate is then used to determine the inflammation suppression rate for each sample, with the rate when plant extracts are not used (GK2 treatment: applying only GK2 and performing LPS stimulation) set to 1. The 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 "no treatment") Inflammation suppression rate for each sample = {(TNF-α amount in "LPS treated" sample - TNF-α amount in each sample) / (TNF-α amount in "LPS treated" sample - TNF-α amount in "untreated" sample)} / Inflammation suppression rate of GK2 treatment Further details will be provided in the examples below.

[0032] The inflammation suppression rate described above is preferably 1.0 or higher, and more preferably 2.0 or higher. Furthermore, the relative expression level of 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 effect using mRNA expression levels of inflammatory cytokines as an indicator. (Example 1-1) In this study, 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, first, 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 that stimulates cells were prepared. Subsequently, 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 this study, the following 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 or 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 neither LPS stimulation was performed. Furthermore, E-MEM medium supplemented with 1% by mass of FBS was used as the cell culture medium.

[0035] [method] 1. Preparation of dipotassium glycyrrhizinate (GK2) solution GK2 (manufactured by Maruzen Pharmaceutical Co., Ltd.), polyoxyethylene hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd., HCO-100), and purified water were dissolved in propylene glycol to a concentration of 1% by mass each to obtain a stock GK2 solution. A GK2 solution was prepared by diluting this stock GK2 solution 2500 times in E-MEM medium supplemented with 1% by mass FBS.

[0036] 2. Preparation of plant extract solution Plant extracts were weighed out to a concentration of 0.1% by mass, and GK2, polyoxyethylene hydrogenated castor oil, and purified water were added to each to a total concentration of 1% by mass. The plant extract stock solution was then 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 in E-MEM medium with 1% by mass FBS.

[0037] 3. Preparation of LPS solution An LPS solution was prepared by diluting Pg-derived LPS (Invivogen) 500-fold in E-MEM medium supplemented with 1% by mass of FBS.

[0038] 4. Preparation of test cells Place culture medium and human oral squamous cell carcinoma cells:Ca9-22 in a 12-well plate. 6 Cells were seeded one cell per well and cultured in a CO2 incubator (CO2: 5%, 37°C) for 24 hours to obtain the test cells.

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

[0040] 6. Measurement of mRNA expression levels (RT-PCR) cDNA was synthesized from RNA extracted from cells using Transcriptor Universal cDNA Master (Roche).

[0041] (Real-time PCR) Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). The process consisted of 50 cycles: 600 seconds at 95°C, followed by 10 seconds at 95°C and 30 seconds at 60°C. The primers used are listed 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] Figure 1 shows the relative expression levels for each sample (relative expression levels) when the relative expression level of IL-6 in the "GK2-treated" group, corrected for GAPDH expression levels, is set to 1 (only each sample and the "GK2-treated" group are shown). Note that the relative expression levels of IL-6 in Figure 1 represent the average of two tests conducted. Furthermore, the following plant extracts were used in Figure 1. • Horsetail: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Horsetail Extract BG • Hawthorn: Manufactured by Ichimaru Falcos, (Product name) Falcorex Hawthorn B • Peony: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Peony Extract BG-JC • Hamamelis: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Hamamelis Extract BG-J • Birch: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Birch Extract BG-JC • Sage: Manufactured by Ichimaru Falcos, (Product name) Falcorex Sage B • Houttuynia cordata: Manufactured by Ichimaru Falcos, (product name) Falcorex 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 amount of IL-6 mRNA expression decreased compared to the "GK2 treatment" without any plant extracts. On the other hand, when Houttuynia cordata extract was added, the relative expression level of IL-6 increased compared to the "GK2 treatment" without any plant extracts. Therefore, it was confirmed that selecting extracts from horsetail, hawthorn, peony, witch hazel, birch, or sage from among plant extracts and adding them to dipotassium glycyrrhizinate improved the anti-inflammatory effect.

[0044] (Examples 1-2) In Example 1-1, the plant extract was a plant extract of horsetail and / or hawthorn, and the final concentration of the plant extract was the concentration shown in Figure 2. The anti-inflammatory effect was evaluated in the same manner as in Example 1-1.

[0045] [result] Figure 2 shows the relative expression levels (for each sample and the "GK2 treatment") of IL-6, with the relative expression level of the "GK2 treatment," corrected for GAPDH expression, set to 1 (only each sample and the "GK2 treatment" are shown). Note that the IL-6 relative expression levels in Figure 2 represent the average of two tests conducted.

[0046] As can be seen from the results in Figure 2, when both horsetail and hawthorn extracts were added in addition to dipotassium glycyrrhizinate, the amount of IL-6 mRNA expression decreased compared to the "GK2 treatment" without any plant extracts. In particular, the amount of IL-6 mRNA expression decreased most significantly when both 0.2 ppm of horsetail extract and 0.2 ppm of hawthorn extract were added. Furthermore, it was found that the amount of IL-6 mRNA expression decreased more when a combination of 0.04 ppm of horsetail extract and 0.04 ppm of hawthorn extract was added than when either horsetail or hawthorn extract was added alone.

[0047] (Examples 1-3) In Example 1-1, 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 shown in Table 1, and the final concentrations of GK2 and each plant extract were set to the concentrations shown in Table 1.

[0048] [result] Table 1 below shows the relative expression levels (IL-6 values) for each sample, with the relative expression level of IL-6 in the "GK2-treated" group, corrected for GAPDH expression levels, set to 1. Note that the IL-6 relative expression levels in Table 1 represent the average of two tests conducted.

[0049] [Table 1]

[0050] As can be seen from the results in Table 1, adding extracts of peony, witch hazel, birch, or sage, in addition to horsetail and hawthorn extracts, resulted in a decrease in IL-6 mRNA expression compared to the "GK2 treatment" without any plant extracts.

[0051] (Examples 1-4) In this study, 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 Aa). Specifically, first, a GK2 solution containing dipotassium glycyrrhizinate (GK2), a plant extract solution containing plant extracts, an LPS solution containing Aa-derived LPS that stimulates cells, and a control solution were prepared. Subsequently, 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 this study, the following 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 or 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 neither LPS stimulation was performed. Furthermore, the cell culture medium used was Keratinocyte-SFM(1×)[+]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, and then filtered and sterilized (hereinafter referred to as K-SFM medium).

[0052] [method] 1. Preparation of dipotassium glycyrrhizinate (GK2) solution GK2 (manufactured by Maruzen Pharmaceutical Co., Ltd.), polyoxyethylene hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd., HCO-100), and purified water were dissolved in propylene glycol to obtain a stock solution of GK2. A GK2 solution was prepared by diluting this stock solution 2500 times with K-SFM medium. 2. Preparation of plant extract solution GK2 (manufactured by Maruzen Pharmaceutical Co., Ltd.), polyoxyethylene hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd., HCO-100), and purified water were dissolved in propylene glycol at a concentration of 1% by mass each, and plant extracts at a concentration of 0.05% or 0.01% by mass to obtain a stock solution of plant extracts. Various plant extract solutions were prepared by diluting this stock solution 2500 times in K-SFM medium. 3. Preparation of LPS solution LPS derived from Aa was diluted in 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 each, and then diluting the solution 2500-fold with K-SFM medium. 5. Preparation of test cells Place culture medium and immortalized oral mucosal epithelial cells:RT7 in a 12-well plate in a 1.0 × 10⁶ well. 5 Cells were seeded in each well and cultured in a CO2 incubator (CO2: 5%, 37°C) for 24 hours to obtain the test cells.

[0053] 6. Induction of inflammation by LPS and exposure of specimens, RNA extraction (1) After removing the culture medium from the test cells, 500 μL of new culture medium was added to each cell and incubated at 37°C for 48 hours. After removing the culture medium, the prepared GK2 solution, plant extract solution, LPS solution, and control solution were added to a total volume of 1 mL as described in (i) to (iv) below, and the cells were left to stand in a CO2 incubator (CO2: 5%, 37°C) for 3 hours before being harvested. (i) 500 μL each of the plant extract solution and LPS solution were added to each sample (sample containing plant extracts). The final concentrations of GK2 and plant extracts were as shown in Table 2. (ii) 500 μL each of GK2 solution and LPS solution were added to the "GK2 treatment" (control sample). (iii) For the "LPS treatment" (control sample), 500 μL each of LPS solution and control solution were added. (iv) For the "untreated" (control) sample, 500 μL each of K-SFM medium and control solution were added. (2) Total RNA was extracted and purified from cells using the RNeasy Mini Kit (QIAGEN).

[0054] 7. Measurement of mRNA expression levels (RT-PCR) RNA extracted from cells was used to synthesize cDNA by RT-PCR using Transcriptor Universal cDNA Master (Roche).

[0055] (Real-time PCR) Real-time PCR was performed using FastStart Essential DNA Green Master (Roche). The process consisted of 45 cycles: 60 seconds at 95°C, followed by 10 seconds at 95°C and 30 seconds at 60°C. The primers used are listed below. ·GAPDH Forward Primer:5'-GCACCGTCAAGGCTGAGAAC-3' Reverse Primer:5'-TGGTGAAGACGCAGTGGA-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] Table 2 shows the relative expression levels (relative expression levels) for each sample, with the relative expression level of IL-6 in the "GK2-treated" group, corrected for GAPDH expression levels, set to 1. Note that the relative expression levels of IL-6 in Table 2 represent the average of three tests conducted. Table 3 shows the relative expression levels (TNF-α values) for each sample, with the relative expression level of TNF-α in the "GK2-treated" group, corrected for GAPDH expression levels, set to 1. Note that the relative expression levels of TNF-α in Table 3 represent 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 decreased compared to the "GK2 treatment" without any plant extracts. Therefore, it was confirmed that selecting an extract from horsetail, hawthorn, peony, witch hazel, birch, or sage and adding it to dipotassium glycyrrhizinate improved the anti-inflammatory effect.

[0060] Example 2: Evaluation of anti-inflammatory effect using TNF-α protein expression level as an indicator. (Example 2-1) In this study, 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. Add 10 nM PMA to RPMI1640 medium supplemented with 1.10% by mass FBS, and inoculate THP-1 cells with 10 5The cells were seeded one per well into a 24-well plate and cultured for 7 days to induce differentiation into macrophage-like organisms. 2. After confirming differentiation induction, the medium was replaced with RPMI1640 medium supplemented with 1% by mass of FBS and incubated for 24 hours. 3. The subject described below was diluted 5000-fold in RPMI1640 medium, and 1 mL of the diluted subject solution was added to the cells. After 2 hours, the diluted subject solution was removed. The cells were then washed once with RPMI1640 medium and stimulated with Pg-derived LPS (0.2 μg / mL, 1% FBS-added medium). 4. Six hours after stimulation, the supernatant was collected and the amount of TNF-α protein was measured by ELISA.

[0062] The inflammation suppression rate was calculated using the following formula, and the inflammation suppression rate for each sample was determined when the inflammation suppression rate for "GK2 treatment" below was set to 1.0. The 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 "no treatment") Inflammation suppression 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" sample)} / Inflammation suppression rate for "GK2 treatment" In the formula, "amount of TNF-α after LPS treatment" refers to the amount of TNF-α when the test is performed using a liquid culture medium instead of the diluted sample solution in method 3 above. Furthermore, "untreated TNF-α levels" refer to the TNF-α levels obtained when the supernatant was used after culturing in 1% FBS RPMI medium for 6 hours without LPS stimulation.

[0063] [Subject] • Sample containing plant extract: GK2 1% by mass + polyoxyethylene hydrogenated castor oil (manufactured by 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: GK2 1% by mass + polyoxyethylene hydrogenated castor oil (manufactured by 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: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Horsetail Extract BG • Hawthorn: Manufactured by Ichimaru Falcos, (Product name) Falcorex Hawthorn B • Peony: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Peony Extract BG-JC • Houttuynia cordata: Manufactured by Ichimaru Falcos, (product name) Falcorex Houttuynia cordata B • Linden: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Linden Extract BG-J • Tea: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Green tea extract BG • Lily: Manufactured by Maruzen Pharmaceutical Co., Ltd., (Product name) Lily Extract BG • Seaweed: Manufactured by 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 suppression rate was higher compared to the "GK2 treatment" without any plant extracts. On the other hand, even with plant extracts, when extracts of Houttuynia cordata, Linden, Tea, Lily, or seaweed were added, the inflammation suppression rate was lower compared to the "GK2 treatment" without any plant extracts.

[0067] (Example 2-2) In this study, 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 culture medium from the test cells, GK2 solution, plant extract solution, LPS solution, and control solution were added as described in (i) to (iv) below. (i) 50 μL each of the plant extract solution and LPS solution were added to each sample (sample containing plant extracts). The final concentrations of GK2 and plant extracts were as shown in Table 5. (ii) 50 μL each of GK2 solution and LPS solution were added to the "GK2 treatment" (control sample). (iii) For the "LPS treatment" (control sample), 50 μL each of LPS solution and control solution were added. (iv) For the "untreated" (control) sample, 50 μL each of K-SFM medium and control solution were 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. The procedure was performed using Human TNF-alpha DuoSet ELISA (manufactured by R&D Systems) according to the manufacturer's specified procedure. 4. The inflammation suppression rate was determined in the same manner as in Example 2-1 above.

[0069] [result] The results are shown in Table 5. The plant extracts used are the same as those used 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 suppression rate was higher compared to the "GK2 treatment" without any plant extracts.

[0072] From the results of the above examples, it was found that a composition containing glycyrrhizic acid or a salt thereof and at least one extract selected from the group consisting of horsetail, hawthorn, peony, witch hazel, birch, and sage has an anti-inflammatory effect.

Claims

1. An oral anti-inflammatory composition with enhanced anti-inflammatory effects, containing glycyrrhizic acid or a salt thereof, an alcoholic extract of horsetail, and at least one extract selected from the group consisting of Houttuynia cordata, Linden, Tea, Lily, and Seaweed.

2. An oral anti-inflammatory composition with enhanced anti-inflammatory effects, containing glycyrrhizic acid or a salt thereof, an alcoholic extract of horsetail, an alcoholic extract of birch, and at least one extract selected from the group consisting of Houttuynia cordata, linden, tea, lily, and seaweed.

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