Expression inhibitor of nitric oxide synthase

Abstract

To identify a component capable of inhibiting an increase in the expression of nitric oxide synthase, and to provide an expression inhibitor of nitric oxide synthase.SOLUTION: An expression inhibitor of nitric oxide synthase comprises, as active ingredients, glycyrrhetinic acid and at least one selected from an extract of Equisetum arvense and an extract of Tilia japonica.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an inhibitor of the expression of nitric oxide synthase. [Background technology]

[0002] Our bodies take in oxygen from the air and generate energy by oxidizing nutrients such as carbohydrates and lipids from food. However, depending on the oxidative reaction environment, excessive oxidation can occur, causing cell damage due to oxidative stress (disruption of redox homeostasis), which can have various adverse effects on health.

[0003] One of the causes of oxidative stress is reactive oxygen species such as superoxide (reactive oxygen), nitric oxide, etc. Nitric oxide is produced by nitric oxide synthase. Nitric oxide synthase includes NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS). Nitric oxide synthase is an enzyme that produces nitric oxide, but it is also known to produce a certain proportion of superoxide. For example, when oxidative stress occurs in the oral cavity, cell damage occurs, leading to stomatitis, periodontitis, etc. It is known that the expression of NOS2 is induced at this time.

[0004] Conventionally, various methods have been investigated for suppressing the excessive production of reactive oxygen species and nitric oxide, which cause oxidative stress. For example, in the field of oral compositions, there have been proposed an oral composition comprising a combination of α-glycosyl-L-ascorbic acid and one or more selected from menthol, carvone, and menthone (Patent Document 1), an oral composition comprising a combination of rutin or a glycoside thereof and one or more flavorings selected from limonene, cineole, isoeugenol, and menthone (Patent Document 2), an oral composition comprising a rutin-containing composition selected from rutin and a glycoside thereof, and one or more selected from citrate, phosphate, carbonate, bicarbonate, and edetate (Patent Document 3), and an oral composition comprising a combination of hyaluronic acid or a non-toxic salt thereof and an antioxidant (Patent Document 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 6-211636 [Patent Document 2] Japanese Patent Application Publication No. 7-165547 [Patent Document 3] Japanese Patent Application Publication No. 7-187974 [Patent Document 4] Japanese Patent Application Publication No. 10-182390 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to identify a component capable of suppressing increased expression of nitric oxide synthase, which is one of the causes of oxidative stress, and to provide an inhibitor of the expression of nitric oxide synthase. [Means for solving the problem]

[0007] As a result of extensive research, the inventors discovered that combining glycyrrhizinic acid with horsetail extract and / or linden extract effectively inhibits the expression of enzymes that produce nitric oxide, thereby completing the present invention.

[0008] That is, the objective of the present invention is achieved by an inhibitor of nitric oxide synthase expression, which comprises glycyrrhizinic acid and at least one of an extract of horsetail and an extract of linden as active ingredients. Effect of the Invention

[0009] According to the present invention, the expression of nitric oxide synthase can be effectively suppressed. In particular, suppressing the expression of NOS2 can suppress the generation of nitric oxide and superoxide, which is expected to reduce oxidative stress. This can reduce the adverse effects on the body when inflammation occurs, and is expected to have preventive and therapeutic effects on various diseases. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph showing the expression level ratio of inducible nitric oxide synthase (NOS2) tested in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiments of the present invention will be described in more detail. In this specification, "ppm" stands for "μg / mL", and 1 ppm is equal to 1 μg / mL.

[0012] The nitric oxide synthase expression inhibitor of the present invention contains glycyrrhizinic acid (hereinafter also referred to as component (A)) and at least one of an extract of horsetail and an extract of linden (hereinafter also referred to as component (B)) as active ingredients.

[0013] <Component (A)> Glycyrrhizic acid (component (A)) is a component known to have anti-inflammatory effects. The (A) component may be used in the form of glycyrrhizinic acid itself or in the form of a salt.Glycyrrhizinic acid may be, for example, β-glycyrrhizinic acid.Glycyrrhizinic acid salts may be, for example, potassium salts, sodium salts, ammonium salts, etc., and specifically, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate, monosodium glycyrrhizinate, disodium glycyrrhizinate, monoammonium glycyrrhizinate, diammonium glycyrrhizinate, etc. Glycyrrhizic acid is known to be contained in plant extracts of the genus Licorice, and may be blended in the form of a plant extract containing glycyrrhizic acid, or commercially available glycyrrhizic acid or glycyrrhizinate may be used.

[0014] The component (A) may be used in one form alone, or in a combination of two or more forms. Among these, from the viewpoint of increasing water solubility and facilitating formulation, it is preferable to use glycyrrhizinate salts, more preferably potassium salts and ammonium salts of glycyrrhizinic acid, and even more preferably dipotassium glycyrrhizinate and monoammonium glycyrrhizinate.

[0015] The amount of component (A) in the nitric oxide synthase expression inhibitor, calculated as glycyrrhizinic acid, is preferably within the range of 0.005 to 3000 ppm (μg / mL) in total. When the content of component (A) is 0.005 ppm or more, the effect of inhibiting the expression of nitric oxide synthase by combining with the horsetail extract and / or the linden extract (component (B)) is exhibited, and when it is 3000 ppm or less, the formulation stability is excellent and the feeling of use is also good. The content of component (A) is more preferably 0.01 ppm or more, even more preferably 0.1 ppm or more, particularly preferably 1 ppm or more, and most preferably 10 ppm or more, and is more preferably 2200 ppm or less, even more preferably 1000 ppm or less, particularly preferably 500 ppm or less, and most preferably 150 ppm or less.

[0016] <(B) component> Horsetail extract (Equisetum arvense extract) is an extract (plant extract) obtained from horsetail (Equisetum arvense), a plant of the Equisetum genus of the Equisetaceae family, which is a type of herbal medicine.

[0017] The horsetail extract may be obtained by extracting the whole plant, stems and / or leaves of the horsetail, but it is preferable to obtain it by extracting the whole plant of the horsetail, and a commercially available product may be used. Methods for obtaining the horsetail extract by extraction include solvent extraction, supercritical extraction, steam distillation, etc.

[0018] Examples of extraction solvents used in the solvent extraction treatment include water and alcohols. Examples of alcohols include methanol, ethanol, propylene glycol, 1,3-butylene glycol, or mixtures thereof, or a 1% urea-containing ethanol solution, or a 1,3-butylene glycol solution containing 1% urea. It is preferable to use an extract obtained by extraction with these solvents, and it is particularly preferable to use an extract obtained by extraction with water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, or a mixture thereof.

[0019] 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.

[0020] Commercially available products of horsetail extract include, for example, "Horsetail Extract BG" (manufactured by Maruzen Pharmaceutical Co., Ltd.), "Horsetail Extract" (manufactured by Maruzen Pharmaceutical Co., Ltd.), and "Sugina Extract" (manufactured by Koei Kogyo Co., Ltd.).

[0021] Linden extract (linden extract) is an extract (plant extract) obtained from linden (Tilia cordata Mill., Tilia platyphyllos Scop.), a plant of the Tilia genus in the Tiliaceae family.

[0022] The linden extract may be obtained by extracting one or more of the leaves, stems, flowers, buds, fruits, peels, and pits of linden, but it is preferable to obtain it by extracting the flowers of linden, and a commercially available product may be used. The method for obtaining the linden extract by extraction can be the same as the extraction method for the horsetail extract described above.

[0023] Commercially available products of Linden extract include, for example, "Linden Extract BG-J" (manufactured by Maruzen Pharmaceutical Co., Ltd.) and "Falcorex Limonium" (manufactured by Ichimaru Pharcos Co., Ltd.).

[0024] The component (B) may be one selected from the group consisting of horsetail extract and linden extract, or two or more of them may be used in combination.

[0025] The (B) component is preferably contained in the nitric oxide synthase expression inhibitor in a total amount of 0.0001 to 100 ppm (μg / mL) calculated as solid content. When the (B) component is 0.0001 ppm or more, the nitric oxide synthase expression inhibitory effect is exerted in combination with glycyrrhizinic acid ((A) component), and when it is 100 ppm or less, the formulation stability is excellent and the feeling of use is also good. The content of component (B) is more preferably 0.001 ppm or more, even more preferably 0.01 ppm or more, and is more preferably 50 ppm or less, even more preferably 25 ppm or less.

[0026] In the nitric oxide synthase expression inhibitor, the (B) component is preferably contained in an amount of 0.00003 to 50 parts by mass, calculated as solid content, per 1 part by mass of the (A) component relative to the (A) component. When the content ratio (B / A) of the (B) component to the (A) component is within the above range, the desired effects of the present invention are obtained, the formulation has excellent stability, and the feeling of use is also good. The content ratio (B / A) of the (B) component to the (A) component is more preferably 0.00005 or more, even more preferably 0.00008 or more, even more preferably 0.0001 or more, and particularly preferably 0.0003 or more, and is more preferably 45 or less, even more preferably 40 or less, even more preferably 35 or less, and particularly preferably 30 or less.

[0027] In the nitric oxide synthase expression inhibitor, the horsetail extract (B1) is preferably contained in an amount of 0.00003 to 50 parts by mass, calculated as solid content, per 1 part by mass of component (A) relative to component (A). When the content ratio of the horsetail extract to component (A) (B1 / A) is within the above range, the desired effects of the present invention are obtained, the formulation has excellent stability, and the feeling of use is also good. The content ratio (B1 / A) of the horsetail extract to the component (A) is more preferably 0.00005 or more, even more preferably 0.00008 or more, even more preferably 0.0001 or more, and particularly preferably 0.0003 or more, and is more preferably 45 or less, even more preferably 40 or less, even more preferably 35 or less, and particularly preferably 30 or less.

[0028] In the nitric oxide synthase expression inhibitor, the linden extract (B2) is preferably contained in an amount of 0.00003 to 50 parts by mass, calculated as solid content, per 1 part by mass of the component (A) relative to the component (A). When the content ratio of the linden extract to the component (A) (B2 / A) is within the above range, the desired effects of the present invention are obtained, the formulation has excellent stability, and the feeling of use is also good. The content ratio of the Linden extract to the (A) component (B2 / A) is more preferably 0.00005 or more, even more preferably 0.00008 or more, even more preferably 0.0001 or more, and particularly preferably 0.0003 or more, and is more preferably 45 or less, even more preferably 40 or less, even more preferably 35 or less, and particularly preferably 30 or less.

[0029] In the nitric oxide synthase expression inhibitor, the glycyrrhizic acid (component (A)) and the horsetail extract and / or the linden extract (component (B)) are preferably contained in a total of 0.0051 ppm or more when the component (A) is calculated as glycyrrhizic acid and the component (B) is calculated as solids. When the nitric oxide synthase expression inhibitor contains a total of 0.0051 ppm or more of the components (A) and (B), the inhibitor exerts an excellent effect as an active ingredient for suppressing the expression of nitric oxide synthase. The total amount of the components (A) and (B) is more preferably 0.0051 to 3100 ppm, even more preferably 0.01 to 2300 ppm, and particularly preferably 10 to 200 ppm.

[0030] The contents of components (A) and (B) in the inhibitor of nitric oxide synthase expression can be measured by standard liquid chromatography methods using liquid chromatography or high performance liquid chromatography.

[0031] The nitric oxide synthase expression inhibitor of the present invention can be obtained by mixing glycyrrhizic acid (component (A)) with a horsetail extract and / or a linden extract (component (B)).

[0032] <Other ingredients> The nitric oxide synthase expression inhibitor of the present invention may contain other optional components, such as solvents, pH adjusters, preservatives, surfactants, dyes, and fragrances, as long as the effects of the present invention are not impaired.

[0033] Examples of the solvent include water such as purified water and ion-exchanged water; lower monohydric alcohols such as ethanol; and polyhydric alcohols such as glycerin, propylene glycol, butylene glycol, and polyethylene glycol.

[0034] Examples of pH adjusters include sodium phosphate salts such as monosodium phosphate and disodium phosphate, sodium citrate, citric acid, glucono-δ-lactone, sodium gluconate, sodium acetate, sodium carbonate, sodium bicarbonate, sodium hydroxide, and hydrates thereof.

[0035] Examples of preservatives include paraoxybenzoic acid esters, sodium benzoate, potassium sorbate, and phenoxyethanol.

[0036] Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Specifically, examples of the nonionic surfactants include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty acid diethanolamides, fatty acid monoglycerides, polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oil, alkyl glucosides, and polyoxyethylene polyoxypropylene block copolymers. Examples of the cationic surfactants include polyoxyethylene alkylamines and fatty acid amides. Examples of the anionic surfactants include sulfate ester salts, α-olefin sulfonates, sulfosuccinates, N-acyl amino acid salts, and acylated methyl taurine salts. Examples of the amphoteric surfactants include betaine acetate amphoteric surfactants and imidazoline amphoteric surfactants.

[0037] Examples of pigments include Blue No. 1, Blue No. 2, Blue No. 201, Yellow No. 4, Yellow No. 5, Yellow No. 202(1), Yellow No. 203, Red No. 3, Red No. 102, Red No. 104, Red No. 105, Green No. 3, and Green No. 201.

[0038] Examples of fragrances include natural essential oils such as peppermint oil, spearmint oil, mint oil, eucalyptus oil, clove oil, thyme oil, rosemary oil, lemon oil, ginger oil, lime oil, cassia oil, cardamom oil, lavender oil, and cinnamon oil; fragrance components such as L-menthol, L-carvone, carvacrol, eugenol, anethole, 1,8-cineole, thymol, vanillin, pinene, 3-L-menthoxypropane-1,2-diol, and methyl salicylate; mixtures of these, natural fragrances, blended fragrances, and synthetic fragrances.

[0039] They may also contain ingredients that are to be applied to the oral cavity or to the skin, such as sweeteners, bactericides, anti-inflammatory agents, tartar prevention agents, enzymes, ion sources, herbal medicines, oils and waxes, hydrocarbons, higher fatty acids, higher alcohols, synthetic ester oils, moisturizers, powders, vitamins, etc.

[0040] Examples of sweeteners include stevioside, rebaudioside, xylitol, erythritol, maltitol, sorbitol, sodium saccharin, sucralose, trehalose, reduced palatinose, aspartame, and acesulfame potassium.

[0041] Examples of the disinfectant include hinokitiol, triclosan, isopropylmethylphenol, thymol, alkyldiaminoethylglycine hydrochloride solution, quaternary ammonium salts such as cetylpyridinium chloride, benzalkonium chloride, and benzethonium chloride.

[0042] Examples of anti-inflammatory agents include aminocaproic acid, allantoin and its derivatives, ascorbic acid and its salts, azulene, azulene sulfonate, dihydrocholesterol, epidihydrocholesterol, tocopherol acetate, tocopherol nicotinate, lysozyme chloride, and tranexamic acid.

[0043] Examples of anti-tartar agents include phosphates, polyphosphates, methoxyethylene maleic anhydride copolymers, zinc chloride, and zinc organic acids.

[0044] Examples of the enzyme include protease, dextranase, amylase, mutanase, and lysozyme.

[0045] Examples of the ion source include fluorine ion sources such as sodium fluoride, monofluorophosphate, and stannous fluoride.

[0046] Examples of herbal medicines include plant extracts other than the above-mentioned horsetail and linden extracts, such as hawthorn extract, witch hazel extract, peony extract, seaweed extract, Phellodendron bark extract, Angelica acutiloba extract, carrot extract, and fennel extract.

[0047] Examples of oils and waxes include 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, etc.

[0048] Examples of the hydrocarbons include liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, vaseline, and microcrystalline wax.

[0049] Examples of higher fatty acids include oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, and undecylenic acid.

[0050] Examples of higher alcohols include cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol.

[0051] Examples of synthetic ester oils include cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, diisopropyl adipate, 2-ethylhexyl sebacate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glycerin di-2-heptylundecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, and pentane erythritol tetra-2-ethylhexanoate.

[0052] Examples of moisturizing agents include sodium hyaluronate, betaine, sodium pyrrolidone carboxylate, lactic acid, and sodium lactate.

[0053] Examples of powders include mica, talc, kaolin, synthetic mica, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate.

[0054] Examples of vitamin preparations include vitamin A or a derivative thereof; B vitamins such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof; E vitamins such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D; vitamin H; pantothenic acid; pantethine; pyrroloquinoline quinone, etc.

[0055] The use of the nitric oxide synthase expression inhibitor of the present invention is not particularly limited as long as it is an application that can enjoy the effect of suppressing the expression of an enzyme that produces nitric oxide, and it can be used in foods and beverages, medicines, quasi-drugs, cosmetics, etc. Medicines, quasi-drugs, and cosmetics include oral preparations, topical skin preparations, etc. Medicines and quasi-drugs include oral preparations, infusions, inhalants, etc.

[0056] The form of administration or ingestion of the nitric oxide synthase expression inhibitor of the present invention is not particularly limited, but for example, oral administration such as oral administration or sublingual administration, oral ingestion, application to epithelial mucosa or skin such as in the oral cavity, pasting, spraying, etc. are preferred.

[0057] The expression inhibitor of nitric oxide synthase of the present invention can be prepared in the form of, for example, liquid, gel, paste, cream, tablet, or other solid form, granule or fine powder, capsule, soft capsule, film, or other form. Among them, liquid preparations are preferred because the active ingredient can be uniformly applied to the epithelial mucosa and skin of the oral cavity. Examples of liquid preparations include mouthwash (also called gargle), liquid dentifrice, mouthwash, mouth spray, lotion, body spray, etc., and when used as an oral preparation, mouthwash, liquid dentifrice, mouthwash, mouth spray, etc. that can be uniformly applied to the oral cavity are preferred.

[0058] In this specification, the term "liquid" refers to a substance that has fluidity and a viscosity at 20°C of The viscosity is 20 mPa·s or less, preferably 10 mPa·s or less. This can be measured using a commercially available rotational viscometer (e.g., a B-type viscometer manufactured by Toki Sangyo Co., Ltd.). .

[0059] Enzymes that produce nitric oxide include NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS). The nitric oxide synthase expression inhibitor of the present invention can inhibit the expression of all of these enzymes, but is particularly effective at inhibiting the expression of NOS2.

[0060] The amount of administration or intake of the nitric oxide synthase expression inhibitor of the present invention is not particularly limited as long as it does not impair the effects of the present invention, and can be appropriately changed depending on various factors such as the type of enzyme to be inhibited, the age and condition of the subject to which it is applied, etc. The preferred amount of administration or intake to obtain the desired effect may be appropriately determined depending on the application of the agent.

[0061] The expression inhibitor of nitric oxide synthase of the present invention can be used as a final product in its original form. It can also be used as an additive for food and beverages, pharmaceuticals, and quasi-drugs. This can impart various effects to food and beverages, pharmaceuticals, and quasi-drugs.

[0062] It is believed that by suppressing oxidative stress with the expression inhibitor of nitric oxide synthase of the present invention, it will be possible to effectively prevent and treat periodontal diseases such as gingivitis and periodontitis, stomatitis, infectious diseases caused by viruses, bacteria, parasites, etc., cancer, myocardial infarction, arteriosclerosis, renal dysfunction, and brain diseases such as Parkinson's disease, Alzheimer's disease, depression, chronic fatigue syndrome, Asperger's syndrome, etc., and it is particularly preferable to use the inhibitor for the prevention and treatment of periodontal diseases such as gingivitis and periodontitis, and stomatitis. EXAMPLES

[0063] The present invention will be further explained below with reference to the following test examples, but is not limited to these examples. The converted amounts of glycyrrhizinic acid in the table below were calculated by analyzing the dipotassium glycyrrhizinate used using high performance liquid chromatography to confirm the content of glycyrrhizinic acid.

[0064] The materials used in the following examples and comparative examples are as follows. Horsetail extract: Maruzen Pharmaceutical Co., Ltd.'s "Horsetail Extract BG" (extracted from the whole horsetail plant using a 50% by volume solution of 1,3-butylene glycol) Linden extract: "Linden Extract BG-J" manufactured by Maruzen Pharmaceutical Co., Ltd. (extracted from linden flowers or leaves with a 50% by volume solution of 1,3-butylene glycol) Birch extract: Maruzen Pharmaceutical Co., Ltd.'s "Birch Extract BG" (extracted from the bark of European birch or its related plants using a 50% by volume solution of 1,3-butylene glycol) Sage extract: "Falcorex Sage B" manufactured by Ichimaru Pharcos Co., Ltd. (extracted from sage flowers, leaves or whole plant with 50% by volume 1,3-butylene glycol solution) Houttuynia cordata extract: "Falcorex Houttuynia cordata B" manufactured by Ichimaru Falcos Co., Ltd. (extracted from the above-ground parts of Houttuynia cordata during the flowering period using a 50% by volume solution of 1,3-butylene glycol) Lipopolysaccharide (LPS): Sigma-Aldrich "Lipopolysaccharide derived from Escherichia coli 026:B6"

[0065] Example 1 The specimen of Example 1 was prepared by adding 0.1 ppm LPS, 40 ppm dipotassium glycyrrhizinate, and 4 ppm horsetail extract as stimulants to RPMI1640 medium supplemented with 3% by mass FBS (Fetal Bovine Serum), and mixing them.

[0066] Example 2 A sample of Example 2 was prepared by adding 0.1 ppm of LPS, 40 ppm of dipotassium glycyrrhizinate, and 4 ppm of Linden extract to RPMI1640 medium containing 3% by mass of FBS and mixing them.

[0067] (Comparative Examples 1 to 9) Samples of Comparative Examples 1 to 9 were prepared in the same manner as in Example 1, except that the components used and their contents were changed as shown in Table 1.

[0068] (Control Example 1) RPMI1640 medium supplemented with 3% by mass of FBS was used.

[0069] (Control Example 2) A sample of Control Example 2 was prepared by adding 0.1 ppm of LPS to RPMI1640 medium containing 3% by mass of FBS and mixing.

[0070] [Table 1]

[0071] The following tests were carried out using the samples of Examples 1 and 2, Comparative Examples 1 to 9, and Control Examples 1 and 2 described above.

[0072] <1. Induction of differentiation of THP-1 cells into macrophages> 1) The cell line used was THP-1 (human monocytic leukemia cell line). 5 THP-1 cells (cells / mL) were placed in a medium (RPMI1640 medium supplemented with 5% FBS by mass) containing 40 nM PMA (phorbol 12-myristate 13-acetate) and cultured in a 6-well plate at 37°C and 5% CO2 for 7 days. 2) The culture supernatant was removed, and RPMI1640 medium supplemented with 3% by mass of FBS was added thereto, and the culture was continued for one day. It was visually confirmed that the cells had taken on a macrophage-like shape.

[0073] 2. Confirmation of the expression level of NOS (Nitric Oxide Synthase) in cells The expression level of NOS2 (iNOS), an inducible nitric oxide synthase (NOS) that is induced by inflammation or stress, was confirmed according to the following 1) to 4). 1) THP-1 cells differentiated into macrophages were stimulated with each sample in duplicate wells, and the cells were harvested one hour later. 2) RNA was extracted from the collected cells using an RNA extraction kit (RNeasy Mini Kit; QIAGEN) according to the protocol. 3) The extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (High-Capacity RNA to cDNA Kit; Thermo). 4) Following the protocol of FastStart Essential DNA Green Master (Roche), real-time PCR was performed using the ΔΔCt method under the following conditions with cDNA as a template to confirm the expression levels of GAPDH and NOS2.

[0074] (Reverse Transcription) Reverse transcription into cDNA of 3) above was carried out according to the following steps (1) to (2). (1) 1 μg of extracted RNA, 10 μL of 2×RT Buffer mix, and 1 μL of 20×RT Enzyme mix were placed in a PCR tube, and RNA-free HO was added to make a total of 20 μL to prepare a reaction solution. (2) The PCR tube containing the reaction solution was placed in a thermal cycler and reacted at 37°C for 60 minutes and at 95°C for 5 minutes, and then kept at 4°C.

[0075] (Reaction solution used for real-time PCR) The reaction solution used in the above 4) real-time PCR was prepared as follows. A total of 20 μL of reaction solution was prepared by measuring out 10 μL of SYBR Green master mix, 0.1 μL of F Primer (100 μM), 0.1 μL of R Primer (100 μM), 7.8 μL of RNase free H2O, and 2 μL of reverse transcribed cDNA into a PCR tube.

[0076] (Primer information) [GAPDH primer] SEQ ID NO: 1: Forward: 5'-GCACCGTCAAGGCTGAGAAC-3' SEQ ID NO: 2: Reverse: 5'-TGGTGAAGACGCCAGTGGA-3' [NOS2 primer] SEQ ID NO: 3: Forward: 5'-ATGGGAGAAGGGGATGAGCT-3' SEQ ID NO: 4: Reverse: 5'-GTCCCAGGTCACATTGGAGG-3'

[0077] The primers having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 were designed based on the human GAPDH gene mRNA sequence (NCBI Nucleotide accession number NM_001256799.3), and the primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 were designed based on the human NOS2 gene mRNA sequence (NCBI Nucleotide accession number NM_000625.4).

[0078] (PCR conditions) 1 cycle at 95℃ for 10 minutes 45 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 40 seconds 1 cycle of 72℃ for 5 minutes

[0079] The results regarding the expression level of NOS2 relative to the expression level of GAPDH are shown in FIG.

[0080] Comparative Examples 1 to 3 showed that when only dipotassium glycyrrhizinate (Comparative Example 1), only horsetail extract (Comparative Example 2), or only linden extract (Comparative Example 3) was used, the NOS2 expression levels were similar to that of Control Example 2, which used LPS stimulation alone, whereas Example 1, which combined dipotassium glycyrrhizinate and horsetail extract, and Example 2, which combined dipotassium glycyrrhizinate and linden extract, showed a significant reduction in NOS2 expression levels, indicating that combining glycyrrhizinic acid with horsetail extract or linden extract had the effect of reducing NOS2 expression levels. On the other hand, as shown in Comparative Examples 4, 6, and 8, the birch extract, sage extract, and Houttuynia cordata extract showed NOS2 expression levels similar to or lower than those of the horsetail extract (Comparative Example 2) and the linden extract (Comparative Example 3), but no effect of reducing the NOS2 expression level was observed even when combined with glycyrrhizinic acid, as compared with Comparative Examples 4 and 5, 6 and 7, and 8 and 9. The birch extract is known to have an effect of suppressing the expression of nitric oxide synthase. This indicates that the effect of reducing the expression level of NOS2 by combining glycyrrhizinic acid with a plant extract is an effect specific to the horsetail extract and the linden extract.

Claims

[Claim 1] An inhibitor of nitric oxide synthase expression, comprising glycyrrhizinic acid and at least one of an extract of horsetail and an extract of linden as active ingredients.

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