Interleukin-37 production promoter and interleukin-37 mRNA expression promoter

Abstract

To discover, from among highly safe natural products, a substance having interleukin-37 production promoting action or interleukin-37 mRNA expression promoting action, and provide an interleukin-37 production promoter and interleukin-37 mRNA expression promoter, including the discovered substance as an active ingredient.SOLUTION: The present invention provides an interleukin-37 production promoter and an interleukin-37 mRNA expression promoter, including at least one selected from the group consisting of glycyrrhizinic acid and derivatives thereof, glycyrrhetinic acid and derivatives thereof, and licorice extract as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an interleukin-37 production promoter and an interleukin-37 mRNA expression promoter. [Background technology]

[0002] Inflammation is a complex reaction that shows symptoms such as redness, edema, fever, pain, itching, and functional impairment. For example, when the skin is exposed to ultraviolet rays or comes into contact with an irritant, inflammatory cytokines are produced in the skin, causing skin inflammation. As a result, the skin tissue is damaged, and various symptoms such as rough skin, redness, edema, and pigmentation occur.

[0003] Interleukin-37 is a member of the interleukin-1 family of cytokines and is known to have an anti-inflammatory effect. Interleukin-37 suppresses inflammation caused by inflammatory cytokines induced by external stimuli such as bacterial infection and exposure to ultraviolet light, and plays a broad defensive role against inflammation.

[0004] Excessive inflammation, which is one of the biological defense responses induced by external stimuli such as bacterial infection and ultraviolet rays, can damage tissues and cells, making it difficult to maintain normal biological responses. Diseases caused by excessive inflammation include allergies, chronic rheumatoid arthritis, and inflammatory bowel disease. In other words, while an inflammatory response to external stimuli is essential, controlling this inflammation so that it does not become excessive and promoting the production of interleukin-37, which has an anti-inflammatory effect, are important in protecting the body from diseases caused by excessive inflammation.

[0005] Conventionally, polypeptides containing variants of interleukin-37, guava leaf extracts, and the like have been known to have the effect of promoting the production of interleukin-37 (see Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2018-522547 A [Patent Document 2] JP 2021-113173 A Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to find a substance having an interleukin-37 production promoting effect or an interleukin-37 mRNA expression promoting effect from among highly safe natural products, and to provide an interleukin-37 production promoting agent and an interleukin-37 mRNA expression promoting agent that contain the substance as an active ingredient. [Means for solving the problem]

[0008] In order to solve the above problems, the interleukin-37 production promoter and interleukin-37 mRNA expression promoter of the present invention are characterized in that they contain at least one active ingredient selected from the group consisting of glycyrrhizic acid and its derivatives, glycyrrhetinic acid and its derivatives, and licorice extract. As the derivative of glycyrrhizinic acid, dipotassium glycyrrhizinate can be used. Effect of the Invention

[0009] According to the present invention, a substance having an interleukin-37 production promoting effect or an interleukin-37 mRNA expression promoting effect has been discovered from among highly safe natural products, and an interleukin-37 production promoting agent and an interleukin-37 mRNA expression promoting agent containing the substance as an active ingredient can be provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] An embodiment of the present invention will now be described in detail. Both the interleukin-37 production promoter and the interleukin-37 mRNA expression promoter of this embodiment contain at least one active ingredient selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract.

[0011] Glycyrrhizic acid derivatives include, for example, glycyrrhizic acid (C 42 H 62 O 16 ) with an alkali. Examples of glycyrrhizinic acid derivatives include dipotassium glycyrrhizinate, trisodium glycyrrhizinate, and monoammonium glycyrrhizinate, and dipotassium glycyrrhizinate is preferably used.

[0012] Dipotassium glycyrrhizinate is obtained by reacting glycyrrhizic acid with potassium hydroxide (KOH) and the like, and glycyrrhizic acid (C 42 H 62 O 16 ) can be obtained by isolating and purifying a plant extract containing glycyrrhizinic acid. The plant extract containing glycyrrhizinic acid includes an extract obtained by using a plant containing glycyrrhizinic acid as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or any of these roughly purified or purified products.

[0013] The glycyrrhetinic acid derivatives can be synthesized by known methods. Examples of the glycyrrhetinic acid derivatives include stearyl glycyrrhetinate, disodium succinyl glycyrrhetinate, and glycyrrhetinyl stearate. For example, stearyl glycyrrhetinate can be obtained by isolating and purifying glycyrrhetinic acid from a plant extract containing glycyrrhetinic acid, and hydrolyzing the glycyrrhetinic acid obtained by this isolation and purification using an aqueous solution of an acid (hydrochloric acid, sulfuric acid, etc.) to obtain glycyrrhetinic acid (C 30 H 46 O4) and then subjecting the glycyrrhetinic acid to an esterification reaction with stearyl alcohol.

[0014] The plant extract containing glycyrrhizic acid can be obtained by a method generally used for extracting plants. An example of a plant containing glycyrrhizic acid is licorice.

[0015] Licorice is a perennial herb belonging to the genus Glycyrrhiza in the family Fabaceae. Licorice includes, for example, Glycyrrhiza glabra, Glycyrrhiza inflata, and Glycyrrhiza uralensis. Any of these species of licorice may be used as the raw material for extraction, but it is particularly preferable to use Glycyrrhiza glabra as the raw material for extraction.

[0016] Examples of licorice parts that can be used as extraction raw materials include above-ground parts such as leaves, branches, bark, trunks, stems, fruits, and flowers, as well as roots and mixtures of these parts, with the roots being preferred.

[0017] A plant extract containing glycyrrhizic acid can be obtained by drying the extraction raw material, pulverizing it directly or using a crusher, and then subjecting it to extraction with an extraction solvent. Drying can be performed in the sun or using a commonly used dryer. In addition, the raw material can be used after pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the plant can be efficiently extracted with a polar solvent.

[0018] As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent, which is preferably used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent.

[0019] Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has been subjected to various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, water that can be used as an extraction solvent in this embodiment also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered physiological saline, etc.

[0020] Examples of hydrophilic organic solvents that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0021] When using a mixture of two or more polar solvents as an extraction solvent, the mixing ratio can be adjusted appropriately. For example, when using a mixture of water and a lower aliphatic alcohol, it is preferable to mix 1-90 parts by volume of the lower aliphatic alcohol with 10 parts by volume of water, when using a mixture of water and a lower aliphatic ketone, it is preferable to mix 1-40 parts by volume of the lower aliphatic ketone with 10 parts by volume of water, when using a mixture of water and a polyhydric alcohol, it is preferable to mix 10-90 parts by volume of the polyhydric alcohol with 10 parts by volume of water.

[0022] The extraction process is not particularly limited as long as the soluble components contained in the extraction raw material can be dissolved in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent of 5 to 15 times the amount (mass ratio) of the extraction raw material, and the soluble components are extracted at room temperature or under reflux heating, and then the extraction residue is removed by filtration to obtain an extract. The solvent is distilled off from the obtained extract to obtain a paste-like concentrate, and the concentrate is further dried to obtain a dry product.

[0023] The method for isolating and purifying glycyrrhizinic acid from the extract obtained as above, the concentrate of the extract, or the dried product of the extract is not particularly limited, and can be performed by a conventional method. For example, the plant extract can be subjected to column chromatography using a porous material such as silica gel or alumina, or a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and eluted with water and alcohol in this order, to obtain glycyrrhizinic acid as a fraction eluted with alcohol.

[0024] Examples of alcohols that can be used as an eluent in column chromatography include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol, and aqueous solutions thereof, but are not particularly limited to these.

[0025] Furthermore, the alcohol fraction obtained by column chromatography may be purified using any organic compound purification means, such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, or column chromatography using an ion exchange resin.

[0026] The glycyrrhizinic acid thus obtained can be reacted with an alkali such as potassium hydroxide (KOH) in a conventional manner to obtain a glycyrrhizinic acid derivative such as dipotassium glycyrrhizinate.

[0027] In addition, glycyrrhetic acid can be obtained by hydrolyzing the glycyrrhizinic acid using an aqueous solution of an acid (hydrochloric acid, sulfuric acid, etc.) in a conventional manner, and glycyrrhetinic acid derivatives such as stearyl glycyrrhetinate can be obtained by esterifying the glycyrrhetinic acid with stearyl alcohol.

[0028] As will be apparent from the examples described below, the glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives and licorice extract obtained as described above have the effect of promoting interleukin-37 production and interleukin-37 mRNA expression, and therefore can be used as active ingredients of interleukin-37 production promoters and interleukin-37 mRNA expression promoters by utilizing these effects.

[0029] The interleukin-37 production promoter or interleukin-37 mRNA expression promoter of the present embodiment may consist of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract, or may be a formulation of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract.

[0030] At least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract can be formulated into any dosage form such as powder, granules, tablets, liquid, etc., according to a conventional method using a pharma- ceutically acceptable carrier such as dextrin, cyclodextrin, and other optional auxiliary agents. In this case, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavorings, and odorants can be used as auxiliary agents. At least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract can be used by blending with other compositions (for example, skin cosmetics, etc.), and can also be used as ointments, external liquids, patches, etc.

[0031] In addition, the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of this embodiment can be used as an active ingredient by blending it with other natural extracts having interleukin-37 production promoting action or interleukin-37 mRNA expression promoting action, if necessary.

[0032] The method of administration of the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of this embodiment generally includes transdermal administration, oral administration, etc., but a method suitable for prevention, treatment, etc. may be appropriately selected depending on the type of disease. The dosage of the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of this embodiment may be appropriately increased or decreased depending on the type and severity of the disease, individual differences between patients, administration method, administration period, etc.

[0033] The interleukin-37 production promoter or interleukin-37 mRNA expression promoter of the present embodiment can prevent, treat or improve contact dermatitis (rash) caused by contact with irritating substances; various skin inflammatory diseases associated with rough skin such as psoriasis, pemphigus vulgaris and others; and skin inflammation such as redness, erythema and pigmentation caused by exposure to ultraviolet light, through the interleukin-37 production promoting effect or interleukin-37 mRNA expression promoting effect possessed by at least one active ingredient selected from the group consisting of glycyrrhizic acid, glycyrrhizic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives and licorice extract. However, the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of the present embodiment can be used for all purposes in which it is meaningful to exert the interleukin-37 production promoting effect or interleukin-37 mRNA expression promoting effect in addition to these uses.

[0034] In addition, the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of the present embodiment has an excellent interleukin-37 production promoting effect or interleukin-37 mRNA expression promoting effect, and is therefore suitable for, for example, incorporation into an external skin preparation or oral composition. In this case, at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetic acid, glycyrrhizinic acid derivatives, and licorice extract may be directly incorporated, or an interleukin-37 production promoter or interleukin-37 mRNA expression promoter formulated from at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhizinic acid, glycyrrhizinic acid derivatives, and licorice extract may be incorporated.

[0035] Here, topical skin preparations are not limited to specific classifications and include a wide range of skin cosmetics, quasi-drugs, medicines, etc. that are used percutaneously. Specific examples include ointments, creams, milky lotions, beauty serums, lotions, packs, foundations, lip balms, bath additives, hair tonics, hair lotions, soaps, body shampoos, etc.

[0036] The amount of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract in the topical skin preparation can be adjusted appropriately depending on the type of topical skin preparation, but a suitable blending ratio is 0.0001 to 10 mass%, and a particularly suitable blending ratio is 0.001 to 1 mass%.

[0037] The oral composition refers to a composition that is unlikely to cause harm to human health and is taken orally or by administration through the digestive tract in normal social life, and is not limited to administrative classifications such as food, medicine, and quasi-drug. Therefore, the "oral composition" in this embodiment broadly includes general food, feed, health food, health functional food (specified health food, nutrient functional food, functional food), quasi-drug, medicine, etc. that are taken orally. The oral composition in this embodiment is preferably an oral composition that can display the favorable action of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract on the oral composition or its packaging, and is particularly preferably a health functional food (specified health food, nutrient functional food, functional food), quasi-drug, or medicine.

[0038] The amount of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract in the oral composition can be appropriately changed in consideration of the purpose of use, symptoms, sex, etc., but it is preferable to make the daily intake of the extract about 1 to 1000 mg for an adult in consideration of the general intake of the oral composition to be added. When the oral composition to be added is in the form of granules, tablets, or capsules, the amount of at least one selected from the group consisting of glycyrrhizinic acid, glycyrrhizinic acid derivatives, glycyrrhetinic acid, glycyrrhetinic acid derivatives, and licorice extract is usually 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, based on the oral composition to be added.

[0039] Furthermore, since the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of this embodiment has excellent interleukin-37 production promoting effect or interleukin-37 mRNA expression promoting effect, it can also be suitably used as a reagent for research on the mechanism of action of these agents.

[0040] In addition, although the interleukin-37 production promoter or interleukin-37 mRNA expression promoter of this embodiment is preferably applied to humans, they can also be applied to animals other than humans as long as their respective functional effects are achieved. EXAMPLES

[0041] The present invention will be described in more detail below with reference to test examples, but the present invention is not limited to the following test examples. In the following test examples, dipotassium glycyrrhizinate (manufactured by Maruzen Pharmaceuticals Co., Ltd.) and glycyrrhetinic acid (manufactured by Maruzen Pharmaceuticals Co., Ltd.) were used as test samples.

[0042] [Test Example 1] Interleukin-37 production promotion test Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsinization. The harvested cells were 2.0 × 10 5 After diluting with KGM to a cell density of 10000000000 cells / mL, 1 mL of each sample was seeded on a 12-well plate and cultured overnight at 37°C under 5% CO2. After culture, the culture medium was discarded, and 1 mL of the test sample dissolved in KGM at the required concentration (see Table 1 below for sample concentration) was added to each well and cultured at 37°C under 5% CO2 for 48 hours. After culture, the medium was removed from each well, washed with 1 mL of PBS(-), and protein was extracted using 100 μL of M-PER (PIERCE). 50 μL of the prepared protein solution was used to quantify interleukin-37 using Human IL-37 / IL-1F7 ELISA Kit PicoKine (Boster), and the remaining 25 μL was used to quantify the protein amount using BCA Protein Assay Kit (PIERCE), and the amount of interleukin-37 per unit protein amount was calculated, and the interleukin-37 production promotion rate (%) was calculated using the following formula.

[0043] Interleukin-37 production promotion rate (%) = A / B x 100 In the formula, A represents "the amount of interleukin-37 per unit protein amount in cells when the test sample is added," and B represents "the amount of interleukin-37 per unit protein amount in cells when the test sample is not added." The results are shown in Table 1.

[0044] [Table 1]

[0045] [Table 2]

[0046] As shown in Tables 1 and 2, it was confirmed that dipotassium glycyrrhizinate and glycyrrhetinic acid have an excellent effect of promoting interleukin-37 production.

[0047] [Test Example 2] Interleukin-37 mRNA expression promoting effect test Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsinization. The harvested cells were 2.0 × 10 5 After diluting with KGM to a cell density of 1000 cells / mL, 1 mL was seeded per well on a 12-well plate and cultured overnight at 37°C under 5% CO2. After culture, the culture medium was discarded, and 1 mL of the test sample dissolved in KGM at the required concentration (see Table 2 below for sample concentration) was added to each well, and cultured at 37°C under 5% CO2 for 48 hours. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (manufactured by Nippon Gene Co., Ltd.). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to be 100 ng / μL.

[0048] Using this total RNA as a template, the expression levels of interleukin-37 and the internal standard YWHAZ mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) with PrimeScript RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.)) and TB Green (registered trademark) Fast qPCR Mix (manufactured by Takara Bio Inc.). The expression level of interleukin-37 was corrected with the expression level of YWHAZ mRNA, and the corrected value was calculated.

[0049] From the obtained corrected values, the interleukin-37 mRNA expression promotion rate (%) was calculated according to the following formula. Interleukin-37 mRNA expression promotion rate (%) = A / B x 100 In the formula, A represents the "corrected value when the sample was added" and B represents the "corrected value when no sample was added." The results are shown in Table 3.

[0050] [Table 3]

[0051] As shown in Table 3, it was confirmed that dipotassium glycyrrhizinate has an excellent effect of promoting interleukin-37 mRNA expression. [Industrial Applicability]

[0052] The interleukin-37 production promoter and interleukin-37 mRNA expression promoter of this embodiment can greatly contribute to the prevention, treatment, or improvement of contact dermatitis (rash) caused by contact with irritating substances; various inflammatory skin diseases such as psoriasis, pemphigus vulgaris, and other skin roughness; and skin inflammation such as redness, erythema, and pigmentation caused by exposure to ultraviolet rays.

Claims

1. An interleukin-37 production promoter comprising at least one active ingredient selected from the group consisting of glycyrrhizic acid and its derivatives, glycyrrhetinic acid and its derivatives, and licorice extract.

2. The interleukin-37 production promoter according to claim 1, characterized in that the derivative of glycyrrhizinic acid is dipotassium glycyrrhizinate.

3. An interleukin-37 mRNA expression promoter comprising at least one active ingredient selected from the group consisting of glycyrrhizic acid and its derivatives, glycyrrhetinic acid and its derivatives, and licorice extract.

4. The interleukin-37 mRNA expression promoter according to claim 3, wherein the derivative of glycyrrhizinic acid is dipotassium glycyrrhizinate.

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