β-defensin-3 mRNA expression promoter

Abstract

To provide a β-defensin-3 mRNA expression promoter that exhibits superior β-defensin-3 mRNA expression promoting action and offers high safety.SOLUTION: A β-defensin-3 mRNA expression promoter includes dipotassium glycyrrhizinate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a β-defensin-3 mRNA expression promoter. [Background technology]

[0002] The skin is the outermost layer of the body and is an important tissue that protects the body from external stimuli, and has a defense function that prevents harmful microorganisms from invading the body. One of these is antimicrobial peptides (AMPs). More than 20 types of AMPs have been identified in the skin, and a representative one is known to be β-Defensin-3. AMPs are produced in epidermal cells, stored in lamellar granules, and released into stratum corneum cells (see, for example, Non-Patent Document 1). AMPs are thought to protect the body from external stimuli by inhibiting the growth of harmful microorganisms as antibacterial agents.

[0003] So far, butcher's broom extract (see, for example, Patent Document 1) and the like are known to have the effect of promoting β-defensin-3 mRNA expression.

[0004] However, there remains a strong demand for new materials that have excellent β-defensin-3 mRNA expression-promoting effects and are highly safe, and therefore can be widely used as ingredients in external compositions such as cosmetics and research reagents, and there is currently a demand for their rapid development. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-104364 A [Non-patent literature]

[0006] [Non-Patent Document 1] Dermatology to read when considering the efficacy of cosmetics, 2020, p.37-39 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to solve the above-mentioned conventional problems and achieve the following object: That is, the present invention aims to provide a β-defensin-3 mRNA expression promoter that has excellent β-defensin-3 mRNA expression promoting activity and is highly safe. [Means for solving the problem]

[0008] As a result of extensive research conducted by the inventors in order to solve the above problems, they discovered that dipotassium glycyrrhizinate has excellent β-defensin-3 mRNA expression promoting effect, is highly safe, and is a useful material for promoting β-defensin-3 mRNA expression.

[0009] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> This is a β-defensin-3 mRNA expression promoter characterized by containing dipotassium glycyrrhizinate. <2> The above composition further comprises an extract of Saxifrage. <1> It is a β-defensin-3 mRNA expression promoter described in . <3> The above-mentioned combination with Saxifrage extract <1> It is a β-defensin-3 mRNA expression promoter described in . Effect of the Invention

[0010] The beta-defensin-3 mRNA expression promoter of the present invention can solve the above-mentioned problems in the conventional art and achieve the above-mentioned objective, thereby providing a beta-defensin-3 mRNA expression promoter that has excellent beta-defensin-3 mRNA expression promoting effect and is highly safe. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (β-Defensin-3 mRNA expression promoter) The β-defensin-3 mRNA expression promoter according to this embodiment contains dipotassium glycyrrhizinate as an active ingredient, and further contains other ingredients as necessary.

[0012] As used herein, promoting the expression of β-defensin-3 mRNA means increasing the expression level of β-defensin-3 mRNA compared to when the β-defensin-3 mRNA expression promoter is not used. The degree of promotion of β-defensin-3 mRNA expression is not particularly limited and can be appropriately selected depending on the purpose.

[0013] The β-defensin-3 mRNA expression promoter is preferably used in combination with saxifrage extract, since it enhances the β-defensin-3 mRNA expression promoting effect and exhibits a more excellent β-defensin-3 mRNA expression promoting effect. The form in which it is used in combination with saxifrage extract is not particularly limited and can be selected appropriately depending on the purpose, and examples include a form in which the β-defensin-3 mRNA expression promoter further contains saxifrage extract, and a form in which the β-defensin-3 mRNA expression promoter is used in combination with saxifrage extract.

[0014] It was not previously known that dipotassium glycyrrhizinate has an excellent β-defensin-3 mRNA expression promoting effect and is useful as a β-defensin-3 mRNA expression promoting agent, and that the β-defensin-3 mRNA expression promoting effect is enhanced when the dipotassium glycyrrhizinate is used in combination with the saxifrage extract, and this is a new discovery by the present inventors.

[0015] <Dipotassium glycyrrhizinate> Dipotassium glycyrrhizinate is C 42 H 60 K 2 O16 It is a compound with a molecular weight of 899.1. Dipotassium glycyrrhizinate used may be prepared by chemical synthesis or may be a commercially available product.

[0016] The method of chemical synthesis is not particularly limited and can be appropriately selected depending on the purpose. For example, there is a method of reacting glycyrrhizinic acid with potassium hydroxide (KOH) or the like.

[0017] The method for obtaining the glycyrrhizinic acid is not particularly limited and can be appropriately selected depending on the purpose. Examples include a method using a commercially available product, and a method for obtaining the glycyrrhizinic acid by at least one of isolation and purification from a plant extract containing glycyrrhizinic acid.

[0018] The plant extract containing glycyrrhizinic acid may be an extract obtained 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 a crude or purified product thereof.

[0019] 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.

[0020] The dipotassium glycyrrhizinate has an excellent effect of promoting the expression of β-defensin-3 mRNA, and thus this effect can be utilized as an active ingredient of the β-defensin-3 mRNA expression promoter.

[0021] <Saxifrage extract> The saxifrage extract is extracted from saxifrage (scientific name: Saxifraga stolonifera).

[0022] Saxifraga stolonifera is an evergreen perennial plant belonging to the genus Saxifragaceae, which is distributed in Honshu, Shikoku, and Kyushu in Japan, and in China overseas, and is easily available in these areas.

[0023] The Saxifrage extract may be prepared from an extraction raw material, or a commercially available product may be used.

[0024] The parts that can be used as the extraction raw material are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include leaves, stems, flowers, buds, seeds, roots, above-ground parts, and mixtures thereof. Among these, the whole plant is preferred. The shape, structure and size of the extraction raw material are not particularly limited and can be appropriately selected depending on the purpose.

[0025] Although the details of the substance contained in saxifrage extract that has the effect of promoting β-defensin-3 mRNA expression are unknown, an extract having the effect of promoting β-defensin-3 mRNA expression can be obtained from saxifrage using extraction methods commonly used for plant extraction, etc.

[0026] In the present invention, the term "extract" includes an extract obtained from an extraction raw material by an extraction process, a diluted or concentrated extract, a dried product obtained by drying an extract, or any of these roughly purified or purified products.

[0027] The above extract can be obtained by drying the extraction raw material, pulverizing it 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. The raw material can also be used after pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the extraction process of the raw material can be efficiently performed with a polar solvent.

[0028] The solvent used for extraction is not particularly limited and can be appropriately selected depending on the purpose. Examples of the solvent include water, a hydrophilic organic solvent, and a mixture thereof. The solvent is preferably used at room temperature or at a temperature below the boiling point of the solvent. Components contained in the extraction raw material that have the effect of promoting β-defensin-3 mRNA expression can be easily extracted by an extraction process using a polar solvent as the extraction solvent.

[0029] The water that can be used as the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. The treatment that is applied to the water is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, the water that can be used as the extraction solvent in this embodiment also includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered physiological saline, etc.

[0030] The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, etc.; lower aliphatic ketones such as acetone, methyl ethyl ketone, etc.; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, glycerin, etc. These may be used alone or in combination of two or more kinds.

[0031] When a mixture of two or more polar solvents is used as an extraction solvent, the mixing ratio can be adjusted appropriately. For example, when a mixture of water and a lower aliphatic alcohol is used as an extraction solvent, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with 10 parts by volume of water. When a mixture of water and a lower aliphatic ketone is used as an extraction solvent, it is preferable to mix 1 to 40 parts by volume of a lower aliphatic ketone with 10 parts by volume of water. When a mixture of water and a polyhydric alcohol is used as an extraction solvent, it is preferable to mix 1 to 90 parts by volume of a polyhydric alcohol with 10 parts by volume of water. Among these, it is preferable to use a mixture of 50% by volume of water and 50% by volume of 1,3-butylene glycol as the extraction solvent.

[0032] 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, extraction can be performed at room temperature or under reflux heating. For example, the extraction raw material is placed in a treatment tank filled with the extraction solvent, and while stirring as necessary, the soluble components are dissolved by leaving it for 30 minutes to 4 hours, and then the solid matter is removed by filtration to obtain an extract. The extraction solvent is distilled off from the obtained extract liquid to obtain a paste-like concentrate, and this concentrate is further dried to obtain a dried product. The extraction conditions are about 1 to 4 hours at 50 to 95 ° C when water is used as the extraction solvent. In addition, when a mixed solvent of water and ethanol is used as the extraction solvent, the extraction conditions are about 30 minutes to 4 hours at 40 to 80 ° C. In addition, when a mixed solvent of water and 1,3-butylene glycol is used as the extraction solvent, the extraction conditions are about 2 hours at 80 to 90 ° C. The above extraction time and extraction temperature are merely examples and may be changed as appropriate. The amount of extraction solvent used is not particularly limited and may be appropriately selected depending on the purpose.

[0033] The extract obtained as described above may be subjected to treatments such as dilution, concentration, drying, purification, etc. according to conventional methods in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude or purified product thereof.

[0034] The obtained extract can be used as it is as an ingredient to be mixed with the β-defensin-3 mRNA expression promoter or as an agent to be used in combination with the β-defensin-3 mRNA expression promoter, but it is preferable to use it in the form of a concentrated liquid or a dried product. When obtaining a dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

[0035] In addition, since saxifrage has a unique odor, it is possible to purify it for the purpose of decolorization, deodorization, etc., to the extent that this does not result in a decrease in its physiological activity, but since it is not used in large quantities when added to cosmetics, there is no practical problem in using it unpurified. Purification can be carried out, for example, by treatment with activated carbon, treatment with adsorption resins, treatment with ion exchange resins, etc.

[0036] The saxifrage extract obtained in the manner described above has an excellent effect of promoting β-defensin-3 mRNA expression, and therefore this effect can be utilized as an ingredient to be incorporated into the β-defensin-3 mRNA expression promoter, or as an active ingredient of an agent to be used in combination with the β-defensin-3 mRNA expression promoter.

[0037] The β-defensin-3 mRNA expression promoter may consist only of dipotassium glycyrrhizinate, or may be a formulation containing dipotassium glycyrrhizinate and, if necessary, the saxifrage extract. When the Saxifrage extract is used, the mass ratio of dipotassium glycyrrhizinate to the Saxifrage extract is not particularly limited and can be appropriately selected depending on the purpose.

[0038] The dipotassium glycyrrhizinate and the Saxifragaceae extract, which is used as necessary, can be formulated into any dosage form, such as powder, granules, or liquid, according to a conventional method using a pharma- ceutically acceptable carrier such as dextrin, cyclodextrin, or any other auxiliary agent. In this case, examples of the auxiliary agent that can be used include excipients, stabilizers, and odorants. Examples of the form of the formulated β-defensin-3 mRNA expression promoter include ointments and external liquids.

[0039] The content of dipotassium glycyrrhizinate and the content of Saxifragaceae extract in the formulated β-defensin-3 mRNA expression promoter are not particularly limited and can be appropriately selected depending on the purpose.

[0040] <Other ingredients> The other components are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the form of use of the β-defensin-3 mRNA expression promoter, and examples of such components include the above-mentioned components that can be used when formulating the formulation. The other components may be used alone or in combination of two or more.

[0041] The content of the other components in the β-defensin-3 mRNA expression promoter is not particularly limited and can be appropriately selected depending on the purpose.

[0042] The method of administering the β-defensin-3 mRNA expression promoter to a subject is not particularly limited and can be appropriately selected depending on the purpose. For example, transdermal administration can be mentioned.

[0043] The dosage, administration period, administration interval, administration site, etc. of the β-defensin-3 mRNA expression promoter for a subject are not particularly limited and can be appropriately selected depending on the purpose.

[0044] The β-defensin-3 mRNA expression promoter can promote the expression of β-defensin-3 mRNA through the β-defensin-3 mRNA expression promoting effect of dipotassium glycyrrhizinate. In addition, the β-defensin-3 mRNA expression promoter can enhance the promotion of β-defensin-3 mRNA expression by using it in combination with the Saxifrage extract, and obtain a more excellent β-defensin-3 mRNA expression promoting effect. This can prevent infectious diseases and strengthen the skin barrier by suppressing the growth of harmful microorganisms. As an example, it can be used to suppress acne. However, the β-defensin-3 mRNA expression promoter can be used for all purposes in which it is meaningful to exert the β-defensin-3 mRNA expression promoting effect in addition to these uses.

[0045] In addition, since the β-defensin-3 mRNA expression promoter has an excellent β-defensin-3 mRNA expression promoting effect, it is suitable for incorporation into cosmetics such as skin cosmetics, scalp cosmetics, hair cosmetics, etc. In this case, dipotassium glycyrrhizinate and, if necessary, Saxifraga saxifrage extract may be incorporated as they are, or a formulation of dipotassium glycyrrhizinate and, if necessary, Saxifraga saxifrage extract may be incorporated.

[0046] Cosmetics that can contain the β-defensin-3 mRNA expression promoter are not particularly limited and can be selected appropriately depending on the purpose, and examples include ointments, creams, emulsions, skin lotions, lotions, beauty serums, packs, foundations, hair tonics, hair lotions, shampoos, rinses, soaps, etc.

[0047] When the β-defensin-3 mRNA expression promoter is incorporated into a cosmetic, the amount to be incorporated is not particularly limited and can be adjusted as appropriate depending on the type of cosmetic, but is preferably about 0.0001 to 10 mass%, and more preferably about 0.001 to 1 mass%, converted into the amount of dipotassium glycyrrhizinate.

[0048] As long as the cosmetic preparation does not interfere with the β-defensin-3 mRNA expression promoting effect of dipotassium glycyrrhizinate, it can be used in combination with the main ingredient, auxiliary agent, or other ingredient used in the manufacture of ordinary cosmetic preparations, such as astringents, germicides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / anti-allergic agents, antioxidants / active oxygen removers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc. By using such combinations, the product becomes more general-purpose, and the synergistic effect with the other active ingredients used in combination can bring about a superior effect than would normally be expected.

[0049] The method for producing the cosmetic preparation is not particularly limited, and any known method can be appropriately selected.

[0050] The β-defensin-3 mRNA expression promoter is preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the β-defensin-3 mRNA expression promoting effect is achieved.

[0051] Furthermore, the β-defensin-3 mRNA expression promoter can also be suitably used as a reagent for research into the mechanism of β-defensin-3 mRNA expression promotion.

[0052] As described above, the β-defensin-3 mRNA expression promoter has an effect of promoting β-defensin-3 mRNA expression. Therefore, the present invention also relates to a method for promoting β-defensin-3 mRNA expression, which comprises administering the β-defensin-3 mRNA expression promoter to a subject. EXAMPLES

[0053] The present invention will be explained in more detail below by showing test examples, production examples and formulation examples, but the present invention is not limited to the following test examples, production examples and formulation examples.

[0054] [Test Example 1] β-Defensin-3 mRNA expression promoting effect test Dipotassium glycyrrhizinate (external glycyrrhizinate dipotassium, Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and a test for the promoting effect of β-defensin-3 mRNA expression was carried out by the following method.

[0055] <Test Method> Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were harvested by trypsinization. The harvested cells were plated in 6-well plates at 3.0 × 10 5 Cells were seeded at 2 mL per well, and incubated at 37°C, 5% CO 2 The mixture was incubated overnight under After culturing, the medium was replaced with a medium (KBM) containing no growth factors. After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KBM at the required concentration (see Table 1 below for the concentration) was added to each well and incubated at 37°C, 5% CO 2 As a control, KBM without the addition of the test sample was cultured in the same manner. After the incubation, the culture medium was discarded, total RNA was extracted using ISOGEN II (Nippon Gene; Cat. no. 311-07361), the amount of each RNA was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL. Using this total RNA as a template, the expression levels of mRNA of β-defensin-3 (h-BD3) and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) with PrimeScript TM This was performed by a two-step real-time RT-PCR reaction using RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green (registered trademark) Fast qPCR Mix (Takara Bio Inc.) The expression level of β-defensin-3 mRNA was calculated by correcting the expression level of GAPDH mRNA. From the obtained results, the β-defensin-3 mRNA expression promotion rate (hereinafter, sometimes referred to as "h-BD3 mRNA expression promotion rate") was calculated using the following formula. The results are shown in Table 1. In the following formula, the β-defensin-3 mRNA expression promotion rate without the addition of the test sample is 100%. β-Defensin-3 mRNA expression promotion rate (%) = A / B x 100 In the above formula, A and B respectively represent the following. A: Correction value when test sample is added B: Corrected value when no test sample was added

[0056] [Table 1]

[0057] As shown in Table 1, it was confirmed that dipotassium glycyrrhizinate has an excellent effect of promoting β-defensin-3 mRNA expression.

[0058] [Test Example 2] β-Defensin-3 mRNA expression promoting effect test The effect of promoting β-defensin-3 mRNA expression when dipotassium glycyrrhizinate and Saxifrage extract were used in combination was examined using the following method. The dipotassium glycyrrhizinate used was the same as in Test Example 1, and the Saxifrage extract used was that produced in Production Example 1 below.

[0059] <Production Example 1> Production of Saxifrage Extract 1,500 mL of 50% by volume butylene glycol was added to 100 g of dried whole saxifrage plant, and extraction was performed by heating at 80 to 90°C for 2 hours in a reflux extractor, followed by hot filtration. The resulting extract was dried to obtain saxifrage extract (10 g).

[0060] <Test Method> Except for using the test samples shown in Table 2 below, the promotion rate of β-defensin-3 mRNA expression was determined in the same manner as in Test Example 1. The results are shown in Table 2.

[0061] [Table 2]

[0062] As shown in Table 2, the combined use of dipotassium glycyrrhizinate and saxifrage extract enhanced the effect of promoting β-defensin-3 mRNA expression.

[0063] [Formulation example 1] A cream having the following composition was prepared by a conventional method. Dipotassium glycyrrhizinate 0.05g (Same as used in Test Example 1) Sophora root extract 0.1g · Scutellaria root extract 0.1g Liquid paraffin 5.0g 4.0g white beeswax Squalane 10.0g Cetanol 3.0g 2.0g Lanolin Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-Butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g · Fragrance 0.1g · Purified water Remainder (total amount is 100g)

[0064] [Formulation example 2] An emulsion having the following composition was prepared by a conventional method. Dipotassium glycyrrhizinate 0.05g (Same as used in Test Example 1) Saxifrage extract (Example 1) 0.05g Jojoba oil - 4.00g 1,3-Butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20E.O.) 2.50g 2.00g olive oil Squalane 2.00g Cetanol 2.00g Glyceryl Monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhetinate 0.10g Phellodendron Bark Extract 0.10g Ginkgo Biloba Extract 0.10g 0.10g conchiolin Phellodendron bark extract 0.10g Chamomile extract 0.10g · Fragrance 0.05g · Purified water Remainder (total amount is 100g)

[0065] [Formulation example 3] A beauty essence having the following composition was prepared by a conventional method. Dipotassium glycyrrhizinate 0.08g (Same as used in Test Example 1) Saxifrage extract (Production Example 1) 0.03g Chamomile extract 0.1g Carrot extract 0.1g 0.3g xanthan gum Hydroxyethylcellulose 0.1g Carboxyvinyl polymer 0.1g 1,3-Butylene glycol 4.0g 2.0g glycerin 0.25g potassium hydroxide · Fragrance 0.01g Preservative (methyl parahydroxybenzoate) 0.15g Ethanol 2.0g · Purified water Remainder (total amount is 100g)

Claims

1. A β-defensin-3 mRNA expression promoter comprising dipotassium glycyrrhizinate.

2. The β-defensin-3 mRNA expression promoter according to claim 1, further comprising an extract of Saxifragaceae.

3. The β-defensin-3 mRNA expression promoter according to claim 1, which is used in combination with an extract of Saxifragaceae.

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