Hair growth and / or hair restoration agent

Abstract

The present invention aims to provide a novel hair growth and / or hair restoration agent containing mushroom extract as an active ingredient. [Solution] A hair growth and / or hair restoration agent containing an extract from a mushroom belonging to the genus Hypsizygus of the family Lyophyllaceae as its active ingredient.

Description

TECHNICAL FIELD

[0001] The present invention relates to a hair growth agent and / or a hair nourishing agent.

BACKGROUND ART

[0002] It has been conventionally known that mushroom extracts are effective for hair loss suppression and hair nourishment. For example, Patent Document 1 describes that a powder composition containing bud powder obtained by pulverizing the buds of Lentinus edodes fruiting bodies has a hair loss suppressing effect. In addition, Patent Document 2 describes that the fruiting bodies, mycelia or mycelial culture solutions of Lyophyllum decastes, Pleurotus eryngii, and Lentinus edodes have effects such as hair growth.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0003]

PATENT DOCUMENT 1

PATENT DOCUMENT 2

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0004] Under the above circumstances, the present invention aims to provide a novel agent for hair growth and / or hair nourishment that contains a mushroom extract as an active ingredient.

MEANS FOR SOLVING THE PROBLEMS

[0005] The present invention for solving the above problems is as follows. [1] A hair growth and / or hair nourishing agent comprising, as an active ingredient, an extract of a mushroom belonging to the genus Hypsizygus in the family Lyophyllaceae.

[0006] [2] The agent according to [1], which is used for inducing differentiation of hair follicle epithelial stem cells and / or enhancing proliferation of dermal papilla cells.

[0007] [3] A hair follicle epithelial stem cell differentiation inducer and / or hair papilla cell proliferation enhancer, comprising an extract of a mushroom belonging to the genus Hypsizygus of the family Lyophyllaceae as the active ingredient.

[0008] [4] The fungus belonging to the genus Hypsizygus of the family Lyophyllaceae is Hypsizygus marmoreus, as described in any of [1] to [3].

[0009] [5] The preparation according to any one of [1] to [4], wherein the mushroom extract belonging to the genus Hypsizygus of the family Lyophyllaceae is an extract of the stem of Hypsizygus marmoreus.

[0010] [6] The agent according to any one of [1] to [5], wherein the extract of the stem of Hypsizygus marmoreus is a hot water extract of the stem.

[0011] [7] The agent according to [6], wherein the hot water extract of the stem is an acidic hot water extract of the stem.

[0012] [8] The agent according to [7], wherein the acidic hot water extract of the stem is the citric acid-containing hot water extract of the stem.

[0013] [9] A method for inducing differentiation of hair follicle epithelial stem cells, comprising applying a composition containing an extract of a mushroom belonging to the genus Hypsizygus of the family Lyophyllaceae as an active ingredient to the hair and / or skin.

[0014]

[10] A method for promoting the proliferation of dermal papilla cells, comprising applying a composition containing an extract of a mushroom belonging to the genus Hypsizygus of the family Lyophyllaceae as an active ingredient to the hair and / or skin. [Effects of the Invention]

[0015] The agent according to the present invention has a hair growth effect and / or a hair nourishing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Figure 1] It is a diagram showing the number of dermal papilla cells counted in Test Example 1. [Figure 2] It is a diagram showing the expression levels of Sox2, Lef1 and versican measured in Test Example 1. [Figure 3] It is a diagram showing the number of dermal papilla cells counted in Test Example 2. [Figure 4] It is a diagram showing the expression levels of Sox2, Lef1 and versican measured in Test Example 2. [Figure 5] It is a diagram showing the number of hair follicle epithelial stem cells counted in Test Example 3. [Figure 6] It is a diagram showing the expression level of CD34 measured in Test Example 3. [Figure 7] It is a diagram showing the results of CD34 immunostaining performed in Test Example 3. [Figure 8] It is a diagram showing the results of AE13 immunostaining performed in Test Example 3. [Figure 9] It is a diagram showing the results of testing the effect of an extract derived from beech shimeji mushroom on hair follicle organs performed in Test Example 4. [Figure 10] It is a diagram showing the results (shaft elongation) of testing the effect of an extract derived from beech shimeji mushroom on hair follicle organs performed in Test Example 4. MODE FOR CARRYING OUT THE INVENTION

[0017] The present invention will be described in detail below. In the embodiments of the present invention, A (numerical value) to B (numerical value) means A or greater and B or less. Furthermore, preferred embodiments and more preferred embodiments illustrated below can be used in appropriate combinations with each other, regardless of expressions such as "for example," "one," "preferred," and "more preferred." In addition, the numerical ranges are illustrative, and ranges obtained by appropriately combining the upper and lower limits of each range and the numerical values ​​of the examples can also be used (for example, if A to B and C to D are written, a combination of A to D or C to B can be used). Furthermore, terms such as "contains" or "includes" may be read as "essentially" or "consists only of."

[0018] This invention is characterized by using a mushroom extract belonging to the genus Hypsizygus of the family Lyophyllaceae as an active ingredient. In the following explanation, "mushrooms belonging to the genus Hypsizygus of the family Lyophyllaceae" may sometimes be written as "mushrooms belonging to the genus Hypsizygus of the family Lyophyllaceae," omitting the scientific name.

[0019] Examples of mushrooms belonging to the genus Hypsizygus in the family Lyophyllum-family include Hypsizygus marmoreus and Hypsizygus ulmarius, but are not limited to these. Preferably, the mushroom belonging to the genus Hypsizygus in the family Lyophyllumaceae is Hypsizygus marmoreus. Hereafter, these will sometimes simply be referred to as "Buna-shimeji" or "Shirotamogitake."

[0020] For mushrooms belonging to the genus Pholiota in the family Lyophyllumaceae, the entire fruiting body may be used for extraction, or parts of the fruiting body such as the cap, stem, or base may be used. In the present invention, the part of the mushroom extract of the genus Pholiota in the family Lyophyllumaceae is preferably the stem end. In other words, the mushroom extract belonging to the genus Pholiota in the family Lyophyllum, according to the present invention, is preferably an extract of the stem of a mushroom belonging to the genus Pholiota in the family Lyophyllum, according to the present invention. Furthermore, while the extraction site may be used directly in the extraction process described later, it is preferable that it be pre-cut or crushed by conventional methods. In addition, the extraction site of mushrooms belonging to the genus Pholiota of the family Lyophyllumaceae, which is to be cut or crushed, may be pre-frozen. In one form, as a pretreatment for the extraction process, the extraction portion of a mushroom extract belonging to the genus Pholiota of the family Lyophyllumaceae can be subjected to a drying process, such as freeze-drying, and a grinding process, in which the dried extraction portion of the mushroom extract belonging to the genus Pholiota of the family Lyophyllumaceae is ground using known equipment, before being subjected to the extraction process. In another configuration, as a pretreatment for the extraction process, the extraction portion of a mushroom belonging to the genus Pholiota in the family Lyophyllumaceae can be subjected to a micronization process, such as by a bead mill, before being used in the extraction process.

[0021] Mushroom extract can be obtained by immersing the extract portion of a mushroom belonging to the genus Pholiota of the family Lyophyllum, which has been pre-treated as needed by shredding, grinding, or micronizing, in an extraction solvent and heating it at 60-100°C. In other words, the extraction process includes at least an immersion step in which the mushroom belonging to the genus Pholiota of the family Lyophyllum is immersed in an extraction solvent, and a heating step in which the extraction solvent in which the mushroom belonging to the genus Pholiota of the family Lyophyllum is immersed is heated. The immersion process and the heating process may be separate processes, or they may be performed in parallel. In other words, in one form, the extract portion of a mushroom belonging to the genus Pholiota in the family Lyophyllumaceae can be immersed in an extraction solvent for a certain period of time, and then the extraction solvent can be directly subjected to a heating process. In another configuration, the extracted portion of a mushroom belonging to the genus Pholiota in the family Lyophyllumaceae can be immersed in an extraction solvent (or dispersed depending on the form of the mushroom) and then immediately subjected to a heating process.

[0022] The extraction temperature is 60-100°C as described above, preferably 70-100°C, and more preferably 80-100°C. Alternatively, the extraction temperature may be 80-90°C or 90-100°C. The extraction time can be adjusted as appropriate, referring to the examples described later. For example, if the extraction temperature is close to 100°C, the extraction time should be increased; if the extraction temperature is close to 60°C, the extraction time should be decreased. Furthermore, "hot water," as described later, refers to water whose temperature falls within the above numerical range. Moreover, "acid-containing hot water (citric acid-containing hot water / hydrochloric acid-containing water)," as described later, refers to acid-containing water (citric acid-containing water / hydrochloric acid-containing water) whose temperature falls within the above numerical range.

[0023] The extraction solvent is preferably water or acid-containing water, and more preferably acid-containing water. In this invention, acid-containing water refers to water that contains an acid. The acid may be either an organic or inorganic acid. Examples of organic acids include, but are not limited to, acetic acid, citric acid, malic acid, or mixtures thereof. Examples of inorganic acids include, but are not limited to, hydrochloric acid, nitric acid, phosphoric acid, or mixtures thereof. Preferably, the acid is citric acid and / or hydrochloric acid. Preferably, the pH of the acid-containing water in which the extracted parts of mushrooms belonging to the genus Pholiota of the family Lyophyllumaceae are immersed or dispersed is 1.0 to 6.0 at 20°C. Preferably, the pH of the acid-containing water in which the extracted parts of mushrooms belonging to the genus Pholiota of the family Lyophyllumaceae are immersed or dispersed is 3.0 to 5.0 at 20°C. When using two or more acids in combination, they should be mixed so that the pH falls within the above numerical range. For example, when using citric acid and hydrochloric acid in combination, the mixture can be prepared such that the amount of hydrochloric acid (mass%) in the water containing the extracted parts of a mushroom belonging to the genus Pholiota of the family Lyophyllum, relative to the amount of citric acid (mass%), is set to 1, and the amount of hydrochloric acid (mass%) in the water containing the extracted parts of a mushroom belonging to the genus Pholiota of the family Lyophyllum, relative to the amount of hydrochloric acid (mass%) is between 0.1 and 10.

[0024] In this case, if the extraction solvent is acid-containing water, the order of the immersion step and the preparation of the acid-containing water in the extraction step is not particularly limited. In other words, in one configuration, the extracted portion of a mushroom belonging to the genus Pholiota of the family Lyophyllum, which has undergone the necessary pretreatment, can be immersed or dispersed in pre-prepared acid-containing water during the immersion process, and the dispersion can then be subjected to the heating process. In another configuration, the extraction portion of a mushroom belonging to the genus Pholiota of the family Lyophyllum, which has undergone the necessary pretreatment, is first immersed or dispersed in water in the immersion step, and then an acid is added to the water to obtain a solution, which can then be used in the subsequent extraction step.

[0025] Considering all of the above, the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, according to the present invention, is preferably a hot water extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum, or an acid-containing hot water extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum. Furthermore, the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, according to the present invention, is more preferably a hot water extract of the stem base of a mushroom belonging to the genus Pholiota of the family Lyophyllum, or an acid-containing hot water extract of the stem base of a mushroom belonging to the genus Pholiota of the family Lyophyllum.

[0026] Furthermore, the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, according to the present invention, is preferably a citric acid-containing hot water extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum, or it is also preferably a hydrochloric acid-containing hot water extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum, Furthermore, the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, according to the present invention, is more preferably a citric acid-containing hot water extract of the stem base of a mushroom belonging to the genus Pholiota of the family Lyophyllum. Alternatively, it is also preferable that it be a hydrochloric acid-containing hot water extract of the stem base of a mushroom belonging to the genus Pholiota of the family Lyophyllum.

[0027] The mushroom extract belonging to the genus Pholiota of the family Lyophyllum, obtained as described above, may be added directly to the hair growth and / or hair tonic, or it may be added to the hair growth and / or hair tonic after a known process, to the extent that it does not impair the effects of the present invention. For example, a filtration step may be performed to filter the obtained mushroom extract belonging to the genus Pholiota of the family Lyophyllum, a concentration step may be performed to concentrate the obtained mushroom extract belonging to the genus Pholiota of the family Lyophyllum, a sterilization step may be performed to kill the obtained mushroom extract belonging to the genus Pholiota of the family Lyophyllum, and a powdering step may be performed to powder the obtained mushroom extract belonging to the genus Pholiota of the family Lyophyllum. Furthermore, all of these processes can be carried out using known equipment and conventional methods. The powdering process is preferably carried out by freeze-drying or spray-drying. If powdering is carried out by spray-drying, a mixture of mushroom extract belonging to the genus Pholiota of the family Lyophyllum, and an excipient such as dextrin may be spray-dried as needed.

[0028] As shown in the examples described later, mushroom extracts belonging to the genus Pholiota of the family Lyophyllum have the effect of inducing the differentiation of hair follicle epithelial stem cells and promoting the cell proliferation of dermal papilla cells. Both hair follicle epithelial stem cells and dermal papilla cells are cells involved in hair growth and development. Therefore, the hair growth and / or hair restoration agent of the present invention is preferably used for inducing differentiation of hair follicle epithelial stem cells and / or promoting proliferation of dermal papilla cells.

[0029] The hair growth and / or hair tonic of the present invention may be used as a topical preparation or as an oral preparation. It may also be used as a pharmaceutical product (hair growth agent) or a quasi-drug (hair tonic).

[0030] When the hair growth and / or hair restoration agent of the present invention is used as a topical preparation, the dosage form is not particularly limited, and an appropriate dosage form such as a cream or liquid can be selected. It may also be used as an aerosol-type hair growth / hair restoration agent in which the liquid is sprayed. Furthermore, it can be appropriately combined with ingredients commonly used in topical preparations, such as bases, moisturizers, fragrances, preservatives, and pH adjusters. The manufacturing method of the topical preparation is also not particularly limited; hair growth and / or hair restoration preparations can be manufactured by conventional methods according to the dosage form.

[0031] In a topical hair growth and / or hair restoration agent, the content (including the solvent) of mushroom extract belonging to the genus Pholiota of the family Lyophyllum is preferably 0.0000001 to 10% by mass, more preferably 0.000001 to 5% by mass, even more preferably 0.00001 to 2% by mass, and still more preferably 0.0001 to 1% by mass. According to the present invention, hair growth and / or hair development can be promoted. If the mushroom extract belonging to the genus Pholiota in the family Lyophyllumaceae is concentrated, it should be adjusted so that it falls within the above numerical range when converted to a straight value.

[0032] There are no particular restrictions on the dosage and administration of topical hair growth and / or hair restoration agents; they can be applied as appropriate in a form that exhibits hair growth / hair restoration effects. For example, it can be applied or sprayed onto the scalp or hair at a dose of 1 mL twice a day, but is not limited to this form. Furthermore, while the scalp or hair may be wet, it is preferable to apply or spray the topical agent of the present invention to a dry scalp or hair.

[0033] When the invention and / or hair growth agent of the present invention is used as an oral preparation, the dosage form is not particularly limited, and any appropriate dosage form can be selected, such as powder, tablets, capsules, liquid (drink, etc.), or jelly (stick jelly, spout container, etc., any form is acceptable). Furthermore, it can be appropriately combined with ingredients commonly used in oral preparations, such as bases, fragrances, flavoring agents, odor masking agents, pH adjusters, preservatives, and excipients. The manufacturing method of the oral preparation is also not particularly limited; hair growth and / or hair restoration agents can be manufactured by conventional methods according to the dosage form.

[0034] In an oral hair growth and / or hair restoration agent, the content (including the solvent) of mushroom extract belonging to the genus Pholiota of the family Lyophyllum is preferably 0.000001 to 100% by mass, more preferably 0.00001 to 80% by mass, even more preferably 0.0001 to 70% by mass, and still more preferably 0.0001 to 50% by mass, relative to the daily intake of the oral agent. According to the present invention, hair growth and / or hair development can be promoted. If the mushroom extract belonging to the genus Pholiota in the family Lyophyllumaceae has undergone a concentration process, it should be adjusted so that it falls within the above numerical range when converted to a straight-out equivalent.

[0035] The dosage and administration of oral hair growth and / or hair restoration agents can be applied as appropriate in a form that particularly exhibits hair growth / hair restoration effects. For example, the oral medication can be taken twice a day, such that the daily intake of mushroom extract belonging to the genus Pholiota of the family Lyophyllum (i.e., the total amount of mushroom extract belonging to the genus Pholiota of the family Lyophyllum) falls within the above numerical range. However, the medication is not limited to this form.

[0036] The present invention may also be used as a hair follicle epithelial stem cell differentiation inducer and / or hair papilla cell proliferation enhancer, comprising a mushroom extract belonging to the genus Pholiota in the family Lyophyllumaceae as an active ingredient. The active ingredient of the hair follicle epithelial stem cell differentiation inducer and / or hair papilla cell proliferation enhancer, the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, and its preferred form as an agent, can be applied to the matters described above for hair growth and / or hair restoration agents.

[0037] The present invention also relates to a method for inducing differentiation of hair follicle epithelial stem cells and a method for promoting proliferation of dermal papilla cells, which includes applying a composition containing an extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum to the hair and / or skin as an active ingredient. Hair follicle epithelial stem cells and dermal papilla cells are both parts of the hair that are involved in hair growth and development. Therefore, according to the present invention, hair growth and / or development can be promoted.

[0038] In the present invention, "applying a composition containing an extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum to the hair and / or skin" means applying a composition containing an extract of a mushroom belonging to the genus Pholiota of the family Lyophyllum to the hair and / or skin in a manner suitable for its dosage form. For example, if a composition containing an extract of a mushroom belonging to the genus Pholiota in the family Lyophyllumaceae is a topical preparation, applying it to the hair and / or skin means applying, dropping, or spraying the composition onto the hair and / or skin (preferably the scalp). For example, if a composition containing an extract of a mushroom belonging to the genus Pholiota in the family Lyophyllumaceae is an oral preparation, applying it to the hair and / or skin means taking the composition orally.

[0039] A preferred embodiment of the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, used in the method for inducing differentiation of hair follicle epithelial stem cells and the method for enhancing proliferation of dermal papilla cells of the present invention, is one to which the above-described matters can be applied.

[0040] As shown in the examples described later, the buna shimeji extract has the effect of promoting cell proliferation of hair papilla cells and increasing the expression levels of Sox2, Lef1, and versican, which are involved in hair formation. Specifically, versican is known to construct the hair follicle stem cell niche through the formation of three-dimensional cell aggregates. Furthermore, Sox2 and Lef1 are known to regulate hair growth through a multi-layered mechanism that includes the construction of stem cell niches, regulation of epithelial-mesenchymal interactions, and integration of morphogenesis signals. Therefore, the agent according to the present invention is preferably an agent for people whose dermal papilla cells have reduced cell proliferation ability. Furthermore, the agent according to the present invention is also preferably an agent for people whose expression levels of one or more selected from Sox2, Lef1, and versican in dermal papilla cells are reduced. Furthermore, it is also preferable that the agent according to the present invention is an agent for improving the expression level of one or more selected from Sox2, Lef1, and versican. Furthermore, it is also preferable that the agent according to the present invention be used as an agent for promoting hair formation. Furthermore, it is also preferable that the agent according to the present invention is an agent for hair loss or thinning hair caused by a decrease in the cell proliferation ability of hair papilla cells.

[0041] Furthermore, as will be shown in the examples described later, the buna shimeji extract has the effect of promoting the differentiation induction of hair follicle epithelial stem cells. Therefore, the agent according to the present invention is preferably an agent for people whose hair follicle epithelial stem cell differentiation ability is reduced. Furthermore, it is preferable that the agent according to the present invention is an agent for hair loss or thinning hair caused by a decrease in the differentiation ability of hair follicle epithelial stem cells. [Examples]

[0042] The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to the following examples.

[0043] <Test Example 1> Effects of Buna Shimeji mushroom extract on dermal papilla cells (DPCs) In this study, the effects of buna shimeji mushroom extract on hair papilla cells were investigated.

[0044] <Preparation> Preparation of Buna Shimeji Extract (1) 40 kg of the stems of buna shimeji mushrooms were freeze-dried using known equipment and then micronized using known equipment. (2) 200g of the finely divided stems of the buna shimeji mushrooms obtained in (1) above were added to 1800g of water (hereinafter this water will also be called "finely divided buna shimeji dispersed water"). The pH of the finely atomized buna shimeji mushroom dispersion was adjusted by adding hydrochloric acid dropwise until it reached 4.5 at 20°C (hereinafter, the dispersion after pH adjustment with acid will also be referred to as the finely atomized buna shimeji mushroom dispersion containing hydrochloric acid). When the pH of the finely atomized buna shimeji mushroom dispersion reached 4.5 at 20°C, 11.9 g of 9 N hydrochloric acid was added. (3) The finely atomized buna shimeji dispersed acid-containing water obtained in (2) above was heated until the liquid temperature reached 80°C to obtain a hydrochloric acid-containing hot water extract of the buna shimeji stems. (4) The hydrochloric acid-containing hot water extract of the stem base of the buna shimeji mushroom obtained in (3) above was cooled to room temperature, filtered and concentrated under reduced pressure using known equipment and methods, and then freeze-dried to obtain a powdered hydrochloric acid-containing hot water extract of the stem base of the buna shimeji mushroom. (5) The hydrochloric acid-containing hot water extract of the stem base of the beech mushroom obtained in (4) above was dissolved in 1×PBS to prepare a 1% beech mushroom extract (hydrochloric acid-containing hot water extract of the stem base of the beech mushroom) stock solution.

[0045] 1. Analysis of the effects of buna shimeji mushroom extract on cell proliferation (1) Dermal papilla cells were isolated and cultured (for 10 days) from the whisker tissue of versican-GFP Tg mice (8 weeks old) or C57BL / 6 mice (WT / 8 weeks old) using a standard method, and their proliferation was confirmed. (2) Appropriate amounts of culture medium (10% FBS + DMEM medium + 1 × P / S) were dispensed into 96-well plates, and the following test substances were added. The components of Control Examples 3-6 and Example 1 were dissolved in PBS, and the component of Control Example 2 was dissolved in DMSO. Four wells were used to culture each of Control Examples 1-7 and Example 1. Test substance Control example 1: No test substance added Control example 2: DMSO (final concentration 0.1% by mass) Control example 3: Minoxidil sulfate (final concentration 1 μM) Control example 4: Putrescine (final concentration 1 μM) Control example 5: Spermidine (final concentration 1 μM) Control example 6: Spermine (final concentration 1 μM) Control Example 7: Water with a pH of 4.5 at 20°C using 9 N hydrochloric acid (extraction solvent in Example 1. The amount added to the culture medium was calculated to be the amount contained when the 1% buna shimeji extract stock solution prepared in <Preparation> above was added at a final concentration of 0.01% by mass). Example 1: Stock solution of extract derived from Buna Shimeji mushroom prepared in the above <Preparation> (final concentration including solvent: 0.0001% by mass)

[0046] (3) The cells that were confirmed to proliferate in (1) above, 3 The seeds were seeded in the 96-well plate prepared in (2) above to a cell / well ratio and cultured for 48 hours. (4) After culturing, the number of cells was counted for each of the control examples 1-7 and examples 1-2. Specifically, each cultured cell was fixed with 4% PFA, the cell nucleus was stained with DAPI using a standard method, and the number of cells in one field of view was counted. The results are shown in Figure 1.

[0047] As shown in Figure 1, the buna shimeji extract from Example 1 was shown to significantly enhance DPC cell proliferation compared to control examples 1-7.

[0048] 2. Analysis of gene expression levels using real-time RT-qPCR (1) Place the DPC (versican-GFP) that was isolated and confirmed to grow in "1.(1)" above into a 24-well plate in 6 × 10 3 Seeds were seeded to a cell / well ratio. The culture medium was the same as that used in "1.(2)" above, and the cells were cultured in the presence of the components of Control Examples 1-3, 7 and Example 1 (the concentrations of each component were the same). (2) After 48 hours of incubation, total RNA was extracted by conventional methods. (3) Reverse transcription was performed using PrimeScript® RT(TaKaRa), and real-time RT-qPCR was performed using SYBR Premix ExTaq(TaKaRa) by a standard method. In real-time RT-qPCR, the expression levels of DPC markers Sox2, Lef1, and versican, as well as β-actin as an endogenous control, were measured, and the expression levels of Sox2, Lef1, and versican were corrected for the expression level of β-actin. The results are shown in Figure 2.

[0049] As shown in Figure 2, in Example 1, where the cells were cultured in the presence of buna shimeji extract, the expression levels of Sox2, Lef1, and versican were significantly increased.

[0050] These results indicate that the buna-shimeji mushroom extract enhances DPC cell proliferation.

[0051] Here, versican is known to construct a hair follicle stem cell niche through the formation of three-dimensional cell aggregates. Furthermore, Sox2 and Lef1 are known to regulate hair growth through a multi-layered mechanism including stem cell niche construction, regulation of epithelial-mesenchymal interactions, and integration of morphogenesis signals. Therefore, these results suggest that the buna shimeji mushroom extract enhances the expression of Sox2, Lef1, and versican, respectively, and induces hair formation.

[0052] <Test Example 2> Examination of the results when extraction is performed using different extraction methods. Except for the fact that in the <Preparation> (1) of Test Example 1 above, the stems of the buna shimeji mushrooms were micronized after being frozen in advance, and in <Preparation> (2) citric acid was used as the acid to prepare micronized buna shimeji dispersed citric acid-containing water with a pH of 3.5 at 20°C, the buna shimeji extract of Example 2 was obtained in the same manner as in <Preparation>. The obtained buna shimeji extract is a citric acid-containing hot water extract of the stems of buna shimeji mushrooms. Furthermore, in the above-mentioned Test Example 1, except that in <Preparation>(1) the stems of the buna shimeji mushrooms that had been frozen in advance were micronized, in <Preparation>(2) citric acid was used as the acid to prepare micronized buna shimeji powder dispersion containing citric acid with a pH of 4.0 at 20°C, and in <Preparation>(4) the citric acid-containing hot water extract of the buna shimeji mushroom stems was cooled, filtered, and concentrated under reduced pressure, then mixed with dextrin and powdered by spray drying, the buna shimeji extract of Example 3 was obtained in the same manner as in <Preparation>. The obtained buna shimeji extract is a citric acid-containing hot water extract of the buna shimeji mushroom stems. The buna shimeji extracts from Examples 2 and 3 were each dissolved in 1×PBS to prepare a 1% buna shimeji extract stock solution.

[0053] In addition to Control Example 1 (no test substance added), Control Example 2 (DMSO), Control Example 7 (water with pH 4.5 at 20°C using 9 N hydrochloric acid), and Example 1, cell counts and gene expression of Sox2, Lef1, and versican were analyzed for the following control examples 8 and 9, and the buna shimeji extracts (final concentration including solvent was 0.0001% by mass each) from Examples 2 and 3, in the same manner as in Test Example 1. The final concentrations in the culture media for Control Example 2, Control Example 7, and Example 1 were the same as those in Test 1. Control example 8: Ornithine (final concentration 10 μM) Control example 9: Ergothioneine (final concentration 100 pM) The cell count results are shown in Figure 3, and the gene expression analysis results are shown in Figure 4.

[0054] As shown in Figure 3, the buna shimeji extracts from Examples 2 and 3 were shown to significantly enhance DPC cell proliferation compared to the control.

[0055] As shown in Figure 4, the beech mushroom extracts from Examples 2 and 3 showed significantly increased expression of Sox2, Lef1, and versican compared to the control.

[0056] These results demonstrate that, regardless of the type of acid used, the buna shimeji extract enhances DPC cell proliferation and improves the expression of Sox2, Lef1, and versican. Furthermore, it was suggested that the buna shimeji extract induces hair formation even when different extraction solvents are used.

[0057] <Test Example 3> Effects of Buna Shimeji extract on hair follicle epithelial stem cells (HFSCs) In this study, the effects of buna shimeji mushroom extract on hair follicle epithelial stem cells were investigated.

[0058] 1. Analysis of the effects of buna shimeji mushroom extract on cell proliferation (1) Dorsal skin tissue was isolated from C57BL / 6 mice (WT / 8 weeks old), treated with 0.25% trypsin (4°C, O / N) according to a standard procedure, and epithelial cells were collected. The collected epithelial cells were confirmed to proliferate by a standard procedure, similar to that in Test Example 1. (2) Appropriate amounts of culture medium (CnT-PR (CELLnTECH), 1×P / S) were dispensed into 96-well plates, and four wells of each were cultured in the presence of the components used in control examples 1-9 and examples 1-3, as used in test examples 1 and 2. The concentrations of each component in the culture medium were the same as in test examples 1 and 2.

[0059] (3) The cells that were confirmed to proliferate in (1) above were divided into 5 × 10 3 The seeds were seeded into the 96-well plate prepared in (2) above to achieve a cell / well ratio, adhered under ON conditions, and then cultured for 72 hours. (4) After culturing, the number of cells was counted for each of the control examples 1-9 and examples 1-3. Specifically, each cultured cell was fixed with 4% PFA, the cell nucleus was stained with DAPI according to the standard method, and the number of cells per field of view was counted. The results are shown in Figure 5.

[0060] As shown in Figure 5, the extract derived from buna shimeji mushrooms significantly suppressed the proliferation of HFSCs.

[0061] 2. Analysis of gene expression levels using real-time RT-qPCR (1) Place the HFSCs that were isolated and confirmed to grow in "2.(1)" above into a 24-well plate in a 3 × 10⁻¹⁶ format. 4 Seeds were seeded to a cell / well ratio. The culture medium was the same as that used in "2.(2)" above, and the cells were cultured in the presence of the components of Control Examples 1-9 and Examples 1-3 (the final concentrations in the medium were the same as in Test Examples 1 and 2). (2) After 72 hours of incubation, total RNA was extracted by conventional methods. (3) Reverse transcription was performed using PrimeScript® RT(TaKaRa), and real-time RT-qPCR was performed using SYBR Premix ExTaq(TaKaRa) by a standard method. In real-time RT-qPCR, the expression levels of CD34, an undifferentiated marker, and β-actin, an endogenous control, were measured, and the CD34 expression level was corrected by dividing it by the β-actin expression level. The results are shown in Figure 6.

[0062] As shown in Figure 6, cells cultured in the presence of buna shimeji extract showed significantly lower CD34 expression levels. In other words, it was suggested that the buna-shimeji extract induces the differentiation of HFSCs.

[0063] 3. Observation of CD34 expression levels by immunohistochemistry. (1) The cells that were confirmed to proliferate in "2.(1)" above, 5 × 10 3 Seeds were seeded into a 96-well plate prepared in the same manner as described in "2.(2)" above, so that the cell / well ratio was maintained, and the plates were cultured for 72 hours. (2) Immunostaining was performed according to standard procedures. Anti-CD34 antibody was used as the primary antibody, and goat anti-rabbit IgG Alexa488 was used as the secondary antibody. (3) After staining, the staining status was observed using a fluorescence microscope. The results are shown in Figure 7.

[0064] As shown in Figure 7, CD34 expression levels were low in cells cultured in the presence of the buna shimeji extract. This suggests that, similar to the results of real-time RT-qPCR, the buna shimeji extract induces the differentiation of HFSCs.

[0065] 4. Observation of AE13 expression levels by immunohistochemistry. Except for using anti-AE13 antibody as the primary antibody and goat anti-mouse IgG Alexa546 as the secondary antibody, HFSC culture and AE13 immunostaining were performed in the same manner as described in "3." above, and the staining status was observed using a fluorescence microscope. Note that AE13 is a differentiation marker. The results are shown in Figure 8.

[0066] As shown in Figure 8, cells cultured in the presence of the buna shimeji extract showed increased expression of AE13. Similar to the results of real-time RT-qPCR and immunostaining, this suggests that the buna shimeji extract induces the differentiation of HFSCs.

[0067] In summary, the results of Test Examples 1 to 3 suggest that the buna shimeji extract enhances the proliferation of dermal papilla cells and induces the differentiation of hair follicle epithelial stem cells. Therefore, buna shimeji extract can be used as a hair growth agent and / or hair tonic.

[0068] <Test Example 4> Experiment on the addition of buna-shimeji mushroom extract using hair follicle organs This study investigated the effects of a buna-shimeji mushroom extract on hair follicle organs.

[0069] (1) Whiskers were isolated from C57BL / 6 mice (WT, 4W-5W), and after confirming the hair follicle organs, each unit was microdissected. (2) The shaft was cut. This day was designated as day 0 of the culture. (3) Dispense an appropriate amount of culture medium (10% FBS + DMEM, 1xP / S) into a 96-well plate, and culture the mustache tissue prepared in (1) above for 72 hours in the presence of the following test substances. Three samples were cultured for each test substance. Test substance Control example 1: No test substance added Control example 3: Minoxidil sulfate (final concentration 1 μM) Control example 5: Spermidine (final concentration 1 μM) Control example 8: Ornithine (final concentration 10 μM) Control example 9: Ergothioneine (final concentration 100 pM) Example 1: Extract from Buna Shimeji mushroom prepared in Test Example 1 (final concentration including solvent: 0.0001% by mass) Example 2: Extract from Buna Shimeji mushroom prepared in Test Example 2 (final concentration including solvent: 0.0001% by mass) Example 3: Extract from Buna Shimeji mushroom prepared in Test Example 2 (final concentration including solvent: 0.0001% by mass) The results are shown in Figures 9 and 10.

[0070] As shown in Figures 9 and 10, the beech mushroom extracts from Examples 1-3 were shown to significantly enhance hair elongation compared to the control example. Therefore, it has been shown that the mushroom extract belonging to the genus Pholiota of the family Lyophyllum, according to the present invention, has hair growth and / or hair restoration effects. [Industrial applicability]

[0071] The present invention can be used as a hair growth agent and / or hair tonic.

Claims

1. A hair growth and / or hair restoration agent containing an extract of a mushroom belonging to the genus Hypsygus of the family Lyophyllaceae as its active ingredient.

2. The agent according to claim 1, used for inducing differentiation of hair follicle epithelial stem cells and / or promoting proliferation of dermal papilla cells.

3. An agent for inducing differentiation of hair follicle epithelial stem cells and / or promoting hair papilla cell proliferation, comprising an extract of a mushroom belonging to the genus Hypsygus of the family Lyophyllacae as the active ingredient.

4. The agent according to any one of claims 1 to 3, wherein the mushroom belonging to the genus Hypsizygus of the family Lyophyllacae is Hypsizygus marmoreus.

5. The agent according to claim 4, wherein the mushroom extract belonging to the genus Hypsygus of the family Lyophyllacae is an extract of the stem of Hypsygus marmoreus.

6. The agent according to claim 5, wherein the stem extract of Hypsygus marmoreus is a hot water extract of the stem.

7. The agent according to claim 6, wherein the hot water extract of the base of the stone is an acid-containing hot water extract of the base of the stone.

8. The agent according to claim 7, wherein the acidic hot water extract of the base of the stone is a citric acid-containing hot water extract of the base of the stone.

9. A method for inducing differentiation of hair follicle epithelial stem cells, comprising applying a composition containing an extract of a mushroom belonging to the genus Hypsizygus of the family Lyophyllaceae as an active ingredient to hair and / or skin.

10. A method for promoting the proliferation of dermal papilla cells, comprising applying a composition containing an extract of a mushroom belonging to the genus Hypsizygus of the family Lyophyllacae as an active ingredient to the hair and / or skin.

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