JP7668046B2 - Composition for activating hair follicle stem cells, composition for inhibiting the expression of wnt3 gene in hair follicle stem cells, and composition for inhibiting the expression of bmp4 gene in hair follicle stem cells - Google Patents

Description

The present invention relates to a composition for activating hair follicle stem cells, a composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and a composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells.

Both the Wnt signaling pathway and the Bmp (Bone Morphogenetic Protein) signaling pathway are composed of multiple factors and transmit signals into cells via receptors on the cell surface. Both of these signaling pathways play important roles in early development, coordinating intercellular signals related to cell differentiation and tissue arrangement, and functioning to appropriately control intercellular interactions.

Wnt3 (Wnt Family Member 3) in the Wnt signaling pathway and Bmp4 (Bone Morphogenetic Protein 4) in the Bmp signaling pathway are known to be involved in maintaining the stemness of hair follicle stem cells (see, for example, Non-Patent Documents 1 and 2).

Hair follicle stem cells remain quiescent while maintaining their stemness until the resting phase. When they transition from the resting phase to the anagen phase, they proliferate and differentiate. When the expression levels of Wnt3 protein and Bmp4 protein decrease, hair follicle stem cells are released from their quiescent state and transition from the resting phase to the anagen phase. In other words, the decrease in Wnt3 protein and Bmp4 protein in hair follicle stem cells induces hair follicle stem cells in the telogen phase to enter the anagen phase, promoting the proliferation of hair follicle stem cells and activating the hair follicle stem cells to promote their differentiation. As a result of the activation of hair follicle stem cells, the proportion of hair matrix cells increases.

Hair growth agents are known as treatments for baldness and male pattern baldness. Various substances are known as active ingredients in hair growth agents, among which are unsaturated fatty acids. In other words, hair growth agents containing unsaturated fatty acids as active ingredients are known (see, for example, Patent Document 1 and Non-Patent Document 3).

On the other hand, royal jelly is a natural product that contains various unsaturated fatty acids. It is known that 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid contained in royal jelly are effective in improving female pattern hair loss (see, for example, Patent Document 2).

JP 2017-81962 A Patent No. 6808737

Plikus MV et al., Nature. 2008 Jan 17;451(7176):340-4. Lim X et al., Proc Natl Acad Sci U S A. 2016 Mar 15;113(11): E1498-505. Brotzu G et al., Dermatol Ther. 2019 Jan;32(1):e12778.

The substances described in Patent Documents 1 and 2 and Non-Patent Document 3 are useful for treating or improving baldness, male pattern baldness, and female pattern baldness. However, there is no mention in these documents of the ability of the active ingredients described therein to activate hair follicle stem cells.

In addition, very little is known about ingredients that are naturally derived and known to be safe yet can activate hair follicle stem cells.

The problem that the present invention aims to solve is to provide a composition for activating hair follicle stem cells, which contains as an active ingredient a component that is naturally derived, has been recognized as safe, and can activate hair follicle stem cells. Furthermore, the problem that the present invention aims to solve is to provide a composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells and a composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells, which can activate hair follicle stem cells.

The inventors of the present invention have conducted extensive research to solve the above problems, and have tried and tested many ingredients, either alone or in appropriate combinations, in an attempt to obtain an active ingredient that activates hair follicle stem cells.

After extensive research, it was finally discovered that a combination of 10-hydroxy-2-decenoic acid and 2-decenedioic acid can activate hair follicle stem cells through their inhibitory effect on the expression of the wnt3 gene and bmp4 gene in hair follicle stem cells. 10-hydroxy-2-decenoic acid and 2-decenedioic acid are ingredients contained in royal jelly and are recognized as safe.

Based on this knowledge, the inventors have succeeded in creating a composition for activating hair follicle stem cells that contains an active ingredient that is naturally derived and recognized as safe, in order to solve the problems of the present invention. The present invention has been completed based on the knowledge first discovered by the inventors and the first successful example achieved.

Thus, from each aspect of the present invention, the following embodiments are provided:
[1] A composition for activating hair follicle stem cells, comprising at least one first active ingredient selected from the group consisting of 10-hydroxy-2-decenoic acid, its derivatives, and salts thereof, and at least one second active ingredient selected from the group consisting of 2-decenedioic acid, its derivatives, and salts thereof.
[2] A composition for suppressing the expression of the wnt3 gene in hair follicle stem cells, or a composition for suppressing the expression of the bmp4 gene in hair follicle stem cells, characterized by comprising at least one first active ingredient selected from the group consisting of 10-hydroxy-2-decenoic acid, its derivatives, and salts thereof, and at least one second active ingredient selected from the group consisting of 2-decenedioic acid, its derivatives, and salts thereof.
[3] The composition described in [1] or [2], which is a food or beverage, a cosmetic, a quasi-drug, or a pharmaceutical.

According to the present invention, hair follicle stem cells can be activated through the suppression of the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells. In addition, the composition for activating hair follicle stem cells, the composition for suppressing the expression of the wnt3 gene in hair follicle stem cells, and the composition for suppressing the expression of the bmp4 gene in hair follicle stem cells of one embodiment of the present invention are highly safe because the active ingredients are related to the components contained in naturally derived royal jelly and their metabolites. Thus, the composition for activating hair follicle stem cells, the composition for suppressing the expression of the wnt3 gene in hair follicle stem cells, and the composition for suppressing the expression of the bmp4 gene in hair follicle stem cells of one embodiment of the present invention can be used for activating hair follicle stem cells in various forms such as food and beverages, cosmetics, pharmaceuticals, and quasi-drugs.

FIG. 1 shows the results of evaluation of the inhibitory effect on the expression of the Wnt3 gene, as described in the Examples below. FIG. 2 shows the results of evaluation of the inhibitory effect on the expression of the Bmp4 gene, as described in the Examples below.

Each aspect of the present invention will be described in detail below, but the present invention is not limited to the items in this section, and various aspects may be adopted as long as the object of the present invention is achieved.

Unless otherwise specified, the terms used in this specification are used in the sense commonly used by those skilled in the art of foods and beverages, cosmetics, pharmaceuticals, and quasi-drugs, and should not be construed as having an unduly restrictive meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

The term "composition" is not particularly limited to the commonly used meaning, but may be, for example, a combination of two or more components. "Component" means a substance used (added) in producing a composition, and is synonymous with substance and (raw) material. A component may exist in a composition in a qualitatively and/or quantitatively maintained or altered state compared to before use.
The term "content" is synonymous with concentration and amount added (amount used), and refers to the ratio of the amount of a component to the total amount of the composition. However, the total amount of the components does not exceed 100%. When a commercially available product is used, the content of the component is preferably the amount of the component contained in the commercially available product, but may be the amount of the commercially available product itself.
"Food and beverages" is a generic term meaning food products, beverages or both.
"Comprise, contain, include" means that elements other than those explicitly stated to be included can be added (same meaning as "comprise at least"), but also encompasses "consist of" and "essentially consists of." That is, "comprise" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or consisting essentially of the explicitly stated elements. Elements include limitations such as ingredients, steps, conditions, and parameters.
"And/or" means any one or any or all combinations of two or more of the associated listed items.
The term "to" in a numerical range includes both the preceding and following numerical values, as well as a range excluding one of those included limits. For example, "0% to 100%" can be 0% or more, 100% or less, or 0% or more and 100% or less. "About" means an amount within ±10% of the quantity following the term. For example, "about 100" means 100±10%, i.e., 90 to 110.

The term "hair follicle stem cell" is not particularly limited to the commonly used meaning, but refers to, for example, epithelial stem cells that are present mainly in the bulge region of hair follicles and have the function of differentiating into hair matrix cells.
"Activation of hair follicle stem cells" refers to any one, two or all three of the following events: promoting differentiation of hair follicle stem cells, promoting proliferation of hair follicle stem cells, and inducing hair follicle stem cells in the anagen phase through the suppression of expression of the wnt3 gene and bmp4 gene in hair follicle stem cells. Therefore, the composition of one aspect of the present invention can also be used as a composition for promoting differentiation of hair follicle stem cells, a composition for promoting proliferation of hair follicle stem cells, and/or a composition for inducing anagen into hair follicle stem cells in the anagen phase.
The "gene expression suppressing effect" refers to at least one effect selected from the group consisting of reducing the expression level of a gene (i.e., the amount of mRNA), reducing the translation level of a gene product (protein) (i.e., the amount of protein), and inhibiting the production of a gene product.

[Summary of the Invention]
One aspect of the present invention is a composition for activating hair follicle stem cells, which is used to activate hair follicle stem cells. Another aspect of the present invention is a composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, which is used to inhibit the expression of the wnt3 gene in hair follicle stem cells. Another aspect of the present invention is a composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells, which is used to inhibit the expression of the bmp4 gene in hair follicle stem cells. The composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one aspect of the present invention are characterized in that they contain two active ingredients. The first active ingredient is at least one ingredient selected from the group consisting of 10-hydroxy-2-decenoic acid and derivatives thereof, and salts thereof. The second active ingredient is at least one ingredient selected from the group consisting of 2-decenedioic acid and derivatives thereof, and salts thereof. In this specification, the first active ingredient and the second active ingredient may be collectively referred to as the active ingredient.

The wnt3 gene is a gene that encodes the Wnt3 protein, which is one of the proteins that constitute the Wnt signaling pathway as is generally known. The bmp4 gene is a gene that encodes the Bmp4 protein, which is one of the proteins that constitute the Bmp signaling pathway as is generally known. As specific examples of the wnt3 gene and the bmp4 gene, the human wnt3 gene is registered under accession number NM_030753.5, and the human bmp4 gene is registered under accession number NM_001202.6.

Hair follicle stem cells in the telogen phase express relatively large amounts of the wnt3 and bmp4 genes without undergoing much proliferation or differentiation. In contrast, hair follicle stem cells in the anagen phase actively proliferate and differentiate, and express relatively small amounts of the wnt3 and bmp4 genes. Thus, when the expression levels of the wnt3 and bmp4 genes decrease, hair follicle stem cells transition from the telogen phase to the anagen phase and begin to proliferate and differentiate (see, for example, Maksim V. Plikus el al, Nature. 2008 January 17; 451(7176): 340-344. doi:10.1038/nature06457; Krzysztof Kobielak et al., PNAS June 12, 2007, vol. 104, no. 24, 10063-10068). As described in the examples below, the active ingredient suppresses the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells. As a result, hair follicle stem cells that were in the resting phase are induced to the anagen phase, where they proliferate and differentiate. Therefore, the active ingredient's effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells activates hair follicle stem cells so that they are induced from the resting phase to the anagen phase, where they proliferate and differentiate. Through these phenomena, the present invention, by including the active ingredient, can take the form of a composition for activating hair follicle stem cells, a composition for suppressing the expression of the wnt3 gene in hair follicle stem cells, and a composition for suppressing the expression of the bmp4 gene in hair follicle stem cells.

[Active ingredient]
10-Hydroxy-2-decenoic acid can be prepared by the conventional method represented by the following formula (I):
(I)
It is a compound having the structure represented by the following formula:

2-Decenedioic acid can be prepared by the conventional method represented by the following formula (II):
(II)
It is a compound having the structure represented by the following formula:

10-hydroxy-2-decenoic acid is metabolized in the human body and converted to 2-decenedioic acid. As for 10-hydroxy-2-decenoic acid, 10-hydroxy-2-decenoic acid itself may be used, or a 10-hydroxy-2-decenoic acid-containing substance containing 10-hydroxy-2-decenoic acid may be used. Similarly, as for 2-decenedioic acid, 2-decenedioic acid itself may be used, or a 2-decenedioic acid-containing substance containing 2-decenedioic acid may be used. When using 10-hydroxy-2-decenoic acid itself and/or 2-decenedioic acid itself, commercially available products may be used, or products chemically synthesized by known methods or extracted from natural products may be used.

The 10-hydroxy-2-decenoic acid-containing material and the 2-decenedioic acid-containing material are not particularly limited as long as they contain 10-hydroxy-2-decenoic acid and 2-decenedioic acid, respectively, but examples include royal jelly, which contains both 10-hydroxy-2-decenoic acid and 2-decenedioic acid.

Royal jelly is a milky white jelly-like substance made by mixing secretions from the hypopharyngeal and mandibular glands of worker honeybees that are 3 to 12 days old. The main physiologically active components in royal jelly include organic acids such as the saturated fatty acids and unsaturated fatty acids that are unique to royal jelly, as well as proteins, amino acids, peptides, lipids, sugars, vitamin B, folic acid, nicotinic acid, pantothenic acid, and other vitamins, and various minerals.

Royal jelly includes raw royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, royal jelly extract, and royal jelly fermentation product. Royal jelly may be produced in any of the following countries: Europe, Oceania, America, Brazil, Japan, China, and other Asian countries.

Dried royal jelly powder is made by drying and powdering fresh royal jelly.
As the drying method, any of the known methods generally used in food processing can be used, such as natural drying such as ventilation drying or sun drying, forced drying in which the food is dried by heating with electricity or the like, freeze drying, etc. Freeze drying is preferred.

Enzyme-treated royal jelly is royal jelly treated with a protein-degrading enzyme (protease). Preferably, it is hypoallergenic enzyme-treated royal jelly in which allergic reactions caused by the proteins contained in royal jelly are suppressed by protease treatment. Therefore, in addition to the protease decomposition products of the proteins contained in royal jelly, enzyme-treated royal jelly may contain organic acids such as the aforementioned saturated fatty acids and unsaturated fatty acids, lipids, sugars, vitamins, and various minerals.

The royal jelly used in the production of enzyme-treated royal jelly is not particularly limited, and examples thereof include raw royal jelly, royal jelly powder obtained by drying and powdering raw royal jelly, and raw royal jelly extracted with water or aqueous ethanol.

Enzyme-treated royal jelly can be produced by treating the royal jelly raw material with an enzyme having at least endopeptidase activity, an enzyme having at least exopeptidase activity, and/or an enzyme having both endopeptidase and exopeptidase activity.

Proteolytic enzymes having at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

Examples of proteolytic enzymes having at least exopeptidase activity include carboxypeptidase, aminopeptidase, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus, Rhizopus, etc.), and pancreatin and pepsin, which also have endopeptidase activity.

Among these various enzymes, preferred examples of enzymes having both exopeptidase activity and endopeptidase activity include peptidase produced by Streptomyces griseus (trade name: Actinase AS), peptidase produced by Aspergillus oryzae (trade name: Protease A, Flavorzyme, Proteax), and peptidase produced by Aspergillus melleus (trade name: Protease P). Preferred examples of enzymes having exoprotease activity include peptidase produced by Aspergillus oryzae (trade name: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), peptidase produced by Aspergillus sojae (trade name: Sternzyme B15024), peptidase produced by the genus Aspergillus (trade name: Coclase P), and peptidase produced by Rhizopus oryzae (trade name: Peptidase R). Further preferred examples of enzymes having endoprotease activity include peptidase produced by Bacillus subtilis (trade name: Orientase 22BF, Nucleisin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutrase), and peptidase produced by the genus Bacillus (trade name: Protamex).

Enzyme treatment to reduce the allergenicity of royal jelly can be carried out, for example, according to the description in JP-A-2007-295919 and JP-A-2007-295920.

Royal jelly extract is obtained by extracting royal jelly (including raw, dried and crushed) with water or aqueous ethanol.

Royal jelly fermentation products can be produced by conventional methods using microorganisms such as yeast and lactic acid bacteria.

Commercially available royal jelly may be used. Examples of commercially available royal jellies include Enzymatically Decomposed Royal Jelly King (manufactured by Yamada Bee Farm), Royal Jelly FD Powder (manufactured by Nakahara Co., Ltd.), Royal Jelly Extract SF (manufactured by Matsuura Pharmaceutical Co., Ltd.), Deproteinized Royal Jelly Powder F (manufactured by Maruzen Pharmaceutical Co., Ltd.), and Deproteinized Royal Jelly Extract (manufactured by API Co., Ltd.).

As described above, 10-hydroxy-2-decenoic acid and 2-decenedioic acid are both types of fatty acids contained in royal jelly. 10-hydroxy-2-decenoic acid and 2-decenedioic acid may be synthetic products or may be derived from natural products such as royal jelly. 10-hydroxy-2-decenoic acid and 2-decenedioic acid derived from royal jelly can be obtained, for example, by extraction and purification from royal jelly using an organic solvent. 10-hydroxy-2-decenoic acid and 2-decenedioic acid may be cis or trans.

The derivatives of 10-hydroxy-2-decenoic acid and 2-decenedioic acid are not particularly limited as long as they have the effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells, and examples thereof include chlorides, esters, amides, acid anhydrides, and glycosides. The derivatives of 10-hydroxy-2-decenoic acid and 2-decenedioic acid may be synthesized from 10-hydroxy-2-decenoic acid and 2-decenedioic acid by a method known in the art, or may be obtained by extraction and purification from natural products such as royal jelly using an organic solvent, etc.

10-Hydroxy-2-decenoic acid and its derivatives, and 2-decenedioic acid and its derivatives may take the form of a salt. These salts are not particularly limited as long as they have the effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells. For example, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine salts, triethylamine salts, cyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, and procaine salts; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts, and isoquinoline salts; tetramethylammonium salts, tetraethylamine salts, and tetraethylamine salts. quaternary ammonium salts such as ammonium salts, benzyltrimethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts, and tetrabutylammonium salts; amino acid salts such as arginine salts, lysine salts, histidine salts, arginine salts, aspartate salts, and glutamate salts; inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, and perchlorate salts; organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, and ascorbate salts; and sulfonate salts such as methanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate.

[Composition for activating hair follicle stem cells]
A composition for activating hair follicle stem cells according to one embodiment of the present invention contains an active ingredient, and is thereby capable of activating hair follicle stem cells.

The content of the active ingredient in the hair follicle stem cell activation composition of one embodiment of the present invention is not particularly limited as long as the hair follicle stem cell activation composition of one embodiment of the present invention has the effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells, but the lower limit, for example, in terms of solid content relative to the total amount of the hair follicle stem cell activation composition is 0.1% by mass or more, 1% by mass or more, 3% by mass or more, 3.5% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, It may be at least 100 mass%, at least 99 mass%, at most 99 mass%, at most 98 mass%, at most 95 mass%, at most 93 mass%, at most 90 mass%, at most 85 mass%, at most 80 mass%. , 75 mass% or less, 70 mass% or less, 65 mass% or less, 60 mass% or less, 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 8 mass% or less, 5 mass% or less, 3 mass% or less, or 1 mass% or less.

The content of the active ingredient is the total amount of the first active ingredient and the second active ingredient. Since both the first active ingredient and the second active ingredient have the effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells, the ratio of the content of the first active ingredient and the content of the second active ingredient in the content of the active ingredients is not particularly limited and may be set appropriately. For example, since the effect of 10-hydroxy-2-decenoic acid in suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells is the same as or greater than that of 2-decenedioic acid, it is preferable that the content of the first active ingredient is the same as or greater than the content of the second active ingredient, and it is more preferable that the content of the first active ingredient is greater than the content of the second active ingredient.

The subject of application of the composition for activating hair follicle stem cells according to one embodiment of the present invention is not particularly limited, and may be, for example, an animal, particularly a mammal, such as a human, dog, cat, cow, horse, pig, sheep, or rabbit, and preferably a human. The subject of application may be a healthy subject, but is preferably a subject in which activation of hair follicle stem cells is desired. Mammalian hair follicles become smaller with age, regardless of gender (see, for example, Hiroyuki Matsumura et al., Science, Vol. 351, ISSUE 6273, p. 575). Hair follicle stem cells are maintained at the time of the onset of male pattern baldness. From this perspective, the age and gender of the subject of application are not particularly limited, and may be, for example, an adult, preferably a middle-aged to elderly male or female, preferably a male, and more preferably a male in the early stages of onset of male pattern baldness.

The route of administration of the composition for activating hair follicle stem cells according to one embodiment of the present invention is not particularly limited, and may be oral or parenteral. Oral administration includes enteral administration, and parenteral administration includes topical administration, for example, transdermal administration.

When the composition for activating hair follicle stem cells according to one embodiment of the present invention is orally administered, the dosage can be appropriately set depending on the active ingredient, the form of the composition, and the application method and amount. For example, when the active ingredient is in the form of royal jelly as a 10-hydroxy-2-decenoic acid-containing substance and a 2-decenedioic acid-containing substance, the amount of active ingredient per day for an adult weighing 60 kg is preferably 10 mg to 30,000 mg, more preferably 100 mg to 20,000 mg, 150 mg to 15,000 mg, 600 mg to 12,000 mg, 1,200 mg to 10,000 mg, or 2,400 mg to 8,000 mg, calculated on a dry solid basis. The dosage can be increased or decreased as appropriate depending on factors such as the health condition of the subject, the administration method, the type of active ingredient, and the combination with other agents.

When the active ingredient is 10-hydroxy-2-decenoic acid or a derivative thereof, or a salt thereof, the dosage of the composition for activating hair follicle stem cells of one embodiment of the present invention is, for example, preferably 1 mg to 200 mg, and more preferably 10 mg to 100 mg, of the total amount of the active ingredient, calculated as dry solids, per day for an adult weighing 60 kg. When the active ingredient is 2-decenedioic acid or a derivative thereof, or a salt thereof, the dosage of the composition for activating hair follicle stem cells of one embodiment of the present invention is, for example, preferably 0.1 mg to 30 mg, and more preferably 1 mg to 15 mg, of the total amount of the active ingredient, calculated as dry solids, per day for an adult weighing 60 kg. The dosage can be increased or decreased as appropriate, depending on factors such as the health condition of the subject, the method of administration, the type of active ingredient, and the combination with other agents.

The composition for activating hair follicle stem cells of one embodiment of the present invention may be administered once a day or in multiple doses, such as twice or three times a day, so long as the effective daily dosage falls within the range described above. The composition for activating hair follicle stem cells of one embodiment of the present invention is preferably administered continuously or intermittently, and more preferably, for example, for one to four weeks, one month or more, six months or more, or one year or more.

When the composition for activating hair follicle stem cells according to one embodiment of the present invention is administered parenterally, the dosage may vary depending on the active ingredient, the site of application, and the range of application. For example, when the active ingredient is royal jelly, the amount applied to the skin is preferably 0.01 mg to 50 mg of the active ingredient, more preferably 0.02 mg to 40 mg, and even more preferably 0.02 mg to 35 mg, or 0.025 mg to 30 mg.

When the active ingredient is 10-hydroxy-2-decenoic acid or a derivative thereof, or a salt thereof, the dosage of the composition for activating hair follicle stem cells of one embodiment of the present invention is, for example, an amount applied to the skin of 0.001 mg to 0.3 mg, and more preferably 0.01 mg to 0.1 mg, as the total amount of the active ingredient. When the active ingredient is 2-decenedioic acid or a derivative thereof, or a salt thereof, the dosage of the composition for activating hair follicle stem cells of one embodiment of the present invention is, for example, an amount applied to the skin of 0.0001 mg to 0.05 mg, and more preferably 0.001 mg to 0.04 mg, as the total amount of the active ingredient.

The composition for activating hair follicle stem cells according to one embodiment of the present invention may contain other ingredients in addition to the active ingredient. The other ingredients are not particularly limited as long as they do not interfere with the solution of the problems of the present invention, and may be, for example, powdered ingredients, liquid oils and fats, solid oils and fats, waxes, hydrocarbon oils, higher fatty acids, higher alcohols, ester oils, silicone oils, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, moisturizers, water-soluble polymers, thickeners, film-forming agents, UV absorbers, sequestering agents, lower alcohols, polyhydric alcohols, sugars, amino acids, organic amines, polymer emulsions, pH adjusters , skin nutrients, vitamins, antioxidants, antioxidant assistants, fragrances, water, preservatives (ethylparaben, butylparaben, etc.), anti-inflammatory agents (glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin, etc.), whitening agents (placenta extract, saxifrage extract, arbutin), various extracts (phellodendron bark, coptis japonica, Lithospermum root, peony root, Swertia japonica, birch, sey , loquat, carrot, aloe, mallow, iris, grape, coix seed, loofah, lily, saffron, cnidium, ginger, hypericum, ononis, garlic, chili pepper, tangerine, angelica, seaweed, etc.), activators (photosensitizers, cholesterol derivatives, etc.), blood circulation promoters (nonyl acid valenylamide, nicotinic acid benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, zingerone, cantharides tincture, Examples of effective anti-inflammatory agents include ichthammol, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclandelate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthine, γ-oryzanol, etc.), antiseborrheic agents (sulfur, thianthol), anti-inflammatory agents (tranexamic acid, thiotaurine, hypotaurine), beekeeping products (propolis, honey, bee hornet's nest, beeswax, pollen, bee venom, etc.).

The hair follicle stem cell activation composition of one embodiment of the present invention can be manufactured by a conventional method by mixing the active ingredient with one or more other ingredients to obtain a desired form. The form of the hair follicle stem cell activation composition of one embodiment of the present invention is not particularly limited as long as it exhibits the active ingredient's effect of suppressing the expression of the wnt3 gene and the bmp4 gene in hair follicle stem cells, but examples of the form include solid, liquid, gel, suspension, cream, sheet, stick, powder, granule, tablet, rod, plate, block, paste, capsule, caplet, aerosol, and foam.

The composition for activating hair follicle stem cells according to one embodiment of the present invention may be a packaged composition filled in a sealable airtight or sealed container. The container is not particularly limited as long as it is an airtight or sealed container, and examples of the container include packaging containers such as single-layer or laminated film bags, retort pouches, vacuum packs, molded containers, bottles, and cans made of metals such as aluminum and steel, paper, plastics such as PET, and glass. The packaged composition can be sold independently and commercially available in the market. Note that a container with a lid used for the purpose of storing food at home is a sealed container, and is strictly distinguished from an airtight container and a sealed container, particularly a sealed container.

The hair follicle stem cell activation effect of the hair follicle stem cell activation composition of one embodiment of the present invention can be confirmed by confirming the suppression of the expression of the wnt3 gene in hair follicle stem cells and the suppression of the expression of the bmp4 gene in hair follicle stem cells. Thus, the hair follicle stem cell activation effect of the hair follicle stem cell activation composition of one embodiment of the present invention may be, for example, to the extent that the expression of the wnt3 gene is suppressed in hair follicle stem cells and the expression of the bmp4 gene is suppressed in hair follicle stem cells, compared to a composition that does not contain an active ingredient (control) under the same conditions.

[Composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells and composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells]
A composition for suppressing the expression of the wnt3 gene in hair follicle stem cells according to one embodiment of the present invention contains an active ingredient, thereby being able to suppress the expression of the wnt3 gene in hair follicle stem cells.A composition for suppressing the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention contains an active ingredient, thereby being able to suppress the expression of the bmp4 gene in hair follicle stem cells.

For the composition for suppressing the expression of the wnt3 gene in hair follicle stem cells according to one embodiment of the present invention and the composition for suppressing the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention, please refer to the above section on [Composition for activating hair follicle stem cells] for information on the type and amount of active ingredient, method and amount of administration, ingredients contained, form, manufacturing method, etc.

The effect of the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells of one embodiment of the present invention on inhibiting the expression of the wnt3 gene in hair follicle stem cells may be to the extent that the expression of the wnt3 gene is inhibited in hair follicle stem cells compared to a composition that does not contain an active ingredient (control) under the same conditions. Similarly, the effect of the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells of one embodiment of the present invention on inhibiting the expression of the bmp4 gene in hair follicle stem cells may be to the extent that the expression of the bmp4 gene is inhibited in hair follicle stem cells compared to a composition that does not contain an active ingredient (control) under the same conditions.

The effect of suppressing the expression of the wnt3 gene in hair follicle stem cells can be confirmed by the method described in the Examples below. For example, a composition for suppressing the expression of the wnt3 gene in hair follicle stem cells according to one embodiment of the present invention preferably has an effect of suppressing the expression of the wnt3 gene in hair follicle stem cells such that the expression level of the wnt3 gene is 90% or less, and more preferably has an effect of suppressing the expression of the wnt3 gene in hair follicle stem cells such that the expression level of the wnt3 gene is 85% or less, compared to a control using DMSO instead of the active ingredient, by the method described in the Examples below.

The effect of suppressing the expression of the bmp4 gene in hair follicle stem cells can be confirmed by the method described in the Examples below. For example, a composition for suppressing the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention preferably has an effect of suppressing the expression of the bmp4 gene in hair follicle stem cells such that the expression level of the bmp4 gene is 90% or less, more preferably has an effect of suppressing the expression of the bmp4 gene in hair follicle stem cells such that the expression level of the bmp4 gene is 80% or less, and even more preferably has an effect of suppressing the expression of the bmp4 gene in hair follicle stem cells such that the expression level of the bmp4 gene is 75% or less, when compared to a control using DMSO instead of the active ingredient, by the method described in the Examples below.

[Food and beverages, cosmetics, quasi-drugs and pharmaceuticals]
The composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention can be used as foods and beverages (particularly foods and beverages intended for health, maintaining or improving health, etc. (e.g. health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), cosmetics, quasi-drugs, medicines, etc. Thus, another aspect of the present invention is foods and beverages, cosmetics, quasi-drugs, or medicines that include, or are themselves, the composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention.

The content of the active ingredient in the food and beverage products, cosmetics, quasi-drugs, and pharmaceuticals according to one embodiment of the present invention is not particularly limited, and may be an effective amount that can achieve the oral or parenteral administration dose of the composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells.

When the food and beverage products, cosmetics, quasi-drugs, and pharmaceuticals of one embodiment of the present invention contain as one component the composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells of one embodiment of the present invention, they can be produced, for example, by adding the composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells of one embodiment of the present invention to an intermediate product in the manufacturing process of these products.

In addition to the active ingredient, the cosmetic product according to one embodiment of the present invention can be appropriately blended with ingredients normally used in cosmetics, such as bactericides, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavorings, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), whitening agents, moisturizers, antioxidants, UV absorbers, powdered ingredients, coloring materials, and various skin nutrients, as needed.

The cosmetic raw material may be in any form, such as a solid, liquid, or paste. The cosmetic may be a medicated cosmetic (i.e., a quasi-drug). Cosmetics include any cosmetic that can be applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, and other parts of animals (including humans).

Cosmetics can take a wide range of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-layer systems, and water-oil-powder three-layer systems.

The use of the cosmetics is also arbitrary. For example, basic cosmetics include face wash, lotion, milky lotion, cream, gel, essence, beauty serum, pack, mask, mist, UV protection cosmetics, etc.; makeup cosmetics include foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc.; nail cosmetics include nail polish, base coat, top coat, nail polish remover, etc.; and other cosmetics include face wash, (paste or liquid) dentifrice, mouth rinse, oral cosmetics, massage agents, cleansing agents, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling products, hair tonics, hair mist, hair foam, hair liquid, hair gel, hair spray, hand cream, hand soap, hair growth agents, antiperspirants, bath agents, etc.

In one embodiment of the present invention, the food and beverage products can contain, in addition to the active ingredient, ingredients that are usually used in food and beverage products, such as minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solubilizers, and humectants.

Food and drink includes any food and drink that can be consumed by animals (including humans). The types of food and drink are not particularly limited, and examples include dairy products; fermented foods (such as yogurt); beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, beverages containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcohol such as fruit wine); spreads (such as custard cream); pastes (such as fruit paste); Western confectionery (chocolate, donuts, pies, cream puffs, gum, gummies, jellies, candies, cookies, cakes, puddings, etc.); Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbets, etc.); foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

The method for producing the food or beverage is not particularly limited, and any known method can be used as appropriate.

A specific example of a food or drink is a supplement. The dosage unit form when used as a supplement is not particularly limited and can be selected appropriately, and examples include chewable tablets, troches, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

The pharmaceutical product of one embodiment of the present invention can be used by mixing, in addition to the active ingredient, other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

In one aspect of the present invention, the active ingredient can be prepared, together with a pharmaceutical acceptable ingredient, into a pharmaceutical formulation in the form of a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsule, pill, powder (dispersed medicine), fine granules, granules, liquid, suspension, emulsion, syrup, paste, injection (including the case where the liquid is prepared by mixing with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use), etc.

The pharmaceutical may be administered locally or systemically. There is no particular limitation on the method of administration, and the pharmaceutical may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

In addition to the active ingredient, the pharmaceutical product of one embodiment of the present invention can be appropriately formulated with pharma- ceutical ingredients that are typically used in pharmaceutical products, such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavorings, and fragrances, and other pharma- ceutical ingredients that are pharma- ceutical acceptable.

Cosmetics and pharmaceuticals according to one aspect of the present invention include quasi-drugs.

The composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention are applied to mammals including humans (preferably humans) so as to be applied to foods and beverages, cosmetics, pharmaceuticals, and quasi-drugs. Thus, the composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention are highly safe because they contain naturally derived ingredients such as royal jelly, which has traditionally been used as a food ingredient, as active ingredients.

[Another aspect of the present invention]
Another aspect of the present invention is a method for activating hair follicle stem cells, comprising applying an active ingredient to a subject to activate hair follicle stem cells in the subject.
Another aspect of the present invention is a method for suppressing expression of the wnt3 gene in hair follicle stem cells, comprising applying an active ingredient to a subject to suppress expression of the wnt3 gene in hair follicle stem cells of the subject.
Another aspect of the present invention is a method for inhibiting expression of the bmp4 gene in hair follicle stem cells, comprising administering an active ingredient to a subject to inhibit expression of the bmp4 gene in hair follicle stem cells of the subject.
Another aspect of the present invention is a method of using an active ingredient to produce a composition for activating hair follicle stem cells, a composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, or a composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be solved.

Using an organoid culture system of whisker tissue, the expression levels of genes involved in hair follicle stem cell activation after 72 hours were evaluated for 10-hydroxy-2-decenoic acid and 2-decenedioic acid. As a result, the expression levels of genes encoding the Wnt3 protein and Bmp4 protein, which are involved in the activation of hair follicle stem cell stemness, were significantly reduced by 10-hydroxy-2-decenoic acid and 2-decenedioic acid. These results demonstrated that 10-hydroxy-2-decenoic acid and 2-decenedioic acid are responsible for activating hair follicle stem cells, such as promoting hair follicle stem cell differentiation, promoting hair follicle stem cell proliferation, and inducing hair follicle stem cells in the anagen phase through their suppressive effect on the expression levels of the wnt3 gene and bmp4 gene.

1. Test Sample
Four types of fatty acids contained in royal jelly were used: 10-hydroxydecanoic acid (manufactured by Combi-Blocks; also referred to as "decanoic acid"), 10-hydroxy-2-decenoic acid (manufactured by Hangzhou Eastbiopharm; also referred to as "decenoic acid"), 2-decenedioic acid (manufactured by Sundia Meditech; also referred to as "decenedioic acid"), and sebacic acid (manufactured by Sigma-Aldrich).

2. Evaluation Method
Using the method described below, we analyzed changes in gene expression levels upon addition of fatty acids to a whisker tissue organoid culture system to evaluate the effects on differentiation markers of hair follicle stem cells.

Mouse whisker follicles and their surrounding tissues (organoids) collected using surgical scissors were placed in a 96-well plate at 37°C in a 5% _CO2 environment. After 24 hours of placement, the wells were filled with DMEM/10% FBS medium (150 μl) containing fatty acids (100 μM), antibiotics (penicillin-streptomycin-amphotericin; PSA), HEPES (10 mM) and glutamine (2 mM). The plate was incubated at 37°C in a 5% _CO2 environment, and tissue pieces were collected after 48 hours.

Total RNA for gene expression analysis was extracted from the collected tissue pieces according to the NucleoSpin® RNA Plus XS (TakaraBio, Sharp U0990C) manual. The extracted pre-RNA was reverse transcribed with PrimeScript™ RT Master Mix (TakaraBio, Sharp RR036B) to prepare cDNA. The expression level of each gene in the cDNA was quantified by qPCR using the primers shown in Table 1 and THUNDERBIRD® Next SYBR® qPCR Mix (TOYOBO, Sharp QPX-201). Beta-actin was used as the reference gene for this test.

All results are shown as mean ± standard deviation. Gene expression levels for each fatty acid were evaluated using Dunnett's test in comparison with the DMSO group as a control group.

3. Evaluation Results
The quantitative results of the expression level of the Wnt3 gene are shown in FIG. 1, and the quantitative results of the expression level of the Bmp4 gene are shown in FIG. 2 (n=3 or 4, *p<0.05; vs. control).

As shown in Figures 1 and 2, when the gene expression levels of whisker tissue organoids when decenoic acid or decenedioic acid was added were compared with those of the control, it was found that the addition of decenoic acid and decenedioic acid reduced the gene expression levels of Wnt3 protein and Bmp4 protein. In particular, it was found that the addition of decenoic acid significantly reduced the gene expression levels of Wnt3 protein and Bmp4 protein, and the addition of decenedioic acid significantly reduced the gene expression level of Bmp4 protein.

These results show that the combination of decenoic acid and decenedioic acid, through its suppressive effect on the expression levels of the wnt3 and bmp4 genes, activates hair follicle stem cells by promoting their differentiation, promoting their proliferation, and inducing hair follicle stem cells in the anagen phase into the resting phase.

The composition for activating hair follicle stem cells, the composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells, and the composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells according to one embodiment of the present invention are naturally derived and contain active ingredients that are recognized as safe, and can activate hair follicle stem cells through the inhibitory effect of the wnt3 gene and the bmp4 gene in hair follicle stem cells. They can be used in various forms such as food and beverages, cosmetics, pharmaceuticals, and quasi-drugs.

Claims (3)

A composition for activating hair follicle stem cells, comprising at least one first active ingredient selected from the group consisting of 10-hydroxy-2-decenoic acid, its acid anhydride and glycoside derivatives, and salts thereof, and at least one second active ingredient selected from the group consisting of 2-decenedioic acid, its acid anhydride and glycoside derivatives, and salts thereof. A composition for inhibiting the expression of the wnt3 gene in hair follicle stem cells or a composition for inhibiting the expression of the bmp4 gene in hair follicle stem cells, characterized by comprising at least one first active ingredient selected from the group consisting of 10-hydroxy-2-decenoic acid, its acid anhydride and glycoside derivatives, and salts thereof, and at least one second active ingredient selected from the group consisting of 2-decenedioic acid, its acid anhydride and glycoside derivatives, and salts thereof. The composition according to claim 1 or 2, which is a food or drink, a cosmetic, a quasi-drug, or a pharmaceutical.