Epidermal stem cell activator

Abstract

To provide a novel epidermal stem cell activator and in particular to provide an epidermal stem cell activator that is effective in reducing skin turnover and in inhibiting skin aging and / or skin thinning.SOLUTION: There is provided an epidermal stem cell activator comprising as an active ingredient at least one selected from a group consisting of royal jelly, 10-hydroxy-2-decenoic acid or its salt, 10-hydroxydecanoic acid or its salt, a decenoic acid or its salt, and a sebacic acid or its salt.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an epidermal stem cell activator.

Background Art

[0002] The skin consists of the epidermis, dermis, and subcutaneous tissue, and mainly has a barrier function against external damage and transepidermal water loss. The outermost epidermis further consists of four layers: the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale, and is mainly composed of epidermal keratinocytes. Epidermal stem cells exist in the stratum basale. After differentiating into progenitor cells of epidermal keratinocytes, epidermal stem cells further differentiate into cells forming the stratum spinosum and stratum granulosum, and finally into cells forming the stratum corneum. It has been reported that with aging, the function of epidermal stem cells decreases, leading to thinning of the skin, delay in skin turnover, and decline in the skin barrier function (Non-Patent Documents 1 to 3).

[0003] Patent Document 1 describes that the supercritical extract of Ganoderma spores can promote the differentiation of epidermal stem cells into epidermal keratinocytes. However, Citation Document 1 does not disclose activating epidermal stem cells.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] The present invention aims to provide a novel epidermal stem cell activator, and in particular, an epidermal stem cell activator that is effective in suppressing the decline in skin turnover, skin aging, and / or skin thinning. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objectives, the present inventors have discovered that royal jelly and several fatty acids contained in royal jelly can activate epidermal stem cells, thereby improving the proliferative capacity of epidermal stem cells while maintaining their stem cell properties, and also suppressing the decline in skin turnover, inhibiting skin aging, and inhibiting skin thinning, leading to the completion of the present invention.

[0008] In other words, the present invention relates to, for example, the following inventions. [1] An epidermal stem cell activator containing at least one selected from the group consisting of royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decendioic acid or a salt thereof, and sebacic acid or a salt thereof as an active ingredient. [2] The epidermal stem cell activator according to [1] that suppresses at least one of the following: a decrease in skin turnover, skin aging, and skin thinning. A cosmetic, food composition, or pharmaceutical composition comprising the epidermal stem cell activator described in [3] [1] or [2]. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a novel epidermal stem cell activator that can be used to suppress the decline in skin turnover, suppress skin aging, and suppress skin thinning. [Brief explanation of the drawing]

[0010] [Figure 1] This is an HE-stained image of three-dimensional cultured epidermis cultured with raw royal jelly or enzymatically hydrolyzed royal jelly in Experimental Example 1. [Figure 2] This graph shows the thickness of three-dimensional cultured epidermis cultured with the addition of raw royal jelly or enzyme-hydrolyzed royal jelly in Experimental Example 1. [Figure 3] This is an immunohistochemical image of three-dimensional cultured epidermis cultured with raw royal jelly or enzyme-hydrolyzed royal jelly in Experimental Example 1. [Figure 4] These are HE-stained images of three-dimensional cultured epidermis cultured with 10-hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, decendioic acid, or sebacic acid in Experimental Example 2. [Figure 5] This graph shows the thickness of three-dimensional cultured epidermis cultured with the addition of 10-hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, decendioic acid, or sebacic acid in Experimental Example 2. [Figure 6] These are immunostained images of three-dimensional cultured epidermis cultured with 10-hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, decendioic acid, or sebacic acid, as shown in Experimental Example 2. [Figure 7] This figure shows the relationship between the number of divisions and the elapsed time of HPEKp cells cultured with the addition of raw royal jelly or enzyme-hydrolyzed royal jelly in Experimental Example 3.

Best Mode for Carrying Out the Invention

[0011] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0012] The epidermal stem cell activator according to this embodiment contains at least one selected from the group consisting of royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decenedioic acid or a salt thereof, and sebacic acid or a salt thereof as an active ingredient.

[0013] Royal jelly is a milky jelly-like substance made by mixing the secretions of worker bees aged 3 to 12 days in honeybees from the hypopharyngeal gland and the major parotid gland. The main bioactive components in royal jelly include, for example, organic acids such as 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid peculiar to royal jelly, as well as proteins, amino acids, peptides, lipids, saccharides, vitamins such as vitamin Bs, folic acid, nicotinic acid, pantothenic acid, and various minerals.

[0014] In this specification, royal jelly may be, for example, raw royal jelly or a processed royal jelly product obtained by processing raw royal jelly.

[0015] Raw royal jelly can be obtained as a beekeeping product, for example, according to a conventional method. The production area of royal jelly is not limited and may be any of Japan, China, Brazil, European countries, Oceania countries, the United States, etc. Raw royal jelly includes frozen royal jelly obtained by freezing the collected royal jelly.

[0016] Examples of royal jelly processed products include royal jelly concentrates or dilutions obtained by concentrating or diluting raw royal jelly, royal jelly powder obtained by drying and pulverizing raw royal jelly, royal jelly organic solvent extracts such as royal jelly ethanol extract obtained by extracting royal jelly with an organic solvent such as ethanol, and enzymatically hydrolyzed royal jelly obtained by treating royal jelly with a proteolytic enzyme. The royal jelly processed product may be subjected to multiple processing steps. The royal jelly may also be enzymatically hydrolyzed and pulverized, resulting in enzymatically hydrolyzed royal jelly powder. The royal jelly processed product is preferably enzymatically treated royal jelly, and more preferably enzymatically treated royal jelly powder.

[0017] Royal jelly concentrate can be obtained, for example, by removing water from raw royal jelly. Royal jelly dilution can be obtained, for example, by adding water to raw royal jelly.

[0018] Royal jelly powder can be obtained by pulverizing raw royal jelly using methods known in the art, such as freeze-drying and spray-drying. Any known drying method used in general food processing can be used, such as natural drying (air-drying or sun-drying), forced drying (heating with electricity, etc.), or freeze-drying. Freeze-drying is preferred. The drying time is not particularly limited; for natural drying (air-drying or sun-drying), it can be about 3 days, and for forced drying (heating with electricity, etc.), it can be about 1 to 3 days at around 50°C. It is generally preferable to dry the royal jelly so that the moisture content is 10% by mass or less, preferably 5% by mass or less. If it is difficult to reduce the moisture content to 10% by mass or less, as in the case of natural drying (air-drying or sun-drying), the royal jelly may be further reduced by freeze-drying. Alternatively, royal jelly powder may be obtained by grinding the royal jelly in a pulverizer (e.g., pin mill, hammer mill, ball mill, jet mill) after freeze-drying or spray-drying.

[0019] Royal jelly organic solvent extracts can be obtained, for example, by extracting raw royal jelly or royal jelly powder using an organic solvent such as ethanol, methanol, propanol, or acetone as a solvent. The extraction time can be appropriately set depending on the form of the raw royal jelly used as the raw material, the type and amount of solvent, the temperature and stirring conditions during extraction, etc. After extraction, solid matter may be removed by filtration, centrifugation, etc. The extracted solution may be used as is, or the solvent may be removed from the solution and used as a concentrated liquid or powder. Royal jelly ethanol extract is preferred as the royal jelly organic solvent extract.

[0020] Enzyme-hydrolyzed royal jelly can be obtained, for example, by treating raw royal jelly or royal jelly powder with a proteolytic enzyme. The proteolytic enzyme is preferably selected from the group consisting of enzymes having endopeptidase activity, enzymes having exopeptidase activity, and enzymes having both endopeptidase and exopeptidase activity. In particular, a peptidase that simultaneously possesses both endopeptidase and exopeptidase activity is preferred. Enzyme treatment using such a peptidase allows for the depolymerization of proteins in a single step, offering the advantages of simple operation and preventing the loss or significant reduction of the physiological activity of beneficial components contained in royal jelly.

[0021] The origin of the proteolytic enzyme is not particularly limited, and peptidases derived from animals, plants, and microorganisms (bacteria, viruses, fungi (molds, yeasts, mushrooms, etc.), algae, etc.) can be widely used.

[0022] Exopeptidases are classified into aminopeptidases and carboxypeptidases. Furthermore, peptidases are sometimes given the terms acidic, neutral, or alkaline depending on their optimal pH, and may be described as, for example, "acidic exopeptidase," "neutral aminopeptidase," and "alkaline endopeptidase."

[0023] Examples of 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.).

[0024] Examples of proteolytic enzymes that possess at least exopeptidase activity include carboxypeptidases, aminopeptidases, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus species, Rhizopus species, etc.), and pancreatin and pepsin, which also possess endopeptidase activity.

[0025] Preferred examples of enzymes possessing both exopeptidase and endopeptidase activity include Streptomyces griseus-produced peptidase (trade name: Actinase AS), Aspergillus oryzae-produced peptidase (trade names: Protease A, Flavorzyme, Proteax, Sumizyme LP-G), and Aspergillus melleus-produced peptidase (trade name: Protease P).

[0026] Furthermore, preferred examples of enzymes possessing exopeptidase activity include Aspergillus oryzae-produced peptidase (product names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), Aspergillus sojae-produced peptidase (product name: Sternzyme B15024), Aspergillus-produced peptidase (product name: Cokurase P), and Rhizopus oryzae-produced peptidase (product name: Peptidase R).

[0027] Furthermore, preferred examples of enzymes possessing endopeptidase activity include peptidase produced by Bacillus subtilis (trade names: Orientase 22BF, Nucleicin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutralase), and peptidase produced by Bacillus species (trade name: Protamex).

[0028] The reaction conditions (amount of proteolytic enzyme used, temperature, pH, reaction time, etc.) when treating raw royal jelly with proteolytic enzymes should be set appropriately according to the type of proteolytic enzyme used.

[0029] Commercially available royal jelly can be used. Specific examples of commercially available royal jelly include, for instance, Enzyme-Decomposed Royal Jelly King (manufactured by Yamada Bee Farm Co., Ltd.).

[0030] As mentioned above, 10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDA) are both fatty acids found in royal jelly. Furthermore, they are metabolized in the body to decendioic acid (2-DA) or sebacic acid (SA). Decendioic acid may also be 2-decendioic acid, and 10H2DA and 2-DA may be cis or trans. 10H2DA, 10HDA, DA, and SA may be synthetic products, or they may be naturally derived, such as from royal jelly. Royal jelly-derived 10H2DA and 10HDA can be obtained, for example, by extraction and purification from royal jelly using an organic solvent.

[0031] The salts of 10H2DA, 10HDA, 2-DA, or SA are not particularly limited, but examples include 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 salt, triethylamine salt, cyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, and procaine salt; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, and isoquinoline salt; tetramethylammonium salt, tetraethylammonium salt, and benzyl Examples include quaternary ammonium salts such as dimethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt; amino acid salts such as arginine salt, lysine salt, histidine salt, arginine salt, aspartate, and glutamate; inorganic salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, and perchlorate; organic salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, and ascorbate; and sulfonates such as methanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate.

[0032] The epidermal stem cell activator according to this embodiment contains at least one selected from the group consisting of royal jelly, 10-hydroxy-2-decenoic acid or its salt, 10-hydroxydecanoic acid or its salt, decendioic acid or its salt, and sebacic acid or its salt as an active ingredient, and therefore has the effect of activating epidermal stem cells. "Activating epidermal stem cells" means improving the proliferation of epidermal stem cells while maintaining their stem cell properties. Furthermore, "stem cell properties" means having the ability to self-replicate and having the ability to differentiate into multiple stem cells. In other words, the epidermal stem cell activator according to this embodiment can also be described as a composition for improving the proliferation of epidermal stem cells while maintaining their stem cell properties.

[0033] The stem cell nature of epidermal stem cells can be confirmed by the expression of undifferentiated markers, and their proliferative capacity can be confirmed by the expression of cell proliferation markers. Examples of undifferentiated markers include p63. Examples of cell proliferation markers include Ki67 and PCNA.

[0034] Activation of epidermal stem cells is sufficient if, for example, when an effective amount of the epidermal stem cell activator according to this embodiment is used on epidermal stem cells, the number of epidermal stem cells expressing undifferentiated markers and cell proliferation markers increases by 10% or more compared to when the epidermal stem cell activator is not used, and preferably increases by 20%, 30%, 50%, 70%, or 100% or more.

[0035] The epidermal stem cell activator according to this embodiment activates epidermal stem cells with the above-mentioned active ingredient, thereby having an effect of suppressing the decline in skin turnover, suppressing skin aging, and / or suppressing skin thinning.

[0036] Skin turnover refers to the turnover of the epidermis, which is important for maintaining homeostasis in the body. However, when epidermal turnover is disrupted due to aging or skin stress, it can cause skin aging symptoms such as thinning of the skin, pigmentation, dullness, uneven skin tone, and fine wrinkles. More specifically, epidermal turnover is maintained by the balance between the proliferation and differentiation of epidermal stem cells, as well as the proliferation of epidermal keratinocytes. Aging or skin stress is thought to induce aging of epidermal stem cells and epidermal keratinocytes, thereby reducing epidermal turnover. In other words, the epidermal stem cell activator according to this embodiment can be expected to suppress the decline in epidermal turnover by activating epidermal stem cells, thereby improving the above-mentioned skin aging symptoms. The effect of suppressing the decline in epidermal turnover can be evaluated, for example, by evaluating the number and proliferative capacity of epidermal stem cells in the basal layer of a three-dimensional skin model, as well as the resulting thickness of the epidermis, or by evaluating the cell proliferative capacity of epidermal keratinocytes in a planar culture system.

[0037] The skin thinning inhibitory effect is sufficient if, for example, when an effective amount of the epidermal stem cell activator according to this embodiment is used on epidermal stem cells, the thickness of the epidermis increases by 10% or more compared to when the epidermal stem cell activator is not used, and preferably increases by 20% or more, 30% or more, 50% or more, 70% or more, or 100% or more. The thickness of the epidermis is not particularly limited, but may be the thickness from the basal layer to the stratum corneum, or the thickness from the basal layer to the granular layer.

[0038] Skin aging refers to the aging of cells in the epidermis (epidermal stem cells and epidermal keratinocytes). Cellular senescence is a state in which cells have stably ceased cell proliferation, induced by factors such as aging, DNA damage, or stress. The characteristics of senescent cells are diverse, including morphological changes, metabolic changes, and changes in gene expression. In the epidermis, the aging of epidermal stem cells or epidermal keratinocytes is considered to be one of the factors that induce age-related skin phenotypes. In other words, the epidermal stem cell activator according to this embodiment is expected to suppress skin aging by activating epidermal stem cells. The inhibitory effect on skin aging can be evaluated using multiple indicators, such as cell doubling time in the epidermis, changes in the cell cycle, or SAβ-galactosidase activity, p16, and p21 gene expression, which specifically increase due to aging in epidermal cells, or telomere length or lamin expression, which decrease due to aging.

[0039] The amount of the active ingredient in the epidermal stem cell activator according to this embodiment may be 0.1% by mass or more and 100% by mass or less on a dry solid content basis relative to the total amount of the epidermal stem cell activator, and may be 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, 80% by mass or more, 85% by mass or more, 90% by mass or more, 93% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass. Furthermore, the content of the above-mentioned active ingredient in the epidermal stem cell activator according to this embodiment may be 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 93% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 60% by mass or less, 55% by mass or less, 50% by mass or less, 45% by mass or less, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 8% by mass or less, 5% by mass or less, 3% by mass or less, or 1% by mass or less, based on the dry solid content of the total amount of the epidermal stem cell activator.

[0040] In the epidermal stem cell activator according to this embodiment, if two or more are selected as active ingredients from the group consisting of royal jelly, 10-hydroxy-2-decenoic acid or its salt, 10-hydroxydecanoic acid or its salt, decendioic acid or its salt, and sebacic acid or its salt, the content of each component may be appropriately adjusted.

[0041] The epidermal stem cell activator of this embodiment can be used, for example, to experimentally activate epidermal stem cells, and can also be administered to humans for the activation of epidermal stem cells in the body. The epidermal stem cell activator of this embodiment may be administered orally or parenterally. Oral administration includes enteral administration, and parenteral administration includes topical administration, and especially includes transdermal administration.

[0042] The dosage of the epidermal stem cell activator of this embodiment when administered orally may vary depending on the active ingredient, the form of the composition, and the method and amount of application. For example, if the active ingredient is royal jelly, the daily dose for an adult weighing 60 kg should be 10 mg to 30,000 mg of royal jelly in terms of dry solid content, 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 of royal jelly.

[0043] Furthermore, if the active ingredient is 10-hydroxy-2-decenoic acid or a salt thereof, the dosage for an adult weighing 60 kg may be 1 mg to 200 mg of 10-hydroxy-2-decenoic acid or a salt thereof per day, preferably 10 mg to 100 mg of 10-hydroxy-2-decenoic acid or a salt thereof. If the active ingredient is 10-hydroxydecanoic acid or a salt thereof, the dosage for an adult weighing 60 kg may be 0.1 mg to 50 mg of 10-hydroxydecanoic acid or a salt thereof per day, preferably 1 mg to 20 mg of 10-hydroxydecanoic acid or a salt thereof. If the active ingredient is 10-decendioic acid or a salt thereof, the dosage for an adult weighing 60 kg may be 0.1 mg to 30 mg of 10-decendioic acid or a salt thereof per day, preferably 1 mg to 15 mg of 10-decendioic acid or a salt thereof. When the active ingredient is sebacic acid or a salt thereof, the dosage for an adult weighing 60 kg per day may be 0.1 mg to 20 mg of sebacic acid or a salt thereof, preferably 0.5 mg to 15 mg of sebacic acid or a salt thereof.

[0044] The dosage can be appropriately increased or decreased depending on factors such as the health condition of the person taking the drug, the method of administration, the type of active ingredient, and the combination with other agents. The epidermal stem cell activator according to this embodiment may be administered once a day, or divided into multiple doses, such as twice a day or three times a day, as long as the effective daily dose is within the range described above. The epidermal stem cell activator of this embodiment provides immediate effects after administration, but continuous administration for 1 to 4 weeks, 1 month or more, 6 months or more, or 1 year or more is preferable because it allows the effects to be sustained more effectively.

[0045] The dosage of the epidermal stem cell activator of this embodiment when administered parenterally may vary depending on the active ingredient, the site of application, and the scope of application. For example, if the active ingredient is royal jelly, the amount applied to the skin may be 0.01 mg to 50 mg of the active ingredient in terms of dry solid content, preferably 0.02 mg to 40 mg, 0.02 mg to 35 mg, or 0.025 mg to 30 mg of the active ingredient.

[0046] Furthermore, if the active ingredient is 10-hydroxy-2-decenoic acid or a salt thereof, the amount applied to the skin may be 0.001 mg to 0.3 mg of the active ingredient, preferably 0.01 mg to 0.1 mg of the active ingredient. If the active ingredient is 10-hydroxydecanoic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.08 mg of the active ingredient, preferably 0.001 mg to 0.06 mg of the active ingredient. If the active ingredient is decendioic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.05 mg of the active ingredient, preferably 0.001 mg to 0.04 mg of the active ingredient. If the active ingredient is sebacic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.03 mg of the active ingredient, preferably 0.001 mg to 0.02 mg of the active ingredient.

[0047] One embodiment of the present invention provides a composition containing the epidermal stem cell activator of the above embodiment as an active ingredient, the composition may be an oral composition or a topical composition, and in particular provides a cosmetic, food composition or pharmaceutical composition containing the epidermal stem cell activator of the above embodiment as an active ingredient. By applying the epidermal stem cell activator of this embodiment, or the cosmetic, food composition or pharmaceutical composition, to humans and allowing it to act on human epidermal stem cells, the epidermal stem cells are activated, thereby providing effects such as suppressing the decline in skin turnover, suppressing skin aging, and / or suppressing skin thinning. As a result, skin beautifying effects and anti-aging effects can be obtained.

[0048] The amount of the active ingredient in the cosmetic, food composition, or pharmaceutical composition containing the epidermal stem cell activator of this embodiment is not particularly limited, and any effective amount that can achieve the oral or parenteral dosage of the epidermal stem cell activator is acceptable.

[0049] The epidermal stem cell activator according to this embodiment, or the cosmetic, food composition, or pharmaceutical composition containing the epidermal stem cell activator, may further contain other components in addition to the above-mentioned active ingredient. Examples of other components include pharmaceutically acceptable components (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents), food-acceptable components (e.g., minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, fragrances, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, humectants), and cosmetic-acceptable components (e.g., whitening agents, moisturizers, antioxidants, oily components, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients).

[0050] The epidermal stem cell activator according to this embodiment, or a food composition or pharmaceutical composition containing an epidermal stem cell activator, may be in any form, such as solid, liquid, or paste, and may be in the form of tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including cases where the injection is prepared as a liquid by mixing it with distilled water or an infusion solution such as an amino acid solution or electrolyte solution at the time of use), topical formulations such as ointments or patches. These various formulations can be prepared, for example, by mixing the active ingredient with other ingredients as needed and molding them into the above-mentioned formulations.

[0051] The food composition is preferably one in which the tertiary function of the food (physical condition regulation function) is emphasized. Examples of foods in which the tertiary function of the food is emphasized include health foods, foods with functional claims, foods with nutritional function claims, nutritional supplements, supplements, and foods for specified health uses.

[0052] The form of the food composition is not particularly limited and may include, for example, beverages (soft drinks such as coffee, juice, and tea beverages, milk beverages, lactic acid bacteria beverages, yogurt beverages, carbonated beverages, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western-style confectionery (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, etc.); Japanese-style confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice pops, sherbet, etc.); food items (curry, beef bowl, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0053] The dosage forms of cosmetics may be, for example, solubilized, emulsified, powdered, oil-based, geled, ointmented, aerosol, water-oil two-layer system, water-oil-powder three-layer system, etc. Examples of cosmetics include basic cosmetics such as facial cleansers, lotions, emulsions, creams, gels, essences, serums, packs, masks, mists, and UV protection cosmetics; makeup cosmetics such as foundations, lipsticks, blushes, eyeshadows, eyeliners, and mascaras; facial cleansers, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, conditioners, hair treatments, hair styling products, hair tonics, hair mists, hair foams, hair liquids, hair gels, hair sprays, hair growth products, antiperspirants, bath additives, mouth rinses, oral cosmetics, toothpastes, hand creams, hand soaps, etc.

[0054] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples. [Examples]

[0055] (Experimental Example 1: Investigation of the ability of raw royal jelly and enzymatically hydrolyzed royal jelly to activate epidermal stem cells using a three-dimensional culture system) [1. Three-dimensional culture of human epidermal keratinocytes] Human epidermal keratinocytes (HPEKp, Juvenile Pooled, CERNTECH), including epidermal stem cells, were cultured in CnT-Prime and Epithelial Culture Medium (CERNTECH) at 37°C under 5% CO2 until subconfluence was reached. The cultured HPEKp were washed once with PBS and detached by treatment with Accutase (registered trademark, Innovative Cell Technologies). The detached HPEKp were suspended in CnT-Prime medium and collected by centrifugation (1,100 rpm, 3 min). The number of cells in the collected HPEKp was counted, suspended in CnT-Prime medium, and used for three-dimensional culture.

[0056] Place a three-dimensional culture insert (Millicell-PCF cell culture insert, filter pore size: 0.4 μm, filter diameter: 12 mm, manufactured by Merck Millipore) in a 60 mm culture dish, and fill each insert with 2 × 10⁶ cells. 5A cell suspension, adjusted to a cell / 400 μL ratio, was seeded inside the insert, and CnT-Prime medium was added to the outside of the insert for 3 days of culture. After 3 days of culture, the media inside and outside the insert were replaced with CnT-PR-3D medium (Cellntech), a three-dimensional culture medium. After another day, only the media inside the insert was removed, and the media outside the insert was replaced to initiate air-liquid-air culture. When replacing the media outside the insert, CnT-PR-3D medium supplemented with fresh royal jelly (NRJ, Yamada Bee Farm Co., Ltd.) or enzymatically hydrolyzed royal jelly (ERJ, Yamada Bee Farm Co., Ltd.) was used. Specifically, NRJ and ERJ were sterilized by passing them through a 0.45 μm filter (Millipore) and then a 0.2 μm filter (Millipore), and then added to CnT-PR-3D medium at a concentration of 100 or 400 μg / mL, and this medium was added to the outside of the insert. The culture medium was changed at 2, 5, 9, 11, and 13 days of culture, and the culture was continued in a medium supplemented with NRJ or ERJ until the end of the culture period.

[0057] [2. Cell fixation] The insert was recovered, and the three-dimensional cultured epidermis obtained in step 1 above was fixed in 4% paraformaldehyde (PFA) fixative, washed with PBS, immersed in 10% sucrose-containing PBS solution, and infiltrated using a shaker. Subsequently, the solution was replaced with 20% sucrose-containing PBS solution and further infiltrated. After that, the solution was replaced with fresh 20% sucrose-containing PBS solution and infiltrated overnight at 4°C. The membrane inside the insert was recovered and immersed in OCT compound (manufactured by Sakura Finetech Japan Co., Ltd.), then placed vertically in a Cryomold No. 1 embedding dish for frozen section preparation, solidified over liquid nitrogen, and embedded using liquid nitrogen.

[0058] [3. Preparation and staining of epidermal frozen sections, and acquisition of images] Using a cryostat (Leica), 10 μm thick epidermal frozen sections were prepared from the three-dimensional cultured epidermis fixed and embedded in step 2 above. These sections were mounted on glass slides and subjected to hematoxylin-eosin staining (HE staining) and immunohistochemistry.

[0059] For HE staining, 10 μm thick frozen sections were prepared, the OCT compound was washed off with tap water, and the nuclei were stained with Meyer hematoxylin stain for 5 minutes. Afterward, the sections were washed with running water for 10 minutes to allow color development. The cytoplasm was stained with 0.1% eosin Y stain for 2 minutes, then dehydrated stepwise with 70%, 80%, and 100% ethanol. Clearing was performed three times for 1 minute each in Remosol (Fujifilm Wako Pure Chemical Industries), and the sections were mounted with Enteran New (Merck Millipore). HE-stained epidermal frozen sections were observed using an all-in-one fluorescence microscope (Keyence), and representative images are shown in Figure 1. The thickness of the three-dimensional cultured epidermis (thickness from the basal layer to the granular layer) was also measured and is shown in Figure 2. 80-110 locations from the acquired images were randomly selected, measured using ImageJ software, and the average value was calculated.

[0060] Immunostaining was performed using antibodies against the three-dimensional cultured epidermal stem cell marker (undifferentiated marker) p63, the cell proliferation marker Ki67, and the epidermal progenitor cell marker Krt14. First, frozen epidermal sections on glass slides were blocked for 1 hour with PBSMT (2% skim milk, PBS containing 0.1% Triton-X100). Then, rabbit anti-Ki67 antibody (NB110-89719, Novus Biologicals), mouse anti-p63 antibody (ab735, abcam), and chicken anti-K14 antibody (906004, BioLegend), diluted 1:500, were added as primary antibodies, and the samples were incubated overnight at 4°C in PBST (PBS containing 0.1% Triton-X100). Next, the sections were washed with PBST, and the following secondary antibodies were added to PBST, each diluted 1:1000: anti-rabbit IgG (A21206, Life Technologies), AF647-labeled anti-mouse IgG (A31571, Life Technologies), and Cy3-labeled anti-chicken IgY (703-165-155, Jackson ImmunoResearch), and the mixture was reacted at room temperature for 1 hour. Further washing with PBST, the sections were mounted with coverslips using the Prolong Antifade Kit with DAPI (P36931, Invitrogen) as a stain-preventing mounting medium. The immunostained epidermal sections were observed using an LSM 980 confocal microscope (ZEISS), and representative images are shown in Figure 3. In Figure 3, "Merge" represents an image of the three fluorescence markers p63, Ki67, and Krt14 superimposed.

[0061] As shown in Figures 1 and 2, it was confirmed that the thickness of the three-dimensional cultured epidermis significantly increased when NRJ or ERJ was added compared to when no NRJ was added. Furthermore, the cells in the three-dimensional cultured epidermis cultured with ERJ and NRJ were densely packed, while the cells in the three-dimensional cultured epidermis without ERJ were only sparsely distributed.

[0062] As shown in Figure 3, in both cases of three-dimensional cultured epidermis cultured with NRJ or ERJ, an increase in p63 and Ki67-positive cells was confirmed compared to the case without the additive. The bright areas in the Merge are Krt14-positive areas, and in both cases of three-dimensional cultured epidermis cultured with NRJ or ERJ, an increase in Krt14-positive cells was confirmed compared to the case without the additive. Furthermore, in both cases of three-dimensional cultured epidermis cultured with NRJ or ERJ, an increase in cells expressing all three markers—p63, Ki67, and Krt14—was confirmed compared to the case without the additive.

[0063] The results above show that adding NRJ or ERJ to culture human epidermal keratinocytes, including epidermal stem cells, increased the thickness of the three-dimensional cultured epidermis and improved the proliferative capacity of the epidermal stem cells while maintaining their stem cell properties. These findings confirm that NRJ or ERJ can activate epidermal stem cells, thereby suppressing the decline in skin turnover and inhibiting skin thinning.

[0064] (Experimental Example 2: Investigation of the ability of fatty acids contained in royal jelly to activate epidermal stem cells using a three-dimensional culture system) During HPEKp culture, the procedure was carried out in the same manner as in Experimental Example 1, except that instead of NRJ or ERJ, 10-hydroxy-2-decenoic acid (10H2DA, Hangzhou Eastbiopharm), 10-hydroxydecanoic acid (10HDA, Combi-Blocks), decendioic acid (2-DA, Sundia MediTech), or sebacic acid (SA, Sigma-Aldrich), which are fatty acids contained in these royal jelly products, were added to the medium outside the insert to a concentration of 80 μM relative to the total volume of the medium. 10H2DA, 10HDA, 2-DA, and SA were dissolved in DMSO and then dissolved in the three-dimensional culture medium. The results of HE staining are shown in Figure 4, the thickness of the three-dimensional cultured epidermis in Figure 5, and the results of immunostaining in Figure 6.

[0065] As shown in Figures 4 and 5, it was confirmed that the thickness of the three-dimensional cultured epidermis significantly increased when 10H2DA, 10HDA, 2-DA, or SA was added to the culture compared to the case without the addition. Furthermore, the increase in the thickness of the three-dimensional cultured epidermis was greatest with 10H2DA, followed by 10HDA, 2-DA, and then SA, with the increase in thickness being almost the same for 10HDA and 2-DA.

[0066] As shown in Figure 6, similar to Experimental Example 1, in all cases of three-dimensional cultured epidermis cultured with or without 10H2DA, 10HDA, 2-DA, or SA, an increase in p63, Ki67, and Krt14-positive cells was confirmed compared to the case without any additives.

[0067] The above results show that adding 10H2DA, 10HDA, 2-DA, or SA to culture human epidermal keratinocytes, including epidermal stem cells, increased the thickness of the three-dimensional cultured epidermis and improved the proliferative capacity of the epidermal stem cells while maintaining their stem cell properties. These findings confirm that 10H2DA, 10HDA, 2-DA, or SA can activate epidermal stem cells, thereby suppressing the decline in skin turnover and inhibiting skin thinning.

[0068] (Experimental Example 3: Evaluation of anti-skin aging ability of raw royal jelly and enzymatically hydrolyzed royal jelly using a replication aging system) Similar to Example 1, human epidermal keratinocytes (HPEKp, Juvenile Pooled, Celntech), including epidermal stem cells, were cultured until subconfluence and then detached. The detached HPEKp was suspended in CnT-Prime medium and collected by centrifugation (1,100 rpm, 3 min). The collected HPEKp was divided into 5 × 10⁶ cells. 3 cells / cm 2The cells were prepared accordingly and seeded in a new petri dish containing CnT-Prime medium for cultivation. During cultivation, CnT-Prime medium supplemented with NRJ (manufactured by Yamada Bee Farm Co., Ltd.) or ERJ (manufactured by Yamada Bee Farm Co., Ltd.) was used. Specifically, NRJ and ERJ were passed through a 0.45 μm filter, then through a 0.2 μm filter for sterilization, and then added to CnT-Prime medium at a concentration of 100 or 10 μg / mL. Culturing was continued in medium supplemented with NRJ and ERJ until the end of the culture. Furthermore, the anti-skin aging ability of HPEKp was evaluated using a curve plotting the cumulative number of cell divisions (Population doubling level, PDL) on a time axis. That is, in each passage, the number of cells at the end of the culture was divided by the number of cells at the start of the culture, and the logarithm was taken to determine the number of divisions. The cumulative number of divisions (PDL) for each passage was then plotted on a time axis for evaluation. The evaluation of HPEKp began when it reached passage number 3. The results are shown in Figure 7.

[0069] As shown in Figure 7, when cultured without additives, the PDL of HPEKp stopped increasing after a certain number of days. This result indicates that the number of cell divisions decreases as cells age. On the other hand, in both cases where NRJ or ERJ was added to the culture, the PDL was higher compared to the unadded case, and the PDL continued to increase even after 30 days.

[0070] Based on these results and the results of Experimental Example 1, it was confirmed that NRJ or ERJ can activate epidermal stem cells, thereby suppressing skin aging.

Claims

[Claim 1] An epidermal stem cell activator for suppressing at least one of the following: decreased epidermal turnover, epidermal aging, and thinning of the epidermis, wherein the royal jelly is raw royal jelly, a concentrate or dilute of raw royal jelly, royal jelly powder, royal jelly ethanol extract, or enzyme-treated royal jelly which is a proteolytic enzyme-treated product of raw royal jelly or royal jelly powder.

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