Dermal stem cell differentiation promoter

Abstract

To discover a novel substance that promotes the differentiation of a dermal stem cell into a dermal fibroblast and provide such a substance as an agent for promoting the differentiation of a dermal stem cell.SOLUTION: An agent for promoting the differentiation of a dermal stem cell into a dermal fibroblast contains extract of a mixture of Hordeum vulgare L. in a germinated state or its extract and Prunus avium L. seeds as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for promoting the differentiation of dermal stem cells into dermal fibroblasts. [Background technology]

[0002] Skin is roughly divided into three layers: epidermis, dermis, and subcutaneous tissue. Dermal fibroblasts exist in the dermis layer, and collagen, elastin, and hyaluronic acid produced by dermal fibroblasts are known to be important components for maintaining firmness, elasticity, and moisture of the skin. In addition, since the cause of wrinkles and sagging is said to be a decrease in these components due to aging and inflammation, dermal fibroblasts can be said to be essential cells for maintaining youthful skin without wrinkles or sagging. Dermal stem cells that produce these dermal fibroblasts exist just below the papillary dermis layer, and they repeatedly proliferate and differentiate as necessary, constantly supplying new dermal fibroblasts to the dermis layer, resulting in constant regeneration of the skin (Non-Patent Document 1). Dermal components such as collagen and elastin are actively produced by dermal fibroblasts produced from dermal stem cells, so it is important to induce differentiation from dermal stem cells to mature dermal fibroblasts in order to maintain healthy dermal tissue.

[0003] In recent years, it has become clear that stem cells present in organs and tissues age (Non-Patent Document 2). Specifically, aging of stem cells refers to a decline in their proliferation and differentiation capabilities, which is thought to be the cause of the decline in the regenerative capabilities of organs and tissues. For example, it has been reported that the number of stem cells present in skin and subcutaneous fat tissue decreases with age, and their differentiation capabilities decline (Non-Patent Documents 3-4). Therefore, a technology that improves the differentiation capabilities of stem cells present in each organ or tissue is considered to be extremely effective for anti-aging (anti-aging) applications such as maintaining tissue homeostasis, repairing and regenerating damaged tissues, and preventing, treating, and improving various diseases. In addition, when considering the application of stem cells to regenerative medicine and regenerative cosmetics, it is important to develop a technology that efficiently induces stem cells to differentiate into cells of the organ or tissue to be regenerated. Furthermore, even if it is possible to induce differentiation into the desired cells, since organs and tissues are constructed three-dimensionally, if this cannot be reproduced, the effect (regeneration of organs and tissues) cannot be exerted even if transplanted into a living body. In other words, if we could develop a technology to culture stem cells outside the body, freely induce differentiation into target cells, and create a three-dimensional structure similar to the organ or tissue we want to regenerate, we could expect a dramatic advance in regenerative medicine and regenerative cosmetics. In particular, skin tissue has a complex three-dimensional structure and is the outermost layer of the human body, making it susceptible to damage from external injuries. In addition, skin tissue is closely related to a person's appearance and beauty, so it is extremely important to advance the regeneration technology for this tissue.

[0004] To date, substances that promote the production of collagen and hyaluronic acid in dermal fibroblasts include a mixture of yeast extract and beech plant extract (Patent Document 1), N-benzoylglycylglycine (Patent Document 2), and extract of seeds of Oriental genus plants (Patent Document 3). However, these do not act on dermal stem cells and promote the induction of differentiation from dermal stem cells to dermal fibroblasts, and in order to promote the production of collagen and hyaluronic acid in a longer-term and more fundamental manner, it is important to promote the differentiation of dermal stem cells and increase the number of dermal fibroblasts. To date, purple barley seed extract (Patent Document 4) has been reported as a substance that has the effect of inducing differentiation of dermal stem cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 11-158054 [Patent Document 2] JP 2014-55116 A [Patent Document 3] WO2012 / 057123 publication [Patent Document 4] JP 2010-22326 A [Non-patent literature]

[0006] [Non-Patent Document 1] Hasebe Y. et al., J. Dermatol. Sci., 2016, Vol. 89, pp. 205-207 [Non-Patent Document 2] Beane OS et al., PLoS One., 2014, Vol. 9, No. 12, e115963 [Non-Patent Document 3] Akamatsu H. et al., J. Dermatol., 2016, Vol. 43, pp. 311-313 [Non-Patent Document 4] Yamada T. et al., J. Dermatol. Sci., 2010, Vol. 58, pp. 36-42 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above circumstances, an object of the present invention is to find a new material that is highly effective in inducing differentiation of dermal stem cells into dermal fibroblasts, and to provide it as an agent for promoting differentiation of dermal stem cells. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above-mentioned problems, the inventors discovered that an extract of germinated purple barley seeds has a greater effect of inducing differentiation of dermal stem cells into dermal fibroblasts than an extract of ungerminated purple barley seeds, and that when used in combination with an extract of sweet cherry seeds, another plant seed, the effect of inducing differentiation of dermal stem cells into dermal fibroblasts is significantly promoted, thereby completing the present invention.

[0009] That is, the present invention includes the following inventions. (1) An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, comprising an extract of germinated purple barley seeds as an active ingredient. (2) A differentiation promoter for dermal stem cells into dermal fibroblasts, comprising an extract of a mixture of germinated purple barley seeds and sweet cherry seeds as an active ingredient. (3) A method for promoting differentiation of dermal stem cells into dermal fibroblasts, comprising the step of culturing dermal stem cells in a medium containing the agent according to (1) or (2). (4) A method for producing dermal fibroblasts, comprising the step of culturing dermal stem cells in a medium containing the agent according to (1) or (2). (5) A composition for promoting differentiation of dermal stem cells into dermal fibroblasts, comprising the agent according to (1) or (2). (6) An anti-aging cosmetic comprising the agent according to (1) or (2). Effect of the Invention

[0010] The differentiation promoter of dermal stem cells into dermal fibroblasts of the present invention can promote the differentiation of dermal stem cells and efficiently induce them into dermal fibroblasts, thereby providing a sufficient supply of components produced by dermal fibroblasts that maintain skin firmness, elasticity, and moisture, such as collagen, elastin, and hyaluronic acid, into the skin, and is effective in improving wrinkles, sagging, and loss of firmness and elasticity caused by aging, ultraviolet rays, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below. 1. Agent for promoting differentiation of dermal stem cells into dermal fibroblasts The differentiation promoter of dermal stem cells into dermal fibroblasts according to the present invention (hereinafter sometimes referred to as "dermal stem cell differentiation promoter") contains, as an active ingredient, an extract of germinated purple barley seeds, or an extract of a mixture of germinated purple barley seeds and sweet cherry seeds.

[0012] In the present invention, "dermal stem cells" refer to cells capable of differentiating into dermal fibroblasts. In the present invention, "promotion of differentiation of dermal stem cells into dermal fibroblasts" refers to activation of differentiation of dermal stem cells into dermal fibroblasts compared to before administration or ingestion of the agent of the present invention.

[0013] Purple barley used in the present invention refers to barley with purple glumes, caryopsis, stems or leaves, and its scientific name is Hordeum vulgare L. Examples of barley varieties include OUC321, CI158, and CI244.

[0014] The purple barley seeds used in the present invention can be obtained by cultivation, can be purchased commercially, or can be obtained from the growing area. Purple barley seeds are usually harvested in the summer.

[0015] In the present invention, germinated purple barley seeds are used as the extraction raw material. Here, "germination" is a term that includes rooting, and "germinated state" refers to a state in which the tip of the young shoot or young root emerges from the seed. In the present invention, "germinated purple barley seeds" include germinated purple barley seeds, malt (seeds obtained by cutting and drying the roots and shoots of germinated barley), germinated young plant bodies (sprouts), etc. Here, germinated seeds generally refer to those having roots, hypocotyls, cotyledons, and endosperm, and young plant bodies (sprouts) generally refer to seeds that have germinated and grown to a state in which they no longer have endosperm.

[0016] The appearance of the germinated purple barley seeds used as the extraction raw material in the present invention is preferably one in which roots extend from the purple barley seeds and new shoots appear, and the whole seed including the roots and new shoots can be used. As for the size of the germinated purple barley seeds, the length of the new shoots is preferably 0.1 mm to 80 mm, more preferably 1 to 50 mm. The length of the roots is preferably 1 mm to 200 mm, more preferably 10 mm to 100 mm.

[0017] The germinated purple barley seeds can be prepared by germinating the purple barley seeds and then drying them. The germination process is carried out by soaking fresh or dried purple barley seeds in water to break dormancy, sowing them in a seedbed, and cultivating them while providing water to prevent the seeds from drying out completely. The temperature of the soaking water is preferably 2 to 40° C., more preferably 10 to 30° C. The soaking time of the seeds is preferably 0.5 to 24 hours, more preferably 2 to 12 hours. The seeding rate is 0.1 to 5 seeds / cm of cultivation area. 2 Examples of the seedbed include nonwoven fabric, towel paper, sponge, vermiculite, perlite, and the like, other than soil. It is more preferable to cover the seedbed with water-absorbed towel paper to keep it moist, and then cover it with food wrap film. The cultivation temperature is preferably 10 to 30°C, and more preferably 20 to 25°C. The cultivation period varies depending on the temperature and light irradiation conditions, but is about 2 to 10 days.

[0018] The drying treatment can be a method commonly used for drying plants. Examples include natural drying (air drying), sun drying, dry heat drying, ventilation drying, hot air drying, spray drying, reduced pressure drying, vacuum drying, etc., and preferably dry heat drying, ventilation drying, and hot air drying. The temperature and time conditions of the drying treatment are not particularly limited, but the temperature is preferably 40 to 70°C, and the time varies depending on the drying temperature, the moisture content of the germinated purple barley seeds, and the total amount to be dried, but is generally in the range of 4 to 24 hours.

[0019] The sweet cherry used in the present invention is also called 'otou', and its scientific name is Prunus avium L. Major varieties include edible varieties such as American cherry, Sato-nishiki, and Napoleon. In the present invention, sweet cherry seeds are used as the extraction raw material, and the seeds refer to seeds that have not germinated (ungerminated seeds).

[0020] In the present invention, for the extraction of germinated purple barley seeds (hereinafter referred to as "germinated purple barley seeds") and sweet cherry seeds, the seeds or a plant containing the seeds may be used as is, or may be subjected to processing such as drying, crushing, shredding, etc. In addition, in the present invention, the extraction method for obtaining the above seed extract may be either a solvent extraction method using water or an organic solvent, or a squeeze extraction method, but the solvent extraction method is preferred.

[0021] The method of extraction using a solvent is not particularly limited, and can be performed by, for example, heating extraction, room temperature extraction, low temperature extraction, stirring extraction, or column extraction. Examples of the solvent used for extraction include water or hot water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water or hot water, lower alcohols, liquid polyhydric alcohols, etc. are preferred. These solvents may be used alone or in combination of two or more. In addition, a solvent having a pH adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0022] The amount of the extraction solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, relative to the extraction raw material (dry weight), but is preferably 100 times or less for convenience of operations such as concentration and isolation after extraction. The extraction temperature and time depend on the type of target plant and solvent used, and may be, for example, 10 to 100°C, preferably 30 to 90°C, and 30 minutes to 24 hours, preferably 1 to 10 hours.

[0023] The extract may be used as it is in the form of an extracted solution, but may be used after being subjected to treatment such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc., as long as the effect of the extract is not adversely affected. Furthermore, the extracted solution may be subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0024] The differentiation promoter of dermal stem cells according to the present invention may contain as an active ingredient the germinated purple barley seed extract obtained as described above, or may contain as an active ingredient an extract of a mixture of germinated purple barley seeds and sweet cherry seeds. The mixture ratio of germinated purple barley seeds and sweet cherry seeds is not limited.

[0025] An extract of germinated purple barley seeds, or an extract of a mixture of germinated purple barley seeds and sweet cherry seeds (hereinafter referred to as "plant seed extract") has the effect of promoting the differentiation of dermal stem cells both at the biological level (in vivo) and at the culture level (in vitro), and therefore the dermal stem cell differentiation promoter of the present invention can be used as a drug for promoting the differentiation of dermal stem cells by administering it to mammals, including humans, and can also be used as a stem cell culture medium additive, research reagent, or medical reagent for promoting the differentiation of dermal stem cells and producing dermal fibroblasts.

[0026] The dermal stem cell differentiation promoter according to the present invention is effective in treating, improving, and preventing diseases or pathologies caused by the failure of dermal fibroblasts to be formed normally due to a decline or failure in the differentiation function of dermal stem cells, since the active ingredient, the plant seed extract, has the effect of promoting the differentiation of dermal stem cells into dermal fibroblasts. Examples of such diseases or pathologies include, but are not limited to, wrinkles, sagging skin, nasolabial folds (nasolabial folds), marionette lines, loss of firmness and elasticity, lack of moisture and luster, stiffness, dullness, solar elastosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, linear atrophy of the skin (skin striae), wounds, burns, bedsores, scars, birthmarks, and liver spots.

[0027] The content of the plant seed extract in the differentiation promoter for dermal stem cells according to the present invention is not particularly limited, but depending on the nature of the extract (liquid extract, concentrate, or dry product), it is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight, relative to the total amount of the agent.

[0028] 2. Method for promoting differentiation of dermal stem cells and method for producing dermal fibroblasts The present invention also relates to a method for promoting differentiation of dermal stem cells, comprising the step of culturing dermal stem cells in a medium containing the above-mentioned agent for promoting differentiation of dermal stem cells, and a method for producing dermal fibroblasts, comprising the step of culturing dermal stem cells in a medium containing the above-mentioned agent for promoting differentiation of dermal stem cells. Dermal fibroblasts produced by inducing differentiation from dermal stem cells in the method according to the present invention can generally be cultured outside the body and then transplanted by direct injection or the like into a wound or a site where tissue is to be regenerated. In other words, dermal fibroblasts produced by the method according to the present invention can be used as a transplant material (cell transplant agent).

[0029] In the method of the present invention, the medium for culturing dermal stem cells and the additives used simultaneously are not particularly limited, and a medium and additives that are generally used for differentiating dermal stem cells into dermal fibroblasts may be used.

[0030] Specifically, media for culturing dermal stem cells include basal media containing components necessary for stem cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, and fatty acids), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), and Hank's balanced salt solution. In addition, in order to increase the cell proliferation rate, the above-mentioned medium may be supplemented with growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc., as well as antibiotics (penicillin, streptomycin, etc.). Each component of the medium is sterilized by an appropriate method before use. Commercially available media in which the above-mentioned components are appropriately added to the basal medium may also be used.

[0031] In addition to the above, it is preferable that serum (e.g., 10% FBS) is contained at a content of 1 to 20%. However, since the components of serum vary depending on the lot, and the effects vary, it is preferable to check the lot before use.

[0032] As a medium for inducing differentiation of dermal stem cells into dermal fibroblasts, a commercially available product can be used, such as Fibroblast Medium (manufactured by Sanko Junyaku Co., Ltd.) and Medium for Normal Human Fibroblasts (manufactured by DS Pharma Biomedical Co., Ltd.).

[0033] According to the above-mentioned dermal stem cell differentiation promoter according to the present invention or the method according to the present invention, the above-mentioned plant seed extract can be provided as a reagent kit for promoting differentiation of dermal stem cells, either alone or separately or mixed with a medium. The kit can include an instruction manual, etc., as necessary. Alternatively, the above-mentioned plant seed extract can be mixed with a medium and provided as a medium for promoting differentiation of dermal stem cells.

[0034] The culture vessel used for culturing dermal stem cells is not particularly limited as long as it is capable of culturing dermal stem cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. The culture vessel may be either cell non-adhesive or cell adhesive, and is appropriately selected depending on the purpose. The cell adhesive culture vessel may be treated with a cell support substrate such as an extracellular matrix in order to improve adhesion to cells. Examples of cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, etc.

[0035] The concentration of the plant seed extract added to the medium used for stem cell culture can be appropriately determined based on the content of the plant seed extract in the differentiation promoter of dermal stem cells according to the present invention described above, and may be, for example, a concentration of 10 to 10,000 μg / mL, preferably 100 to 5,000 μg / mL, calculated as the dry matter of the plant seed extract. Furthermore, these extracts may be added to the medium periodically during the stem cell culture period.

[0036] The culture conditions for stem cells may be the same as those normally used for culturing stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably 36 to 37°C. The CO2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. The medium is preferably replaced once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately changed and set within a range in which stem cells can survive and grow.

[0037] In vitro determination of whether differentiation of dermal stem cells into dermal fibroblasts has been promoted can be performed by a method commonly used by those skilled in the art, and can be evaluated by whether the expression level of dermal fibroblast marker genes is significantly higher at the mRNA or protein level in stem cells cultured in the presence of the differentiation promoter of dermal stem cells according to the present invention than in stem cells cultured in the absence of the differentiation promoter of dermal stem cells according to the present invention. Examples of dermal fibroblast marker genes include, but are not limited to, COL1A1 (type I collagen α1), COL1A2 (type I collagen α2), COL3A1 (type III collagen α1), S100A4 (S100 calcium binding protein), αSMA (alpha-smooth muscle actin), HAS1 (hyaluronic acid synthase-1), ELN (elastin), HYAL3 (hyaluronidase 3), and galectin 9.

[0038] 3. Composition for promoting differentiation of dermal stem cells into dermal fibroblasts When the differentiation promoter of dermal stem cells according to the present invention is administered to a living body, it can be administered as it is, but it can also be provided by being incorporated into various compositions such as cosmetics, quasi-drugs, medicines, food and beverages, etc. together with appropriate additives within a range that does not impair the effects of the present invention. In particular, it is preferable to provide it by being incorporated into a composition for external use on the skin.

[0039] In addition, it is particularly preferable that the skin topical composition is an anti-aging cosmetic. Here, "anti-aging" refers to suppressing the decline in the function and activity of skin cells due to various internal or external factors such as aging and photoaging, as well as dryness, ultraviolet rays, excessive skin washing, hormonal imbalance, stress, and lifestyle habits (sleep and smoking habits), leading to a youthful and healthy skin condition with even texture, firmness, and luster. Specifically, it refers to preventing or improving wrinkles, sagging, dullness, loss of firmness, hardening, etc. of the skin by suppressing the decline in the differentiation ability of dermal stem cells.

[0040] When the differentiation promoter of dermal stem cells according to the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of an aqueous solution, a solubilized solution, an emulsion, a powder, a powder dispersion, an oil solution, a gel, an ointment, an aerosol, a water-oil two-layer system, or a water-oil-powder three-layer system. In addition, the cosmetics or quasi-drugs can be produced by appropriately blending various ingredients, additives, bases, etc. that are normally used in skin external compositions together with the differentiation promoter of dermal stem cells according to the type of the composition, in accordance with a method known in the art. The form may be any of a liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients included in the skin topical composition include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, palmitic acid, glyceryl stearate ... These include: isopropyl myristate, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0041] Examples of types of cosmetics and quasi-drugs include lotions, milky lotions, gels, beauty serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, bath additives, body lotions, body shampoos, hair shampoos, hair conditioners, and hair growth agents.

[0042] When the differentiation promoter of dermal stem cells according to the present invention is incorporated into a pharmaceutical product, it can be mixed with pharmacologically and pharmacy-acceptable additives and formulated into various preparations in a formulation form suitable for application to the affected area. As pharmacologically and pharmacy-acceptable additives, a formulation base material or carrier, excipient, diluent, binder, lubricant, coating agent, disintegrant or disintegration aid, stabilizer, preservative, preservative, bulking agent, dispersant, wetting agent, buffer, solubilizer or solubilization aid, isotonicity agent, pH regulator, propellant, colorant, sweetener, flavoring agent, flavoring agent, flavoring agent, etc., appropriately selected according to the dosage form and application, may be added appropriately to prepare various formulation forms that can be administered orally or parenterally, systemically or locally, by various known methods. When the pharmaceutical product of the present invention is provided in each of the above forms, it can be manufactured by a method commonly used by those skilled in the art, for example, the method shown in each article of the General Provisions for Preparations [2] of the Japanese Pharmacopoeia.

[0043] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs, and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drips, suppositories, ointments, lotions, sprays, transdermal absorption agents, transmucosal absorption agents, and patches. In addition, the pharmaceutical may be a dried product that is redissolved when used, and in the case of an injection preparation, it is provided in the form of a unit-dose ampule or a multi-dose container.

[0044] When the differentiation promoter of dermal stem cells according to the present invention is used as a pharmaceutical for treating, improving, and preventing the above-mentioned skin diseases and pathological conditions, the form suitable for use is an external preparation, for example, an ointment, cream, gel, liquid, patch (poultice, plaster), foam, spray, and aerosol. An ointment refers to a homogeneous semi-solid external preparation, and includes an oleaginous ointment, an emulsion ointment, and a water-soluble ointment. A gel refers to an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. A liquid refers to a liquid external preparation, and includes a lotion, a suspension, an emulsion, a liniment, and the like.

[0045] For oral administration formulations, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, starch, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used, but are not limited to these.

[0046] Preparations for parenteral administration may contain, but are not limited to, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum water, vegetable oil, etc.; isotonicity agents such as glucose, sodium chloride, D-mannitol, etc.; pH adjusters such as inorganic acids, organic acids, inorganic bases, or organic bases, etc.

[0047] The pharmaceutical of the present invention functions as a preventive drug for suppressing the onset of the above-mentioned skin diseases and / or as a therapeutic drug for improving the condition to a normal state. Since the active ingredient of the pharmaceutical of the present invention is derived from a natural product, it is very safe and has no side effects, and therefore, when used as a pharmaceutical for treating, improving, and preventing the above-mentioned diseases, it can be orally or parenterally administered in a wide range of dosages to mammals such as humans, mice, rats, rabbits, dogs, and cats.

[0048] The content of the dermal stem cell differentiation promoter in the cosmetic, pharmaceutical, or quasi-drug of the present invention is not particularly limited, but is preferably 0.001 to 30 wt %, more preferably 0.01 to 10 wt %, calculated as the dry matter of the above plant seed extract relative to the total weight of the formulation (composition). The above amounts are merely examples, and may be appropriately set or adjusted in consideration of the type and form of the composition, the general amount used, efficacy, and effects. In addition, the method of adding the active ingredient in the formulation may be added in advance or during production, and may be appropriately selected in consideration of workability.

[0049] The dosage of the pharmaceutical of the present invention can be appropriately determined depending on the type of disease, the age, sex, weight, severity of symptoms, etc. For example, when orally administered to an adult, the daily dosage is 0.1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 300 mg of the plant seed extract.

[0050] The differentiation promoter of dermal stem cells of the present invention can also be incorporated into food and drink. In the present invention, the term "food and drink" refers to general food and drink, as well as foods that can be taken for the purpose of maintaining or promoting health other than pharmaceuticals, such as health foods, functional foods, health functional foods, or special purpose foods. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements, etc. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act, and include specific health foods and nutritional functional foods that can display specific health effects, nutritional component functions, disease risk reduction, etc., as well as functional labeling foods that can display the contents of the scientific evidence-based functionality notified to the Commissioner of the Consumer Affairs Agency. Special purpose foods also include foods for sick people, foods for the elderly, foods for infants, foods for pregnant women, etc. that display that they are suitable for specific subjects or patients with specific diseases. The differentiation promoter of dermal stem cells of the present invention can be suitably used in the above-mentioned health foods, etc., in that it can be taken continuously on a daily basis, especially when it is necessary to take it for a long period of time to improve and prevent various symptoms such as wrinkles, sagging, firmness, and loss of elasticity associated with aging. Here, indications of specific health benefits or nutritional functions etc. attached to foods and beverages may be made on display items such as product containers, packaging, instructions, package inserts, product flyers and pamphlets, and product advertisements in newspapers, magazines etc.

[0051] Furthermore, when the food and drink of the present invention is used for mammals other than humans, it can be used in the sense including pet food and feed.

[0052] The form of the food or drink may be any form suitable for consumption, such as solid, liquid, granule, powder, capsule, cream, or paste. In particular, in the case of the above-mentioned health foods, etc., the form is preferably, for example, tablet, round, capsule, powder, granule, fine granule, lozenge, liquid (including syrup, milk, and suspension).

[0053] Types of food and beverages include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock foods, oils and fats and processed oils and fats, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated concentrates and powders for adjusting such beverages.

[0054] The food and drink of the present invention may be appropriately mixed with additives that are usually used according to the type of food and drink. Any additives that are acceptable under the Food Sanitation Act can be used as additives, and examples of such additives include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and preservatives.

[0055] When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding a plant seed extract in the normal production process of the food or drink. In addition, in the case of health foods, it is sufficient to follow the production method of the above-mentioned pharmaceutical products. For example, in the case of tablet-type supplements, additives such as excipients are added to the plant seed extract, mixed, and molded under pressure with a tablet press or the like. In the case of capsule-type supplements, it can be produced by filling a liquid, suspension, paste, powder, or granular food composition containing the plant seed extract into a capsule, or by encapsulating and molding with a capsule base. In addition, other materials (for example, minerals such as iron and potassium, vitamins such as vitamin C, vitamin B2, vitamin B6, folic acid, dietary fiber, etc.) can also be added as necessary.

[0056] The amount of plant seed extract to be incorporated in the food or beverage of the present invention may be any amount that is sufficient to exert the effect of promoting differentiation of dermal stem cells, and may be appropriately determined taking into consideration the general intake amount of the target food or beverage, the form of the food or beverage, efficacy and effects, taste, preference, cost, etc.

[0057] When the food or drink of the present invention is ingested for the purpose of preventing or improving the above-mentioned disease or pathology, the intake amount varies depending on the condition of the subject to be ingested, the intake form, the intake amount, etc., but is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg of plant seed extract per day for an adult. The above amount may be ingested in one sitting, or may be ingested in several sittings (2 to 4 sittings). The food or drink of the present invention is preferably packaged or filled in a container such as a bag or bottle in an amount to be ingested in one sitting as a guide for the intake amount. EXAMPLES

[0058] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, % in the examples refers to % by weight.

[0059] [Example 1] (1) Preparation of germinated purple barley seeds Germinated purple barley seeds used as the extraction raw material were prepared as follows: 200 g of purple barley seeds (dried) were soaked in water for 6 hours, sown on paper towels moistened with water, covered with paper towels and food wrap, and kept moist at room temperature for 4 days to germinate, and then dry-dried at 60°C for 5 hours to obtain 194 g of germinated purple barley seeds.

[0060] (2) Extract manufacturing example In the production of the following extracts, when the extraction raw material was germinated purple barley seeds, the germinated purple barley seeds prepared in (1) were used, and when the sweet cherry seeds were used, ungerminated seeds were used.

[0061] (Production Example 1) Production of hot water extract of germinated purple barley seeds 200 mL of purified water was added to 10 g of germinated purple barley seeds, and the mixture was extracted at 95 to 100°C for 2 hours. The mixture was then filtered, and the filtrate was concentrated and freeze-dried to obtain 0.98 g of a hot water extract of germinated purple barley seeds.

[0062] (Production Example 2) Production of 50% ethanol extract of germinated purple barley seeds 10 g of germinated purple barley seeds were added to 200 mL of 50% ethanol aqueous solution and soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.00 g of 50% ethanol extract of germinated purple barley seeds.

[0063] (Production Example 3) Production of 100% ethanol extract of germinated purple barley seeds 10 g of germinated purple barley seeds were added to 200 mL of ethanol and soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.24 g of 100% ethanol extract of germinated purple barley seeds.

[0064] (Production Example 4) Production of hot water extract of sweet cherry seeds 200 mL of purified water was added to 10 g of sweet cherry seeds, and the mixture was extracted at 95 to 100° C. for 2 hours. The mixture was then filtered, and the filtrate was concentrated and freeze-dried to obtain 1.33 g of a hot water extract of sweet cherry seeds.

[0065] (Production Example 5) Production of 50% ethanol extract of sweet cherry seeds 200 mL of 50% ethanol aqueous solution was added to 10 g of sweet cherry seeds, and the seeds were soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.50 g of 50% ethanol extract of sweet cherry seeds.

[0066] (Production Example 6) Production of 100% ethanol extract of sweet cherry seeds 10 g of sweet cherry seeds were added with 200 mL of ethanol and soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.29 g of 100% ethanol extract of sweet cherry seeds.

[0067] (Production Example 7) Production of a hot water extract of an equal mixture of germinated purple barley seeds and sweet cherry seeds 200 mL of purified water was added to 10 g of an equal mixture of germinated purple barley seeds and sweet cherry seeds, and the mixture was extracted at 95 to 100°C for 2 hours. The mixture was then filtered, and the filtrate was concentrated and freeze-dried to obtain 1.77 g of a hot water extract of the mixture of germinated purple barley seeds and sweet cherry seeds.

[0068] (Production Example 8) Preparation of a 50% ethanol extract of an equal mixture of germinated purple barley seeds and sweet cherry seeds 10 g of an equal mixture of germinated purple barley seeds and sweet cherry seeds was added with 200 mL of 50% ethanol solution and soaked at room temperature for 7 days for extraction. The resulting extract was filtered, concentrated, and freeze-dried to obtain 1.21 g of 50% ethanol extract of the mixture of germinated purple barley seeds and sweet cherry seeds.

[0069] (Production Example 9) Production of 100% ethanol extract of an equal mixture of germinated purple barley seeds and sweet cherry seeds 10g of an equal mixture of germinated purple barley seeds and sweet cherry seeds was added with 200mL of ethanol and soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.52g of 100% ethanol extract of the mixture of germinated purple barley seeds and sweet cherry seeds.

[0070] (Comparative Production Example 1) Production of hot water extract of ungerminated purple barley seeds 200 mL of purified water was added to 10 g of ungerminated purple barley seeds, and the mixture was extracted at 95 to 100°C for 2 hours. The mixture was then filtered, and the filtrate was concentrated and freeze-dried to obtain 0.90 g of a hot water extract of ungerminated purple barley seeds.

[0071] (Comparative Production Example 2) Production of 50% ethanol extract of ungerminated purple barley seeds 200mL of 50% ethanol was added to 10g of ungerminated purple barley seeds, and the seeds were soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.12g of 50% ethanol extract of ungerminated purple barley seeds.

[0072] (Comparative Production Example 3) Production of 100% ethanol extract of ungerminated purple barley seeds 200 mL of ethanol was added to 10 g of ungerminated purple barley seeds, and the seeds were soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.33 g of 100% ethanol extract of ungerminated purple barley seeds.

[0073] (Comparative Production Example 4) Production of a hot water extract of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds 200 mL of purified water was added to 10 g of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds, and the mixture was extracted at 95 to 100°C for 2 hours. The mixture was then filtered, and the filtrate was concentrated and freeze-dried to obtain 2.21 g of a hot water extract of the mixture of ungerminated purple barley seeds and sweet cherry seeds.

[0074] (Comparative Production Example 5) Production of a 50% ethanol extract of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds 10 g of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds was added with 200 mL of 50% ethanol solution and soaked at room temperature for 7 days for extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.11 g of 50% ethanol extract of the mixture of ungerminated purple barley seeds and sweet cherry seeds.

[0075] (Comparative Production Example 6) Production of a 100% ethanol extract of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds 200mL of ethanol was added to 10g of an equal mixture of ungerminated purple barley seeds and sweet cherry seeds, and the mixture was soaked at room temperature for 7 days to perform extraction. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.64g of a 100% ethanol extract of the mixture of ungerminated purple barley seeds and sweet cherry seeds.

[0076] [Example 2] Evaluation of differentiation-promoting effect of dermal stem cells Experiments to evaluate the differentiation-promoting effect of the extracts produced in Example 1 (Production Examples 1 to 9, Comparative Production Examples 1 to 6) on dermal stem cells were carried out as follows.

[0077] Commercially available human dermal fibroblasts (manufactured by Toyobo Co., Ltd.) were used as dermis-derived cells, and dermal stem cells were isolated using NGFR (nerve growth factor receptor: Genbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-093383 A. Dermal stem cells maintained in α-MEM medium (manufactured by Thermo Co., Ltd.) containing 15% FBS were isolated at a concentration of 2 × 10 4 The cells were seeded in a 24-well plate (Falcon) so that each cell was 100 μg / mL. After 24 hours of culture, the cells were attached, and each extract was added to a final concentration of 100 μg / mL, and cultured for 72 hours. After the culture was completed, the cells were fixed with 4% PFA / PBS, permeabilized with 0.2% Triron-X-100 (Wako) / PBS, and then treated with anti-S100A4 antibody, anti-COL1A1 antibody, anti-αSMA antibody (all Novus) as marker protein antibodies for dermal fibroblasts, and fluorescently labeled secondary antibodies, and immunostained. In addition, the nuclei of each cell were labeled by DAPI staining. Then, the expression level of each protein per cell (fluorescence intensity / cell number) was calculated using a fluorescent microscope and image analysis software (ImageJ). For comparison, the expression levels of each protein in cells cultured in a culture medium without the extract were used as a control, and the expression levels of each protein in cells cultured in a culture medium containing 10 ng / mL TGFβ1 (Pepro Tech), which is generally used as a differentiation inducer from dermal stem cells to dermal fibroblasts, were used as a positive control. The expression levels of marker proteins (S100A4, COL1A1, αSMA) in the absence of the test substance (control) were set to 100%, and the relative expression levels (%) of the same proteins (S100A4, COL1A1, αSMA) in cells cultured with the extract were calculated and evaluated. The test results are shown in Table 1 below.

[0078] [Table 1]

[0079] As shown in Table 1, the germinated purple barley seed extract (Production Examples 1-3) had a higher dermal stem cell differentiation promoting effect than TGFβ1, which is commonly used as a differentiation inducer from dermal stem cells to dermal fibroblasts, and the ungerminated purple barley seed extract (Comparative Production Examples 1-3). Furthermore, a remarkable synergistic effect was observed in the dermal stem cell differentiation promoting effect in the extract of a combination of germinated purple barley seeds and sweet cherry seeds (Production Examples 7-9). It should be noted that the sweet cherry seed extract alone (Production Examples 4-6) did not have a higher effect than the differentiation promoting effect of TGFβ1, and when the combination was with ungerminated purple barley seeds, the differentiation promoting effect was improved compared to that of the single extract, but no remarkable synergistic effect was obtained (Comparative Production Examples 4-6). [Industrial Applicability]

[0080] The differentiation promoter of dermal stem cells into dermal fibroblasts of the present invention can efficiently induce differentiation of dermal stem cells into dermal fibroblasts in vivo or ex vivo. Therefore, the present invention can be used in the fields of manufacturing cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and pathologies caused by functional decline or insufficiency of dermal stem cells, and in the field of manufacturing transplant materials.

Claims

1. An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, comprising as an active ingredient a hot water extract, an aqueous ethanol extract, or an ethanol extract of germinated purple barley seeds.

2. A differentiation promoter for dermal stem cells into dermal fibroblasts, comprising as an active ingredient a hot water extract, an aqueous ethanol extract, or an ethanol extract of a mixture of germinated purple barley seeds and sweet cherry seeds.

3. A method for promoting differentiation of dermal stem cells into dermal fibroblasts, comprising the step of culturing dermal stem cells in a medium containing the agent according to claim 1 or 2.

4. A method for producing dermal fibroblasts, comprising a step of culturing dermal stem cells in a medium containing the agent according to claim 1 or 2.

5. A composition for promoting differentiation of dermal stem cells into dermal fibroblasts, comprising the agent according to claim 2.

6. An anti-aging cosmetic comprising the agent according to claim 2.

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