Agent for promoting differentiation of skin stem cells

Abstract

To discover a novel naturally derived material with high safety that acts on skin stem cells to promote their differentiation, thereby simply and efficiently regenerating skin cells and provide the discovered material as a differentiation promoter for skin stem cells.SOLUTION: The present invention provides an agent for promoting the differentiation of skin stem cells into skin cells, containing Zanthoxylum extract as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] The skin has a three-layer structure, broadly divided into the epidermis, dermis, and subcutaneous tissue. The epidermis, which is the outermost layer of the skin, is mainly composed of keratinocytes. The epidermis is composed of multiple layers (basal layer, spinous layer, granular layer, and stratum corneum) of keratinocytes at different stages of maturation. Keratinocytes divide in the basal layer, which is the lowest layer of the epidermis, and as they mature, they migrate to the upper layers, keratinize, and eventually fall off (keratinization or turnover). Keratinocyte stem cells exist in the basal layer, and they repeatedly proliferate and differentiate as needed, constantly supplying new cells to the epidermis, and as a result, the skin is constantly regenerating. Therefore, in order to normalize turnover and exert a stronger epidermal barrier function, it is important to induce stem cells to differentiate into the stratum corneum, the outermost layer.

[0003] On the other hand, 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. The dermal stem cells that produce these dermal fibroblasts are present just below the papillary dermis, 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 fibroblasts produced from dermal stem cells, so it is important to induce differentiation from dermal stem cells to mature fibroblasts in order to maintain healthy dermal tissue.

[0004] In recent years, it has become clear that stem cells present in organs and tissues age (Non-Patent Document 2). Stem cell aging refers to a decline in proliferation and differentiation capabilities, and damage from ultraviolet rays and oxidative stress is thought to be the cause. Therefore, technology that maintains and 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 essential to develop substances and technologies that efficiently control the differentiation of stem cells into target cells. In particular, skin tissue has a complex three-dimensional structure and is located in the outermost layer of the human body, making it susceptible to damage from external injuries. In addition, it is a tissue that is greatly related to a person's appearance and beauty, and it is extremely important to advance the regeneration technology of this tissue.

[0005] To date, factors that have been reported to promote the differentiation of epidermal stem cells into keratinocytes include extract from seeds of Acanthus erecta (Patent Document 1), and extract from seeds of purple barley (Patent Document 2) as factors that promote the differentiation of dermal stem cells into dermal fibroblasts. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2010-22327 A [Patent Document 2] JP 2010-22326 A [Non-patent literature]

[0007] [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 Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above circumstances, the object of the present invention is to discover a new, highly safe, naturally-derived material that can act on skin stem cells to promote their differentiation and regenerate skin cells simply and efficiently, and to provide it as a differentiation promoter for skin stem cells. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the inventors have found that an extract of Japanese pepper has the effect of promoting the differentiation of epidermal stem cells into epidermal keratinocytes, the effect of promoting the differentiation of dermal stem cells into dermal fibroblasts, and the effect of recovering the inhibition of differentiation of epidermal stem cells and dermal stem cells caused by oxidative stress, thereby completing the present invention.

[0010] That is, the present invention includes the following. (1) An agent for promoting the differentiation of skin stem cells into skin cells, containing pepper extract as an active ingredient. (2) The differentiation promoter described in (1), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (3) The differentiation promoter described in (1), wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts. (4) A composition for skin regeneration comprising the agent according to any one of (1) to (3). (5) The composition for skin regeneration described in (4), wherein the composition is a cosmetic, a quasi-drug, a medicine, or a food or beverage. (6) A method for promoting differentiation of skin stem cells into skin cells, comprising a step of culturing skin stem cells in a medium containing an extract of Japanese pepper. (7) The method according to (6), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (8) The method according to (6), wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts. Effect of the Invention

[0011] The differentiation promoter of skin stem cells of the present invention can promote the differentiation of epidermal stem cells and dermal stem cells, and can efficiently induce them into epidermal keratinocytes and dermal fibroblasts, respectively. Epidermal keratinocytes are involved in the barrier function and moisture retention function of the skin, and also provide sufficient supplies of collagen, elastin, hyaluronic acid, and other components that maintain the firmness, elasticity, and moisture of the skin produced by dermal fibroblasts, into the skin. Therefore, the differentiation promoter of skin stem cells of the present invention is effective in treating, improving, and preventing various skin symptoms (skin diseases such as atopic dermatitis and dry skin, reduced barrier function and turnover, age spots, wrinkles, sagging, reduced firmness and elasticity, etc.) caused by dryness, ultraviolet rays, aging, oxidative stress, etc., and can greatly contribute to the fields of regenerative medicine, regenerative cosmetics, and anti-aging. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. 1. Skin stem cell differentiation promoter The agent for promoting differentiation of skin stem cells into skin cells according to the present invention (hereinafter, sometimes referred to as "agent for promoting differentiation of skin stem cells") contains an extract of Japanese pepper as an active ingredient.

[0013] In the present invention, "skin stem cells" refer to epidermal stem cells present in the epidermis and / or dermal stem cells present in the dermis. "Epidermal stem cells" refer to cells capable of differentiating into epidermal keratinocytes, and "dermal stem cells" refer to cells capable of differentiating into dermal fibroblasts. Thus, "skin cells" in the present invention include epidermal keratinocytes and / or dermal fibroblasts. In the present invention, the origin of the skin stem cells is not limited, and the effect can be exerted on skin stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs.

[0014] The Japanese pepper (Zanthoxylum piperitum) used in the present invention is a deciduous shrub of the genus Zanthoxylum in the family Rutaceae, and grows wild in large quantities in Japan. It is also the source plant of the herbal medicine "Zanthoxylum" (Japanese name: Sansho). Examples of Japanese pepper that can be used in the present invention include Zanthoxylum piperitum (L.) DC.f.inerme Makino), Zanthoxylum piperitum (L.) DC forma inerme (Makino) Makino), Zanthoxylum schinifolium, and Zanthoxylum piperitum (L.) DC forma brevispinosum Makino), which are all members of the genus Zanthoxylum in the family Rutaceae. Among these, Zanthoxylum piperitum (L.) DC forma brevispinosum Makino is preferred.

[0015] In the present invention, the extract of Japanese pepper refers to an extract of a part of the plant body such as the flower, fruit, peel, stem, leaf, branch, root, seed, etc., or the whole plant body, or a mixture thereof, but the parts used as the extraction raw material in the present invention are preferably the peel, fruit, or seed. The peel (powdered Japanese pepper) that is commercially available as a seasoning can be used.

[0016] The extraction method for obtaining an extract from Japanese pepper is not particularly limited, and may be, for example, a heated extraction method, or a room temperature or cold extraction method. The extraction solvent may be water or hot water, or a mixture of water and an organic solvent. As the organic solvent, 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.), etc. may be used, but water-soluble organic solvents such as ethanol, methanol, acetone, n-propanol, t-butanol, propylene glycol, and 1,3-butylene glycol are preferred, and one or more of these may be used in combination. Among them, a water-ethanol mixed polar solvent (30 to 70 v / v % aqueous ethanol solution) and water are more preferable, and a 50 v / v % aqueous ethanol solution is even more preferable. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0017] The amount of the 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 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. More specifically, for example, a Japanese pepper extract can be obtained by adding water to the Japanese pepper peel and performing hot water extraction at 95 to 100°C. Alternatively, a Japanese pepper extract can be obtained by adding a lower alcohol (e.g., ethanol, etc.) to the Japanese pepper peel and performing extraction at room temperature (e.g., 5 to 35°C).

[0018] The extract may be used as it is in the form of an extracted solution, but if necessary, it may be used after being subjected to treatments such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc., within the scope that does not affect the effect of the extract. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0019] The thus obtained Zanthoxylum extract has the effect of promoting the differentiation of epidermal stem cells into epidermal keratinocytes and the effect of promoting the differentiation of dermal stem cells into dermal fibroblasts at the living body level (in vivo) or at the culture level (ex vivo), and can therefore be used as an active ingredient of a differentiation promoter for skin stem cells. The differentiation promoter for skin stem cells of the present invention can be incorporated or applied to medicines, quasi-drugs, cosmetics, etc. as a drug for promoting the differentiation of epidermal stem cells or dermal stem cells by administering it to mammals, including humans. Furthermore, the differentiation promoter for skin stem cells of the present invention can be used in vitro as an additive for stem cell culture medium, a research reagent, or a medical reagent for promoting the differentiation of epidermal stem cells or dermal stem cells and producing epidermal keratinocytes or dermal fibroblasts.

[0020] The skin stem cell differentiation promoter of the present invention contains an extract of Japanese pepper as an active ingredient, which has the effect of promoting the differentiation of epidermal stem cells and dermal stem cells, and is therefore effective in treating, improving, and preventing diseases or pathologies caused by the failure of epidermal keratinocytes or dermal fibroblasts to be formed normally due to a decrease or failure in the differentiation ability of epidermal stem cells or dermal stem cells. Examples of diseases or pathologies caused by the failure of epidermal keratinocytes to be formed normally due to a decrease or failure in the differentiation ability of epidermal stem cells include atopic dermatitis, psoriasis (accompanied by erythema, scales, and desquamation), delayed healing of burns and wounds, rough skin, dry skin, sensitive skin, keratinocyte hyperplasia, melasma, age spots, dullness, and open pores. In addition, examples of diseases or pathological conditions caused by the failure to form normal dermal fibroblasts due to a decrease or failure in the differentiation ability of dermal stem cells include wrinkles, sagging, 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, etc. In addition, the cause of the decrease or failure in the differentiation ability of epidermal stem cells or dermal stem cells is not important, but examples include oxidative stress, which is the excessive production of active oxygen due to various internal or external factors such as aging, ultraviolet rays, air pollution, physical stimuli, diseases, drugs, lifestyle habits (sleep and smoking habits), excessive exercise, and mental and physical stress.

[0021] The content of Japanese pepper extract in the skin stem cell differentiation promoter of the present invention varies depending on the nature of the extract (liquid extract, concentrate, or dried product) and is not particularly limited; however, for example, it is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight, calculated as the dry product, relative to the total amount of the drug.

[0022] 2. Composition for skin regeneration When the differentiation promoter of skin stem cells of the present invention is used in a living body, it can be used as it is, but it can also be blended with appropriate additives within the scope of the effect of the present invention in various compositions such as cosmetics, medicines, quasi-drugs, food and beverages, etc., and provided as a composition for skin regeneration. In particular, it is preferable to provide it by blending it in a composition for external use on the skin.

[0023] 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. The cosmetics or quasi-drugs may 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 their types, in accordance with methods 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.

[0024] 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.

[0025] When the differentiation promoter of skin 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, antiseptic, 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 manufacturing method commonly used by those skilled in the art, for example, a manufacturing method shown in each article of the General Provisions for Preparations [2] of the Japanese Pharmacopoeia.

[0026] 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.

[0027] When the differentiation promoter of skin stem cells according to the present invention is used as a pharmaceutical for treating, improving, and preventing the above-mentioned skin diseases and pathologies, the form suitable for use is an external preparation, for example, an ointment, cream, gel, liquid, patch (poultice, plaster), foam, spray, aerosol, etc. 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, etc.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 pepper extract.

[0032] The content of the differentiation promoter of skin stem cells 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-mentioned Zanthoxylum 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 other factors. 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.

[0033] The differentiation promoter of skin stem cells of the present invention can also be added to 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 improving health other than medicines, 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, and supplements. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act, and include foods for specified health uses and foods with nutritional functions that can display specific health effects, functions of nutritional components, reduction of disease risks, etc., as well as functional 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., which display that they are suitable for specific subjects or patients with specific diseases. The differentiation promoter of skin stem cells of the present invention can be suitably used in the above-mentioned health foods, etc., because it can be taken continuously on a daily basis, particularly when long-term administration is required for the improvement and prevention of various symptoms associated with a decrease in skin barrier function, such as atopic dermatitis, dry skin, rough skin, wrinkles, sagging skin, and loss of firmness and elasticity associated with aging. Here, the indication of specific health benefits and nutritional component functions, etc., attached to foods and beverages can be displayed on product containers, packaging, instructions, package inserts, and other labels, product flyers and pamphlets, and product advertisements in newspapers, magazines, etc.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] 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.

[0038] When the food and drink of the present invention is a general food and drink, it can be produced by including a process of adding an extract of Japanese pepper in the normal production process of the food and drink. In addition, in the case of health foods, it is sufficient to follow the production method of the above-mentioned medicines. For example, in the case of tablet-type supplements, it can be produced by adding additives such as excipients to the extract of Japanese pepper, mixing, and molding by applying pressure with a tableting machine 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 extract of Japanese pepper 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.

[0039] The amount of pepper extract to be incorporated in the food and beverage of the present invention may be any amount that is sufficient to exert the effect of promoting the differentiation of skin stem cells, and may be appropriately determined taking into consideration the general intake amount of the target food and beverage, the form of the food and beverage, efficacy and effects, taste, preference, cost, etc.

[0040] 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 pepper 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.

[0041] 3. Method for promoting differentiation of skin stem cells The present invention also relates to a method for promoting differentiation of skin stem cells into skin cells, comprising a step of culturing skin stem cells in a medium containing an extract of Japanese pepper. The method includes a method for promoting differentiation of epidermal stem cells into epidermal keratinocytes and a method for promoting differentiation of dermal stem cells into dermal fibroblasts. In the method according to the present invention, epidermal keratinocytes produced by inducing differentiation from epidermal stem cells and dermal fibroblasts produced by inducing differentiation from dermal stem cells 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. That is, the epidermal keratinocytes and dermal fibroblasts produced by the method according to the present invention can be used as transplantation materials (cell transplant agents).

[0042] In the method of promoting differentiation of skin stem cells of the present invention, the medium for culturing skin stem cells and the additives used at the same time are not particularly limited, and any medium and additives commonly used for the proliferation of skin stem cells (epidermal stem cells or dermal stem cells) may be used.

[0043] Specifically, the medium for culturing skin stem cells is a basic medium containing components necessary for the survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, 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), Hank's balanced salt solution, etc., to which at least one differentiation induction or promotion factor according to the cells to be differentiated is added. Examples of differentiation induction factors from epidermal stem cells to epidermal keratinocytes include calcium salts, lysophosphatidic acid (LPA), etc. Examples of differentiation induction factors from dermal stem cells to dermal fibroblasts include TGFβ, etc. Furthermore, in order to increase the proliferation rate of stem cells, the above-mentioned medium may contain, as necessary, 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 supplements, N2 supplements, ITS supplements, etc., and may also contain antibiotics (penicillin, streptomycin, etc.). Each component of the medium is sterilized by an appropriate method before use.

[0044] 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.

[0045] A commercially available medium may be used for culturing skin stem cells. Examples of commercially available medium include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, Toyobo's MF medium, and Sigma's Hank's balanced salt solution. In addition, a differentiation induction medium containing a differentiation induction factor for a target cell is commercially available, and these commercially available mediums may be used. For example, an example of a medium for inducing differentiation of epidermal cells is CnT-Prime 3D Barrier Culture Medium (CELLn TEC). Examples of a medium for inducing differentiation of dermal cells are Fibroblast Medium (Sanko Junyaku), and a medium for normal human fibroblasts (DS Pharma Biomedical).

[0046] According to the above-mentioned differentiation promoter of skin stem cells according to the present invention or the method of the present invention, the extract of Japanese pepper can be provided as a reagent kit for promoting differentiation of skin stem cells, either alone or separately or mixed with a medium. The kit can include an instruction manual, etc., as necessary. Alternatively, the extract of Japanese pepper can be mixed with a medium and provided as a medium for promoting differentiation of skin stem cells.

[0047] The culture vessel used for culturing skin stem cells is not particularly limited as long as it is capable of culturing 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.

[0048] The concentration of Zanthoxylum zerumbet extract added to the medium used for culturing skin stem cells can be appropriately determined according to the content of Zanthoxylum zerumbet extract in the differentiation promoter of skin stem cells according to the present invention described above, and may be, for example, 10 to 10,000 μg / mL, preferably 100 to 5,000 μg / mL, calculated as the dry matter of the extract. Furthermore, Zanthoxylum zerumbet extract may be periodically added to the medium during the culturing period of stem cells.

[0049] The culture conditions for skin 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 about 36 to 37°C. The CO2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. The medium is preferably changed once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately changed within a range in which the stem cells can survive and grow.

[0050] The differentiation of epidermal stem cells into epidermal keratinocytes can be confirmed, for example, by determining whether the expression level of epidermal keratinocyte marker genes at the mRNA level or protein level is significantly increased in cells cultured in the presence of Zanthoxylum japonicum extract compared to the expression level at the start of culture in cells cultured in the absence of Zanthoxylum japonicum extract. Similarly, the differentiation of dermal stem cells into dermal fibroblasts can be confirmed, for example, by determining whether the expression level of dermal fibroblast marker genes at the mRNA level or protein level is significantly increased in cells cultured in the presence of Zanthoxylum japonicum extract compared to the expression level at the start of culture in cells cultured in the presence of Zanthoxylum japonicum extract compared to the expression level at the start of culture in cells cultured in the absence of Zanthoxylum japonicum extract. Examples of epidermal keratinocyte marker genes include, but are not limited to, FLG (filaggrin), IVL (involucrin), KRT10 (keratin 10), LOR (loricrin), OCLN (occludin), CLDN (claudin), etc. In addition, 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), FAP (fibroblast activation protein), HAS1 (hyaluronic acid synthase-1), ELN (elastin), HYAL3 (hyaluronidase 3), and galectin 9. At the mRNA level, for example, a method of confirmation by RT-PCR, quantitative PCR, or northern blotting using primers or probes specific to each of the above marker genes can be mentioned. At the protein level, for example, an immunological method such as ELISA, flow cytometry, or western blotting using an antibody specific to the protein encoded by each of the above marker genes can be mentioned.

[0051] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these.

[0052] [Example 1] Production example of pepper extract An extract of Zanthoxylum nigra was prepared as follows. (Production Example 1) Production of hot water extract of grape pepper seeds 200 mL of water was added to 10 g of dried grape pepper seeds, and extraction was carried out for 2 hours at 95 to 100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.3 g of a hot water extract of grape pepper seeds.

[0053] (Production Example 2) Production of 50% ethanol extract of grape pepper seeds 10 g of dried grape pepper seeds were soaked in 200 mL of 50% (v / v) ethanol aqueous solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of 50% ethanol extract of grape pepper seeds.

[0054] (Production Example 3) Production of ethanol extract of grape pepper seeds 10 g of dried grape pepper seeds were soaked in 200 mL of ethanol solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.6 g of ethanol extract of grape pepper seeds.

[0055] (Production Example 4) Production of hot water extract of grape pepper peel 200 mL of water was added to 10 g of dried grape pepper skin, and extraction was carried out for 2 hours at 95 to 100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.3 g of a hot water extract of grape pepper skin.

[0056] (Production Example 5) Production of 50% ethanol extract of grape pepper peel 10 g of dried grape pepper peel was immersed in 200 mL of 50% (v / v) ethanol aqueous solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of 50% ethanol extract of grape pepper peel.

[0057] (Production Example 6) Production of ethanol extract of grape pepper peel 10 g of dried grape pepper peel was immersed in 200 mL of ethanol solution at room temperature for 4 days. The obtained extract was filtered and then concentrated to dryness using an evaporator to obtain 0.5 g of ethanol extract of grape pepper peel.

[0058] (Production Example 7) Production of hot water extract of grape pepper fruit 200 mL of water was added to 10 g of dried grape pepper fruit (including the skin and seeds) and extracted for 2 hours at 95-100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.5 g of a hot water extract of grape pepper fruit.

[0059] (Production Example 8) Production of 50% ethanol extract of grape pepper fruit 10 g of dried grape pepper fruit (including skin and seeds) was immersed in 200 mL of 50% (v / v) ethanol solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of 50% ethanol extract of grape pepper fruit.

[0060] (Production Example 9) Production of ethanol extract of grape pepper fruit 10 g of dried grape pepper fruit (including skin and seeds) was immersed in 200 mL of ethanol solution at room temperature for 4 days. The obtained extract was filtered and then concentrated to dryness using an evaporator to obtain 0.5 g of ethanol extract of grape pepper fruit.

[0061] [Example 2] (Experimental Example 1) Evaluation of the differentiation-promoting effect of Japanese pepper extract on epidermal stem cells Commercially available normal human adult epidermal keratinocytes (Kurabo) were used as epidermis-derived cells, and epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Gebank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-055721 A. The above-mentioned epidermal stem cells were maintained in Humedia-KG2 medium (Kurabo) until the cell number reached 5 × 105 The cells were seeded on a 12-well plate (Falcon) so that each well was 100 μg / mL. After 24 hours of culture, the cells were allowed to attach, and then the test substances (Zanthoxylum chinense extracts of Production Examples 1 to 9) were added to a final concentration of 50 μg / mL, and the cells were allowed to attach for 72 hours. In addition, after the cells were attached, the test substances were not added, and 1.5 mM CaCl2 was added, and the cells were cultured for 72 hours to induce differentiation, and used as a positive control. After the culture was completed, mRNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction was performed on this mRNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of FLG (filaggrin), IVL (involucrin), and KRT10 (keratin 10) were analyzed. After initial denaturation at 95°C for 2 minutes, PCR was performed using the following primer set, with 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Other procedures were performed according to the prescribed method.

[0062] (Flag primer set) 5'-TCGAAGGAGCCAAAAATATAAAACAG-3' (SEQ ID NO: 1) 5'-GAATTCCAATAGAAGGATAATAGAGAAAGATG-3' (SEQ ID NO: 2)

[0063] (Primer set for IVL) 5'-CCATCAGGAGCCAAATGAAACAG-3' (SEQ ID NO: 3) 5'-GCTCGACAGGCACCTTCTG-3' (SEQ ID NO: 4)

[0064] (Primer set for KRT10) 5'-ACTGAAGAGCTGGCCTATCTGAA-3' (SEQ ID NO:5) 5'-CATCACCAGTGGACACATTTCG-3' (SEQ ID NO: 6)

[0065] (Primer set for GAPDH (internal standard)) 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 7) 5'-TCTTCTGGGTGGCAGTGATG-3' (SEQ ID NO: 8)

[0066] The expression of each gene, FLG, IVL, and KRT10, was evaluated by calculating the expression levels of FLG, IVL, and KRT10 in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH, which is an internal standard, and setting the FLG gene expression level / GAPDH gene expression level, IVL gene expression level / GAPDH gene expression level, and KRT10 gene expression level / GAPDH gene expression level as 100, and calculating the relative expression levels of the FLG, IVL, and KRT10 genes in cells cultured with the addition of the test substance. The test results are shown in Table 1 below.

[0067] [Table 1]

[0068] As shown in Table 1, it was confirmed that the extract of Japanese pepper has the effect of promoting the differentiation of epidermal stem cells even without the differentiation inducer (CaCl2). In particular, the differentiation promoting effect was high when the 50% ethanol extract of Japanese pepper peel (Production Example 5) was used.

[0069] (Experimental Example 2) Evaluation of the differentiation-promoting effect of Japanese pepper extract on dermal stem cells 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: Gebank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-093383 A. The above dermal stem cells were maintained in 10% FBS-containing DMEM medium (manufactured by Nacalai Co., Ltd.) until the cell count reached 2 × 10 4The cells were seeded on a 12-well plate (Falcon) so that each well was 10 μg / mL. After 24 hours of culture, the cells were attached, and then the test substance (Zanthoxylum chinense extract of Production Examples 1 to 9) was added to a final concentration of 50 μg / mL, and the cells were cultured for 48 hours. In addition, after the cells were attached, the test substance was not added, and TGFβ, which is generally used as a differentiation inducer for fibroblasts, was added at 10 ng / mL (Pepro Tech), and the cells were cultured for 48 hours to induce differentiation, and used as a positive control. After the culture was completed, mRNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction was performed on this mRNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of COL1A1 (Collagen Type I Alpha 1 Chain) and FAP (Fibroblast Activation Protein Alpha) were analyzed. After initial denaturation at 95°C for 2 minutes, PCR was performed using the following primer set, with 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Other procedures were performed according to the prescribed method.

[0070] (Primer set for COL1A1) 5'-GCTACCCAACTTGCCTTCATG-3' (SEQ ID NO: 9) 5'-TTCTTGCAGTGGTAGGTGATGTTC-3' (SEQ ID NO: 10)

[0071] (Fast Amplification Primer Set) 5'-CTAATTCAAGTGTATGGTGGTCCC-3' (SEQ ID NO: 11) 5'-CCAGTGATGAAACGTATCCTCC-3' (SEQ ID NO: 12)

[0072] (Primer set for GAPDH (internal standard)) 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 7) 5'-TCTTCTGGGTGGCAGTGATG-3' (SEQ ID NO: 8)

[0073] The expression of the COL1A1 and FAP genes was evaluated by calculating the expression levels of COL1A1 and FAP in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH, which is an internal standard, as COL1A1 gene expression level / GAPDH gene expression level and FAP gene expression level / GAPDH gene expression level, which were set at 100, and calculating the relative gene expression levels of COL1A1 and FAP in cells cultured with the addition of the test substance. The test results are shown in Table 2 below.

[0074] [Table 2]

[0075] As shown in Table 2, it was confirmed that the extract of Japanese pepper has the effect of promoting the differentiation of dermal stem cells even without a differentiation-inducing factor (TGFβ). In particular, the differentiation-promoting effect was high when a 50% ethanol extract of Japanese pepper peel (Production Example 5) was used.

[0076] (Experimental Example 3) Evaluation of the effect of grape pepper extract on epidermal stem cell differentiation disorder caused by hydrogen peroxide It is known that oxidative stress accumulates in skin tissue due to the effects of aging and ultraviolet rays. The effects of reactive oxygen on differentiation induction of epidermal stem cells were analyzed, and the effectiveness of applying an extract of Japanese pepper was evaluated. Commercially available normal human adult epidermal keratinocytes (manufactured by Kurabo Industries, Ltd.) were used as epidermis-derived cells, and epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Gebank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-055721 A. The above-mentioned epidermal stem cells were maintained in Humedia-KG2 medium (manufactured by Kurabo Industries, Ltd.) until the cell number reached 5 × 10 5The cells were seeded on a 12-well plate (Falcon) so that each well was 100 μg / mL. After 24 hours of culture, the cells were attached, and then 1.5 mM CaCl2 was added to induce differentiation. Hydrogen peroxide was added as oxidative stress at 100 mM, and the test substance (Zanthoxylum chinense extract of Production Examples 1 to 9) was added to a final concentration of 50 μg / mL, and the cells were cultured for 72 hours. After the cells were attached, the cells were cultured for 72 hours without adding hydrogen peroxide or the test substance, and used as a positive control. After the culture was completed, mRNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction was performed on this mRNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of FLG (Filaggrin), IVL (Involucrin), and KRT10 (Keratin10) were analyzed in the same manner as in Experimental Example 1.

[0077] The expression of each gene, FLG, IVL, and KRT10, was evaluated by calculating the expression levels of FLG, IVL, and KRT10 in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH, which is an internal standard, and setting the FLG gene expression level / GAPDH gene expression level, IVL gene expression level / GAPDH gene expression level, and KRT10 gene expression level / GAPDH gene expression level as 100, and calculating the relative expression levels of the FLG, IVL, and KRT10 genes in cells cultured with / without the addition of the test substance. The test results are shown in Table 3 below.

[0078] [Table 3]

[0079] As shown in Table 3, differentiation of epidermal stem cells was inhibited by oxidative stress (hydrogen peroxide) (differentiation induction / hydrogen peroxide / no extract added), but when Japanese pepper extract (Production Examples 1-9) was added at the same time, it was confirmed that the inhibition of differentiation of epidermal stem cells caused by oxidative stress was improved. In particular, the use of a 50% ethanol extract of Japanese pepper peel (Production Example 5) was more effective.

[0080] (Experimental Example 4) Evaluation of the effect of grape pepper extract on hydrogen peroxide-induced dermal stem cell differentiation disorder It is known that oxidative stress accumulates in skin tissue due to the effects of aging and ultraviolet rays. The effects of reactive oxygen on differentiation induction of dermal stem cells were analyzed, and the effectiveness of applying an extract of Japanese pepper was evaluated. 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: Gebank 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 10% FBS-containing DMEM medium (manufactured by Nacalai Co., Ltd.) were isolated at 2 × 10 4The cells were seeded on a 12-well plate (Falcon) so that each well was 1000 cells. After 24 hours of culture, TGFβ (Pepro Tech), which is generally used as a differentiation inducer for fibroblasts, was added at 10 ng / mL to induce differentiation, and hydrogen peroxide was added as an oxidative stress at 50 mM, and the test substance (Zanthoxylum chinense extract of Production Examples 1 to 9) was added at a final concentration of 50 μg / mL, and the cells were cultured for 48 hours. After the cells were engrafted, cells were cultured for 48 hours without adding hydrogen peroxide or the test substance as a positive control. After the culture was completed, RNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction was performed on this RNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of COL1A1 and FAP were analyzed in the same manner as in Experimental Example 2.

[0081] The expression of the COL1A1 and FAP genes was evaluated by calculating the expression levels of COL1A1 and FAP in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH, which is an internal standard, and setting the COL1A1 gene expression level / GAPDH gene expression level and the FAP gene expression level / GAPDH gene expression level as 100, and calculating the relative gene expression levels of COL1A1 and FAP in cells cultured with / without the addition of the test substance (with differentiation induction).These test results are shown in Table 4 below.

[0082] [Table 4]

[0083] As shown in Table 4, dermal stem cell differentiation was inhibited by oxidative stress (hydrogen peroxide) (differentiation induction / hydrogen peroxide / no extract added), but it was confirmed that the simultaneous addition of Japanese pepper extract (Production Examples 1-9) improved the inhibition of dermal stem cell differentiation caused by oxidative stress. In particular, the use of a 50% ethanol extract of Japanese pepper peel (Production Example 5) was more effective.

[0084] (Experimental Example 5) Evaluation of the effect of grape pepper extract on epidermal stem cell differentiation disorder caused by stretching stimulation Skin tissue is stretched by facial expressions, speech, etc., and it is known that stretching stimuli cause oxidative stress. The effect of stretching stimuli on differentiation induction of epidermal stem cells was analyzed, and the effectiveness of applying pepper extract was evaluated. Commercially available normal human adult epidermal keratinocytes (Kurabo) were used as epidermis-derived cells, and epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an indicator according to the method described in JP 2017-055721 A. The above-mentioned epidermal stem cells maintained in Humedia-KG2 medium (Kurabo) were placed in collagen gel (Nitta Gelatin) at 1 × 10 6The cells were seeded on a stretch chamber (STREX) at 1000 cells / mL, and Humedia-KG2 medium was added and cultured for 24 hours. After 24 hours, 1.5 mM CaCl2 was added to induce differentiation, and the test substance (Zanthoxylum chinense extract of Production Examples 1 to 9) was added to a final concentration of 50 μg / mL, and cultured for 24 hours while stretching the gel at a stretching ratio of 1.4 times the length using a stretching device (STREX). In addition, cells cultured for 24 hours without adding the test substance and without stretching were used as a positive control. After the end of the culture, RNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction using this RNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of FLG, IVL, and KRT10 were analyzed in the same manner as in Example 1.

[0085] The expression of each gene of FLG, IVL, and KRT10 was evaluated by calculating the expression levels of FLG, IVL, and KRT10 in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH as an internal standard, where FLG gene expression level / GAPDH gene expression level, IVL gene expression level / GAPDH gene expression level, and KRT10 gene expression level / GAPDH gene expression level were set to 100, and the relative expression levels of FLG, IVL, and KRT10 genes in cells cultured with / without the addition of the test substance (with differentiation induction) were calculated. The test results are shown in Table 5 below.

[0086] [Table 5]

[0087] As shown in Table 5, differentiation of epidermal stem cells was inhibited by stretch stimulation (differentiation induction / stretch stimulation / no extract added), but it was confirmed that the inhibition of differentiation of epidermal stem cells by stretch stimulation was improved when Japanese pepper extract (Production Examples 1 to 9) was added at the same time. In particular, the use of 50% ethanol extract of Japanese pepper peel (Production Example 5) was more effective.

[0088] (Experimental Example 6) Evaluation of the effect of grape pepper extract on dermal stem cell differentiation disorder caused by stretching stimulation Skin tissue is stretched by facial expressions, speech, etc., and it is known that stretching stimuli can cause oxidative stress. The effect of stretching stimuli on differentiation induction of dermal stem cells was analyzed, and the effectiveness of applying pepper extract was evaluated. 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: Geanbank 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 10% FBS-containing DMEM medium (manufactured by Nacalai Co., Ltd.) were placed in collagen gel (manufactured by Nitta Gelatin Co., Ltd.) at 1 × 10 6The cells were seeded on a stretch chamber (STREX) at 1000 cells / mL, and 10% FBS-containing DMEM medium was added and cultured for 24 hours. After 24 hours, TGFβ, which is generally used as a differentiation inducer for fibroblasts, was added at 10 ng / mL (Pepro Tech) to induce differentiation, and the test substance (Zanthoxylum chinense extract of Production Examples 1 to 9) was added to a final concentration of 50 μg / mL, and the gel was stretched at a stretching ratio of 1.4 times the length of the gel using a stretching device (STREX) and cultured for 24 hours. In addition, cells cultured for 24 hours without adding the test substance and without stretching were used as a positive control. After the culture was completed, RNA was isolated and extracted using RNAIso+ (Takara Bio). After cDNA synthesis by reverse transcription reaction was performed on this RNA using High Capacity RNA to cDNA Kit (Thermo), PCR reaction was performed using SYBR Select Master Mix (Thermo), and the gene expression levels of COL1A1 and FAP were analyzed in the same manner as in Experimental Example 2.

[0089] The expression of the COL1A1 and FAP genes was evaluated by calculating the expression levels of COL1A1 and FAP in cells (control) cultured without the addition of the test substance (without differentiation induction) as a ratio to the expression level of GAPDH, which is an internal standard, as COL1A1 gene expression level / GAPDH gene expression level and FAP gene expression level / GAPDH gene expression level, which were set at 100, and calculating the relative gene expression levels of COL1A1 and FAP in cells cultured with / without the addition of the test substance (with differentiation induction).These test results are shown in Table 6 below.

[0090] [Table 6]

[0091] As shown in Table 6, the differentiation of dermal stem cells was inhibited by stretch stimulation (differentiation induction / stretch stimulation / no extract added), but it was confirmed that the inhibition of differentiation of dermal stem cells by stretch stimulation was improved when Japanese pepper extract (Production Examples 1 to 9) was added at the same time. In particular, the use of 50% ethanol extract of Japanese pepper peel (Production Example 5) was more effective. [Industrial Applicability]

[0092] The differentiation promoter of skin stem cells of the present invention can promote the differentiation of skin stem cells in vivo or ex vivo, and thus the present invention can be used in the fields of cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and pathologies caused by impaired or insufficient function of epidermal stem cells or dermal stem cells, and in the fields of transplantation materials for regenerative medicine and regenerative beauty.

Claims

1. An agent that promotes the differentiation of skin stem cells into skin cells, containing pepper extract as an active ingredient.

2. The differentiation promoter according to claim 1 , wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

3. The differentiation promoter according to claim 1 , wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts.

4. A composition for skin regeneration comprising the agent according to any one of claims 1 to 3.

5. The composition for skin regeneration according to claim 4 , wherein the composition is a cosmetic, a quasi-drug, a medicine, or a food or beverage.

6. A method for promoting differentiation of skin stem cells into skin cells, comprising a step of culturing skin stem cells in a medium containing an extract of Japanese pepper.

7. The method of claim 6 , wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

8. The method of claim 6 , wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts.

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