Differentiation promoter for skin stem cells

Abstract

To discover a highly safe novel material of natural origin that acts on skin stem cells to promote their differentiation and achieves regeneration of skin cells in a convenient and efficient manner, and to provide the material as a differentiation promoter for skin stem cells.SOLUTION: The present invention provides a differentiation promoter for skin stem cells into skin cells, comprising an extract of Althaea officinalis 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 Art

[0002] The skin is roughly divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. Among them, the outermost epidermis is mainly composed of keratinocytes (epidermal keratinocytes). The epidermis is composed of multiple layers (basal layer, spinous layer, granular layer, stratum corneum) consisting of keratinocytes at different maturation stages. Keratinocytes divide in the basal layer, the lowest layer of the epidermis, and as they mature, they migrate to the upper layers, become keratinized, and eventually peel off (keratinization or turnover). The stem cells of keratinocytes exist in the basal layer and repeat proliferation and differentiation as needed, constantly supplying new cells to the epidermis. As a result, the skin is constantly regenerating. Therefore, in order to normalize turnover and exhibit a stronger epidermal barrier function, it is important to direct the differentiation of stem cells to the outermost stratum corneum.

[0003] On the other hand, dermal fibroblasts exist in the dermis layer. Collagen, elastin, and hyaluronic acid produced from dermal fibroblasts are known as important components for maintaining the firmness, elasticity, and moisture of the skin. In addition, it has been reported that these components decrease due to aging and inflammation, which are the causes of wrinkles and sagging. Therefore, dermal fibroblasts can be said to be essential cells for maintaining young-looking skin without wrinkles and sagging. The dermal stem cells that produce these dermal fibroblasts exist directly below the dermal papillary layer and repeat proliferation and differentiation as needed, constantly supplying new dermal fibroblasts to the dermis layer. As a result, the skin is constantly regenerating (Non-Patent Document 1). Since dermal components such as collagen and elastin are actively produced from fibroblasts generated from dermal stem cells, it is important to direct the differentiation of dermal stem cells into mature fibroblasts for maintaining a healthy dermal tissue.

[0004] In recent years, it has been clarified that stem cells existing in organs and tissues age (Non-Patent Document 2). Aging of stem cells refers to a decrease in proliferation ability and differentiation ability, and damage such as ultraviolet rays and oxidative stress is considered to be the cause. Therefore, a technology for maintaining and improving the differentiation ability of stem cells existing in each organ and 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 beauty, the development of substances and technologies for efficiently controlling the differentiation of stem cells into target cells is essential. In particular, the skin tissue has a complex three-dimensional structure and is located on the outermost layer of the human body, so it is a tissue that is easily damaged by 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 improve the regeneration technology of this tissue.

[0005] So far, as factors that promote the differentiation of epidermal stem cells into keratinocytes, extracts from the seeds of Hantakai (Patent Document 1) and as factors that promote the differentiation of dermal stem cells into dermal fibroblasts, extracts from the seeds of purple wheat (Patent Document 2) etc. have been reported.

[0006] Althaea (scientific name: Althaea officinalis) is a perennial plant belonging to the genus Althaea in the family Malvaceae. Although it has been reported to have effects such as whitening effect (Patent Document 3) and anti-inflammatory effect (Patent Document 4), its effect of promoting the differentiation of skin stem cells is not known.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Literature

[0008]

Non-Patent Literature 1

Non-Patent Literature 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] In view of the above circumstances, an object of the present invention is to find a new natural product-derived material with high safety that acts on skin stem cells to promote their differentiation and can simply and efficiently regenerate skin cells, and to provide it as a skin stem cell differentiation promoter.

Means for Solving the Problems

[0010] As a result of intensive research to solve the above problems, the present inventors have found that the extract of Althaea has an effect of promoting the differentiation from epidermal stem cells to epidermal keratinocytes, an effect of promoting the differentiation from dermal stem cells to dermal fibroblasts, and an effect of restoring the inhibition of the differentiation of epidermal stem cells and dermal stem cells by oxidative stress, and have thus completed the present invention.

[0011] That is, the present invention includes the following. (1) A skin stem cell differentiation promoter to skin cells containing an extract of Althaea as an active ingredient. (2) A skin stem cell differentiation promoter to skin cells containing an extract of Althaea cultivated by irradiating with a combination of two types of artificial light having different wavelength ranges as an active ingredient. (3) The skin stem cell differentiation promoter to skin cells according to (2), wherein the two types of artificial light having different wavelength ranges are blue light having a wavelength range of 400 to 515 nm and red light having a wavelength range of 570 to 730 nm. (4) The ratio of the photosynthetic photon flux density (PPFD) of the blue light to the red light is 4:1 to 2:1, and the promoter for promoting the differentiation of the skin stem cells into skin cells according to (3). (5) The skin stem cells are epidermal stem cells and / or dermal stem cells, and the promoter for promoting the differentiation of the skin stem cells into skin cells according to (1) or (2). (6) The skin cells are epidermal keratinocytes and / or dermal fibroblasts, and the promoter for promoting the differentiation of the skin stem cells into skin cells according to (1) or (2). (7) A composition for skin regeneration containing the promoter for promoting the differentiation of the skin stem cells into skin cells according to (1) or (2). (8) The composition is a cosmetic, quasi-drug, pharmaceutical, or food or drink, and the composition for skin regeneration according to (7). (9) A method for promoting the differentiation of skin stem cells into skin cells, which includes the step of culturing skin stem cells in a medium containing an extract of Althaea. (10) The skin stem cells are epidermal stem cells and / or dermal stem cells, and the method according to (9). (11) The skin cells are epidermal keratinocytes and / or dermal fibroblasts, and the method according to (9).

Advantages of the Invention

[0012] The promoter for promoting the differentiation of the 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 (keratinocytes) and dermal fibroblasts, respectively. Epidermal keratinocytes are involved in the barrier function and water retention function of the skin, and components such as collagen, elastin, and hyaluronic acid produced by dermal fibroblasts that maintain the firmness, elasticity, and moisture of the skin are sufficiently supplied in the skin. Therefore, the promoter for promoting the differentiation of the skin stem cells of the present invention is effective for the treatment, improvement, and prevention of various skin symptoms (such as skin diseases such as atopic dermatitis and dry skin, reduction of barrier function and turnover, spots, wrinkles, dullness, reduction of firmness and elasticity, etc.) caused by dryness, ultraviolet rays, aging, oxidative stress, etc., and can greatly contribute to the fields of regenerative medicine, regenerative cosmetology, and anti-aging.

Modes for Carrying Out the Invention

[0013] The present invention will be described in detail below. 1. Agent for promoting differentiation of skin stem cells The agent for promoting the 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 Althaea as an active ingredient.

[0014] 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. Therefore, "skin cells" in the present invention include epidermal keratinocytes and / or dermal fibroblasts. In the present invention, the origin of skin stem cells is not limited, and it can exert an effect on skin stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

[0015] Althaea (scientific name: Althaea officinalis) used in the present invention is a perennial plant belonging to the genus Althaea in the family Malvaceae, and is also called by other names such as Marshmallow. It has cold tolerance and is widely distributed from Europe to North Africa and West Asia. The leaves are green, ovate-heart-shaped with serrations, and have stellate hairs.

[0016] In the present invention, the extract of Althaea refers to an extract of a part of the plant body such as the flowers, seeds, leaves, stems, roots of Althaea or the whole plant body (whole herb), or a mixture thereof. However, the parts used as extraction raw materials in the present invention are preferably the roots and leaves. Also, for extraction, the plant body may be used as it is, or treatments such as drying, pulverizing, and cutting into small pieces may be performed.

[0017] In a preferred embodiment of the present invention, as the extraction material of hollyhock, hollyhock cultivated by irradiating a combination of two types of artificial light with different wavelength ranges is used. As the cultivation method of hollyhock, it can be carried out by cultivation using soil or hydroponics. In the case of hydroponics, after sowing the seeds, in the state of having taken root, it can be subjected to hydroponics. The cultivation is preferably carried out in a facility where the temperature, light, and carbon dioxide concentration are controlled. The cultivation temperature is 15 to 30 °C, preferably 20 to 25 °C. The cultivation period varies depending on the conditions for irradiating the above artificial light, but it can generally be harvested in about 10 to 30 days. It is also possible to cultivate for a longer period.

[0018] The light source for irradiating artificial light may be any light source that can selectively irradiate a specific wavelength range, and examples include optical semiconductor elements such as light-emitting diodes (LEDs) and laser diodes, and LEDs are preferred.

[0019] As the two types of artificial light with different wavelength ranges for irradiating hollyhock, blue light with a wavelength range of 400 to 515 nm and red light with a wavelength range of 570 to 730 nm are preferred, and blue light with a wavelength range of 430 to 460 nm and red light with a wavelength range of 630 to 680 nm are more preferred. These lights are most preferably irradiated simultaneously. The wavelength at this time refers to the maximum wavelength (peak wavelength) of the irradiation spectrum. As long as it is a light source having such a wavelength peak, those made independently or commercially available can also be used. In addition to the above two types of artificial light, light sources such as sunlight and fluorescent lamps can also be used. When using light sources such as sunlight and fluorescent lamps, an optical filter may be used so that the above wavelengths can be selectively irradiated.

[0020] The amount of light irradiated is expressed as the photosynthetic photon flux density (PPFD), and in the case of irradiating by combining two types of light emitters, it means the total amount of light. The amount of light is preferably 10 to 300 μmol·m -2 s -1 and more preferably 50 to 200 μmol·m -2 s -1is more preferable. In the case of a light amount outside this range, growth disorders or poor growth may occur. The irradiation is preferably performed from a position 10 to 50 cm above the top of the althaea. The irradiation time can be appropriately changed according to the characteristics and purposes of the plants, but it is preferably 6 hours or more per day, and more preferably 12 to 24 hours.

[0021] The ratio of the photosynthetic photon flux density (PPFD) of blue light to red light can be selected according to purposes such as yield and effectiveness, but the ratio of blue light:red light is preferably 4:1 to 2:1, and more preferably 2:1.

[0022] In the present invention, the extraction material for obtaining the extract of althaea may be althaea cultivated under natural light, or althaea cultivated by irradiating a combination of two types of artificial light with different wavelength ranges as described above. The extraction method for obtaining the extract from althaea is not particularly limited, and for example, a heat extraction method may be used, or a normal temperature or cold temperature extraction method may also be used. As the extraction solvent, water or hot water, or a mixed solvent of water and an organic solvent can be used. 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.) and the like can be used, but water-soluble organic solvents such as ethanol, methanol, acetone, n-propanol, t-butanol, propylene glycol, and 1,3-butylene glycol are preferable, and one or more of these may be mixed and used. Among them, water or hot water, a water-ethanol-based mixed polar solvent (30 to 70 v / v% ethanol aqueous solution) is more preferable, and hot water is even more preferable. Further, an acid or an alkali can be added to the above extraction solvent to use a solvent adjusted in pH.

[0023] There is no particular limitation on the amount of the solvent used. For example, it may be 10 times or more, preferably 20 times or more, based on the extraction raw material (dry weight), but it is preferably 100 times or less for the convenience of operations when performing concentration or isolation after extraction. Also, the extraction temperature and time depend on the type of solvent used. For example, it can be 10 to 100 °C, preferably 30 to 90 °C. In the case of room temperature extraction, it can be 1 to 2 weeks, and in the case of heat extraction, it can be 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, for example, by adding water to the roots and leaves of hollyhock and performing hot water extraction at 95 to 100 °C, an extract of hollyhock can be obtained. Alternatively, by adding a lower alcohol (such as ethanol, etc.) to the roots and leaves of hollyhock and performing extraction at room temperature (for example, 5 to 35 °C), an extract of hollyhock can be obtained.

[0024] The extract may be used as the extracted solution as it is, but if necessary, within the range that does not affect its effect, it may be used after performing treatments such as concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc. 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.

[0025] The extract of hollyhock thus obtained has the effect of promoting the differentiation from epidermal stem cells to epidermal keratinocytes or the differentiation from dermal stem cells to dermal fibroblasts at the in vivo level (in the living body) or the in vitro culture level. Therefore, it can be used as an active ingredient of a skin stem cell differentiation promoter. The skin stem cell differentiation promoter of the present invention can be formulated and applied to pharmaceuticals, 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. Also, the skin stem cell differentiation promoter of the present invention can be used as an additive for a stem cell culture medium for promoting the differentiation of epidermal stem cells or dermal stem cells and producing epidermal keratinocytes or dermal fibroblasts, a research reagent, or a medical reagent in vitro.

[0026] The skin stem cell differentiation promoter of the present invention contains an extract of Althaea officinalis as an active ingredient, which has the effect of promoting the differentiation of epidermal stem cells and dermal stem cells. Therefore, it is effective for treating, improving, and preventing diseases or conditions caused by reduced or defective differentiation ability of epidermal stem cells or dermal stem cells, resulting in the abnormal formation of normal epidermal keratinocytes or dermal fibroblasts. Diseases or conditions caused by reduced or defective differentiation ability of epidermal stem cells, resulting in the abnormal formation of normal epidermal keratinocytes, include, for example, atopic dermatitis, psoriasis (accompanied by erythema, scales, and desquamation), delayed wound healing of burns and injuries, rough skin, dry skin, sensitive skin, hyperkeratosis, melasma, freckles, dullness, enlarged pores, etc. Diseases or conditions caused by reduced or defective differentiation ability of dermal stem cells, resulting in the abnormal formation of normal dermal fibroblasts, include, for example, wrinkles, eye bags, nasolabial folds, marionette lines, loss of firmness and elasticity, lack of moisture and gloss, roughness, dullness, solar elastosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytosis, linear atrophic dermatosis (cutaneous striae), wounds, burns, pressure ulcers, scars, moles, etc. Also, regardless of the cause of reduced or defective differentiation ability of epidermal stem cells or dermal stem cells, for example, oxidative stress caused by excessive production of reactive oxygen species 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 stress can be mentioned.

[0027] The content of the extract of Althaea officinalis in the skin stem cell differentiation promoter of the present invention varies depending on the properties of the extract (extract solution, concentrate, or dried product) and is not particularly limited. For example, in terms of the dried product, it is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight, based on the total amount of the drug.

[0028] 2. Composition for Skin Regeneration When using the skin stem cell differentiation promoter of the present invention in vivo, it can be used as it is, but within the range that does not impair the effects of the present invention, it can be formulated into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, foods and drinks, etc. together with appropriate additives, and provided as a composition for skin regeneration. In particular, it is preferably provided by formulating it into a composition for external use on the skin.

[0029] When formulating the skin stem cell differentiation promoter according to the present invention into cosmetics or quasi-drugs, the dosage form can be any of an aqueous solution system, solubilized system, emulsion system, powder system, powder dispersion system, oil solution system, gel system, ointment system, aerosol system, water-oil two-layer system, or water-oil-powder three-layer system, etc. Further, the cosmetics and quasi-drugs can be manufactured according to known methods in the art by selecting and appropriately formulating various components, additives, bases, etc. usually used in skin external compositions together with the differentiation promoter of dermal stem cells according to their types. The form can be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of the components to be formulated in the skin external composition include fats and oils (such as olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (such as lanolin, beeswax, carnauba wax, etc.), hydrocarbons (such as liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (such as myristyl alcohol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (such as isopropyl myristate, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (such as citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidonecarboxylic acid, etc.), saccharides (such as maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extract components, various surfactants, moisturizers, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances, etc.

[0030] Examples of types of cosmetics and quasi-drugs include, for example, lotions, milky lotions, gels, beauty essences, general creams, sunscreen creams, packs, masks, facial washes, toilet soaps, foundations, face powders, bath agents, body lotions, body shampoos, hair shampoos, hair conditioners, hair growth agents, and the like.

[0031] When formulating the skin stem cell differentiation promoter according to the present invention into a pharmaceutical, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharmaceutically acceptable additives, depending on the dosage form and use, appropriately selected pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegrant aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffering agents, solubilizers or solubilizing aids, isotonic agents, pH adjusters, propellants, coloring agents, sweeteners, flavoring agents, fragrances, etc. can be appropriately added, and it can be prepared into various dosage forms that can be administered orally or parenterally, systemically or locally, by various known methods. When providing the pharmaceutical of the present invention in the above-mentioned various forms, it can be manufactured by production methods commonly used by those skilled in the art, for example, the production methods shown in each article of the General Rules of Preparations of the Japanese Pharmacopoeia [2].

[0032] The form of the pharmaceutical of the present invention is not particularly limited, but examples include oral preparations such as tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs, etc., and parenteral preparations such as injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories, ointments, lotions, sprays, transdermal absorption agents, transmucosal absorption agents, patches, etc. Also, it may be a dry product that is redissolved when used, and in the case of an injectable preparation, it is provided in the form of a unit dose ampoule or a multi-dose container.

[0033] When the promoter for differentiating skin stem cells according to the present invention is used as a pharmaceutical for treating, improving, and preventing the above skin diseases and conditions, a suitable form is a topical preparation, for example, an ointment, a cream, a gel, a liquid, a patch (a poultice, a plaster), a foam, a spray, an aerosol, etc. An ointment refers to a homogeneous semi-solid topical preparation, including an oily ointment, an emulsion ointment, and a water-soluble ointment. A gel refers to a topical preparation in which a water-insoluble component's water-containing compound is suspended in an aqueous liquid. A liquid refers to a liquid topical preparation, including a lotion, a suspending agent, an emulsion, a liniment, etc.

[0034] For oral administration preparations, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegrant aids such as carboxymethyl cellulose, carboxymethyl cellulose calcium, starch, or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropyl methylcellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat, etc. can be used, but are not limited thereto.

[0035] For parenteral administration preparations, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, vegetable oil, etc.; isotonic agents such as glucose, sodium chloride, D-mannitol, etc.; pH adjusters such as inorganic acids, organic acids, inorganic bases, or organic bases, etc. can be used, but are not limited thereto.

[0036] The pharmaceutical of the present invention functions as a prophylactic agent for suppressing the onset of the above skin diseases and / or as a therapeutic agent for improving 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. Therefore, when used as a pharmaceutical for treating, improving, and preventing the aforementioned diseases, it can be administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, and cats in a wide range of dosages.

[0037] The dosage of the pharmaceutical of the present invention can be appropriately determined according to the type of disease, the age, sex, weight, and degree of symptoms of the administration subject. 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 as the extract of Althaea.

[0038] The content of the skin stem cell differentiation promoter in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but in terms of the total weight of the formulation (composition), in terms of the dried product of the above extract of Althaea, 0.001 to 30% by weight is preferred, and 0.01 to 10% by weight is more preferred. The above amounts are merely examples, and they may be appropriately set and adjusted in consideration of the type and form of the composition, the general usage amount, the efficacy and effect, etc. Also, regarding the method of adding the active ingredient in the formulation, it may be added in advance or during the manufacturing process, and it may be appropriately selected considering workability.

[0039] In addition, the skin stem cell differentiation promoter of the present invention can be incorporated into foods and drinks. In the present invention, foods and drinks are used to mean, in addition to general foods and drinks, foods that can be ingested for the purpose of maintaining and promoting health other than pharmaceuticals, for example, health foods, functional foods, health functional foods, or special-purpose foods. Health foods include foods provided under names such as dietary supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Law or the Food Promotion Law, and include foods for specified health uses and nutritional functional foods that can display specific health effects, functions of nutritional components, reduction of disease risks, etc., as well as functional display foods that can display the content reported to the Commissioner of the Consumer Affairs Agency regarding functionality based on scientific grounds. Special-purpose foods include foods for patients, foods for the elderly, foods for infants, foods for pregnant women, etc., which display that they are suitable for specific target persons or patients with specific diseases. The skin stem cell differentiation promoter of the present invention is particularly suitable for use in the above-mentioned health foods, etc. in that it can be continuously ingested daily when long-term ingestion is required for the improvement and prevention of various symptoms such as atopic dermatitis, dry skin, rough skin, wrinkles associated with aging, sagging, and loss of firmness and elasticity accompanying the decline of the skin barrier function. Here, the display of specific health effects, functions of nutritional components, etc. attached to foods and drinks can be made on display items such as product containers, packages, instruction manuals, attached documents, product flyers and pamphlets, and advertisements for products in newspapers and magazines.

[0040] Furthermore, when the food or drink of the present invention is used for mammals other than humans, it can be used to mean pet food and feed.

[0041] The form of the food or drink may be any form suitable for consumption, for example, solid, liquid, granular, pellet, powder, capsule, cream, or paste. In particular, as the shape in the case of the above-mentioned health foods, etc., for example, tablet, round, capsule, powder, granule, fine granule, lozenge, liquid (including syrup, milk, suspension) etc. are preferable.

[0042] Examples of food and drink products include, but are not limited to, breads, noodles, confectioneries, dairy products, processed fishery and livestock foods, oils and fat-processed foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, milk drinks, etc.), concentrated stock solutions of such beverages, and powders for adjustment.

[0043] The food and drink products of the present invention may be appropriately blended with additives commonly used according to their types. Any additives that are acceptable under the Food Sanitation Law can be used. Examples 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, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0044] When the food and drink products of the present invention are general food and drink products, they can be produced by including a step of adding the extract of althaea in the normal manufacturing process of the food and drink products. In the case of health foods, it may be in accordance with the manufacturing method of the above-mentioned pharmaceuticals. For example, in the case of tablet supplements, additives such as excipients are added to and mixed with the extract of althaea, and then molded by applying pressure with a tableting machine or the like. In the case of capsule supplements, they can be produced by filling a liquid, suspension, paste, powder, or granular food composition containing the extract of althaea into capsules or coating and molding them with capsule bases. Also, other materials (for example, minerals such as iron and potassium, vitamins such as vitamin C, vitamin B2, and vitamin B6, folic acid, dietary fiber, etc.) can be added as necessary.

[0045] The blending amount of the extract of althaea in the food and drink products of the present invention may be any amount that can exhibit the effect of promoting the differentiation of skin stem cells, but it may be appropriately set in consideration of the general intake amount of the target food and drink products, the form of the food and drink products, efficacy and effects, taste, palatability, and cost.

[0046] When the food or drink of the present invention is ingested for the purpose of preventing or improving the aforementioned diseases or conditions, the intake amount varies depending on the condition of the subject to be ingested, the intake form, the intake amount, etc. As the extract of Althaea, for adults per day, it is 0.1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 300 mg. The above amount may be ingested at one time, or may be ingested in several (2 to 4) divided doses. It is preferable that the food or drink of the present invention is packaged or filled in a container such as a single bag or bottle with the amount of food or drink to be ingested at one time as a guide for the intake amount.

[0047] 3. Method for promoting differentiation of skin stem cells The present invention also relates to a method for promoting the differentiation of skin stem cells into skin cells, which includes the step of culturing skin stem cells in a medium containing an extract of Althaea. The extract of Althaea used here may be an extract of Althaea cultivated under normal natural light, or may be an extract of Althaea cultivated by irradiating a combination of two types of artificial light with different wavelength ranges as described above. This method includes a method for promoting the differentiation of epidermal stem cells into epidermal keratinocytes and a method for promoting the 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 transplanted directly by injection or the like to the wound area or the site where tissue regeneration is desired after culturing in vitro. That is, the epidermal keratinocytes and dermal fibroblasts produced by the method according to the present invention can be used as transplantation materials (cell transplantation agents).

[0048] In the method for promoting the differentiation of skin stem cells of the present invention, the medium for culturing skin stem cells and the additives used simultaneously are not particularly limited, and media and additives generally used for the proliferation of skin stem cells (epidermal stem cells or dermal stem cells) may be used.

[0049] Specifically, for the medium for culturing skin stem cells, a basal 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 (Hank's solution), etc., is used, and at least one differentiation-inducing or promoting factor corresponding to the cells for the purpose of differentiation induction is added. Examples of the differentiation-inducing factor from epidermal stem cells to epidermal keratinocytes include calcium salts, lysophosphatidic acid (LPA), etc. Examples of the differentiation-inducing factor from dermal stem cells to dermal fibroblasts include TGFβ, etc. In addition, in order to increase the proliferation rate of stem cells, growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), etc., 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. may be added to the above medium as needed, and antibiotics (penicillin, streptomycin, etc.) may also be added. Each component of the medium is sterilized by a suitable method before use.

[0050] In addition, other than the above, it is preferable that serum (for example, 10% FBS) is contained at a content rate of 1 to 20%. However, since the components of serum vary depending on the lot and there are variations in its effects, it is preferable to use it after lot checking.

[0051] As for the medium for culturing skin stem cells, commercially available products can also be used. Examples of commercially available media include the mesenchymal stem cell basal medium manufactured by Invitrogen, the mesenchymal stem cell basal medium manufactured by Sanko Junyaku Co., Ltd., the MF medium manufactured by Toyobo Co., Ltd., and Hank’s balanced salt solution manufactured by Sigma. In addition, differentiation induction media supplemented with differentiation inducing factors for the target cells are commercially available, and these commercially available media may also be used. For example, as the epidermal cell differentiation induction medium, CnT-Prime 3D Barrier Culture Medium (manufactured by CELLn TEC) and the like can be mentioned. As the dermal cell differentiation induction medium, Fibroblast Medium (manufactured by Sanko Junyaku Co., Ltd.), the medium for normal human fibroblasts (manufactured by DS Pharma Biomedical Co., Ltd.) and the like can be mentioned.

[0052] In accordance with the skin stem cell differentiation promoter according to the present invention or the method according to the present invention described above, the extract of Arteia can be provided alone, separately from the medium or mixed with the medium as a reagent kit for promoting the differentiation of skin stem cells. The kit can include an instruction manual and the like as necessary. Alternatively, the extract of Arteia described above can be mixed with the medium and provided as a medium for promoting the differentiation of skin stem cells.

[0053] The incubator used for culturing skin stem cells is not particularly limited as long as it can culture stem cells. Examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, and the like. The incubator may be non-cell adhesive or cell adhesive, and is appropriately selected according to the purpose. As the cell adhesive incubator, for the purpose of improving the adhesiveness with cells, those treated with a cell support substrate such as an extracellular matrix may be used. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, and the like.

[0054] The addition concentration of the extract of Althaea to the medium used for culturing skin stem cells can be appropriately determined according to the content of the extract of Althaea in the skin stem cell differentiation promoter according to the present invention described above. However, in terms of the dry matter of the extract, for example, concentrations of 10 to 10,000 μg / mL, preferably 100 to 5,000 μg / mL can be mentioned. Also, during the culture period of the stem cells, the extract of Althaea may be periodically added to the medium.

[0055] The culture conditions for skin stem cells may follow the normal conditions 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%. It should be noted that it is preferable to change the medium once every 2 to 3 days, and it is more preferable to change it every day. The above culture conditions can also be appropriately adjusted and set within the range where the stem cells can survive and proliferate.

[0056] 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 in cells cultured in the presence of an extract of Althaea is significantly increased at the mRNA level or protein level compared to the expression level at the start of culture in cells cultured in the absence of the extract of Althaea. 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 in cells cultured in the presence of an extract of Althaea is significantly increased at the mRNA level or protein level compared to the expression level at the start of culture in cells cultured in the absence of the extract of Althaea. 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. 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 (hyaluronan synthase-1), ELN (elastin), HYAL3 (hyaluronidase 3), galectin 9, etc. At the mRNA level, methods such as RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific for each of the above marker genes can be mentioned for confirmation. At the protein level, immunological methods such as ELISA, flow cytometry, and Western blotting using antibodies specific for the proteins encoded by each of the above marker genes can be mentioned.

Example

[0057] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited thereto.

[0058] [Example 1] (1) Experimental materials and growth conditions Althaea seeds were sown in vermiculite containing moisture, germinated in the dark at a temperature of 22 - 25°C, cultivated under fluorescent lights at 22 - 26°C, and seedlings were raised. Subsequently, the young shoots were wrapped in sponge, and using a hydroponic cultivation device, at room temperature of 21 - 25°C for 24 hours, from a position 30 cm directly above the plants, blue LEDs (peak wavelength 450 nm) and red LEDs (peak wavelength 660 nm) were simultaneously irradiated, and cultivation was carried out such that the total photosynthetic photon flux density of the blue LED and the red LED was 100 μmol·m -2 s -1 −2·s−1, and the light quantity ratio of blue light to red light was set to 4:1, 3:1, or 2:1. During cultivation, the light quantity ratio was not changed. Also, cultivation under sunlight was carried out. In all cases, after 4 weeks of cultivation, harvesting was done, and by drying with warm air at about 60°C, dry matter of Althaea was obtained.

[0059] (2) Production example of Althaea extract (1) The Althaea cultivated under sunlight obtained in (1) was designated as "Althaea (sunlight cultivation)", and the Althaea cultivated with the light quantity ratio of blue light to red light set to 4:1, 3:1, and 2:1 were designated as "Althaea (blue:red = 4:1 cultivation)", "Althaea (blue:red = 3:1 cultivation)", and "Althaea (blue:red = 2:1 cultivation)", respectively. Using the roots and leaves of Althaea cultivated under each of these conditions as extraction materials, an Althaea extract was produced as follows.

[0060] (Production example 1) Preparation of hot water extract of Althaea (sunlight cultivation) 200 mL of water was added to 10 g of the dry matter of Althaea (sunlight cultivation), and extraction was carried out at 95 - 100°C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.0 g of the hot water extract of Althaea (sunlight cultivation).

[0061] (Production example 2) Preparation of 50% ethanol extract of Althaea (sunlight cultivation) 10 g of the dry matter of Althaea (sunlight cultivation) was immersed in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days for extraction. After the obtained extract was filtered, it was concentrated to dryness with an evaporator to obtain 1.5 g of the 50% ethanol extract of Althaea (sunlight cultivation).

[0062] (Production Example 3) Preparation of Ethanol Extract of Althea (Sunlight Cultivation) 10 g of the dried product of Althea (sunlight cultivation) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 0.5 g of the ethanol extract of Althea (sunlight cultivation).

[0063] (Production Example 4) Preparation of Hot Water Extract of Althea (Cultivation with Blue:Red = 4:1) 200 mL of water was added to 10 g of the dried product of Althea (cultivation with blue:red = 4:1), and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.6 g of the hot water extract of Althea (cultivation with blue:red = 4:1).

[0064] (Production Example 5) Preparation of 50% Ethanol Extract of Althea (Cultivation with Blue:Red = 4:1) 10 g of the dried product of Althea (cultivation with blue:red = 4:1) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 1.2 g of the 50% ethanol extract of Althea (cultivation with blue:red = 4:1).

[0065] (Production Example 6) Preparation of Ethanol Extract of Althea (Cultivation with Blue:Red = 4:1) 10 g of the dried product of Althea (cultivation with blue:red = 4:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 0.4 g of the ethanol extract of Althea (cultivation with blue:red = 4:1).

[0066] (Production Example 7) Preparation of Hot Water Extract of Althea (Cultivation with Blue:Red = 3:1) 200 mL of water was added to 10 g of the dried product of Althea (cultivation with blue:red = 3:1), and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.2 g of the hot water extract of Althea (cultivation with blue:red = 3:1).

[0067] (Production Example 8) Preparation of 50% Ethanol Extract of Althaea (cultivated with blue:red = 3:1) 10 g of the dried product of Althaea (cultivated with blue:red = 3:1) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 1.0 g of 50% ethanol extract of Althaea (cultivated with blue:red = 3:1).

[0068] (Production Example 9) Preparation of Ethanol Extract of Althaea (cultivated with blue:red = 3:1) 10 g of the dried product of Althaea (cultivated with blue:red = 3:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 0.4 g of ethanol extract of Althaea (cultivated with blue:red = 3:1).

[0069] (Production Example 10) Preparation of Hot Water Extract of Althaea (cultivated with blue:red = 2:1) 200 mL of water was added to 10 g of the dried product of Althaea (cultivated with blue:red = 2:1), and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.3 g of hot water extract of Althaea (cultivated with blue:red = 2:1).

[0070] (Production Example 11) Preparation of 50% Ethanol Extract of Althaea (cultivated with blue:red = 2:1) 10 g of the dried product of Althaea (cultivated with blue:red = 2:1) was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness by an evaporator to obtain 1.2 g of 50% ethanol extract of Althaea (cultivated with blue:red = 2:1).

[0071] (Production Example 12) Preparation of Ethanol Extract of Althaea (cultivated with blue:red = 2:1) 10 g of the dried product of Althaea (cultivated with a blue:red ratio of 2:1) was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness using an evaporator to obtain 0.3 g of the ethanol extract of Althaea (cultivated with a blue:red ratio of 2:1).

[0072] [Example 2] [Experimental Example 1] Evaluation of the effect of promoting differentiation of the extract of Althaea on epidermal stem cells Using commercially available normal human adult epidermal keratinocytes (manufactured by Kracie) as epidermal-derived cells, epidermal stem cells were isolated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index according to the method described in JP 2017-055721 A. The above epidermal stem cells maintained in Humedia-KG2 medium (manufactured by Kracie) were seeded in a 12-well plate (manufactured by Falcon) so that the number of cells became 5×10 5 cells. After culturing for 24 hours and allowing the cells to attach, the test substance (the extract of Althaea in Production Examples 1 to 12) was added so that the final concentration became 50 μg / mL, and the cells were cultured for 72 hours. Also, after the cells attached, cells that were cultured for 72 hours with 1.5 mM of CaCl2 added without adding the test substance to induce differentiation were used as a positive control. After culturing, mRNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio). For this mRNA, after cDNA synthesis by reverse transcription reaction using High Capacity RNA to cDNA Kit (manufactured by Thermo), PCR reaction was carried out using SYBR Select Master Mix (manufactured by Thermo), and the gene expression levels of FLG (filaggrin), IVL (involucrin), and KRT10 (keratin 10) were analyzed. The PCR reaction was performed with an initial denaturation at 95°C for 2 minutes, and then 40 cycles were carried out with 95°C for 15 seconds and 60°C for 60 seconds as one cycle using the following primer sets. Other operations were carried out according to the defined methods.

[0073] [Primer set for FLG] 5'-TCGAAGGAGCCAAAAATATAAAACAG-3’ (SEQ ID NO: 1) 5'-GAATTCCAATAGAAGGATAATAGAGAAAGATG-3’(SEQ ID NO: 2)

[0074] (Primer set for IVL) 5'-CCATCAGGAGCCAAATGAAACAG-3’(SEQ ID NO: 3) 5'-GCTCGACAGGCACCTTCTG-3’(SEQ ID NO: 4)

[0075] (Primer set for KRT10) 5'-ACTGAAGAGCTGGCCTATCTGAA-3’(SEQ ID NO: 5) 5'-CATCACCAGTGGACACATTTCG-3’(SEQ ID NO: 6)

[0076] (Primer set for GAPDH (internal standard)) 5'-TGCACCACCAACTGCTTAGC-3'(SEQ ID NO: 7) 5'-TCTTCTGGGTGGCAGTGATG-3'(SEQ ID NO: 8)

[0077] The expression levels of each of the FLG, IVL, and KRT10 genes were calculated as the ratios of the expression levels of FLG, IVL, and KRT10 in cells cultured without the test substance (without differentiation induction) (control) to the expression level of GAPDH as the internal standard, i.e., 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, with the values for cells cultured with the test substance being calculated and evaluated. The results of these tests are shown in Table 1 below.

[0078]

Table 1

[0079] As shown in Table 1, it was confirmed that the extract of Althaea has the effect of promoting the differentiation of epidermal stem cells even without a differentiation inducer (CaCl2). When compared under the same cultivation conditions, the differentiation-promoting effect was higher when using a hot water extract. In addition, the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher differentiation-promoting effect than the non-irradiated extract of Althaea, and the effectiveness was particularly high when the light intensity ratio of blue light to red light was 2:1.

[0080] (Experimental Example 2) Evaluation of the differentiation-promoting effect of the extract of Althaea on dermal stem cells Using commercially available human skin fibroblasts (manufactured by Toyobo Co., Ltd.) as dermal-derived cells, dermal stem cells were isolated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index according to the method described in JP 2017-093383 A. The above dermal stem cells maintained in DMEM medium containing 10% FBS (manufactured by Nacalai) had a cell number of 2×10 4They were seeded in a 12-well plate (manufactured by Falcon) so as to be [X] cells. After culturing for 24 hours and allowing the cells to attach, the test substances (extracts of Arteia of Production Examples 1 to 12) were added so that the final concentration became 50 μg / mL, and then cultured for 48 hours. Also, after the cells attached, without adding the test substances, cells induced to differentiate by adding 10 ng / mL of TGFβ (manufactured by Pepro Tech), which is generally used as a differentiation inducer for fibroblasts, and culturing for 48 hours were used as the positive control. After the culture was completed, mRNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). After performing cDNA synthesis by reverse transcription reaction on this mRNA using the High Capacity RNA to cDNA Kit (manufactured by Thermo), a PCR reaction was carried out using SYBR Select Master Mix (manufactured by Thermo), and the gene expression levels of COL1A1 (Collagen Type I Alpha 1 Chain) and FAP (Fibroblast Activation Protein Alpha) were analyzed. The PCR reaction was performed with an initial denaturation at 95°C for 2 minutes, and then 40 cycles were carried out with 95°C for 15 seconds and 60°C for 60 seconds as one cycle using the following primer sets. Other operations were carried out according to the defined methods.

[0081] (Primer set for COL1A1) 5'-GCTACCCAACTTGCCTTCATG-3’ (SEQ ID NO: 9) 5'-TTCTTGCAGTGGTAGGTGATGTTC-3’(SEQ ID NO: 10) (Primer set for FAP) 5'-CTAATTCAAGTGTATGGTGGTCCC-3’(SEQ ID NO: 11) 5'-CCAGTGATGAAACGTATCCTCC-3’(SEQ ID NO: 12) (Primer set for GAPDH (internal standard)) 5'-TGCACCACCAACTGCTTAGC-3'(SEQ ID NO: 7) 5'-TCTTCTGGGTGGCAGTGATG-3'(SEQ ID NO: 8)

[0082] The expression of each gene of COL1A1 and FAP was calculated as the ratio of the expression levels of COL1A1 and FAP in cells cultured without the test substance (without differentiation induction) (control) to the expression level of GAPDH, which was used as an internal standard, i.e., COL1A1 gene expression level / GAPDH gene expression level and FAP gene expression level / GAPDH gene expression level. These ratios were set to 100. In contrast, the relative gene expression levels of COL1A1 and FAP in cells cultured with the test substance were calculated and evaluated. The results of these tests are shown in Table 2 below.

[0083]

Table 2

[0084] As shown in Table 2, it was confirmed that the extract of Althaea has the effect of promoting the differentiation of dermal stem cells even without a differentiation inducer (TGFβ). When compared under the same cultivation conditions, the differentiation-promoting effect was higher when using a hot water extract. In addition, the extract of Althaea irradiated with a combination of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher differentiation-promoting effect than the non-irradiated extract of Althaea, and the effectiveness was particularly high when the light intensity ratio of blue light to red light was 2:1.

[0085] (Experimental Example 3) Evaluation of the effect of the extract of Althaea on the impairment of epidermal stem cell differentiation by hydrogen peroxide It is known that oxidative stress accumulates in skin tissue due to factors such as aging and ultraviolet rays. The effect of reactive oxygen species on the induction of epidermal stem cell differentiation was analyzed, and the effectiveness of the extract of Althaea was evaluated when it was allowed to act. Commercially available normal human adult epidermal keratinocytes (manufactured by Kurabo Industries Ltd.) were used as epidermal-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 index according to the method described in JP 2017-055721 A. The above-mentioned epidermal stem cells maintained in Humedia-KG2 medium (manufactured by Kurabo Industries Ltd.) were adjusted to a cell number of 5×10 5They were seeded into a 12-well plate (manufactured by Falcon) so as to be [X] cells. After culturing for 24 hours and allowing the cells to attach, 1.5 mM of CaCl2 was added to induce differentiation. As oxidative stress, 100 mM of hydrogen peroxide and the test substances (extracts of Arteia of Production Examples 1 to 12) were added so that the final concentration became 50 μg / mL, and the cells were cultured for 72 hours. Also, after the cells attached, cells cultured for 72 hours without adding hydrogen peroxide and the test substances were used as a positive control. After the culturing was completed, mRNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). For this mRNA, after cDNA synthesis by reverse transcription reaction using a High Capacity RNA to cDNA Kit (manufactured by Thermo), a PCR reaction was carried out using SYBR Select Master Mix (manufactured by 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.

[0086] The expression of each gene of FLG, IVL, and KRT10 was calculated as the ratio of the expression levels of FLG, IVL, and KRT10 in cells cultured without the test substance (without differentiation induction) (control) to the expression level of GAPDH as an internal standard, i.e., 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, with these values being set to 100. In contrast, the values of the relative gene expression levels of FLG, IVL, and KRT10 in cells cultured with / without the test substance were calculated and evaluated. The results of these tests are shown in Table 3 below.

[0087]

Table 3

[0088] As shown in Table 3, the differentiation of epidermal stem cells was inhibited by oxidative stress (hydrogen peroxide) (with differentiation induction / with hydrogen peroxide / without extract addition). However, when the extracts of Althaea (Production Examples 1 to 12) were added simultaneously, it was confirmed that the inhibition of epidermal stem cell differentiation by oxidative stress was improved. When compared under the same cultivation conditions, the effect of improving the inhibition of differentiation was higher when using hot water extracts. In addition, the extracts of Althaea irradiated with a combination of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher effect of improving the inhibition of differentiation than the non-irradiated extracts of Althaea. In particular, the effectiveness was high when the light intensity ratio of blue light to red light was 2:1.

[0089] (Experimental Example 4) Evaluation of the effect of Althaea extract on the impairment of dermal stem cell differentiation by hydrogen peroxide It is known that oxidative stress accumulates in skin tissue due to the effects of aging, ultraviolet rays, etc. The effect of reactive oxygen species on the induction of dermal stem cell differentiation was analyzed, and the effectiveness of the Althaea extract was evaluated when it was allowed to act. Using commercially available human skin fibroblasts (manufactured by Toyobo Co., Ltd.) as dermal-derived cells, dermal stem cells were isolated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index according to the method described in JP 2017-093383 A. Dermal stem cells maintained in DMEM medium containing 10% FBS (manufactured by Nacalai) were 2×10 4They were seeded into a 12-well plate (manufactured by Falcon) so that the number of cells was [specific number]. After culturing for 24 hours and the cells had attached, TGFβ (10 ng / mL, manufactured by Pepro Tech), which is generally used as a differentiation inducer for fibroblasts, was added to induce differentiation. Hydrogen peroxide at 50 mM was added as oxidative stress, and the test substances (extracts of Arteia in Production Examples 1 to 12) were added so that the final concentration was 50 μg / mL, and then cultured for 48 hours. Also, after the cells had attached, cells cultured for 48 hours without adding hydrogen peroxide and the test substances were used as a positive control. After the culture was completed, RNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). For this RNA, after cDNA synthesis by reverse transcription reaction using High Capacity RNA to cDNA Kit (manufactured by Thermo), PCR reaction was carried out using SYBR Select Master Mix (manufactured by Thermo), and the gene expression levels of COL1A1 and FAP were analyzed in the same manner as in Experimental Example 2.

[0090] The expression of each gene of COL1A1 and FAP was calculated as the ratio of the expression levels of COL1A1 and FAP in cells cultured without the test substance (without differentiation induction) (control) to the expression level of GAPDH, which is an internal standard, COL1A1 gene expression level / GAPDH gene expression level, FAP gene expression level / GAPDH gene expression level was set to 100. In contrast, the values of the relative gene expression levels of COL1A1 and FAP in cells cultured with / without the test substance (with differentiation induction) were calculated and evaluated. The results of these tests are shown in Table 4 below.

[0091]

Table 4

[0092] As shown in Table 4, the differentiation of dermal stem cells was inhibited by oxidative stress (hydrogen peroxide) (with differentiation induction / with hydrogen peroxide / without extract added), but when the extract of Althaea (Production Examples 1 to 12) was added simultaneously, it was confirmed that the inhibition of dermal stem cell differentiation by oxidative stress was improved. When compared under the same cultivation conditions, the effect of improving the inhibition of differentiation was higher when using the hot water extract. In addition, the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher effect of improving the inhibition of differentiation than the extract of Althaea without irradiation, and the effectiveness was particularly high when the light intensity ratio of blue light to red light was 2:1.

[0093] (Experimental Example 5) Evaluation of the effect of the extract of Althaea on epidermal stem cell differentiation disorder caused by stretching stimulation The skin tissue is subjected to stretching stimulation due to facial expression changes, speech, etc., and it is known that stretching stimulation becomes oxidative stress. The influence of stretching stimulation on the differentiation induction of epidermal stem cells was analyzed, and the effectiveness when the extract of Althaea was allowed to act was evaluated. Commercially available normal human adult epidermal keratinocytes (manufactured by Kracie Pharmaceuticals) were used as epidermal-derived cells, and epidermal stem cells were separated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index according to the method described in JP-A-2017-055721. The above epidermal stem cells maintained in Humedia-KG2 medium (manufactured by Kracie Pharmaceuticals) were placed in a collagen gel (manufactured by Nitta Gelatin) at 1×10 6Cells were seeded at a density of cells / mL on a stretch chamber (manufactured by STREX), and Humedia-KG2 medium was added and cultured for 24 hours. After 24 hours, 1.5 mM of CaCl2 was added to induce differentiation, and the test substances (extracts of Arteia from Production Examples 1 to 12) were added so that the final concentration was 50 μg / mL. The cells were cultured for 24 hours in a stretched state with a stretch ratio of 1.4 times the gel length using a stretching device (manufactured by STREX). 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+ (manufactured by Takara Bio Inc.). For this RNA, cDNA synthesis by reverse transcription reaction was performed using a High Capacity RNA to cDNA Kit (manufactured by Thermo), and then a PCR reaction was carried out using SYBR Select Master Mix (manufactured by Thermo). The gene expression levels of FLG, IVL, and KRT10 were analyzed in the same manner as in Example 1.

[0094] The expression of each gene of FLG, IVL, and KRT10 was calculated as the ratio of the expression levels of FLG, IVL, and KRT10 in cells cultured without adding the test substance (without differentiation induction) (control) to the expression level of GAPDH as an internal standard, i.e., 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, which were set to 100. On the other hand, the relative gene expression levels of FLG, IVL, and KRT10 in cells cultured with / without adding the test substance (with differentiation induction) were calculated and evaluated. The results of these tests are shown in Table 5 below.

[0095]

Table 5

[0096] As shown in Table 5, the differentiation of epidermal stem cells was suppressed by stretching stimulation (with differentiation induction / with stretching stimulation / without extract addition), but when the extract of Althaea (Production Examples 1 to 12) was added simultaneously, it was confirmed that the suppression of epidermal stem cell differentiation by stretching stimulation was improved. When compared under the same cultivation conditions, the effect of improving the suppression of differentiation was higher when using the hot water extract. In addition, the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher effect of improving the suppression of differentiation than the extract of Althaea without irradiation, and the effectiveness was particularly high when the light intensity ratio of blue light to red light was 2:1.

[0097] (Experimental Example 6) Evaluation of the effect of the extract of Althaea on the impairment of dermal stem cell differentiation by stretching stimulation The skin tissue is subjected to stretching stimulation due to facial expression changes and speech, etc., and it is known that stretching stimulation becomes oxidative stress. The influence of stretching stimulation on the induction of dermal stem cell differentiation was analyzed and the effectiveness was evaluated when the extract of Althaea was allowed to act. Using commercially available human skin fibroblasts (manufactured by Toyobo Co., Ltd.) as dermal-derived cells, dermal stem cells were isolated using NGFR (nerve growth factor receptor: Geanbank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index according to the method described in JP-A-2017-093383. Dermal stem cells maintained in DMEM medium containing 10% FBS (manufactured by Nacalai) were placed in a collagen gel (manufactured by Nitta Gelatin) at 1×10 6Cells were seeded at a density of cells / mL on a stretch chamber (manufactured by STREX), and DMEM medium containing 10% FBS was added, followed by culturing for 24 hours. After 24 hours, TGFβ (10 ng / mL, manufactured by Pepro Tech), which is generally used as a differentiation inducer for fibroblasts, was added to induce differentiation. The test substances (extracts of Arteia in Production Examples 1 to 12) were added so that the final concentration was 50 μg / mL, and the cells were cultured for 24 hours in a stretched state with a stretch ratio of 1.4 times the gel length using a stretching device (manufactured by STREX). 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+ (manufactured by Takara Bio Inc.). For this RNA, after cDNA synthesis by reverse transcription reaction using a High Capacity RNA to cDNA Kit (manufactured by Thermo), a PCR reaction was carried out using SYBR Select Master Mix (manufactured by Thermo), and the gene expression levels of COL1A1 and FAP were analyzed in the same manner as in Experimental Example 2.

[0098] The expression of each gene of COL1A1 and FAP was calculated as the ratio of the expression levels of COL1A1 and FAP in cells cultured without adding the test substance (without differentiation induction) (control) to the expression level of GAPDH, which is an internal standard, and the COL1A1 gene expression level / GAPDH gene expression level and FAP gene expression level / GAPDH gene expression level were set to 100. In contrast, the relative gene expression levels of COL1A1 and FAP in cells cultured with / without adding the test substance (with differentiation induction) were calculated and evaluated. The results of these tests are shown in Table 6 below.

[0099]

Table 6

[0100] As shown in Table 6, the differentiation of dermal stem cells was suppressed by stretching stimulation (with differentiation induction / with stretching stimulation / without extract addition), but when the extract of Althaea (Production Examples 1 to 12) was added simultaneously, it was confirmed that the suppression of dermal stem cell differentiation by stretching stimulation was improved. When compared under the same cultivation conditions, the effect of improving the suppression of differentiation was higher when using a hot water extract. In addition, the extract of Althaea irradiated with a combination of artificial light of blue light with a wavelength of 450 nm and red light with a wavelength of 660 nm had a higher effect of improving the suppression of differentiation than the non-irradiated extract of Althaea, and the effectiveness was particularly high when the light intensity ratio of blue light to red light was 2:1.

Industrial Applicability

[0101] The promoter for promoting the differentiation of skin stem cells of the present invention can promote the differentiation of skin stem cells in vivo or in vitro. Therefore, the present invention can be used in the fields of manufacturing cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and conditions caused by functional decline or failure of epidermal stem cells and dermal stem cells, and in the field of manufacturing transplantation materials for regenerative medicine and regenerative aesthetics.

Claims

1. A promoter for promoting the differentiation of skin stem cells into skin cells, containing an extract of hollyhock as an active ingredient.

2. A promoter for promoting the differentiation of skin stem cells into skin cells, containing an extract of hollyhock cultivated by irradiating with a combination of two types of artificial light having different wavelength ranges as an active ingredient.

3. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 2, wherein the two types of artificial light having different wavelength ranges are blue light with a wavelength range of 400 to 515 nm and red light with a wavelength range of 570 to 730 nm.

4. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 3, wherein the ratio of the photosynthetic photon flux density (PPFD) of the blue light and the red light is 4:1 to 2:

1.

5. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 1 or 2, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

6. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 1 or 2, wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts.

7. A composition for skin regeneration, containing the promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 1 or 2.

8. The composition for skin regeneration according to Claim 7, wherein the composition is a cosmetic, quasi-drug, pharmaceutical, or food or drink product.

9. A method for promoting the differentiation of skin stem cells into skin cells, including the step of culturing skin stem cells in a medium containing an extract of hollyhock.

10. The method according to Claim 9, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

11. The method according to Claim 9, wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts.

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