Differentiation promoter for skin stem cells

Abstract

To discover a highly safe material derived from natural products that acts on skin stem cells and promotes their differentiation, thereby enabling easy and efficient regeneration of skin cells, and to provide the discovered material as a differentiation promoter for skin stem cells.SOLUTION: A differentiation promoter for skin stem cells into skin cells comprises an extract of Rosa roxburghii 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) of keratinocytes at different maturation stages. 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 peel off (keratinization or turnover). The stem cells of keratinocytes are present 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 are present in the dermis layer. Collagen, elastin, and hyaluronic acid produced from dermal fibroblasts are known as important components for maintaining skin firmness, elasticity, and moisture. 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 are present 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 revealed that stem cells existing in organs and tissues age (Non-Patent Document 2). The aging of stem cells refers to the decline 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 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 aesthetics, 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. It is also 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] So far, as factors that promote the differentiation of epidermal stem cells into keratinocytes, an extract of the seeds of Rosa roxburghii (Patent Document 1) has been reported, and as a factor that promotes the differentiation of dermal stem cells into dermal fibroblasts, an extract of the seeds of purple wheat (Patent Document 2) has been reported.

[0006] Rosa roxburghii (scientific name: Rosa roxburghii) is a plant of the genus Rosa in the family Rosaceae, and it has been reported to have effects such as preventing or improving freckles (Patent Document 3) and promoting the production of bleomycin hydrolase (Patent Document 4), but nothing is known about its effect of promoting the differentiation of skin stem cells.

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, highly safe material derived from natural products 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 Rosa multiflora var. cathayensis 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 differentiation inhibition 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 into skin cells, containing a solvent extract of Rosa multiflora var. cathayensis as an active ingredient. (2) A skin stem cell differentiation promoter into skin cells, containing a solvent extract of the filtration residue of the aqueous extract of Rosa multiflora var. cathayensis as an active ingredient. (3) The skin stem cell differentiation promoter into skin cells according to (2), wherein the temperature of the aqueous extraction is 20°C or lower, and the solvent extract of the filtration residue is an extract extracted at a temperature 20°C or higher than the temperature of the aqueous extraction. (4) The differentiation promoter of skin stem cells into skin cells according to (1) or (2), wherein the solvent is one or more selected from the group consisting of water, lower alcohols, and liquid polyhydric alcohols. (5) The differentiation promoter of skin stem cells into skin cells according to (1) or (2), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (6) The differentiation promoter of skin stem cells into skin cells according to (1) or (2), wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts. (7) A skin regeneration composition comprising the differentiation promoter of skin stem cells into skin cells according to (1) or (2). (8) The skin regeneration composition according to (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, comprising the step of culturing skin stem cells in a medium containing an extract of Rosa multiflora var. cathayensis. (10) The method according to (9), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (11) The method according to (9), wherein the skin cells are epidermal keratinocytes and / or dermal fibroblasts. [Effect of the Invention]

[0012] The differentiation promoter of skin stem cells of the present invention can promote the differentiation of epidermal stem cells and dermal stem cells, and 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 skin firmness, elasticity, and moisture are sufficiently supplied into the skin. Therefore, the differentiation promoter of 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, freckles, 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] Hereinafter, the present invention will be described in detail. 1. Skin Stem Cell Differentiation Promoter The skin stem cell to skin cell differentiation promoter according to the present invention (hereinafter may be referred to as "skin stem cell differentiation promoter") contains an extract of Rosa roxburghii 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 (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 effects on skin stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

[0015] The Rosa roxburghii used in the present invention is a deciduous shrub of the genus Rosa in the family Rosaceae. It is a variety of rose native to China named after the moon on the sixteenth night because its petals are not completely circular and some are missing. In the present invention, in addition to the original species, varieties, hybrids, etc. of Rosa roxburghii can also be used.

[0016] In the present invention, the part of Rosa roxburghii used for extraction may be a part of the plant body such as flowers, fruits, pericarp, stems, leaves, branches, roots, seeds, etc., the whole plant body (whole herb), or a mixture thereof, but fruits are preferred. 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 addition, as the fruits of the Rosa multiflora var. cathayensis, it is preferable to use those with a major axis length in the range of 1 cm to 10 cm, and more preferably those with a major axis length in the range of 1 cm to 4 cm. Also, the fruits to be used can be fresh, or dried products such as natural dried products or sun-dried products can be used.

[0018] In addition, in a preferred embodiment of the present invention, the filtration residue of the aqueous extract of Rosa multiflora var. cathayensis is used as the extraction material. The water used for the aqueous extraction is not particularly limited, and examples include tap water, distilled water, deionized water, purified water, etc. Also, the water used for the aqueous extraction may be water with pH adjusted by adding an acid or an alkali. Regarding the amount of water used for the aqueous extraction, there is no particular limitation. For example, it is 3 times or more, preferably 5 times or more, more preferably 10 times or more based on the extraction raw material (dry weight). However, in the present invention, it is preferably 100 times or less for the convenience of the operation of filtering the aqueous extract of Rosa multiflora var. cathayensis to collect the residue. The filtration is not particularly limited, and it can be performed using filter paper, mesh, sieve, etc. The temperature and time of the aqueous extraction can be appropriately selected according to the pressure during extraction, etc. The temperature is, for example, 20°C or lower, preferably 2 to 15°C, more preferably 5 to 10°C, and the time is, for example, 10 to 30 hours, preferably 15 to 25 hours. In the present invention, the operation of the above-mentioned aqueous extraction of Rosa multiflora var. cathayensis, filtering the obtained aqueous extract to separate it into a filtrate and a residue, and collecting the residue is sometimes referred to as "pretreatment".

[0019] In the present invention, the extraction material may be untreated Rosa multiflora var. cathayensis or Rosa multiflora var. cathayensis subjected to the above-mentioned pretreatment. The Rosa multiflora var. cathayensis subjected to the pretreatment (filtration residue of the aqueous extract of Rosa multiflora var. cathayensis) may be dried before being subjected to extraction, or may be directly used for extraction without drying.

[0020] As the extraction solvent for obtaining an extract from the above-mentioned extraction material, water or hot water, or a mixed solvent of water and an organic solvent can be used. As the organic solvent, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) and liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.) can be used, and one or more of these can be mixed and used. Among them, a water-ethanol-based mixed polar solvent (an ethanol aqueous solution of 30 to 70 v / v%) or ethanol is preferable, and an ethanol aqueous solution of 50 v / v% is more preferable. In addition, an acid or an alkali can be added to the above-mentioned extraction solvent to use a solvent with adjusted pH.

[0021] The extraction temperature is a temperature below the boiling point of the solvent used for extraction, and is not particularly limited. For example, it may be heat extraction or normal temperature extraction. The extraction time is 1 to 2 weeks in the case of normal temperature extraction, and 30 minutes to 24 hours, preferably 1 to 10 hours, in the case of heat extraction, but it can be appropriately adjusted according to conditions such as the type of extraction solvent and extraction temperature.

[0022] In the present invention, when using the filtration residue of the water extract of Rosa multiflora var. cathayensis as the extraction material, the extraction temperature is preferably 20°C or higher than the temperature of water extraction. Among them, when using water as the extraction solvent, it is preferably 70°C or higher, more preferably 80°C or higher, and most preferably 90°C or higher than the temperature of water extraction.

[0023] The extract may be used as the extracted solution as it is, but if necessary, within a range that does not affect its effect, it may be used after performing treatments such as concentration (concentration by an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, and ethanol precipitation. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, and freeze drying, and used as a dried product.

[0024] The solvent extract of Rosa multiflora var. carnea obtained in this manner, and the solvent extract of the filtration residue of the aqueous extract of Rosa multiflora var. carnea (hereinafter, these are collectively referred to as the "extract of Rosa multiflora var. carnea") have the effect of promoting the differentiation from epidermal stem cells to epidermal keratinocytes at the in vivo (inside the living body) or in vitro (in culture) level, and the effect of promoting the differentiation from dermal stem cells to dermal fibroblasts. Therefore, it can 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 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. Further, the differentiation promoter for skin stem cells of the present invention can also 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, and a medical reagent in vitro.

[0025] The skin stem cell differentiation promoter of the present invention contains an extract of Rosa multiflora var. cathayensis 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 impaired 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 impaired 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 healing of burns and wounds, rough skin, dry skin, sensitive skin, hyperkeratosis, melasma, freckles, dullness, enlarged pores, etc. Diseases or conditions caused by reduced or impaired 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. In addition, regardless of the cause of reduced or impaired 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.

[0026] The content of the extract of Rosa multiflora var. cathayensis in the skin stem cell differentiation promoter of the present invention varies depending on the properties of the extract (extract, 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.

[0027] 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. However, within the scope that does not impair the effects of the present invention, it can be formulated into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, food and drinks, etc., together with appropriate additives, and provided as a composition for skin regeneration. In particular, it is preferably formulated into a composition for external use on the skin.

[0028] 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 produced 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 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, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances, etc.

[0029] 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.

[0030] 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, buffers, solubilizers or solubilizing aids, isotonic agents, pH adjusters, propellants, colorants, 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 produced 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].

[0031] 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., injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories, ointments, lotions, sprays, transdermal absorption agents, transmucosal absorption agents, patches, and other parenteral preparations. Also, it may be a dry product that is redissolved when used, and in the case of injectable preparations, it is provided in the form of unit-dose ampoules or multi-dose containers.

[0032] When the skin stem cell differentiation promoter according to the present invention is used as a pharmaceutical for treating, improving, and preventing the skin diseases and conditions, a suitable form is a topical preparation, for example, ointments, creams, gels, liquids, patches (poultices, plasters), foams, sprays, aerosols, etc. An ointment refers to a homogeneous semi-solid topical preparation and includes oleaginous ointments, emulsion ointments, and water-soluble ointments. 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 and includes lotions, suspensions, emulsions, liniments, etc.

[0033] 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.

[0034] For parenteral administration preparations, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, vegetable oil, etc.; tonicity 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.

[0035] 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. When used as a pharmaceutical for the treatment, improvement, and prevention of the aforementioned diseases, it can be orally or parenterally administered to mammals such as humans, mice, rats, rabbits, dogs, and cats in a wide range of dosages.

[0036] 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 Rosa multiflora var. cathayensis.

[0037] 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 dry matter of the above extract of Rosa multiflora var. cathayensis, 0.001 to 30% by weight is preferable, and 0.01 to 10% by weight is more preferable, based on the total weight of the formulation (composition). 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 in consideration of workability.

[0038] In addition, the skin stem cell differentiation promoter of the present invention can also be incorporated into foods and drinks. In the present invention, foods and drinks are used in the meaning that, in addition to general foods and drinks, they include 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 use, nutritional functional foods, and functional display foods that can display specific health effects, functions of nutritional components, reduction of disease risks, etc., and can display the content reported to the Commissioner of the Consumer Affairs Agency regarding the 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 indicate 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 on a daily basis when it is necessary to take it over a long period of time 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 the containers, packages, instructions, and attached documents of the products, leaflets and pamphlets of the products, and advertisements of the products in newspapers and magazines, etc.

[0039] Furthermore, when the foods and drinks of the present invention are used for mammals other than humans, they can be used in the meaning including pet foods and feeds.

[0040] The form of the foods and drinks may be any form suitable for ingestion, for example, solid, liquid, granular, pellet, powdery, capsule, cream, or paste form. In particular, as the shape in the case of the above-mentioned health foods, etc., for example, tablet, round, capsule, powdery, granular, fine granular, lozenge, liquid (including syrup, milk, and suspension) forms are preferable.

[0041] 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 and processed oil and fat products, 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.

[0042] The food and drink products of the present invention may be appropriately blended with additives commonly used according to their types. Any additive that is acceptable under the Food Sanitation Law can be used. Examples of 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, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0043] When the food and drink products of the present invention are general food and drink products, they can be manufactured by including a step of adding the extract of Rosa multiflora Thunb. var. cathayensis Rehd. in the normal manufacturing process of the food and drink products. In the case of health foods, it may be carried out according to the manufacturing method of the above-mentioned pharmaceuticals. For example, in the case of tablet-shaped supplements, additives such as excipients are added to and mixed with the extract of Rosa multiflora Thunb. var. cathayensis Rehd., and then molded by applying pressure with a tableting machine or the like. In the case of capsule-shaped supplements, they can be manufactured by filling a liquid, suspension, paste, powder, or granular food composition containing the extract of Rosa multiflora Thunb. var. cathayensis Rehd. into capsules or coating and molding with capsule bases. Further, 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.

[0044] The blending amount of the extract of Rosa multiflora Thunb. var. cathayensis Rehd. 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, and 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.

[0045] 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 state of the subject to be ingested, the intake form, the intake amount, etc. As the extract of Rosa multiflora var. cathayensis, for adults, it is 0.1 to 1000 mg per day, 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.

[0046] 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 Rosa multiflora var. cathayensis. The extract of Rosa multiflora var. cathayensis used here may be an extract of untreated Rosa multiflora var. cathayensis, or may be an extract of Rosa multiflora var. cathayensis subjected to the aforementioned pretreatment. 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 by direct injection or the like to the site where the wound or tissue is desired to be regenerated 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).

[0047] 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.

[0048] 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, etc., is used, and at least one differentiation-inducing or promoting factor corresponding to the cells for the purpose of differentiation induction is added thereto. 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 growth 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.

[0049] 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 performing lot checking.

[0050] 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, the MF medium manufactured by Toyobo, and the 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), the medium for normal human fibroblasts (manufactured by DS Pharma Biomedical) and the like can be mentioned.

[0051] In accordance with the skin stem cell differentiation promoter according to the present invention or the method according to the present invention, the extract of Rosa multiflora Thunb. var. carnea Thory 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 above extract of Rosa multiflora Thunb. var. carnea Thory can be mixed with the medium and provided as a medium for promoting the differentiation of skin stem cells.

[0052] The incubator used for culturing skin stem cells is not particularly limited as long as it can culture stem cells. For example, flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles and the like can be mentioned. The incubator may be non-cell adhesive or cell adhesive, and is appropriately selected according to the purpose. For the purpose of improving the adhesiveness with cells, the cell adhesive incubator may be treated with a cell support substrate such as an extracellular matrix. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

[0053] The addition concentration of the Rosa multiflora extract to the medium used for culturing skin stem cells can be appropriately determined according to the content of the Rosa multiflora extract in the skin stem cell differentiation promoter according to the present invention described above. However, in terms of the dried 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 Rosa multiflora extract may be periodically added to the medium.

[0054] The culture conditions of 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%. In addition, 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 varied and set within the range where the stem cells can survive and proliferate.

[0055] The differentiation of epidermal stem cells into epidermal keratinocytes can be confirmed by determining whether the expression level of epidermal keratinocyte marker genes 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 presence of an extract of Rosa multiflora Thunb. var. carnea Thory, as compared to cells cultured in the absence of the extract of Rosa multiflora Thunb. var. carnea Thory. Similarly, the differentiation of dermal stem cells into dermal fibroblasts can be confirmed by determining whether the expression level of dermal fibroblast marker genes 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 presence of an extract of Rosa multiflora Thunb. var. carnea Thory, as compared to cells cultured in the absence of the extract of Rosa multiflora Thunb. var. carnea Thory. 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 of confirmation include, for example, RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific to each of the above marker genes. At the protein level, immunological methods such as ELISA, flow cytometry, and Western blotting using antibodies specific to the proteins encoded by each of the above marker genes can be mentioned.

Example

[0056] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited thereto.

[0057] [Example 1] Preparation of extract of Rosa multiflora Thunb. var. carnea Thory (Production Example 1) Preparation of a hot water extract of Rosa multiflora var. cathayensis without pretreatment To 10 g of the dried product of the fruit of Rosa multiflora var. cathayensis, 20 times the amount of water was added, and extraction was carried out at 95 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.2 g of a hot water extract of Rosa multiflora var. cathayensis (without pretreatment / hot water extract of Rosa multiflora var. cathayensis fruit).

[0058] (Production Example 2) Preparation of a 50% ethanol extract of Rosa multiflora var. cathayensis without pretreatment To 10 g of the dried product of the fruit of Rosa multiflora var. cathayensis, it was immersed in 20 times the amount of 50% ethanol aqueous solution at 25 °C for 7 days for extraction. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.7 g of a 50% ethanol extract of Rosa multiflora var. cathayensis (without pretreatment / 50% ethanol extract of Rosa multiflora var. cathayensis fruit).

[0059] (Production Example 3) Preparation of an ethanol extract of Rosa multiflora var. cathayensis without pretreatment To 20 g of the dried product of the fruit of Rosa multiflora var. cathayensis, it was immersed in 20 times the amount of ethanol at 25 °C for 7 days for extraction. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.3 g of an ethanol extract of Rosa multiflora var. cathayensis (without pretreatment / ethanol extract of Rosa multiflora var. cathayensis fruit).

[0060] (Production Example 4) Preparation of a hot water extract of Rosa multiflora var. cathayensis with pretreatment To 10 g of the dried product of the fruit of Rosa multiflora var. cathayensis, 10 times the amount of water was added, and extraction was carried out at 5 °C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours and no further extraction occurred). After the obtained extract was filtered through filter paper, the residue was dried, 20 times the amount of water was added to the obtained dried residue, and extraction was carried out at 95 °C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.3 g of a hot water extract of Rosa multiflora var. cathayensis (with pretreatment / hot water extract of Rosa multiflora var. cathayensis fruit).

[0061] (Production Example 5) Preparation of a 50% ethanol extract of Rosa multiflora var. cathayensis with pretreatment To 10 g of the dried fruit of Rosa multiflora var. cathayensis, 10 times the amount of water was added, and extraction was carried out at 5°C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours, and no further extraction occurred). After filtering the obtained extract through filter paper, the residue was dried, and the obtained dried residue was immersed in 20 times the amount of 50% ethanol aqueous solution at 25°C for 7 days for extraction. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 0.4 g of a 50% ethanol extract of Rosa multiflora var. cathayensis (with pretreatment / 50% ethanol extract of Rosa multiflora var. cathayensis fruit).

[0062] (Production Example 6) Preparation of ethanol extract of Rosa multiflora var. cathayensis with pretreatment To 20 g of the dried fruit of Rosa multiflora var. cathayensis, 10 times the amount of water was added, and extraction was carried out at 5°C for 24 hours (the increase in the solid content concentration of the extract stopped within 6 hours, and no further extraction occurred). After filtering the obtained extract through filter paper, the residue was dried, and the obtained dried residue was immersed in 20 times the amount of ethanol at 25°C for 7 days for extraction. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 0.2 g of an ethanol extract of Rosa multiflora var. cathayensis (with pretreatment / ethanol extract of Rosa multiflora var. cathayensis fruit).

[0063] [Example 2] (Experimental Example 1) Evaluation of the promoting effect of Rosa multiflora var. cathayensis extract on the differentiation of epidermal stem cells As epidermal-derived cells, commercially available normal human adult epidermal keratinocytes (manufactured by Kracie Pharmaceuticals) were used, 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 epidermal stem cells maintained in Humedia-KG2 medium (manufactured by Kracie Pharmaceuticals) were adjusted to a cell number of 5×10 5They were seeded in 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, the test substances (extracts of Rosa multiflora Thunb. var. cathayensis Rehd. et Wils. from Production Examples 1 to 6) were added so that the final concentration was 50 μg / mL, and then cultured for 72 hours. Also, after the cells had attached, cells that were induced to differentiate by adding 1.5 mM of CaCl2 without adding the test substance and culturing for 72 hours were used as the positive control. After the culture 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 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 FLG (filaggrin), IVL (involucrin), and KRT10 (keratin 10) were analyzed. The PCR reaction was initially denatured at 95°C for 2 minutes, and then 40 cycles were performed 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.

[0064] (Primer set for FLG) 5'-TCGAAGGAGCCAAAAATATAAAACAG-3’ (SEQ ID NO: 1) 5'-GAATTCCAATAGAAGGATAATAGAGAAAGATG-3’(SEQ ID NO: 2)

[0065] (Primer set for IVL) 5'-CCATCAGGAGCCAAATGAAACAG-3’(SEQ ID NO: 3) 5'-GCTCGACAGGCACCTTCTG-3’(SEQ ID NO: 4)

[0066] (Primer set for KRT10) 5'-ACTGAAGAGCTGGCCTATCTGAA-3’(SEQ ID NO: 5) 5'-CATCACCAGTGGACACATTTCG-3’(SEQ ID NO: 6)

[0067] (Primer set for GAPDH (internal standard)) 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 7) 5'-TCTTCTGGGTGGCAGTGATG-3' (SEQ ID NO: 8)

[0068] 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 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 the values for the cells cultured without the test substance (without differentiation induction) (control) set to 100. Then, the relative gene expression levels of FLG, IVL, and KRT10 in the cells cultured with the test substance were calculated and evaluated. The results of these tests are shown in Table 1 below.

[0069]

Table 1

[0070] As shown in Table 1, it was confirmed that the extract of Rosa multiflora Thunb. has the effect of promoting the differentiation of epidermal stem cells even without a differentiation inducer (CaCl2). When 50% ethanol was used as the extraction solvent, the differentiation-promoting effect was high, and particularly for the extract of Rosa multiflora Thunb. with pretreatment, a higher differentiation-promoting effect was obtained.

[0071] (Experimental Example 2) Evaluation of the effect of the extract of Rosa multiflora Thunb. on the differentiation of dermal stem cells Using commercially available human dermal 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) were adjusted to a cell number of 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 allowing the cells to attach, the test substances (extracts of Rosa multiflora Thunb. from Production Examples 1 to 6) 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, TGFβ (10 ng / mL, manufactured by Pepro Tech), which is generally used as a differentiation inducer for fibroblasts, was added and cultured for 48 hours to induce differentiation, and the cells were used as a positive control. After the culture was completed, mRNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). For this mRNA, after performing cDNA synthesis by reverse transcription reaction 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 initially denatured at 95°C for 2 minutes, and then using the following primer sets, 40 cycles were performed with 95°C for 15 seconds and 60°C for 60 seconds as one cycle. Other operations were carried out according to the defined methods.

[0072] (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)

[0073] 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. The value of 100 was set for these ratios. 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.

[0074]

Table 2

[0075] As shown in Table 2, it was confirmed that the extract of Rosa multiflora Thunb. var. cathayensis Rehd. has the effect of promoting the differentiation of dermal stem cells even without a differentiation inducer (TGFβ). When 50% ethanol was used as the extraction solvent, the effect of promoting differentiation was high, and particularly for the extract of Rosa multiflora Thunb. var. cathayensis Rehd. with pretreatment, a higher effect of promoting differentiation was obtained.

[0076] (Experimental Example 3) Evaluation of the effect of the extract of Rosa multiflora Thunb. var. cathayensis Rehd. 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 Rosa multiflora Thunb. var. cathayensis Rehd. 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 indicator according to the method described in JP 2017-055721 A. The above 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 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, 1.5 mM of CaCl₂ was added to induce differentiation. As oxidative stress, 100 mM of hydrogen peroxide and a test substance (extracts of Rosa multiflora Thunb. var. cathayensis Rehd. from Production Examples 1 to 6) were added so that the final concentration was 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 substance were used as a positive control. After completion of the culture, mRNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). For this mRNA, after performing 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.

[0077] 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, 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 adding the test substance were calculated and evaluated. The results of these tests are shown in Table 3 below.

[0078]

Table 3

[0079] Note: The value of "[X]" in the translation of is not provided in the original text, so it remains as "[X]" in the translation.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), but when the extract of Rosa multiflora Thunb. (Production Examples 1 to 6) was added simultaneously, it was confirmed that the inhibition of epidermal stem cell differentiation by oxidative stress was improved. When 50% ethanol was used as the extraction solvent, the effect of improving the inhibition of differentiation was high, and particularly for the extract of Rosa multiflora Thunb. subjected to pretreatment, a higher effect of improving the inhibition of differentiation was obtained.

[0080] (Experimental Example 4) Evaluation of the effect of the extract of Rosa multiflora Thunb. 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 extract of Rosa multiflora Thunb. 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-A-2017-093383. Dermal stem cells maintained in DMEM medium containing 10% FBS (manufactured by Nacalai) were 2×10 4They were seeded in a 12-well plate (manufactured by Falcon) so as to be 12 wells. After culturing for 24 hours and the cells had attached, TGFβ, which is generally used as a differentiation inducer for fibroblasts, was added at 10 ng / mL (manufactured by Pepro Tech) to induce differentiation, and hydrogen peroxide at 50 mM as oxidative stress and the test substances (extracts of Rosa multiflora var. cathayensis of Production Examples 1 to 6) were added so that the final concentration became 50 μg / mL, followed by culturing 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 completion of the culture, RNA was isolated and extracted using RNAiso+ (manufactured by Takara Bio Inc.). For this RNA, after performing 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.

[0081] 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 as an internal standard, i.e., COL1A1 gene expression level / GAPDH gene expression level, FAP gene expression level / GAPDH gene expression level, with these set to 100. In contrast, the values of 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 4 below.

[0082]

Table 4

[0083] As shown in Table 4, the differentiation of dermal stem cells was suppressed by oxidative stress (hydrogen peroxide) (with differentiation induction / with hydrogen peroxide / without extract addition), but when the extract of Rosa multiflora Thunb. (Production Examples 1 to 6) was added simultaneously, it was confirmed that the suppression of dermal stem cell differentiation by oxidative stress was improved. When 50% ethanol was used as the extraction solvent, the effect of improving the suppression of differentiation was high, and in particular, for the extract of Rosa multiflora Thunb. subjected to pretreatment, a higher effect of improving the suppression of differentiation was obtained.

[0084] (Experimental Example 5) Evaluation of the effect of the extract of Rosa multiflora Thunb. on epidermal stem cell differentiation disorder caused by stretching stimulation 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 Rosa multiflora Thunb. was allowed to act was evaluated. As epidermal-derived cells, commercially available normal human adult epidermal keratinocytes (manufactured by Kracie Pharmaceuticals) were used, 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 2017-055721 A. 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 [number of cells] per mL on a stretch chamber (manufactured by STREX), and Humedia-KG2 medium was added, followed by culturing for 24 hours. After 24 hours, 1.5 mM of CaCl2 was added to induce differentiation, and the test substances (extracts of Rosa multiflora Thunb. var. cathayensis Rehd. from Production Examples 1 to 6) were added to a final concentration of 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). Also, 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 was performed by reverse transcription reaction 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.

[0085] 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 induction of differentiation) (control) to the expression level of GAPDH, which was used as an internal standard, and the values of 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. In contrast, the relative gene expression values of FLG, IVL, and KRT10 in cells cultured with / without adding the test substance (with induction of differentiation) were calculated and evaluated. The results of these tests are shown in Table 5 below.

[0086]

Table 5

[0087] 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). However, when the extract of Rosa multiflora Thunb. (Production Examples 1 to 6) was added simultaneously, it was confirmed that the suppression of epidermal stem cell differentiation by stretching stimulation was improved. When 50% ethanol was used as the extraction solvent, the effect of improving the suppression of differentiation was high. In particular, for the extract of Rosa multiflora Thunb. subjected to pretreatment, a higher effect of improving the suppression of differentiation was obtained.

[0088] (Experimental Example 6) Evaluation of the effect of the extract of Rosa multiflora Thunb. on the impairment of dermal stem cell differentiation 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 effect of stretching stimulation on the induction of dermal stem cell differentiation was analyzed, and the effectiveness of the extract of Rosa multiflora Thunb. was evaluated when it was allowed to act. As dermal-derived cells, commercially available human skin fibroblasts (manufactured by Toyobo Co., Ltd.) were used, and dermal 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-2017-093383. 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, which is generally used as a differentiation inducer for fibroblasts at a concentration of 10 ng / mL (manufactured by Pepro Tech), was added to induce differentiation. The test substances (extracts of Rosa multiflora Thunb. var. carnea Thory from Production Examples 1 to 6) were added to achieve a final concentration of 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, cDNA synthesis was performed by reverse transcription reaction 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 COL1A1 and FAP were analyzed in the same manner as in Experimental Example 2.

[0089] 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 values 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.

[0090]

Table 6

[0091] 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). However, when the extract of Rosa multiflora Thunb. var. cathayensis Rehd. (Production Examples 1 to 6) was added simultaneously, it was confirmed that the suppression of dermal stem cell differentiation by stretching stimulation was improved. When 50% ethanol was used as the extraction solvent, the effect of improving the suppression of differentiation was high. In particular, for the extract of Rosa multiflora Thunb. var. cathayensis Rehd. subjected to pretreatment, a higher effect of improving the suppression of differentiation was obtained.

Industrial Applicability

[0092] The differentiation promoter for 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 insufficiency 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 obtained by solvent extraction of Rosa multiflora var. carnea Thory as an active ingredient.

2. A promoter for promoting the differentiation of skin stem cells into skin cells, containing an extract obtained by solvent extraction of the filtration residue of the aqueous extract of Rosa multiflora var. carnea Thory as an active ingredient.

3. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 2, wherein the temperature of the aqueous extraction is 20°C or lower, and the extract obtained by solvent extraction of the filtration residue is an extract extracted at a temperature 20°C or higher than the temperature of the aqueous extraction.

4. The promoter for promoting the differentiation of skin stem cells into skin cells according to Claim 1 or 2, wherein the solvent is one or more selected from the group consisting of water, lower alcohols, and liquid polyhydric alcohols.

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 Rosa multiflora var. carnea Thory.

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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