Proliferation promoter and undifferentiated state maintenance agent for skin stem cells

Abstract

The objective is to discover a novel substance that has high proliferation-promoting activity for skin stem cells and to provide it as a proliferation-promoting agent for skin stem cells. [Solution] A skin stem cell proliferation promoter and undifferentiated state maintenance agent comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a skin stem cell growth promoter and an undifferentiated state maintenance agent.

Background Art

[0002] The skin is roughly divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. Among these, the epidermis, which is the outermost layer, is mainly composed of epidermal keratinocytes. The epidermal tissue consists of four different cell layers: the basal layer, the spinous layer, the granular layer, and the stratum corneum, in order from the bottom layer. Epidermal keratinocytes divide in the basal layer, move upward, lose their ability to divide, differentiate, reach the stratum corneum, and differentiate into corneocytes. The stratum corneum is the final differentiation product of epidermal keratinocytes and plays an important role in the barrier function against various environmental factors (such as ultraviolet rays and dryness) from the outside, and then eventually peels off from the skin. This process of proliferation and differentiation of epidermal keratinocytes is called turnover.

[0003] Epidermal keratinocytes maintain epidermal homeostasis through this turnover process, where cells are periodically regenerated. The starting point of this turnover is epidermal stem cells located in the basal layer, and they are thought to play a crucial role in this process. Epidermal stem cells specifically express high levels of marker genes such as ITGA6, ITGB1, and CD271 as undifferentiated cells. Cells that detach from the skin's basement membrane migrate to the upper layers and mature into keratinocytes while expressing differentiation markers such as KRT10, FLG, and IVL. In recent years, it has been reported that the number of these epidermal stem cells decreases with age (Non-Patent Literature 1), and that INHBA / Activin-A is a factor that suppresses the proliferation of epidermal stem cells (Non-Patent Literature 2). On the other hand, dermal fibroblasts are essential cells for maintaining youthful skin free from wrinkles and sagging. Dermal stem cells, which give rise to these dermal fibroblasts, are located directly beneath the dermal papillary layer. They proliferate and differentiate as needed, constantly supplying new dermal fibroblasts to the dermis, resulting in continuous regeneration of dermal tissue (Non-Patent Literature 3). Since dermal components such as collagen and elastin are actively produced by dermal fibroblasts derived from dermal stem cells, dermal stem cells, like epidermal stem cells, play an important role in skin regeneration. However, it has been shown that their number decreases with age (Non-Patent Literature 1). Skin aging is caused by a decline in the regenerative capacity of these skin stem cells and a lengthening of cell turnover. Therefore, if we can target these skin stem cells, especially epidermal stem cells which are the starting point of cell turnover, and dermal stem cells which are the source of dermal fibroblasts that produce dermal components such as collagen and elastin, and promote the proliferation of epidermal and dermal cells, thereby suppressing the decrease in stem cells due to aging, it is thought that we can promote more fundamental and sustainable anti-aging and regeneration of the skin. Furthermore, it is believed that culturing epidermal stem cells and dermal stem cells in vitro will enable the efficient acquisition of cultured skin, which is expected to be extremely beneficial in transplant medicine for burns and wounds, as well as in cosmetic applications.However, although there are reports on methods for promoting cell proliferation in epidermal keratinocytes (keratinocytes) and dermal fibroblasts (Patent Document 1, Patent Document 2, etc.), the proliferation promotion methods and their mechanisms specific to epidermal stem cells and dermal stem cells have not been generalized, and at present, there is no clear prospect for practical application.

[0004] Wurfbainia vera (Amomum krervanh) is a perennial herb belonging to the genus Amomum of the Zingiberaceae family. The crude drug Wurfbainia vera (white cardamom) is the dried mature fruit of Amomum kravanh or Amomum cardamomum. Wurfbainia vera is used as a crude drug, traditional Chinese medicine, and spice. It has been reported that the extract of Wurfbainia vera has an effect of promoting prostaglandin production (Patent Document 3) and an effect of extending neuronal processes (Patent Document 4). Myrtus communis is an evergreen shrub belonging to the genus Myrtus of the Myrtaceae family. It has been reported that the extract of the leaves and / or fruits of Myrtus communis has an effect of suppressing melanin production and has depigmenting activity (Patent Document 5), the essential oil (myrtle oil) of Myrtus communis has an effect of promoting fat accumulation by directly acting on adipose tissue (Patent Document 6), and the extract of Myrtus communis has a ceramidase inhibitory effect and can be used as a ceramide amount regulator (Patent Document 7). Ligusticum sinense is a perennial herb belonging to the genus Ligusticum of the Apiaceae family. The crude drug Ligusticum sinense (Chinese lovage) is the dried root and rhizome of this plant. It has been reported that the extract of Ligusticum sinense has a whitening effect due to tyrosinase inhibitory activity (Patent Document 8), an effect of promoting blood circulation in the skin (Patent Document 9), an effect of promoting lipolysis (Patent Document 10), etc.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] [Non-Patent Document 1] Akamatsu H. et al., Journal of Dermatology. 2016;Vol.43,No.3,pp.311-313, Age-related decrease in CD271+ cells in human skin [Non-Patent Document 2] Kawagishi-Hotta M. et al.,J Dermatol Sci. 2022;106(3), pp.150-158, Increase in inhibin beta A / Activin-A expression in the human epidermis and the suppression of epidermal stem / progenitor cell proliferation with aging [Non-Patent Document 3] Hasebe Y. et al., J. Dermatol. Sci., 2016,Vol.89, pp.205-207 [Summary of the Invention] [Problems that the invention aims to solve]

[0007] In view of the circumstances described above, the present invention aims to find a new substance that has high proliferation-promoting activity for skin stem cells and to provide it as a proliferation-promoting agent for skin stem cells. [Means for solving the problem]

[0008] As a result of diligent research to solve the above problems, the inventors of this invention have discovered that extracts from one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* have an excellent proliferation-promoting effect on skin stem cells while maintaining their undifferentiated state, and have completed the present invention.

[0009] In other words, the present invention encompasses the following inventions. (1) A skin stem cell proliferation promoter comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* as an active ingredient. (2) The skin stem cell proliferation promoter according to (1), wherein the extract of two or more plants is a mixture of an extract of Myrtus communis and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense. (3) The skin stem cell proliferation promoter according to (1) or (2), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (4) An agent for maintaining the undifferentiated state of skin stem cells, comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum erythrostictum* as an active ingredient. (5) The undifferentiated state maintenance agent for skin stem cells according to (4), wherein the extract of two or more plants is a mixture of an extract of Amaranthus erythrosora and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense. (6) The undifferentiated state maintenance agent for skin stem cells according to (4) or (5), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (7) An expression promoter for undifferentiated genes in skin stem cells, comprising an extract of one or more plants selected from Paeonia suffruticosa, Cinnamomum camphora, and Ligusticum chuanxiong as an active ingredient. (8) The expression promoter for undifferentiated genes in skin stem cells according to (7), wherein the extract of the two or more plants is a mixture of an extract of Paeonia suffruticosa and an extract of one or two plants selected from Cinnamomum camphora and Ligusticum chuanxiong. (9) The expression promoter for undifferentiated genes in skin stem cells according to (7) or (8), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (10) A composition for promoting the proliferation of skin stem cells, comprising the proliferation promoter for skin stem cells according to (1) or (2). (11) A method for culturing skin stem cells, comprising the step of culturing skin stem cells in a medium containing an extract of one or more plants selected from Paeonia suffruticosa, Cinnamomum camphora, and Ligusticum chuanxiong. (12) The method for culturing skin stem cells according to (11), wherein the extract of the two or more plants is a mixture of an extract of Paeonia suffruticosa and an extract of one or two plants selected from Cinnamomum camphora and Ligusticum chuanxiong. (13) The method for culturing skin stem cells according to (11) or (12), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. [Advantages of the Invention]

[0010] According to the present invention, there are provided a proliferation promoter and an undifferentiated state maintenance agent for skin stem cells that can efficiently proliferate epidermal stem cells and dermal stem cells while maintaining undifferentiation. Therefore, the proliferation promoter and undifferentiated state maintenance agent for skin stem cells of the present invention are 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, sagging, and reduction of firmness and elasticity) caused by dryness, ultraviolet rays, aging, etc., and can greatly contribute to the fields of regenerative medicine, regenerative cosmetology, and anti-aging. [Embodiments for Carrying Out the Invention]

[0011] Hereinafter, the present invention will be described in detail. 1. Skin Stem Cell Proliferation Promoter and Undifferentiated State Maintenance Agent The skin stem cell proliferation promoter and undifferentiated state maintenance agent according to the present invention contains, as an active ingredient, an extract of one or more plants selected from Bupleurum falcatum, Glycyrrhiza glabra, and Ligusticum chuanxiong.

[0012] In the present invention, each extract of Bupleurum falcatum, Glycyrrhiza glabra, and Ligusticum chuanxiong may be used alone, but it is preferable to use two or three of them in combination because the proliferation promoting effect and undifferentiated state maintaining effect on skin stem cells are enhanced. When using two or three of them in combination, the combination is not limited, but a mixture of an extract of Bupleurum falcatum and one or two extracts selected from Glycyrrhiza glabra and Ligusticum chuanxiong is preferable. Specifically, a mixture of Bupleurum falcatum extract and Glycyrrhiza glabra extract, a mixture of Bupleurum falcatum extract and Ligusticum chuanxiong, and a mixture of Bupleurum falcatum extract, Glycyrrhiza glabra extract, and Ligusticum chuanxiong can be mentioned. Also, the mixing ratio of each extract in the above mixture is not limited, but it is preferable that they are equal amounts (1:1 in the case of a mixture of two extracts, 1:1:1 in the case of a mixture of three extracts).

[0013] In the present invention, the "skin stem cell" is not particularly limited as long as it is a stem cell present in the epidermis, dermis, and subcutaneous adipose tissue. In the present invention, the "epidermal stem cell" refers to a cell capable of differentiating into epidermal keratinocytes (keratinocytes), and the "dermal stem cell" refers to a cell capable of differentiating into dermal fibroblasts. Also, the "adipose stem cell" refers to a cell capable of differentiating into adipocytes, chondrocytes, osteoblasts, etc. The origin of the skin stem cell 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.

[0014] (Bupleurum falcatum) The plant used in this invention, Wurfbainia vera (Amomum krervanh), is a perennial herbaceous plant belonging to the genus Amomum in the family Zingiberaceae. It is native to southwestern India and is mainly cultivated in tropical regions. Wurfbainia vera is a close relative of cardamom, the world's oldest spice, and is also called white cardamom. Wurfbainia vera is the source plant for the herbal medicine Wurfbainia vera (also known as Takotsu, Byakku, or Byakku). The plant used in this invention may be of the same genus or a closely related plant, and its place of origin is not a factor. In the present invention, the extract of *Hypericum frutescens* refers to an extract of a part of the plant, such as its leaves, stems, fruits, pericarp, flowers, flower buds, seeds, roots, rhizomes, etc., or the entire plant (whole plant), or a mixture thereof. However, in the present invention, the part used as the raw material for extraction is preferably the fruit, which is used as the crude drug "Hypericum frutescens (white bean stalk)".

[0015] (Myrtus communis) The myrtle (scientific name: Myrtus communis) used in this invention is an evergreen shrub native to the Mediterranean coast, belonging to the genus Myrtus in the family Myrtaceae. In this invention, the myrtle extract refers to an extract of a part of the plant, such as its flowers, fruits, seeds, leaves, branches, or roots, or the entire plant (whole plant), or a mixture thereof. However, in this invention, the whole plant is preferred as the raw material for extraction.

[0016] (Ligustichthys sinense) Ligusticum sinense, used in this invention, is a perennial herb belonging to the Apiaceae family and is a medicinal plant native to China. Ligusticum sinense is the source plant for the crude drug Kouhon, which is made from the dried roots and rhizomes of Ligusticum sinense and is used as an analgesic for headaches and other ailments. It contains phthalides, which play a part in its medicinal effects. The flowers are arranged in a plural inflorescence characteristic of the Apiaceae family, and white, umbrella-shaped flowers bloom from summer to early autumn. In this invention, the extract of Ligusticum sinense refers to an extract of a part of the plant, such as its flowers, fruits, seeds, leaves, stems, rhizomes, or roots, or the whole plant (whole plant), or a mixture thereof. However, in this invention, the parts used as extraction raw materials are preferably the roots and rhizomes, which are used as crude drugs.

[0017] (Extraction methods for each plant material) For the extraction of the above-mentioned Myrtus communis, Myrtus communis, and Ligustrum sinense, the plant material may be used as is, or it may be processed by drying, crushing, or finely chopping.

[0018] The extraction method for extracts from Myrtus communis, Myrtus communis, and Ligustrum sinense is not particularly limited and may be any of the following: a heat extraction method (e.g., 40-100°C), a room temperature extraction method (e.g., 15-25°C), or a low temperature extraction method (e.g., 0-15°C). Examples of solvents used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are used, and more preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol are used. These solvents may be used individually or as a mixture of two or more. Particularly preferred extraction solvents include water, a water-ethanol mixed polar solvent, or a water-1,3-butylene glycol mixed polar solvent. Among these, it is preferable to contain 20 to 100% by weight of ethanol or 1,3-butylene glycol, and most preferably 50 to 100% by weight. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0019] Using the solvent described above, *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* are subjected to solvent extraction. There are no particular limitations on the amount of solvent used; it should be 5 times or more, preferably 10 times or more, relative to the dry weight of the extraction material. However, for convenience in operations such as concentration or isolation after extraction, it is preferable to use 100 times or less. The extraction temperature and time can be appropriately selected depending on the type of solvent used and the pressure during extraction.

[0020] The above extract may be used as is, but if necessary, it may be used after treatment such as concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization and deodorization with activated carbon, ethanol precipitation, etc., to the extent that the effects of the present invention are achieved. Furthermore, the extracted solution may be treated by concentration to dryness, spray drying, freeze-drying, etc., and used as a dried product.

[0021] The extracts of Myrtus communis, Myrtus communis, and Ligustrum sinense obtained in this manner have the effect of proliferating skin stem cells at the biological level (in vivo) or at the culture level (extra vivo). Therefore, the skin stem cell proliferation promoter of the present invention can be incorporated into and applied to pharmaceuticals, quasi-drugs, cosmetics, food and beverages, etc., as a drug to proliferate skin stem cells when administered to mammals including humans (monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats, pigs, etc.).

[0022] Furthermore, extracts of *Myrtus communis*, *Myrtus nipponicus*, and *Ligustrum lucidum* have the effect of maintaining the undifferentiated state of skin stem cells and promoting the expression of undifferentiated genes in skin stem cells, and can therefore be used as agents for maintaining the undifferentiated state of skin stem cells and as agents for promoting the expression of undifferentiated genes in skin stem cells. Examples of undifferentiated genes in skin stem cells include CD271, PROCR, and NANOG. In this invention, "promotion of undifferentiated gene expression" means promoting the expression of the above undifferentiated genes at the biological or culture level. In addition, in this invention, "promotion of undifferentiated gene expression" means promoting mRNA expression and protein expression of the above undifferentiated genes. Furthermore, extracts of *Myrtus communis*, *Myrtus communis*, and *Ligustrum lucidum* can also be used as culture medium additives for promoting the proliferation of skin stem cells and for producing skin stem cells, as well as as research reagents and medical reagents.

[0023] The skin stem cell proliferation promoter of the present invention is effective in treating, improving, and preventing diseases or conditions caused by the inability to form epidermal keratinocytes or dermal fibroblasts normally due to decreased or impaired proliferative capacity of epidermal stem cells or dermal stem cells. Diseases or conditions caused by the inability to form epidermal keratinocytes normally due to decreased or impaired proliferative capacity of epidermal stem cells include, for example, atopic dermatitis, psoriasis (accompanied by erythema, scaling, and desquamation), delayed healing of burns and wounds, rough skin, dry skin, sensitive skin, keratinization, melasma, age spots, dullness, and enlarged pores. Furthermore, diseases or conditions resulting from the inability to form dermal fibroblasts normally due to decreased or impaired proliferative capacity of dermal stem cells include, for example, wrinkles, sagging, nasolabial folds, marionette lines, loss of firmness and elasticity, lack of moisture and luster, roughness, dullness, photoelastic fibrosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, linear cutaneous atrophy (striata), wounds, burns, pressure ulcers, scars, and birthmarks.

[0024] The amount of the above-mentioned plant extract in the skin stem cell proliferation promoter of the present invention is not particularly limited, but depending on the properties of the extract (extract, concentrate, or dried product), for example, 0.0001 to 10% by weight is preferred, and 0.001 to 1% by weight is more preferred. If it is less than 0.0001% by weight, the effect may not be sufficiently exhibited. On the other hand, if it exceeds 10% by weight, the enhancement of the effect is unlikely to be observed and it is uneconomical.

[0025] When administering the skin stem cell proliferation promoter of the present invention into a living organism, it can be administered as is. However, it can also be incorporated into various compositions such as cosmetics, quasi-drugs, pharmaceuticals, and food and beverages with appropriate additives, as long as the effects of the present invention are not impaired, and provided as a composition for promoting the proliferation of skin stem cells. The pharmaceuticals of the present invention also include drugs used in animals, i.e., veterinary drugs.

[0026] When the skin stem cell proliferation promoter of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of the following: aqueous solution, solubilized, emulsified, powder, powder dispersion, oil-liquid, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, such cosmetics and quasi-drugs can be manufactured by selecting and appropriately incorporating various components, additives, bases, etc., commonly used in topical skin compositions, along with the skin stem cell proliferation promoter of the present invention, according to the methods known in the art. The form may be any of the following: liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients in topical skin compositions include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), and esters (isopropyl myristate, palmitic acid, etc.). Examples of ingredients include isopropyl tinate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, humectants, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0027] Examples of cosmetics and quasi-drugs include lotions, emulsions, gels, serums, general creams, sunscreens, face masks, facial cleansers, cosmetic soaps, foundations, face powders, bath additives, body lotions, and body shampoos.

[0028] When incorporating the skin stem cell proliferation promoter of the present invention into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations suitable for application to the affected area. Pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and application, formulation bases or carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances, etc., which can be appropriately added to prepare various formulations that can be administered orally or parenterally systemically or topically by various known methods. When providing the pharmaceutical product of the present invention in any of the above forms, it can be manufactured by methods commonly used by those skilled in the art, such as those indicated in the individual articles of the General Provisions for Formulations of the Japanese Pharmacopoeia [2].

[0029] The form of the pharmaceutical product of the present invention is not particularly limited, but examples include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injectable preparations (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, suppositories, ointments, lotions, eye drops, sprays, transdermal preparations, transmucosal preparations, and patches. It may also be provided as a dried product that is redissolved before use, and in the case of injectable preparations, it is provided in the form of unit dose ampoules or multi-dose containers.

[0030] Oral formulations may use, but are not limited to, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, starch, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium dioxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

[0031] Parenteral formulations may use, but are not limited to, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, and vegetable oil; isotonic agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, or organic bases.

[0032] When the skin stem cell proliferation promoter of the present invention is used as a pharmaceutical for treating, improving, and preventing skin-related injuries and diseases, the most suitable form is a topical formulation, such as ointments, creams, gels, liquids, patches (poultices, plasters), foams, sprays, and aerosols. Ointments refer to homogeneous, semi-solid topical formulations and include oily ointments, emulsion ointments, and water-soluble ointments. Gels refer to topical formulations in which a water-insoluble water-holding compound is suspended in an aqueous solution. Liquids refer to liquid topical formulations and include lotions, suspensions, emulsions, liniments, and the like.

[0033] The pharmaceutical product of the present invention functions as a preventive agent to suppress the onset of the above-mentioned diseases and / or as a therapeutic agent to improve them to a normal state. When the skin stem cell proliferation promoter of the present invention is used as a pharmaceutical product for the treatment, improvement, and prevention of 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.

[0034] The dosage of the pharmaceutical product of the present invention can be appropriately determined according to the type of disease, the age, sex, weight, and severity of symptoms of the recipient. For example, when administered orally to an adult, the daily dose is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg, of the plant extract.

[0035] The content of the skin stem cell proliferation promoter of the present invention in the cosmetics, quasi-drugs, and pharmaceuticals of the present invention is not particularly limited, but it is preferably 0.001 to 30% by weight, and more preferably 0.01 to 10% by weight, relative to the total weight of the formulation (composition). The above amounts are merely examples and should be set and adjusted as appropriate considering the type and form of the composition, the general amount used, efficacy and effects, etc. Furthermore, the method of adding the active ingredient in formulation may be added in advance or added during manufacturing, and the appropriate method should be selected considering workability.

[0036] Furthermore, the skin stem cell proliferation promoter of the present invention can also be incorporated into food and beverages. Providing it in the form of food and beverages makes it easy to ingest the active ingredients of the present invention on a daily or continuous basis. In the present invention, "food and beverages" is used to mean not only general food and beverages, but also foods other than pharmaceuticals that can be consumed for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or foods for special dietary uses. Health foods include foods provided under names such as nutritional supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act and include Foods for Specified Health Uses and Foods with Nutrient Function Claims, which can display specific health effects, the functions of nutritional components, and reduction of disease risk. The form of food and beverages may be any form suitable for consumption, such as solid, liquid, granular, granular, powder, capsule, cream, or paste.

[0037] Examples of food and beverage products include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock products, oils and fats and processed oils and fats products, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated liquids and powders for adjusting such beverages.

[0038] The food and beverages of the present invention may contain additives that are commonly used depending on their type. Any additive that is permissible from a food hygiene standpoint can be used, but 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, flavorings, colorings, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0039] The amount of the above-mentioned plant extract in the food and beverage of the present invention should be an amount that can exert a skin stem cell proliferation-promoting effect, but it should be set appropriately considering the general intake amount of the food and beverage, the form of the food and beverage, efficacy and effects, taste, palatability, and cost.

[0040] 2. Method for culturing skin stem cells The present invention also relates to a method for culturing skin stem cells, comprising the step of culturing skin stem cells in a medium containing an extract of one or more plants selected from mulberry, myrtle, and ligustycum sinense.

[0041] In the culture method of the present invention, commercially available epidermal keratinocytes and fibroblasts, preferably primary cultured epidermal keratinocytes and fibroblasts, can be used as skin stem cells. Stem cells and progenitor cells can be isolated from these cells using FACS or the like, with the expression of a marker to evaluate the undifferentiated nature of stem cells as an indicator. Alternatively, immortalized cells that proliferate indefinitely while maintaining stem cell properties may be used as skin stem cells. The immortalization method is not limited to any method that immortalizes cultured cells without inducing cell death, but examples include introducing an immortalization gene into skin stem cells. Here, "immortalization gene" refers to a gene that immortalizes cells and gives them the ability to proliferate indefinitely. Examples include the telomerase reverse transcriptase (TERT) gene, genes that regulate telomerase expression or activity (e.g., Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.). The telomerase reverse transcriptase (TERT) gene is preferred, and the human telomerase reverse transcriptase (hTERT) gene is more preferred.

[0042] In the culture method of the present invention, the culture medium for culturing skin stem cells and the additives used simultaneously are not particularly limited, and any culture medium and additives commonly used for the proliferation of skin stem cells (epidermal stem cells, dermal stem cells, adipose stem cells) may be used.

[0043] Specifically, the culture medium used for culturing skin stem cells includes basic media containing components necessary for stem cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, and fatty acids), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), and Hank's balanced salt solution. Furthermore, to increase the rate of cell proliferation, the above culture medium may contain, as needed, growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27 supplement, N2 supplement, ITS supplement, etc., and may also contain antibiotics (penicillin, streptomycin, etc.). Each component of the culture medium should be sterilized by an appropriate method before use.

[0044] In addition to the above, it is preferable that the product contains serum (e.g., 10% FBS) at a concentration of 1-20%. However, since the composition of serum varies depending on the lot and its effectiveness can vary, it is preferable to use the product after performing a lot check.

[0045] Commercial culture media can be used to culture skin stem cells. Examples of commercially available media include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hank's balanced salt solution.

[0046] In accordance with the skin stem cell proliferation promoter or culture method of the present invention described above, the above-mentioned plant extract can also be provided alone, separately from or mixed with a culture medium, as a reagent kit for promoting the proliferation of skin stem cells. The kit may include instructions for use, if necessary. Alternatively, the above-mentioned plant extract can be mixed with a culture medium and provided as a culture medium for promoting the proliferation of skin stem cells.

[0047] The culture vessel used for culturing skin stem cells is not particularly limited as long as it is capable of culturing stem cells, but examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. The culture vessel may be cell-non-adherent or cell-adherent, and can be appropriately selected according to the purpose. Cell-adherent culture vessels may be treated with cell-supporting substrates such as extracellular matrix to improve adhesion to cells. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0048] The concentration of the above-mentioned plant extract added to the culture medium used for culturing skin stem cells can be appropriately determined in accordance with the content of the above-mentioned plant extract in the skin stem cell proliferation promoter according to the present invention described above, but for example, concentrations of 1 to 1000 μg / mL, preferably 10 to 400 μg / mL, are possible. In addition, the above-mentioned plant extract may be added to the culture medium periodically during the stem cell culture period.

[0049] The culture conditions for skin stem cells can follow the usual conditions used for stem cell culture, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30-40°C, preferably about 36-37°C. The CO2 gas concentration is, for example, about 1-10%, preferably about 2-5%. The culture medium is preferably changed once every 2-3 days, and more preferably daily. The culture conditions can also be adjusted as appropriate within a range in which the stem cells can survive and proliferate.

[0050] The promotion of skin stem cell proliferation can be evaluated, for example, by determining whether the number of cells in a skin stem cell proliferation promoter cultured in the presence of the skin stem cell proliferation promoter according to the present invention is significantly increased compared to stem cells cultured in the absence of the skin stem cell proliferation promoter according to the present invention. The number of cells can be measured using a commercially available cell counting kit, for example, by the MTT method or WST method. If the number of stem cells cultured for a predetermined time in the presence of the skin stem cell proliferation promoter according to the present invention is greater than the relative ratio (control) when cultured in the absence of the skin stem cell proliferation promoter according to the present invention, it can be determined that the proliferation of skin stem cells has been promoted.

[0051] The culture method of the present invention allows for the efficient proliferation of skin stem cells. Furthermore, skin stem cells produced by this culture method can generally be transplanted directly into wounds or areas where tissue regeneration is desired after being cultured in vitro. In other words, skin stem cells produced by the culture method of the present invention can be used as transplant material (cell transplant agent). [Examples]

[0052] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0053] [Example 1] In production examples 1, 4, and 7, extracts of *Myrtus communis* were prepared as follows: in production examples 2, 5, and 8, extracts of *Myrtus communis* were prepared as follows: in production examples 3, 6, and 9, extracts of *Riguschikumushinense* were prepared as follows: mature fruits were used for *Myrtus communis*, the whole plant for *Myrtus communis*, and the roots and rhizomes (kouhon) of *Riguschikumushinense*.

[0054] (Manufacturing Example 1) Preparation of Hot Water Extract of Amaranthus erythrosora 10 g of dried mulberry fruit was added to 200 mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 0.4 g of hot water extract of mulberry.

[0055] (Production Example 2) Preparation of Myrtus communis hot water extract 10 g of dried whole myrtle plant was added to 200 mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 2.3 g of myrtle hot water extract.

[0056] (Production Example 3) Preparation of a hot water extract of Ligustichodium sinense 10 g of dried roots and rhizomes of Ligustichum sinense were added to 200 mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 3.3 g of hot water extract of Ligustichum sinense.

[0057] (Manufacturing Example 4) Preparation of a 50% ethanol extract of *Aster savatieri* 10 g of dried mulberry fruit was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the contents. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.5 g of 50% ethanol extract of mulberry.

[0058] (Manufacturing Example 5) Preparation of a 50% ethanol extract of Myrtus communis 10 g of dried whole myrtle plants were immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the extract. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 1.2 g of 50% ethanol extract of myrtle.

[0059] (Manufacturing Example 6) Preparation of a 50% ethanol extract of Ligustichodium sinense 10 g of dried roots and rhizomes of Ligustichum sinense were immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the extract. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 3.1 g of a 50% ethanol extract of Ligustichum sinense.

[0060] (Manufacturing Example 7) Preparation of Ethanol Extract of Amaranthus erythrosora 10 g of dried mulberry fruit was immersed in 200 mL of ethanol at room temperature for 7 days to extract the contents. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.02 g of mulberry ethanol extract.

[0061] (Manufacturing Example 8) Preparation of Ethanol Extract of Myrtus communis 10 g of dried whole myrtle plants were steeped in 200 mL of ethanol at room temperature for 7 days to extract the extract. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.5 g of myrtle ethanol extract.

[0062] (Production Example 9) Preparation of ethanol extract of Ligustichodium sinense 10 g of dried roots and rhizomes of Ligustichum sinense were immersed in 200 mL of ethanol at room temperature for 7 days to extract the extract. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.88 g of ethanol extract of Ligustichum sinense.

[0063] [Example 2] Evaluation of the proliferation-promoting effect of each extract on skin stem cells For the Epidermal stem cells, commercially available normal human epidermal keratinocytes (Kurabo Industries Ltd.) maintained in HuMedia-KG2 medium (Kurabo Industries Ltd.) were used in the experiment. For the Dermal stem cells, commercially available normal human fibroblasts (Kurabo Industries Ltd.) maintained in DMEM medium containing 10% FBS (Nacalai Tesque Corporation) were used in the experiment. These cells were cultured in 10 cm dishes, harvested with trypsin-EDTA, and then immunostained with the stem cell marker anti-CD271 antibody (Origene). From the stained cells, the CD271-positive cell fraction was isolated using FACS Melody (Becton Dickinson, Inc.), and epidermal stem cells and dermal stem cells were obtained.

[0064] The collected epidermal stem cells and non-epidermal stem cells were each placed in 96-well plates (FALCON) in a 3x10⁶ arrangement. 4Seeds were sown at a rate of 1 / well. The following day, each sample (extracts from Production Examples 1-9 and mixtures of two or three of them) was added to the culture medium so that the final concentration of each sample was 10 μg / mL, and the culture was incubated for 48 hours. For the mixed samples, in the case of a two-sample mixture, a mixture of each sample at 50% by weight was used, and in the case of a three-sample mixture, a mixture of each sample at 33.3% by weight was used, and each mixture was added to the culture medium so that the final concentration of each mixture was 10 μg / mL.

[0065] The culture medium was replaced with a chromogenic reagent (CCK8: Dojindo Co., Ltd.), and the cell proliferation-promoting effect was evaluated by measuring the relative number of epidermal and dermal stem cells cultured under the sample-added conditions compared to the number of epidermal and dermal stem cells cultured under the sample-free conditions (control) (set as 100%). The results of these tests are shown in Table 1 below.

[0066] [Table 1]

[0067] As shown in Table 1, extracts of *Myrtus communis*, *Myrtus nigra*, and *Ligustrum lucidum* all showed excellent proliferation-promoting effects on skin stem cells (epidermal stem cells and / or dermal stem cells). Furthermore, a significant synergistic effect on proliferation-promoting effects on skin stem cells was observed when *Myrtus communis* extract was used in combination with one or two of the *Myrtus communis* and *Ligustrum lucidum* extracts. On the other hand, these extracts did not show proliferation-promoting effects on non-epidermal stem cells and non-dermal stem cells, which are CD271-negative cell fractions.

[0068] [Example 3] Evaluation of the effect of each extract on maintaining the undifferentiated state of skin stem cells In Example 2, the epidermal stem cells and dermal stem cells were each placed in 12-well plates (FALCON) in a 1x10⁶ arrangement. 5Seeds were sown at a rate of 1 / well. The following day, each sample (extracts from Production Examples 1-9 and mixtures of two or three of them) was added to the culture medium to a final concentration of 10 μg / mL, and the culture was incubated for 24 hours. For the mixed samples, in the case of a two-sample mixture, a mixture of 50% by weight of each sample was used, and in the case of a three-sample mixture, a mixture of 33.3% by weight of each sample was used, and each mixture was added to the culture medium to a final concentration of 10 μg / mL.

[0069] After culturing, the cells were washed twice with PBS(-), and RNA was extracted from the cells using Trizol Reagent (Invitrogen). The extracted RNA was reverse transcribed into cDNA using a 2-STEP Real-Time PCR Kit (Applied Biosystems). Real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was then performed using an ABI7300 (Applied Biosystems) with the following primer set. In addition to the expression level of CD271, an undifferentiated marker used for sorting, the expression levels of PROCR, another epidermal stem cell marker, and NANOG, another dermal stem cell marker, were evaluated as indicators of undifferentiation. Other procedures were performed according to the prescribed method.

[0070] Primer set for CD271: 5'-CATCCTGGCTGCTGTGGTT-3'(Sequence ID 1) 5'-TGCAGCTGTTCCACCTCTTG-3'(Sequence ID 2) PROCR Primer Set: 5'-GTCTTCTTCGAAGTGGCTGTG-3'(Sequence ID 3) 5'-TTGTTTGGCTCCCTTTCGTG-3'(Sequence ID 4) Primer set for NANOG: 5'-CCTTCCTCCATGGATCTGCTT-3'(Sequence ID 5) 5'-AAGTGGGTTGTTTGCCTTTGG-3'(Sequence ID 6) Primer set for 18srRNA (internal standard): 5'-CCGAGCCGCCTGGATAC-3'(Sequence ID 7) 5'-CAGTTCCGAAAACCAACAAAATAGA-3'(Sequence ID 8)

[0071] The expression of CD271, PROCR, and NANOG (hereinafter referred to as "undifferentiated marker genes") was evaluated by calculating the relative expression level of undifferentiated marker genes (undifferentiated marker gene expression level / 18srRNA gene expression level), which was calculated as the ratio of the mRNA expression level of undifferentiated marker genes in cells without the added sample to the expression level of the internal standard 18s ribosomal RNA (18srRNA), with the value set to 100. In contrast, the relative expression level of undifferentiated marker genes in cells cultured with the added sample was calculated and evaluated. The results of these tests are shown in Table 2 below.

[0072] [Table 2]

[0073] As shown in Table 2, extracts of *Myrtus communis*, *Myrtus nigra*, and *Ligustrum erythrostictum* all showed excellent undifferentiated state-maintaining effects on skin stem cells (epidermal stem cells and / or dermal stem cells). Furthermore, a significant synergistic effect was observed in the undifferentiated state-maintaining effect of skin stem cells when *Myrtus communis* extract was used in combination with one or two of the *Myrtus communis* and *Ligustrum erythrostictum* extracts. On the other hand, these extracts did not show an undifferentiated state-maintaining effect on non-epidermal stem cells and non-dermal stem cells, which are CD271-negative cell fractions. [Industrial applicability]

[0074] The skin stem cell proliferation promoter of the present invention can promote proliferation of skin stem cells in vivo or in vitro while maintaining their undifferentiated state. Therefore, the present invention can be used in the field of manufacturing cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and conditions caused by dysfunction or failure of epidermal stem cells and dermal stem cells, as well as in the field of manufacturing transplant materials for regenerative medicine and regenerative beauty.

Claims

1. A skin stem cell proliferation promoter comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* as an active ingredient.

2. The skin stem cell proliferation promoter according to claim 1, wherein the extracts of two or more plants are a mixture of an extract of Myrtus communis and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense.

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

4. An agent for maintaining the undifferentiated state of skin stem cells, comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum sinense* as an active ingredient.

5. The undifferentiated state-maintaining agent for skin stem cells according to claim 4, wherein the extracts of two or more plants are a mixture of an extract of Myrtus communis and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense.

6. The undifferentiated state-maintaining agent for skin stem cells according to claim 4 or 5, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

7. An expression promoter for undifferentiated genes in skin stem cells, comprising an extract of one or more plants selected from *Myrtus communis*, *Myrtus communis*, and *Ligustrum erythrostictum* as an active ingredient.

8. The expression promoter of undifferentiated genes in skin stem cells according to claim 7, wherein the extract of two or more plants is a mixture of an extract of Myrtus communis and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense.

9. The skin stem cell expression promoter in skin stem cells according to claim 7 or 8, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

10. A composition for promoting the proliferation of skin stem cells, comprising the skin stem cell proliferation promoter described in claim 1 or 2.

11. A method for culturing skin stem cells, comprising the step of culturing skin stem cells in a culture medium containing an extract of one or more plants selected from mulberry, myrtle, and ligustycum sinense.

12. The method for culturing skin stem cells according to claim 11, wherein the extracts of two or more plants are a mixture of an extract of Myrtus communis and an extract of one or two plants selected from Myrtus communis and Ligustrum sinense.

13. The method for culturing skin stem cells according to claim 11 or 12, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

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