JP2019094268A - Undifferentiated state maintenance agent for hair follicle stem cells - Google Patents

Abstract

To find a novel factor that can be easily introduced into a living body and maintain the function of hair follicle stem cells to provide as a drug for preventing and/or ameliorating hair loss fundamentally and efficiently.SOLUTION: The present invention provides an undifferentiated state maintenance agent for Hair follicle stem cells, a promotor for gene expression of extracellular matrix protein, an agent for preventing and/or improving alopecia, as well as a hair composition, which are characterized by containing heat-treated-Panax ginseng extract after a steam heating treatment as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to an agent for maintaining undifferentiated state of hair follicle stem cells and use thereof.

  In recent years, due to various factors such as aging, stress and ultraviolet rays, hair problems such as thinning and hair loss are increasing. Hair covers the body surface and is one of the appendages of the skin, and among the hair, hair is a very important cosmetic factor. Therefore, thinning hair and hair loss have a negative impact on the psychological aspect, and become a factor that greatly reduces the quality of life (Quality of Life, QOL) (Non-Patent Document 1).

  The hair has a complex structure consisting of multiple types of cells, but is mainly produced by hair follicle stem cells present in the bulge region. From the telogen to the growth phase of the hair cycle, hair follicle stem cells present in this bulge region undergo cell division to form hair mother cells, and hair mother cells are formed from the hair mother cells.

  Although the detailed mechanism of thinning hair and hair loss has not been elucidated, recent research has shown that in conditions such as thinning hair and hair loss, hair follicle stem cells age with age, the ability to self-replicate is lost, and shrinkage of hair follicles occurs It is thought that hair follicle stem cells are damaged by environmental stress and the like for many years, resulting in failure of function or failure of maintenance mechanism. In recent years, research has also been conducted on the fine environment (stem cell niche) around stem cells, and it is also important that type 17 collagen, which is an extracellular matrix present around hair follicle tissue, is important in maintaining hair follicle stem cell function. It became clear (nonpatent literature 2). Furthermore, FGF2 and FGF7 produced from dermal papilla cells are known to be involved in hair regeneration and homeostasis maintenance (Non-patent Document 3).

  Therefore, for the amelioration of hair loss symptoms associated with aging etc., preventing the deterioration of hair follicle stem cells responsible for hair regeneration and homeostasis maintenance and restoring the function, and extracellular matrix around the stem cells It is considered important to properly improve environmental factors such as However, the above-described factors which have been reported so far are difficult to use on a daily basis because the means for control and introduction in vivo are complicated. Therefore, elucidation of new factors to replace them is desired.

  Panax ginseng (Scientific name: Panax ginseng CAMey) is a perennial plant belonging to the genus Ekogidae Ginseng (carrot) is a dried ginseng root, which contains ginsenosides of saponin, etc., tonic, sedative, It is used in traditional Chinese medicine that has anti-fatigue, cardiac and diuretic effects. Ginseng (carrot) has also been reported to have hypoglycemic action (Patent Document 1), angiotensin II activity inhibitory action (Patent Document 2), etc. alone or in combination with other plant extracts. However, the undifferentiated maintenance effect of hair follicle stem cells has never been known so far.

Japanese Patent Application Laid-Open No. 6-237735 JP, 2013-119534, A

Sawant N et al., Androgenetic Alopecia: Quality-of-life and Associated Lifestyle Patterns, Int J Trichology. 2010 Jul; 2 (2): 81-5 Matsumura H et al., Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. Science. 2016 Feb 5; 351 (6273) Rosenquist TA et al., Fibroblast growth factor signaling in the hair growth cycle: expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle. Dev Dyn. 1996 Apr; 205 (4): 379-86.

  In view of the above-mentioned situation, the present invention finds a new factor which can be easily introduced into a living body and can maintain the function of hair follicle stem cells, and prevents and / or prevents alopecia fundamentally and / or efficiently. The task is to provide as a drug for improvement.

  As a result of intensive studies to solve the above problems, the present inventors have found that the extract of Ginseng extracted after being subjected to pretreatment such as heat drying after steam heat treatment is present in the bulge region of the hair follicle. It has been found that hair follicle stem cells are maintained in an undifferentiated state and have an action to suppress differentiation, and that they have an action to promote the expression of extracellular matrix protein gene in hair follicle stem cells. Therefore, the extract of ginseng treated with the above-mentioned pretreatment can prevent the loss or depletion of hair follicle stem cells serving as a source of hair follicles, which are organs producing hair, and adjust the extracellular environment around hair follicle stem cells. It is effective for radical prevention and / or amelioration of alopecia. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) An agent for maintaining an undifferentiated state of stem cells, which comprises as an active ingredient an extract of heat-treated dried ginseng after steam heat treatment.
(2) The agent for maintaining an undifferentiated state of stem cells according to (1), wherein the stem cells are hair follicle stem cells.
(3) A preventive and / or ameliorating agent for alopecia characterized by containing an extract of heat-dried treated ginseng as an active ingredient after steam heat treatment.
(4) An expression promoter for an extracellular matrix protein gene, which comprises as an active ingredient an extract of heat-dried ginseng after steam heat treatment.
(5) The agent in any one of (1)-(4) whose said ginseng is dried or raw ginseng.
(6) A composition for hair comprising the agent for preventing and / or ameliorating alopecia according to (3).

  According to the present invention, an agent for maintaining the undifferentiated state of hair follicle stem cells and an agent for preventing and / or improving alopecia, which comprise the extract of heat-dried treated ginseng as an active ingredient after steam heat treatment, are provided. After steam heat treatment, the heat-dried extract of ginseng can prevent the loss or depletion of hair follicle stem cells, and adjust the extracellular environment around hair follicle stem cells necessary for hair follicle stem cell growth and maintenance. It is effective for radical prevention and / or amelioration of alopecia.

  The agent for maintaining undifferentiated state of stem cells of the present invention contains heat-treated and dried ginseng after steam heating treatment as an active ingredient. By applying the agent for maintaining undifferentiated state of stem cells according to the present invention to stem cells of mammals including humans, the undifferentiated state of stem cells can be maintained and differentiation can be suppressed. The stem cells to which the agent for maintaining an undifferentiated state of stem cells according to the present invention is applied are not particularly limited as long as they meet the object of the present invention, for example, embryonic stem cells (ES cells); bone marrow, blood, skin (epidermis) , Dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues somatic stem cells present; stem cells artificially produced by gene transfer etc. (artificial pluripotent Stem cells (iPS cells), but exert more effect on hair follicle stem cells. The agent for maintaining the undifferentiated state of stem cells according to the present invention can be applied to all mammalian stem cells as long as they have the same characteristics with respect to the directionality of the differentiation of stem cells, the process of differentiation and the like. For example, the agent for maintaining the undifferentiated state of stem cells according to the present invention is effective against mammalian stem cells such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats and pigs. Can be demonstrated.

  According to a preferred embodiment of the present invention, the stem cells are hair follicle stem cells, and the hair follicle stem cell undifferentiated state maintenance agent of the present invention comprises heat-treated and dried ginseng after steam heating treatment as an active ingredient, hair It can maintain the undifferentiated state of the hair follicle stem cells in the bulge region in the hair follicle that is the niche of the follicle stem cells (the ecologically appropriate place of the stem cells) and suppress the differentiation.

  In the present invention, “maintenance of the undifferentiated state of hair follicle stem cells” means maintenance of the undifferentiated nature of hair follicle stem cells and maintenance of functionality in the intra-hair follicle bulge region.

  The Panax ginseng (scientific name: Panax ginseng CAMey, aka: ginseng, ginseng, ginseng) used in the present invention is a perennial plant belonging to the genus Araliaceae, Arachiaceae (Panax). Ginseng, scientific name: Ginseng Radix) is an original plant. The dried ginseng root is used as a herbal medicine, but due to the difference in its production method, the skin of the root is peeled off and the dried "white ginseng" is steamed and dried with the skin being covered. It is divided roughly into "red ginseng".

  In the present invention, ginseng can be used as part of a plant or whole plant such as its leaves, stems, fruits, peels, flowers, flower buds, seeds, whole grass, roots, rhizomes, or any mixture of them. However, the root is preferred, and the lateral root portion of the root is more preferred.

  In the present invention, as ginseng, either dried ginseng or raw ginseng can be used, but dried ginseng is preferable. In the case of dried ginseng, those dried to a water content of 20% or less, preferably 10% or less are preferable. The water content can be measured using a method such as loss on drying of the Japanese Pharmacopoeia. The drying method is not particularly limited as long as the method is usually used as a method for drying a plant and the water content is in the above range, but for example, natural drying (air drying), sun drying, ventilation drying, hot air drying , Spray drying, vacuum drying, vacuum drying and the like.

  In the present invention, before the above-described dried ginseng or raw ginseng is extracted, steam heat treatment and heat drying treatment corresponding to a restoration treatment (processing to dry steaming) performed at the time of processing of a herbal medicine are performed. The steam heating process is a process of heating an object under a high humidity atmosphere, for example, under an atmosphere of humidity of 80% or more, using hot water, saturated steam, superheated steam, reduced pressure (vacuum) steam or the like as a heat medium. Steam heating may be performed by bringing the heat medium into direct contact with the object, or the object may be heated indirectly through a heat exchanger. The heating may be performed under normal pressure or under pressure. As conditions of steam heat processing, 70-180 ° C is preferred and 100-150 ° C is more preferred. Although time changes with temperature, 1 to 15 hours are preferable, 2 to 10 hours are more preferable, and 2 to 6 hours are further more preferable. These conditions of temperature and time are only examples and can be suitably changed by mutual relation of temperature and time. Further, the steam heat treatment in the present invention may be carried out using a continuous or batch type steamer (steamer), an autoclave or the like.

  Since steam-heated ginseng contains water, it is heated and dried. As temperature of heat-drying, 40-85 ° C is preferred, and 50-70 ° C is more preferred. As a drying method, ventilation drying, hot air drying, microwave drying or the like can be used. The drying time (heating time) varies depending on the heating temperature, the water content of the ginseng after steam heat treatment, and the total amount to be dried, and can not be specified, but is in the range of about 6 to 24 hours.

  After steam heat treatment as described above, the heat-treated dried ginseng is extracted. The extraction method is not particularly limited, and can be carried out by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. As the organic solvent, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1, 3-butylene glycol, propylene glycol, glycerin, etc.) , Ketones (acetone, methyl ethyl ketone etc.), acetonitrile, esters (ethyl acetate, butyl acetate etc.), hydrocarbons (hexane, heptane, liquid paraffin etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether etc.) Be Among them, polar solvents such as lower alcohols and liquid polyhydric alcohols are preferred, and water-soluble organic solvents such as ethanol, 1,3-butylene glycol and propylene glycol are more preferred, and one or more of these may be used. . Particularly preferred extraction solvents include water or mixed polar solvents of water-ethanol type. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more with respect to the above-mentioned ginseng (dry weight), but in the case of concentration or isolation after extraction For convenience of operation, it is preferably 100 times or less. Moreover, although extraction temperature and time are based on the kind of solvent to be used, they can be illustrated at, for example, 10 to 100 ° C., preferably 30 to 90 ° C., for 30 minutes to 24 hours, preferably 1 to 10 hours. In addition, the extract may be used as it is in the extracted solution, but if necessary, concentration (organic solvent, concentration by vacuum concentration, concentration by membrane concentration, etc.), dilution, filtration, to the extent that the effect is not affected. It may be used after processing such as decolorization with activated carbon, deodorization, ethanol precipitation and the like. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, lyophilization and the like, and used as a dried product.

  After steam heat treatment as described above, the extract of heat-treated ginseng (hereinafter sometimes referred to as "extract of Shuji ginseng") is differentiated at the biological level or at the culture level in hair follicle stem cell differentiation Since it has the effect | action to suppress an undifferentiated state, it can be used as an undifferentiated state maintainer of a hair follicle stem cell. In addition, the agent for maintaining the undifferentiated state of hair follicle stem cells of the present invention can also be referred to as an agent for suppressing differentiation of hair follicle stem cells or an agent for maintaining hair follicle stem cell functions.

  The extract of the modified ginseng can inhibit the reduction or depletion of the hair follicle stem cells that are the origin of the hair by suppressing the differentiation of the hair follicle stem cells in the bulge region in the hair follicle, and the gene expression of extracellular matrix protein in the hair follicle stem cells As it can be promoted to adjust the extracellular environment around hair follicle stem cells, it can be used as an active ingredient of a hair loss preventing and / or improving agent, an expression promoting agent of extracellular matrix protein gene.

  In the present invention, “preventing and / or ameliorating alopecia” includes preventing hair loss and thin hair generation, improving the degree (number and range) of hair loss and thin hair, decreasing the rate of hair loss or thin hair progression, hair loss and thin hair And the reduction of the reduction of the gloss and elasticity of the hair. In addition, the preventive and / or ameliorating effect of alopecia includes both the case of showing a direct action mechanism to hair and the case of showing a percutaneous action mechanism on the head.

  In the present invention, alopecia refers to a condition in which part or all of the hair is removed and part or all of the scalp is seen through, for example, due to aging, disease, various stress such as ultraviolet light or overwork. . Alopecia, for example, male pattern alopecia (AGA), diffuse alopecia (FAGA), alopecia areata, senile alopecia, seborrheic alopecia, epileptic alopecia, postpartum (postpartum) alopecia , Mechanical (compression or traction) alopecia, congenital alopecia, alopecia after burns or trauma, drug alopecia with anti-cancer drug, scarring alopecia There may be included, but not limited to, baldness, hair follicle folliculitis, papillary dermatitis of the head, etc.), trichotyllania (hair loss) and the like.

  Moreover, the preventive and / or ameliorating agent for alopecia of the present invention can be used as it is, but it is mixed with an appropriate additive and the like within the range that does not impair the effect of the present invention. It can be in the form of a composition such as cosmetics. Among them, a hair composition formulated in a formulation suitable for use on the scalp and hair is preferred.

  The hair composition may be prepared according to methods known in the art, selecting various components, additives, bases and the like usually used in the composition for external use for skin according to the types thereof, and combining them appropriately. it can. Ingredients to be blended, additives, base materials, for example, diluents (refined water, ethanol, etc.), fats and oils (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, vaseline etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid etc.), higher alcohols (myristyl alcohol, Setanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol etc., esters (isopropyl myristate, isopropyl palmitate, 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.), surfactant, silicone oil, moisturizer, thickener, UV light Absorbents, sequestering agents, refreshing agents, antioxidants, stabilizers, preservatives, anti-inflammatory agents, antiseptics, bactericides, fragrances, coloring agents and the like can be mentioned.

In addition, the above-mentioned composition for hair may contain a component conventionally known as a component of a hair-growing agent and a hair-growing agent, as long as the effect of the present invention is not adversely affected. For example, extract of plant extract such as cerebrum extract, citrus extract, vitamin B ₆ , vitamin E and derivatives thereof, vitamins such as biotin, pantothenic acid and derivatives thereof, glycyrrhizinic acid and derivatives thereof, nicotinate ester, serine, methionine, etc. Amino acids, ceforanthine, capronium chloride, minoxidil, nicorandil, acetylcholine derivatives, cyclosporins, female hormone agents such as estradiol, and the like, and mixtures thereof can be mentioned.

  In the present invention, the composition for hair generally refers to those used for scalp and hair, and any composition that can be applied to scalp and hair may be used, and there is no particular limitation on the dosage form. For example, any of liquid, emulsion, cream, gel, paste, spray and the like may be used. Specific product forms include creams, lotions, emulsions, ointments, gels, hair shampoos, hair rinses, hair treatments, hair conditioners, scalp treatments, hair sprays, hair packs, hair essences, hair tonics, hair liquids, hair mousses, etc. It can be mentioned.

  The content of the extract of modified ginseng in the composition for hair in the form of these products can not be specified because it varies depending on the form, but in general, relative to the total weight of the composition, it is from 0.0001 to It is 20% by weight (w / w), preferably 0.001 to 10% by weight (w / w). About the method of addition of Shuji ginseng, it may be added in advance or may be added during the production, and it may be appropriately selected in consideration of workability.

  "Extracellular matrix (extracellular matrix)" refers to a fibrillar or mesh-like structure present on the outside (between cells) of cells and controls cell shape retention and function (proliferation, differentiation, migration, etc.) Play an important role. In the present invention, "extracellular matrix protein" refers to a protein constituting an extracellular matrix, and includes collagen, fibrillin, elastin, laminin, fibronectin, tenascin, entactin, proteoglycan (decorin, versican, perlecan, aggrecan, etc.) More specifically, type VII collagen (COL7A1), type XVII collagen (COL17A1), laminin 332 (LAMA3, LAMB3, etc.) constituting the basement membrane of skin basement membrane and skin appendages (hair follicle, sweat gland, sebaceous gland etc.) LAMC 2), laminin 511 (LAMA5, LAMB1, LAMC1), decorin and the like.

  The extract of Shujin ginseng can also be used as a medium additive to maintain the undifferentiated state of hair follicle stem cells. Hair follicle stem cells cultured in a medium to which the medium additive has been added can survive for a certain period of time while maintaining their undifferentiated nature. The undifferentiated state maintenance period of cells can be appropriately changed according to the purpose of culture, the type of basal medium, the culture temperature and the like. For example, when an extract of Shujin ginseng is added to a culture medium and cultured under normal culture conditions, hair follicle stem cell undifferentiated at least 1 week or more, preferably at least 2 weeks or more, more preferably at least 1 month or more. It is possible to maintain the state. Thus, the present invention also provides a method for maintaining an undifferentiated state of hair follicle stem cells, which comprises the step of culturing hair follicle stem cells in a medium to which an extract of modified ginseng has been added. The method can be used, for example, to elucidate the mechanism of hair follicle stem cell maintenance and differentiation, the mechanism of thinning hair and / or hair loss, and the mechanism of action of an anti-hair loss agent.

  As a medium used for culturing hair follicle stem cells, a basal medium generally used for culturing conventional epithelial cells can be used except for the addition of an extract of modified ginseng. For example, a basal medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins and fatty acids) necessary for the survival and proliferation of hair follicle stem cells, such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Ham's F12, Ham's F12 K (Kaighn's modified of Ham's F12), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minim Essential Medium (Glasgow MEM), Hanks' solution (Hank's balanced salt solution), MCDB 153 medium and the like can be mentioned. In addition, growth media (epithelial growth factor (EGF), cholera toxin, basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), Stem Cell Factor (SCF), etc.) , Hydrocortisone etc.) is preferably added. Furthermore, if necessary, the medium may be tumor necrosis factor (TNF), vitamins, interleukins, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement And antibiotics may be contained.

  In addition to the above components, preferably, bovine pituitary extract (BPE), serum or serum substitute is contained in the medium at a content of 0.1 to 20% (v / v). The serum or serum substitute may be any known one. For example, serum includes fetal bovine serum (FBS, FCS) and the like, and serum substitutes include KSR and the like. However, since serum has different components due to differences in lots and variations in its effects, it is preferable to use after performing lot check.

  Media for epithelial cells containing components necessary for culturing epithelial cells (for example, insulin, hydrocortisone, epidermal growth factor, etc.) are commercially available, and these commercially available media may be used. For example, Keratinocyte-SFM medium (Thermo Fisher Scientific), Humedia-KG2 medium (KURABO), CnT-PR medium (CellnTec), MCDB153 medium (Sigma), serum-free medium for normal human keratinocytes (DS Pharma) Biomedical Inc.) can be used.

  The incubator used for culturing hair follicle stem cells is not particularly limited as long as it can culture hair follicle stem cells, and, for example, flasks, petri dishes, dishes, multiwell plates (eg, 6 wells, 12 wells, 24 wells) , 96 wells), chamber slides, tubes, trays, culture bags, roller bottles and the like. As materials for the incubator, polymer materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, nitrocellulose, cellulose acetate, cellulose acetate, polyethylene terephthalate, etc. Glass, ceramics, magnetic particles, metals and the like can be mentioned.

  The incubator may be noncell-adhesive or adherent, and is appropriately selected according to the purpose. The culture vessel for cell adhesion may be one coated with a substrate for supporting cells by extracellular matrix or the like for the purpose of improving adhesion to cells. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

  The addition concentration of the extract of modified ginseng to the culture medium used for hair follicle stem cell culture should be appropriately determined according to the content of the extract of ginseng in the undifferentiated state maintenance agent of hair follicle stem cells according to the present invention described above. The concentration is, for example, 10 to 10000 μg / mL, preferably 100 to 5000 μg / mL in terms of the amount of extract of Shuji ginseng. Also, during the stem cell culture period, extracts of modified ginseng may be periodically added to the medium.

The culture conditions of hair follicle stem cells may 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 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. In addition, it is preferable to change a culture medium once every two to three days, and it is more preferable to carry out every day. The culture conditions can also be set by appropriately changing the stem cells within the viable and proliferative range.

  The undifferentiated state maintenance of hair follicle stem cells is, for example, compared to the hair follicle stem cells cultured in the absence of the hair follicle stem cell undifferentiated state maintenance agent according to the present invention, the undifferentiated hair follicle stem cells according to the present invention In the stem cells cultured in the presence of the state maintenance agent, the expression level of the stem cell undifferentiation marker gene is evaluated whether it is significantly maintained at the mRNA or protein level to the same level as the expression level at the start of culture. be able to. Examples of hair follicle stem cell undifferentiated marker genes include KRT15 (Keratin, type I cytoskeletal 15) and the like.

  As a method of measuring the hair follicle stem cell undifferentiated marker gene expression level, methods such as RT-PCR, quantitative PCR, northern blotting, etc. using primers and probes specific for the hair follicle stem cell undifferentiated marker gene are used at the mRNA level, for example. Also, at the protein level, for example, immunological methods such as ELISA using a specific antibody for a protein encoded by the hair follicle stem cell undifferentiated marker gene, flow cytometry, western blotting and the like can be mentioned.

  As a result of measurement of the expression level, it is determined in the presence of the hair follicle stem cell undifferentiated marker gene expression level in the hair follicle stem cell at the start of culture (100% undifferentiated state) and the presence of the hair follicle stem cell undifferentiated state maintenance agent of the present invention. The relative ratio to the expression level of the hair follicle stem cell undifferentiated marker gene in hair follicle stem cells after long-term culture is the same phase contrast when cultured in the absence of the hair follicle stem cell undifferentiated state maintenance agent of the present invention (control It can be determined that the undifferentiated state of hair follicle stem cells can be maintained when the size is larger.

  Hereinafter, the present invention will be more specifically described by way of examples. However, the present invention is not limited to these.

Example 1 Production Example of Extract of Panax Ginseng Extract of Panax ginseng was produced as follows. Production Examples 2-a to 2-e were carried out in the same manner as Production Example 2 except that the temperature of steam heating was changed.

Preparation Example 1 Preparation of Hot Water Extract of Raw Ginseng Root (Steam Heating: 105 ° C., Heating and Drying: 50 ° C.) After steaming of freshly harvested ginseng (water content 80%) after harvest at 105 ° C. for 8 hours And dried at 50 ° C. (compatible with the Japanese Pharmacopoeia "red ginseng"). 800 mL of purified water is added to 40 g of the dried ginseng, and extracted for 2 hours at 95-100 ° C., followed by filtration, the filtrate is concentrated to dryness, and the steam heat treatment (105 ° C.) of fresh ginseng root 17.5 g of hot water extract was obtained.

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 105 ° C., Heat Drying: 50 ° C.) After steaming dried Ginseng root (water content 9%) at 105 ° C. for 8 hours, It was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted for 2 hours at 95-100 ° C., followed by filtration, and the filtrate is concentrated, lyophilized and steam-heated (105 ° C.) dried ginseng root 18.7 g of a hot water extract of

Preparation Example 3 Preparation of 50% ethanolic extract of dried ginseng root (vapor heating: 105 ° C., heating and drying: 70 ° C.) After steaming dried ginseng root (water content 9%) at 105 ° C. for 8 hours And dried at 70 ° C. After adding 500 mL of purified water and 500 mL of ethanol to 100 g of the dried ginseng, and extracting for 7 days at room temperature, the filtrate is concentrated to dryness and steam heated (105 ° C.) of dried ginseng root 43 g of 50% ethanol extract was obtained.

Preparation Example 4 Preparation of 1,3-Butylene Glycol Extract of Dried Panax ginseng Leaves and Stems (Steam Heating: 100 ° C., Heating and Drying: 60 ° C.) 100 Dried Panax Ginseng Leaves and Stems (10% Water Content) After steaming at 4 ° C for 4 hours, it was dried at 60 ° C. 200 mL of 1,3-butylene glycol is added to 20 g of the dried ginseng, and extracted for 7 days at room temperature, followed by filtration and steam heating (100 ° C.) of dried ginseng leaves and stems of 1,3-butylene glycol 141 g of extract was obtained.

(Comparative Production Example 1) Preparation of hot water extract of fresh ginseng root (without steam heating treatment) 800 mL of purified water is added to 40 g of dried fresh ginseng root after harvest at 65 ° C., and 95 to 100 After 2 hours of extraction at ° C., the filtrate was concentrated and lyophilized to give 12.5 g of a hot water extract of Ginseng root.

Comparative Preparation Example 2 Preparation of 50% Ethanol Extract of Raw Ginseng Root (Without Steam Heating Treatment) 500 mL of purified water and 500 mL of ethanol were added to 100 g of dried ginseng root after harvest at 65 ° C. After 7 days of extraction at room temperature, the filtrate was concentrated to dryness to obtain 15.1 g of a 50% ethanol extract of Ginseng roots.

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 70 ° C., Heated Drying: 50 ° C.) Dried ginseng root (water content 9%) was steamed at 70 ° C. for 8 hours Thereafter, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted at 95-100 ° C. for 2 hours, followed by filtration, the filtrate is concentrated, lyophilized and steam-heated (70 ° C.) dried ginseng root 17.2 g of a hot water extract of

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 100 ° C., Heat Drying: 50 ° C.) Dried ginseng root (water content 9%) was steamed at 100 ° C. for 8 hours Thereafter, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted at 95-100 ° C. for 2 hours, followed by filtration, the filtrate is concentrated, lyophilized, and steam-heated (100 ° C.) dried ginseng root 18.1 g of a hot water extract of

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 121 ° C., Heated Drying: 50 ° C.) Dried ginseng root (water content 7%) was steamed at 121 ° C. for 8 hours Thereafter, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted at 95-100 ° C. for 2 hours, followed by filtration, the filtrate is concentrated, freeze-dried and steam-heated (121 ° C.) dried ginseng root 17.6 g of a hot water extract of

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 150 ° C., Heated Drying: 50 ° C.) Dried ginseng root (water content 9%) was steamed at 150 ° C. for 8 hours Thereafter, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted for 2 hours at 95-100 ° C., followed by filtration, the filtrate is concentrated, freeze-dried and steam-heated (150 ° C.) dried ginseng root 19.0 g of a hot water extract of

Preparation Example 2 Preparation of Hot Water Extract of Dried Ginseng Root (Steam Heating: 180 ° C., Heated Drying: 50 ° C.) Dried ginseng root (water content 9%) was steamed at 180 ° C. for 8 hours Thereafter, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried ginseng, and extracted for 2 hours at 95-100 ° C., followed by filtration, the filtrate is concentrated, lyophilized and steam-heated (180 ° C.) dried ginseng root 18.6 g of a hot water extract of

[Example 2] Evaluation of maintenance effect of undifferentiated state on hair follicle stem cells of extract of Panax ginseng and expression promoting effect of extracellular matrix gene Undifferentiation of hair follicle stem cells using each extract of Panax ginseng prepared in Example 1 The evaluation of the sex maintenance state effect and the expression promoting effect of the extracellular matrix protein gene (hereinafter referred to as "the extracellular matrix gene") was performed.

(Experimental Example 1) Evaluation of maintenance effect of undifferentiated state on hair follicle stem cells (1) Culture of human hair follicle stem cells Human hair is collected by tweezers and tissues including bulge region of hair follicle tissue are collected using a female etc. did. After washing with PBS (−), treatment with trypsin (manufactured by BD Biosciences) was performed. Thereafter, cells were isolated and recovered using Cell Trainer (manufactured by FALCON). The recovered cells were seeded on a culture plate, and maintained until confluence using KG2 medium (manufactured by KURABO). Confluent cells were collected and replated on the same culture plate, and then engrafted and proliferated cells were used as hair follicle stem cells in the following test.

(2) Evaluation of the undifferentiated nature of hair follicle stem cells when adding ginseng extract The extract of ginseng is caused to act on hair follicle stem cells, and the extract of ginseng affects how undifferentiated of hair follicle stem cells It was evaluated. For undifferentiated cells, 1 × 10 ⁴ hair follicle stem cells cultured in KG2 medium (manufactured by KURABO) are seeded on a 24-well culture plate, and the extract of Panax ginseng (Production Example 1 to 4, Production Example 2-2 a to 2-e, Comparative Production Examples 1 and 2) are added and cultured so that the final concentration is 100 μg / ml, hair follicle stem cells are collected 48 hours after addition of the extract of Ginseng, and hair follicles The undifferentiated nature of stem cells was analyzed using the gene expression level of KRT15 (Keratin, type I cytoskeletal 15) as an index.

  The gene expression analysis of KRT15 was performed as follows. Hair follicle stem cells after addition of a ginseng extract were washed twice with PBS (−), and RNA was extracted from the cells with Trizol Reagent (manufactured by Invitrogen). After reverse transcription of the extracted RNA to cDNA using a 2-STEP real time PCR kit (manufactured by Applied Biosystems), real time PCR (95 ° C: 15 seconds) using the following primer set by ABI 7300 (manufactured by Applied Biosystems) At 60 ° C. for 30 seconds, and 40 cycles were performed, and the gene expression level of KRT15 was measured. Other operations were carried out according to the prescribed method.

Primer set for KRT15 (hair follicle stem cell undifferentiated marker):
5'- GATGCTGCTTGACATAAAGACAG-3 '(SEQ ID NO: 1)
5'- ACCTG TCC ATCC ACT GACT CT TC-3 '(SEQ ID NO: 2)
Primer set for 18srRNA (internal standard):
5'-CCGAGCCCGCCTGGATAC-3 '(SEQ ID NO: 3)
5'-CAGTTCCGAAAACCAACAAAATAGA-3 '(SEQ ID NO: 4)

  The undifferentiated state maintenance effect of hair follicle stem cells is calculated by expressing the expression level of KRT15 mRNA in hair follicle stem cells cultured without adding ginseng extract as a percentage of the expression level of 18s ribosomal RNA (18srRNA) as an internal standard. The value of the KRT15 gene relative expression level (KRT15 gene expression level / 18srRNA gene expression level) is 1, and the value of the gene relative expression level of KRT15 in hair follicle stem cells cultured by adding the extract of Ginseng Calculated and evaluated. The results are shown in Tables 1 and 2 below.

  As shown in Table 1, hair follicle stem cells cultured in a medium to which the extract of Panax ginseng (Production Examples 1 to 4) subjected to heating and drying was added after steam heating was used. Compared with the extracts (Comparative Production Examples 1 and 2), the expression of the undifferentiated marker KRT15 was significantly increased, and it was shown to have an excellent undifferentiated maintenance effect of hair follicle stem cells. Moreover, the said effect was higher when using ginseng (Product Examples 2 to 4) dried before the steam heat treatment than using raw ginseng (Product Example 1). In addition, as shown in Table 2, the undifferentiated maintenance effect of hair follicle stem cells was particularly remarkable when the temperature of steam heat treatment was in the range of 100 to 150 ° C. (Production Example 2-b, 2-c, 2- d).

(Experimental Example 2) Expression promoting effect of extracellular matrix gene of Ginseng extract (1) Culture of human hair follicle stem cells Human hair is collected by tweezers and tissue including bulge region of hair follicle tissue is obtained using a female etc. It was collected. After washing with PBS (−), treatment with trypsin (manufactured by BD Biosciences) was performed. Thereafter, cells were isolated and recovered using Cell Trainer (manufactured by FALCON). The recovered cells were seeded on a culture plate, and maintained until confluence using KG2 medium (manufactured by KURABO). Confluent cells were collected and replated on culture plates, and then engrafted and proliferated cells were used as hair follicle stem cells in the following tests.

(2) Evaluation of the expression level of extracellular matrix gene when adding ginseng extract The extract of ginseng is made to act on hair follicle stem cells and how the extract of ginseng is related to the expression of extracellular matrix gene in hair follicle stem cells It was evaluated whether it was related to

  The test was performed as follows. Extracts of Panax ginseng (Production Examples 1 to 4; Comparative Production Examples 1 and 2) were added to hair follicle stem cells to a final concentration of 100 μg / ml. Extract of Panax ginseng added, cells after 48 hours were collected, extracellular matrix gene expression levels of hair follicle stem cells were expressed as COL7A1, COL17A1, LAMA3, LAMB3, LAMC2, LAMC2, LAMA5, LAMB1, LAMC1, DCN Was analyzed as an index.

  Gene expression analysis was performed as follows. The hair follicle stem cells after addition of the extract of Panax ginseng were washed twice with PBS (−), and then RNA was extracted from the cells with Trizol Reagent (manufactured by Invitrogen). After reverse transcription of the extracted RNA into cDNA using a 2-STEP real time PCR kit (manufactured by Applied Biosystems), real time PCR (95 ° C .: 15 seconds, 60 ° C .: 30 seconds) by ABI 7300 (manufactured by Applied Biosystems) 40 cycles, and the gene expression levels of COL7A1, COL17A1, LAMA3, LAMB3, LAMC2, LAMC5, LAMB1, LAMC1, DCN were measured. Other operations were carried out according to the prescribed method.

Primer set for COL7A1:
5'-CACTGGAGCCACAGCTTACA-3 '(SEQ ID NO: 5)
5'-GCTGACTCCACCTTCGAGAC-3 '(SEQ ID NO: 6)
Primer set for COL17A1:
5'-TCCTGTCCAACTCAGAAACC-3 '(SEQ ID NO: 7)
5'-ACCTGGATCACCTTTGTCAC-3 '(SEQ ID NO: 8)
Primer set for LAMA3:
5'-AACCAAAAGTGCTCGGAGAC-3 '(SEQ ID NO: 9)
5'-TCAGTAGGTGGTAAGCCCAAATC-3 '(SEQ ID NO: 10)
Primer set for LAMB3:
5'- GGGAGACCCCCACATTCAA-3 '(SEQ ID NO: 11)
5'-GCTGTGTGTTCTGCTCGTTCTT-3 '(SEQ ID NO: 12)
Primer set for LAMC2:
5'-GGATGAGAATCCTGACATTGAGTGT-3 '(SEQ ID NO: 13)
5'-GTCGTGCGGATCGTTGTAGA-3 '(sequence number 14)
Primer set for LAMA5:
5'-CAGCACTGCACTAGAAGAGG-3 '(SEQ ID NO: 15)
5'-CTTGCTTGTCTCGTCTTGTGTC-3 '(SEQ ID NO: 16)
Primer set for LAMB1:
5'-ATCC CAAAGTTCTCAAGTCCT-3 '(SEQ ID NO: 17)
5'-GTCCATTACATTTACATCTGCCCAC-3 '(SEQ ID NO: 18)
Primer set for LAMC1:
5'-CTTCTGTCTGTACAAAACGCTG-3 '(SEQ ID NO: 19)
5'-CCATCCTTCATCAATCTGAAAGG-3 '(SEQ ID NO: 20)
Primer set for DCN:
5'-TTCTGCCCACCTGGACACA-3 '(SEQ ID NO: 21)
5'-GACCGGGTTGCTGAAAAGAC-3 '(SEQ ID NO: 22)
Primer set for 18srRNA (internal standard):
5'-CCGAGCCCGCCTGGATAC-3 '(SEQ ID NO: 3)
5'-CAGTTCCGAAAACCAACAAAATAGA-3 '(SEQ ID NO: 4)

  The expression of extracellular matrix gene (hereinafter referred to as "target gene") is 18s ribosomal RNA (18srRNA), which is an internal standard for the expression level of mRNA of each target gene in hair follicle stem cells cultured without adding ginseng extract Each value of target gene relative expression level (target gene expression level / 18srRNA gene expression level) calculated as a ratio to expression level of 1) is 1, and each of hair follicle stem cells cultured by adding extract of Panax ginseng The value of the target gene relative expression level was calculated and evaluated. The results are shown in Table 3 below.

  As shown in Table 3, hair follicle stem cells cultured in a medium to which the extract of Panax ginseng (Production Examples 1 to 4) subjected to heating and drying was added after steam heating was used. Compared with the extracts (Comparative Production Examples 1 and 2), the expression levels of COL7A1, COL17A1, LAMA3, LAMB3, LAMC2, LAMC2, LAMA5, LAMB1, LAMC1, DCN are higher, showing an excellent expression promoting effect on extracellular matrix genes The Moreover, the said effect was higher when using ginseng (Product Examples 2 to 4) dried before the steam heat treatment than using raw ginseng (Product Example 1).

  The present invention can be used in the field of manufacturing medicines, quasi-drugs or cosmetics intended for the prevention and / or amelioration of alopecia.

Claims (6)

  An agent for maintaining an undifferentiated state of stem cells, comprising an extract of heat-dried processed ginseng as an active ingredient after steam heat treatment.   The agent for maintaining the undifferentiated state of stem cells according to claim 1, wherein the stem cells are hair follicle stem cells.   An agent for the prevention and / or amelioration of alopecia, which comprises an extract of heat-dried treated ginseng as an active ingredient after steam heat treatment.   An expression promoter for an extracellular matrix protein gene, which comprises as an active ingredient an extract of heat-dried ginseng after steam heat treatment.   The agent according to any one of claims 1 to 4, wherein the ginseng is dried or raw ginseng.   A hair composition comprising the agent for preventing and / or ameliorating alopecia according to claim 3.