JP2020182415A - Agent for promoting differentiation of dermal stem cell - Google Patents

Abstract

To discover a novel material for promoting the differentiation of a dermal stem cell into a dermal fibroblast, and provide it as an agent for promoting the differentiation of a dermal stem cell.SOLUTION: An agent for promoting the differentiation of a dermal stem cell into a dermal fibroblast contains, as an active ingredient, supercritical extract of Reishi mushroom spore, or a mixture of supercritical extract of Reishi mushroom spore and extract of one or more crude drugs selected from Citrus unshiu peel, ginger, plantain, licorice, Platycodon, hawthorn, cinnamon bark, and carrot.SELECTED DRAWING: None

Description

The present invention relates to an agent that promotes the differentiation of dermal stem cells into dermal fibroblasts.

The skin has a three-layer structure of epidermis, dermis, and subcutaneous tissue. Dermal fibroblasts are present in the dermis layer, and collagen, elastin, and hyaluronic acid produced by dermal fibroblasts are known as important components for maintaining the firmness, elasticity, and moisturization of the skin. Dermal fibroblasts are essential for maintaining youthful skin without wrinkles and tarmi, as aging and inflammation reduce these components as a cause of wrinkles and tarmi. It can be called a cell. The dermal stem cells that produce these dermal fibroblasts are located just below the papillary dermis layer and repeatedly proliferate and differentiate as needed to constantly supply new dermal fibroblasts to the dermal layer, resulting in constant regeneration of the skin. Is repeated (Non-Patent Document 1). Since dermal components such as collagen and elastin are actively produced from fibroblasts produced from dermal stem cells, it is important to induce differentiation from dermal stem cells to mature fibroblasts in order to maintain healthy dermal tissue. is important.

In recent years, it has become clear that stem cells existing in organs and tissues are aging (see Non-Patent Document 2). Specifically, aging of stem cells is a decrease in proliferative ability and differentiation ability, and is considered to be a cause of a decrease in regenerative ability of organs and tissues. For example, it has been reported that the number of stem cells present in the skin and subcutaneous adipose tissue decreases with aging and the differentiation ability decreases (see Non-Patent Documents 3 to 4). Therefore, technologies for improving the differentiation ability of stem cells existing in each organ / tissue are used for anti-aging (anti-aging) such as maintenance of tissue homeostasis, repair / regeneration of damaged tissues, prevention / treatment / improvement of various diseases, etc. It is considered to be extremely effective. In addition, when considering the application of stem cells to regenerative medicine and regenerative beauty, it is important to develop a technique for efficiently inducing differentiation of stem cells into cells of organs and tissues to be regenerated, and further, it was possible to induce differentiation into target cells. Even so, since organs and tissues are constructed three-dimensionally, if this cannot be reproduced, even if they are transplanted into a living body, their effects (regeneration of organs and tissues) cannot be exhibited. In other words, if we can develop a technology for culturing stem cells in vitro, freely inducing differentiation into target cells, and constructing three-dimensionally similar to the organ or tissue to be regenerated, a leap forward in regenerative medicine and regenerative beauty in the future. Development can be expected. In particular, the skin tissue has a complicated three-dimensional structure and is provided in the outermost layer of the human body, so that it is easily damaged by an external injury. In addition, it is an organization that is greatly related to human appearance and beauty, and it is extremely important to advance the regeneration technology of this organization.

So far, as substances that promote the production of collagen and hyaluronic acid in dermal fibroblasts, a mixture of yeast extract and beech plant extract (Patent Document 1), N-benzoylglycylglycine (Patent Document 2), and Konotegashiwa Extracts of seeds of genus plants (Patent Document 3) and the like are known. However, they act on dermal stem cells and do not promote the induction of differentiation from dermal stem cells to dermal fibroblasts. An extract of purple wheat seed (Patent Document 4) has been reported as a substance having an effect of inducing differentiation of dermal stem cells into dermal fibroblasts.

Japanese Unexamined Patent Publication No. 11-158054 Japanese Unexamined Patent Publication No. 2014-55116 WO2012 / 057123 Japanese Unexamined Patent Publication No. 2010-22326

Hasebe Y. et al., J. Dermatol. Sci., 2016, Vol. 89, pp. 205-207 Beane O.S. et al., PLoS One., 2014, Vol. 9, 12, e115963 Akamatsu H. et al., J. Dermatol., 2016, Vol. 43, pp. 311-313 Yamada T. et al., J. Dermatol. Sci., 2010, Vol. 58, pp. 36-42

An object of the present invention is to find a novel substance that promotes the differentiation of dermal stem cells into dermal fibroblasts in view of the above circumstances, and to provide the substance as a differentiation promoting agent for dermal stem cells.

As a result of diligent research to solve the above problems, the present inventors have obtained a supercritical extract of Mannentake spores or a mixture of a supercritical extract of Mannentake spores and an extract of other crude drugs into dermal fibers of dermal stem cells. We have found that it has an excellent promoting action on differentiation into blast cells, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, which contains a supercritical extract of Ganoderma lucidum spores as an active ingredient.
(2) (A) Supercritical extract of dermis spores and (B) Extract of one or more crude drugs selected from (B) Chinpi, Shokyo, Obako, Kanzo, Kikyo, Hawthorn, Keihi, and Carrot. An agent for promoting the differentiation of hawthorn stem cells into dermal fibroblasts, which contains a mixture of and as an active ingredient.
(3) To dermal fibroblasts of dermal stem cells containing a mixture of a supercritical extract of Mannentake spores and an extract of one or two crude drugs selected from chimpi and gypsum as an active ingredient. Differentiation promoter.
(4) A method for promoting differentiation of dermis stem cells into dermal fibroblasts, which comprises a step of culturing dermis stem cells in a medium containing the agent according to any one of (1) to (3).
(5) A method for producing dermal fibroblasts, which comprises a step of culturing dermal stem cells in a medium containing the agent according to any one of (1) to (3).
(6) A composition for promoting differentiation of dermal stem cells into dermal fibroblasts, which contains the agent according to any one of (1) to (3).

Since the agent for promoting the differentiation of dermal stem cells into dermal fibroblasts of the present invention can promote the differentiation of dermal stem cells and efficiently induce dermal fibroblasts, collagen produced by dermal fibroblasts, Ingredients such as erastin and hyaluronic acid that maintain the firmness, elasticity, and moisture of the skin are sufficiently supplied into the skin, and are effective in improving wrinkles, tarmi, firmness, and loss of elasticity due to aging and ultraviolet rays.

Hereinafter, the present invention will be described in detail.
1. 1. Agent for promoting differentiation of dermal stem cells into dermal fibroblasts The agent for promoting differentiation of dermal stem cells into dermal fibroblasts according to the present invention (hereinafter, may be referred to as “dermis stem cell differentiation promoting agent”) is Mannentake spores. Supercritical extract of Mannentake spore oil (also referred to as Mannentake spore oil) or supercritical extract of Mannentake spore, and one or two selected from chimpi, ginger, oyster, kanzo, kikyo, sanzashi, keihi, and carrot. It contains a mixture with the above extract of crude drug as an active ingredient.

In the present invention, the "dermal stem cell" refers to a cell capable of differentiating into dermal fibroblasts. In the present invention, "promotion of differentiation of dermal stem cells into dermal fibroblasts" means that differentiation of dermal stem cells into dermal fibroblasts is activated as compared with before administration or ingestion of the agent of the present invention. To say.

Ganoderma is a basidiomycete belonging to the genus Ganoderma of the family Ganodermataceae and is used in the crude drug "Reishi". Ganoderma lucidum (Ganoderma lucidum), Ganoderma lucidum (Ganoderma lucidum), Ganoderma lucidum (Ganoderma lucidum), Ganoderma lucidum (Ganoderma lucidum) ), Ganoderma lucidum (Ganoderma lucidum) and Ganoderma lucidum (Ganoderma lucidum) are described to exist. In addition, as a kind of red ganoderma, antler ganoderma is also known. The "Ganoderma spore" used in the present invention is not particularly limited as long as it is a spore of Ganoderma genus Ganoderma of the family Ganoderma, for example, red ganoderma, black ganoderma, purple ganoderma, blue ganoderma, and yellow spirit. Ganoderma lucidum and black ganoderma (Ganoderma lucidum) and black ganoderma (Ganoderma sinense, Ganoderma japonicum, Ganoderma atrum) are more preferable.

Ganoderma lucidum spores are brown powdery substances that appear on the umbrella when Ganoderma lucidum matures. In the present invention, Ganoderma lucidum spores include spores and spore spores in which a plurality of spores are endogenous. The recovered Ganoderma lucidum spores may be used as they are for the extraction of Ganoderma lucidum spores, but it is preferable to perform a wall breaking treatment for disrupting the cell wall of the spores by a physical force. The method for treating the rupture wall is not particularly limited, but can be, for example, atomization treatment, roll press treatment, grinding treatment, ultra-high pressure microsteam treatment, and other mechanical methods usually used industrially. When ruptured spores are used, those obtained by any of the above methods may be used, or commercially available products may be used.

In the present invention, the method for extracting Mannentake spores is not particularly limited as long as it is a method in which a fluid in a supercritical state (supercritical fluid) is brought into contact with Mannentake spores, but the solvent can be safely and easily removed. Therefore, an extraction method using carbon dioxide in a supercritical state (supercritical carbon dioxide) is preferable. The supercritical carbon dioxide refers to carbon dioxide that has become a fluid state under the conditions of a temperature of 31 ° C. or higher and a pressure of 7 MPa or higher, and in the present invention, the supercritical state includes a state in the vicinity thereof.

As the extraction conditions with carbon dioxide in the supercritical state, the temperature is preferably 31 to 100 ° C., more preferably 31 to 80 ° C., further preferably 31 to 60 ° C., and the pressure is preferably 7 to 100 MPa, 7 to 50 MPa. Is preferable, and 7 to 30 MPa is more preferable. Among them, the temperature is particularly preferably 31 to 80 ° C. and the pressure is 7 to 50 MPa, and the temperature is most preferably 31 to 60 ° C. and the pressure is 7 to 30 MPa. The amount of supercritical carbon dioxide supplied during extraction is preferably 5 to 500 parts by weight, more preferably 10 to 100 parts by weight, based on 1 part by weight of Ganoderma lucidum spores (dry matter equivalent). The extraction time is preferably 30 minutes to 24 hours, more preferably 1 to 10 hours. Further, an organic solvent can be used as a co-solvent (entrainer). Examples of the co-solvent (entrainer) include ethanol, acetone and the like. Of these, ethanol is preferable from the viewpoint of safety.

Extraction with carbon dioxide in a supercritical state can be performed, for example, by continuously blowing carbon dioxide under the above extraction conditions. Next, the carbon dioxide fluid containing the extract of Ganoderma lucidum spores is guided to a separation tank and separated by a commonly used method such as a method of lowering the pressure or a method of changing the temperature. At this time, the separation tank can be filled with an adsorbent capable of adsorbing the extracted solute, a medium (solvent, base) capable of dissolving or dispersing, and the like, and appropriate separation according to the extraction conditions. Means can be adopted. The separated carbon dioxide can be transported to a liquefaction tank for reuse.

"Chinpi" (Japanese name: Chenpi, scientific name: AURANTII NOBILIS PERICARPIUM) is the mature pericarp of Satsuma mandarin (scientific name: Citrus unshiu Marcowicz or Citrus reticulata Blanco), which is an evergreen shrub of the Rutaceae family. In the pharmacy), it is mainly used as a stomachic medicine. The extraction raw material used in the present invention is preferably the peel of Satsuma mandarin, which is used as the crude drug "chinpi".

"Ginger" (Japanese name: ginger, scientific name: ZINGIBERIS RHIZOMA) is the rhizome of ginger (scientific name: Zingiber officinale Roscoe), which is a perennial plant of the ginger family (Zingiberaceae), and sometimes the peripheral skin is removed. It is mainly used as a stomachic medicine in (Japanese Pharmacy). "Ginger" is a raw dried ginger rhizome, and "Kankyo" is a steamed and dried ginger rhizome. The extraction raw material used in the present invention is preferably the rhizome of ginger, which is used as the crude drug "Ginger".

"Plantago" (Japanese name: Plantain), also known as: Shazensou, scientific name:
PLANTAGINIS HERBA) is a whole plantago (scientific name: Plantago asiatica Linne), which is a perennial plant of the plantain family (Plantaginaceae), and is mainly used as an expectorant in crude drugs (Japanese Pharmacopoeia). As for plantains, the Japanese native species are plantain (Plantago asiatica), plantain camtschatica (Plantago camtschatica), plantago japonica (Plantago japonica), plantago lanceolata (Plantago lanceolata), and their closely related naturalized species, plantain psyllium (Plantago major) and psyllium. (Plantago virginica) and the like, and may be a plant of the same genus or related plants thereof. The extraction raw material used in the present invention is preferably the whole plantago asiatica, which is used as the crude drug "Shazensou".

"Glycyrrhiza vulgaris" (Japanese name: licorice, scientific name: GLYCYRRHIZAE RADIX) is a perennial herb of the legume family (Fabaceae), the root or running stem (stron), and sometimes the peripheral skin is removed (skin). It is a licorice (licorice), and is mainly used as an analgesic and antispasmodic (gastrointestinal) and sputum in crude drugs (Japanese Pharmacy). Examples of licorice include licorice (scientific name: Glycyrrhiza uralensis), licorice (scientific name: Glycyrrhiza glabra), licorice (scientific name: Glycyrrhiza echinata), and the like, or related plants thereof. The extraction raw material used in the present invention is preferably licorice root or stalk (stron), which is used as the crude drug "licorice (licorice)". Licorice is a crude drug name (Japanese Pharmacopoeia) and a plant name at the same time.

"Kikyo" (Japanese name: Kikyo, scientific name: PLATYCODI RADIX) is the root of Kikyo (scientific name: Platycodon grandiflorus A. De Candolle), which is a perennial plant of the Bellflower family (Campanulaceae), and is a dried one (raw dried bellflower). ) And dried (bleached bellflower) except for cork skin. It is mainly used as an expectorant in crude drugs (Japanese Pharmacopoeia). The extraction raw material used in the present invention is preferably the root of Japanese bellflower (raw dried bellflower) or the root without cork skin (bleached bellflower), which is used as the crude drug "Platycodon grandiflorum". In addition, Kikyo is a crude drug name (Japanese Pharmacopoeia) and a plant name at the same time.

"Hawthorn" (Japanese name: Yamasuko, scientific name: CRATAEGI FRUCTUS) is a false fruit of the Rosaceae hawthorn (Crataegus cuneata Siebold et Zuccarini) or hawthorn (Crataegus pinnatifida Bunge var. Major NE Brown). It is cut vertically or horizontally, and is mainly used as a digestive and absorption aid in hawthorn (Japanese Pharmacy). The extraction raw material used in the present invention is preferably hawthorn fruit (fake fruit), which is used as the crude drug "Hawthorn (Hawthorn)". Hawthorn is a crude drug name (Japanese Pharmacopoeia) and a plant name at the same time.

"Cinnamon" (Japanese name: cinnamon bark, scientific name: CINNAMOMI CORTEX) excludes part of the bark or peripheral bark of Lauraceae Tonkinnikkei (cassia) (scientific name: Cinnamomum cassia Blume) or other similar plants. It is mainly used as a stomachic medicine in crude drugs (Japanese cinnamon). The extraction raw material used in the present invention is preferably the bark of Nikkei, which is used as the crude drug "Cinnamon bark".

"Ginseng" (Japanese name: ginseng, scientific name: GINSENG RADIX) excludes the fine roots of Panax ginseng CA Meyer (scientific name: Panax ginseng CA Meyer, also known as Korean ginseng, medicinal ginseng), which is a perennial plant of the Araliaceae family. The root or this is lightly boiled, and is mainly used for health tonics and stomachic medicines in raw medicine (Japanese Pharmacy). Panax ginseng may be a plant of the same genus or related plants thereof, for example, Panax japonicus (scientific name: Panax japonicus CAMey), Panax notoginseng (scientific name: Panax notoginseng), Panax notoginseng (scientific name: Panax quinquefolius), Siberian ginseng (scientific name: Eleutherococcus senticosus). ) Etc. can be mentioned. The extraction raw material used in the present invention is preferably ginseng root, which is used as a crude drug "carrot".

In the present invention, the above-mentioned extraction raw materials may be used as they are for the extraction of chimpi, sardine, sardine, licorice, licorice, hawthorn, cinnamon, and carrot, and they are dried, crushed, shredded, and the like. May be good.

The extraction method is not particularly limited, and may be, for example, a heat extraction method, or a normal temperature or cold temperature extraction method. Examples of the solvent used for extraction include water or hot water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), and liquid polyhydric alcohols (1,3). -Butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl) Ether, tetrahydrofuran, propyl ether, etc.) and the like. Among these solvents, water or hot water, lower alcohols, liquid polyhydric alcohols and the like are preferable. These solvents may be used alone or in admixture of two or more. Further, it is also possible to use a solvent whose pH is adjusted by adding an acid or an alkali to the above extraction solvent.

The amount of the extraction solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more the amount of the extraction raw material (dry weight), but in the case of concentration or isolation after extraction. It is preferably 100 times or less for convenience of operation. The extraction temperature and time will vary depending on the target plant and the type of solvent used, and for example, 10 to 100 ° C., preferably 30 to 90 ° C., 30 minutes to 24 hours, preferably 1 to 10 hours will be exemplified. Can be done.

The extract may be used as it is in the extracted solution, but if necessary, concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, etc., within a range that does not affect the effect. It may be used after being subjected to treatments such as decolorization with activated carbon, deodorization, and ethanol precipitation. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze drying and the like, and used as a dried product.

The dermis stem cell differentiation promoter according to the present invention may contain the supercritical extract of Ganoderma lucidum spores obtained as described above as an active ingredient, and the supercritical extract of Ganoderma lucidum spores
The active ingredient may contain a mixture of one or more herbal extracts selected from chimpi, ginger, sardine, licorice, licorice, hawthorn, cinnamon, and carrot. The crude drug extracts used in combination with the supercritical extract of Mannentake spores are chimpi extract, ginger extract, barb extract, kanzo extract, kyo extract, sanzashi extract, keihi extract, and carrot extract. Any one of the above may be used, but two or more are preferable, and three are more preferable. Of these, chenpi extract and ginger extract are preferable. When two or more types are used in combination, the combination and mixing ratio are not limited.

Supercritical extract of Mannentake spores, or supercritical extract of Mannentake spores and one or more crude drug extracts selected from chimpi, ginger, oyster, kanzo, kikyo, sanzashi, keihi, and carrot Since the mixture with and (hereinafter referred to as "herbal extract") has an action of promoting the differentiation of dermal stem cells at the biological level (in vivo) or at the culture level (in vitro), the differentiation promotion of the dermal stem cells of the present invention is promoted. The agent is a medium for culturing stem cells for promoting the differentiation of dermal stem cells by administering to mammals including humans, and for promoting the differentiation of dermal stem cells and producing dermal fibroblasts. It can also be used as an additive, research reagent, and medical reagent.

In the dermis stem cell differentiation promoting agent according to the present invention, since the above-mentioned biopharmaceutical extract, which is an active ingredient, has an action of promoting the differentiation of dermis stem cells into dermal fibroblasts, it is normal due to a decrease or failure of the differentiation function of the dermis stem cells. It is effective in treating, ameliorating, and preventing diseases or conditions caused by the non-formation of dermal fibroblasts. Such diseases or conditions include, for example, wrinkles, tarmi, stretch marks (nasal lip groove), marionette lines, decreased elasticity and elasticity, lack of moisture and luster, stiffness, dullness, sun elastic fibrosis, scleroderma. , Fibrosarcoma, pigmented psoriasis, cutaneous histiocytoma, stretch marks (skin streaks), wounds, burns, decubitus, scars, mother's spots, chloasma, etc., but not limited to these.

The content of the crude drug extract in the dermis stem cell differentiation promoting agent according to the present invention is not particularly limited, but depending on the properties of the extract (extract, concentrate, or dried product), for example, with respect to the total amount of the drug. It is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight.

2. A method for promoting differentiation of dermal stem cells, a method for producing dermal fibroblasts The present invention also comprises a method for promoting differentiation of dermal stem cells, which comprises a step of culturing the dermal stem cells in a medium containing the above-mentioned agent for promoting differentiation of dermal stem cells. The present invention relates to a method for producing dermal fibroblasts, which comprises a step of culturing dermal stem cells in a medium containing the above-mentioned dermal stem cell differentiation promoting agent. The dermal fibroblasts produced by inducing differentiation from dermal stem cells in the method according to the present invention can generally be cultured in vitro and then transplanted directly into a wound or a site where tissue is to be regenerated by injection or the like. is there. That is, the dermal fibroblasts produced by the method according to the present invention can be used as a transplant material (cell transplant agent).

In the method according to the present invention, the medium for culturing dermal stem cells and the additive used at the same time are not particularly limited, and the medium generally used for differentiating dermal stem cells into dermal fibroblasts and Additives may be used.

Specifically, the medium for culturing dermal stem cells is a basal medium containing components necessary for the survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), for example, Dulvecco. 's Modified Eagle's Medium (D-MEM), Minimum Nutrient Medium (MEM), RPMI 1640, Basic Medium Eagle (BME), Dulvecco's Modern Eagle's Medium-Med Glassgo Minimum Essential Medium (Grasgow MEM), Hank's balanced salt solution, and the like are used. In addition, in order to increase the growth rate of cells, the above medium contains growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and tumor necrosis factor (TNF), if necessary. ), Vitamin, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc. Often, antibiotics (penicillin, streptomycin, etc.) and the like may be added. Each component of the medium is sterilized and used by a suitable method. A commercially available medium in which each of the above components is appropriately added to the basal medium can also be used.

In addition to the above, it is preferable that serum (for example, 10% FBS) is contained at a content of 1 to 20%. However, since the components of serum differ depending on the lot and the effect varies, it is preferable to use the serum after performing a lot check.

According to the above-mentioned dermis stem cell differentiation promoter according to the present invention or the method according to the present invention, the above crude drug extract can be used alone or separately from the medium or mixed with the medium to promote the differentiation of dermis stem cells. It can also be provided as a reagent kit of. The kit may include an instruction manual or the like, if necessary. Alternatively, the above crude drug extract can be mixed with a medium and provided as a medium for promoting differentiation of dermal stem cells.

The incubator used for culturing dermal stem cells is not particularly limited as long as it can cultivate dermal stem cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. Can be mentioned. The incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the intended purpose. As the cell adhesion incubator, those treated with a cell support substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

The concentration of the crude drug extract added to the medium used for stem cell culture can be appropriately determined according to the content of the crude drug extract in the above-mentioned dermal stem cell differentiation promoter according to the present invention, but the crude drug extract In terms of dry matter, for example, a concentration of 10 to 10000 μg / mL, preferably 100 to 5000 μg / mL can be mentioned. In addition, these extracts may be added to the medium on a regular basis during the stem cell culture period.

The stem cell culture conditions 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 about 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%. The medium is preferably changed once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately varied and set within a range in which stem cells can survive and proliferate.

Whether or not the differentiation of dermal stem cells into dermal fibroblasts has been promoted can be determined in vitro by a method usually performed by those skilled in the art. For example, the dermal stem cell differentiation promoting agent according to the present invention. The expression level of the dermal fibroblast marker gene is significantly higher at the mRNA level or the protein level in the stem cells cultured in the presence of the differentiation promoter of the dermal stem cells according to the present invention, as compared with the stem cells cultured in the absence of. It can be evaluated by whether it is high or not. Examples of dermal fibroblast marker genes include COL1A1 (type I collagen α1), COL1A2 (type I collagen α2), COL3A1 (type III collagen α1), HAS1 (hyaluronan synthase-1), ELN (elastin), and so on. HYAL3 (hyaluronidase 3), galectin 9 (galectin 9), etc., but are not limited to these.

3. 3. Composition for promoting differentiation of dermal stem cells into dermal fibroblasts When the dermal stem cell differentiation promoting agent according to the present invention is administered in vivo, it can be administered as it is, but the effect of the present invention is not impaired. It can be provided by blending it with various compositions such as cosmetics, quasi-drugs, pharmaceuticals, foods and drinks together with appropriate additives within the range. In particular, it is preferable to provide it by blending it with a composition for external use on the skin.

When the dermal stem cell differentiation promoting agent according to the present invention is blended in cosmetics or non-pharmaceutical products, the dosage form is aqueous solution system, solubilization system, emulsification system, powder system, powder dispersion system, oil solution system, gel. It may be any of a system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil-powder three-layer system, and the like. In addition, for the cosmetics and quasi-drugs, various components, additives, bases and the like usually used in the external composition for skin are selected according to the type and appropriately blended together with the differentiation promoting agent for dermal stem cells. , Can be produced according to a method known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray and the like. Examples of the components of the composition for external use on the skin include fats and oils (olive oil, palm oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, honeybee, carnauba wax, etc.), and hydrocarbons (carnauba wax, etc.) Liquid paraffin, squalane, squalane, vaseline, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.) , Esters (isopropyl myristate, isopropyl palmitate, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citrate, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid) Acids, etc.), sugars (martitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and hydrolyzates of proteins, amino acids and their salts, vitamins, plant / animal extracts, various surface activities Examples thereof include agents, moisturizers, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, and fragrances.

Types of cosmetics and non-pharmaceutical products include, for example, lotions, milky lotions, gels, beauty essences, general creams, sunscreen creams, facial masks, facial cleansers, cosmetic soaps, foundations, face powders, bathing agents, body lotions, etc. Examples include body shampoos, hair shampoos, hair conditioners, and hair restorers.

When the dermal stem cell differentiation promoting agent according to the present invention is blended in a pharmaceutical product, it is mixed with a pharmacologically and pharmacologically acceptable additive and formulated into various preparations in a preparation form suitable for application to the affected area. Can be transformed into. Pharmacologically and pharmacologically acceptable additives include formulation substrates and carriers, excipients, diluents, binders, preservatives, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegrants, stabilizers, preservatives, preservatives, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH regulators, propellants , Coloring agents, sweeteners, flavoring agents, fragrances and the like may be appropriately added to prepare various formulations that can be orally or parenterally administered systemically or topically by various known methods. When the pharmaceutical product of the present invention is provided in each of the above forms, it can be produced by a manufacturing method usually used by those skilled in the art, for example, the manufacturing method shown in each article of the general formulation [2] formulation of the Japanese Pharmacopoeia.

The form of the pharmaceutical product of the present invention is not particularly limited, but for example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspending agents, and elixirs. Oral agents such as drugs, injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), infusions, suppositories, ointments, lotions, sprays, transdermal absorbents. , Transmucosal absorbents, parenteral agents such as patches, and the like. It may also be a dry product that is redissolved upon use, and in the case of injectable formulations, it is provided in the form of unit dose ampoules or multidose containers.

A suitable form for using the dermal stem cell differentiation promoting agent according to the present invention as a pharmaceutical product for treating, ameliorating, or preventing the skin disease or pathological condition is an external preparation, for example, an ointment, a cream, or a gel. Examples thereof include agents, liquids, patches (pap agents, plaster agents), foam agents, spray agents, and spray agents. The ointment refers to a homogeneous semi-solid external preparation, and includes an oily ointment, an emulsion ointment, and a water-soluble ointment. A gel agent refers to an external preparation in which a water-holding compound, which is a water-insoluble component, is suspended in an aqueous solution. The liquid agent refers to a liquid external preparation, and includes a lotion agent, a suspension agent, an emulsion, a liniment agent, and the like.

Formulations for oral administration include, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; carboxymethyl cellulose, carboxymethyl cellulose calcium, etc. Disintegrants or disintegrants such as starch or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc. Coating agents such as hydroxypropylmethyl cellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as vaseline, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used. Not limited to these.

Solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, myoban water, and vegetable oil; isotonic agents such as glucose, sodium chloride, and D-mannitol; inorganic acids , Organic acids, inorganic bases, pH adjusters such as organic bases, and the like can be used, but the present invention is not limited thereto.

The pharmaceutical product of the present invention functions as a preventive agent for suppressing the onset of the skin disease and / or as a therapeutic agent for improving the normal state. Since the active ingredient of the pharmaceutical product of the present invention is derived from a natural product, it is extremely safe and has no side effects. Therefore, when used as a therapeutic, ameliorating, or preventive drug for the above-mentioned diseases, humans, mice, rats, and rabbits , Dogs, cats and other mammals can be administered orally or parenterally in a wide range of doses.

The content of the dermis stem cell differentiation-promoting agent in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but is converted into the dried product of the above crude drug extract with respect to the total weight of the preparation (composition). , 0.001 to 30% by weight, more preferably 0.01 to 10% by weight. The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, general usage amount, efficacy / effect, and the like. Further, the method of adding the active ingredient in the formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

The dose of the drug of the present invention can be appropriately determined according to the type of disease, the age, sex, body weight, degree of symptoms, and the like of the administration target. For example, when orally administered to an adult, the daily dose is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg as a crude drug extract.

In addition, the dermal stem cell differentiation promoter of the present invention can also be added to foods and drinks. In the present invention, foods and drinks include general foods and drinks, as well as foods other than pharmaceuticals that can be ingested for the purpose of maintaining or improving health, such as health foods, functional foods, health functional foods, or special purpose foods. It is used in the meaning of including. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements and the like. Health functional foods are defined by the Food Sanitation Law or the Food Promotion Law, and are based on specified health foods and nutritionally functional foods that can display specific health effects, functions of nutritional components, reduction of disease risk, etc., and scientific evidence. Includes foods with functional claims that can display the contents notified to the Commissioner of the Consumer Affairs Agency. In addition, special-purpose foods include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, and the like, which indicate that they are suitable for a specific target person or a patient having a specific disease. The dermal stem cell differentiation promoter of the present invention is continuously administered on a daily basis, especially when it is necessary to take it for a long period of time for improvement and prevention of various symptoms such as wrinkles, tarmi, elasticity and elasticity associated with aging. It can be suitably used for the above-mentioned health foods and the like because it can be ingested. Here, the indications such as specific health effects and functions of nutritional components attached to foods and drinks are indications such as product containers, packaging, manuals, attached documents, product leaflets and pamphlets, newspapers and magazines, etc. It can be an advertisement for a product of.

Further, when the food or drink of the present invention is used for mammals other than humans, it can be used in the sense of including pet food and feed.

The form of the food or drink may be any of edible forms such as solid, liquid, granular, granular, powdery, capsule-like, cream-like, and paste-like. In particular, in the case of the above-mentioned health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, and liquids (including syrup, milky, and suspension) Etc. are preferable.

The types of foods and drinks include breads, noodles, confectionery, dairy products, processed marine and livestock foods, fats and oils, processed fats and oils, seasonings, and various beverages (soft drinks, sparkling drinks, beauty drinks, nutritional drinks, fruit drinks). , Milk beverages, etc.) and concentrated stock solutions of the beverages, preparation powders, etc., but are not limited thereto.

The food and drink of the present invention may appropriately contain additives that are usually used depending on the type of food and drink. As the additive, any additive that is acceptable under the Food Sanitation Law can be used, and for example, sweeteners such as starch, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid and malic acid. , Acidulants such as citric acid; Excipients such as dextrin and starch; Binding agents, diluents, fragrances, coloring agents, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidants and preservatives.

When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding a crude drug extract in a normal manufacturing process of the food or drink. In the case of health foods, the above-mentioned method for manufacturing pharmaceuticals may be followed. For example, in the case of tablet-shaped supplements, additives such as excipients are added to and mixed with the crude drug extract, and a tableting machine or the like is used. It can be manufactured by molding under pressure. Capsular supplements are produced by filling capsules with liquid, suspension, paste, powder, or granular food compositions containing herbal extracts, or by encapsulating with a capsule base. be able to. Further, it other materials as needed (e.g., iron, minerals such as potassium, vitamin C, vitamin B _2, vitamin such as vitamin B _6, folic acid, dietary fiber, etc.) also be added.

The blending amount of the crude drug extract in the food or drink of the present invention may be an amount capable of exerting the differentiation promoting action of dermal stem cells, but the general intake amount of the target food or drink, the form of the food or drink, the efficacy / effect, and the presentation It may be set appropriately in consideration of taste, taste, cost and the like.

When the food or drink of the present invention is ingested for the purpose of preventing or ameliorating the above-mentioned diseases or pathological conditions, it varies depending on the condition of the subject to be ingested, the form of ingestion, the amount of food intake, etc., but as a crude drug extract, one day for an adult It is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg. The above amount may be ingested once, or may be ingested in several times (2 to 4 times). In the food and drink of the present invention, it is preferable that the amount of food and drink to be ingested at one time is packaged or filled in a container such as a bag or a bottle in order to use it as a guideline for the amount of intake.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

[Example 1]
(Production Example 1) Supercritical Extract of Mannentake Spores 1 kg of Mannentake spores is placed in an extraction tank with an internal volume of 5 L, and supercritical carbon dioxide (temperature 60 ° C., pressure 25 MPa, carbon dioxide supply 15 m ³ ) is added to this. It was supplied for .5 hours and led to a separation tank (temperature 40 ° C., pressure 4 MPa) connected to the extraction tank to separate carbon dioxide and the extract, and 15.9 g of a supercritical extract of Mannentake spores was obtained.

(Production Example 2) Production of hot water extract of chinpi 800 mL of purified water was added to 100 g of dried chenpi peel (chinpi), extracted at 95 to 100 ° C. for 2 hours, filtered, and the filtrate was concentrated. , Freeze-dried to obtain 6.1 g of hot water extract of chinpi.

(Production Example 3) Production of hot water extract of ginger 800 mL of purified water is added to 100 g of dried ginger rhizome (ginger), extracted at 95 to 100 ° C. for 2 hours, filtered, and the filtrate is concentrated. Then, it was freeze-dried to obtain 3.7 g of a hot water extract of ginger.

(Production Example 4) Production of hot water extract of plantain Add 800 mL of purified water to 100 g of dried plantain plant (Plantago asiatica), extract at 95-100 ° C. for 2 hours, filter, and concentrate the filtrate. , Freeze-dried to obtain 5.5 g of hot water extract of plantain.

(Production Example 5) Production of hot water extract of kanzo Add 800 mL of purified water to 100 g of dried kanzo root, extract at 95-100 ° C for 2 hours, filter, concentrate the filtrate, and freeze-dry. 5.1 g of hot water extract of Kanzo was obtained.

(Production Example 6) Production of hot water extract of Japanese bellflower Add 800 mL of purified water to 100 g of dried Japanese bellflower root, extract at 95-100 ° C. for 2 hours, filter, concentrate the filtrate, and freeze-dry. 5.1 g of hot water extract of Platycodon grandiflorum was obtained.

(Production Example 7) Production of hot water extract of hawthorn 800 mL of purified water is added to 100 g of dried hawthorn fake fruit, extracted at 95 to 100 ° C. for 2 hours, filtered, the filtrate is concentrated, and freeze-dried. Then, 2.6 g of a hot water extract of hawthorn was obtained.

(Production Example 8) Production of hot water extract of Chinese cinnamon 800 mL of purified water is added to 100 g of dried cinnamon bark (Cinnamon bark), extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate is concentrated. Freeze-drying gave 2.1 g of a hot water extract of cinnamon cinnamon.

(Production Example 9) Production of hot water extract of carrot 800 mL of purified water is added to 100 g of dried ginseng root (carrot), extracted at 95 to 100 ° C. for 2 hours, filtered, and the filtrate is concentrated. It was lyophilized to give 6.7 g of a hot water extract of carrots.

[Example 2] Evaluation of differentiation promoting effect of dermal stem cells The differentiation promoting effect of the extract produced in Example 1 (Production Examples 1 to 9) and the mixture of the extracts (Production Examples 10 to 18) on dermis stem cells. The evaluation experiment was conducted as follows.

Commercially available skin fibroblasts (manufactured by Toyo Spinning Co., Ltd.) are used as dermis-derived cells, and NGFR (nerve growth factor receptor: GenBank number: Nucleotide NM_002507.3; Protein NP_002498) is used according to the method described in JP-A-2017-093383. Dermal stem cells were isolated using .1) as an index. The above dermal stem cells maintained in 15% FBS Minimum Essential Media alpha medium (manufactured by Gibco) were seeded on a 6-well plate (manufactured by Falcon) so that the number of cells was 1 × 10 ⁵ . Next, the above medium was replaced with 1% FBS Minimum Essential Media alpha medium (manufactured by Gibco), which is a differentiation medium, and the test substance (each extract of Production Examples 1 to 18) was adjusted to a final concentration of 100 μg / mL. It was added and cultured for 24 hours. In the case of a mixture of extracts, they were added so that the ratio of each extract was the same so that the total concentration of the extracts had the above concentration.

After 24 hours of culturing, the cells were collected, washed twice with PBS (-), and RNA was extracted from the cells by Trizol Reagent (manufactured by Invitrogen). After reverse transcribing the extracted RNA to cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95 ° C: 15) using the following primer set using ABI7300 (manufactured by Applied Biosystems). For 40 cycles) at 60 ° C. for 30 seconds, gene expression of dermal fibroblast markers COL1A2 (type I collagen) and COL3A1 (type III collagen) was confirmed. Other operations were performed according to the prescribed method.

(Primer set for COL1A2 (type I collagen) gene)
5'-GCTACCCAACTTGCCTTCATG-3' (SEQ ID NO: 1)
5'-TTCTTGCAGTGGTAGGTGATGTTC-3' (SEQ ID NO: 2)
(COL3A1 (type III collagen) primer set)
5'-GGTTTTGCCCCGTATTATGGA-3' (SEQ ID NO: 3)
5'-GTGAAGTCATAATCTCATCGGTGTTG-3' (SEQ ID NO: 4)
(Primer set for 18s rRNA (internal standard))
5'-CCGAGCCGCCTGGATAC-3' (SEQ ID NO: 5)
5'-CAGTTCCGAAAACCAACAAAATAGA-3' (SEQ ID NO: 6)

The differentiation-promoting effect of dermal stem cells was calculated by calculating the expression level of COL1A2 and COL3A1 mRNA in cells to which no extract was added as the ratio to the expression level of 18s ribosomal RNA (18srRNA), which is an internal standard, and relative expression of COL1A2 and COL3A1 genes. The value of the amount (COL1A2, COL3A1 gene expression level / 18srRNA gene expression level) was set to 1, and the values of the gene relative expression levels of COL1A2 and COL3A1 in the cells cultured with the addition of the extract were calculated and evaluated. .. The results of these tests are shown in Table 1 below.

As shown in Table 1, supercritical extract of Mannentake spore, extract of chimpi, extract of ginger, extract of barb, extract of kanzo, extract of kikyo, extract of sanzashi, extract of keihi , And the carrot extract were all found to have an excellent effect of promoting the differentiation of dermal stem cells (Production Examples 1 to 9). Further, in the combined use of the supercritical extract of Mannentake spores and the extract of other crude drugs, a mixture of the supercritical extract of Mannentake spores (Production Example 1) and the extract of chimpi (Production Example 2) (Production Example 10). , A mixture of a supercritical extract of Mannentake spores (Production Example 1) and an extract of Drosophila (Production Example 3) (Production Example 11), a supercritical extract of Mannentake spores (Production Example 1) and an extract of chimpi (Production Example 1). A remarkable synergistic effect was observed in the mixture (Production Example 18) of Production Example 2) and the extract of Drosophila (Production Example 3) with respect to the effect of promoting the differentiation of dermal stem cells.

The agent for promoting the differentiation of dermal stem cells into dermal fibroblasts of the present invention can efficiently induce the differentiation of dermal stem cells into dermal fibroblasts in vivo or in vitro. Therefore, the present invention can be used in the fields of manufacturing cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and pathological conditions caused by dysfunction or failure of dermal stem cells, and in the field of manufacturing transplant materials.

Claims (6)

An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, which contains a supercritical extract of Ganoderma spores as an active ingredient. A mixture of (A) a supercritical extract of dermis spores and (B) an extract of one or more crude drugs selected from (B) chimpi, ginger, oyster, kanzo, kikyo, sanzashi, keihi, and carrot. An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, which contains the active ingredient. An agent for promoting the differentiation of dermal stem cells into dermal fibroblasts, which comprises a mixture of a supercritical extract of Mannentake spores and an extract of one or two crude drugs selected from chimpi and gypsum as an active ingredient. .. A method for promoting differentiation of dermal stem cells into dermal fibroblasts, which comprises a step of culturing dermal stem cells in a medium containing the agent according to any one of claims 1 to 3. A method for producing dermal fibroblasts, which comprises a step of culturing dermal stem cells in a medium containing the agent according to any one of claims 1 to 3. A composition for promoting differentiation of dermal stem cells into dermal fibroblasts, which comprises the agent according to any one of claims 1 to 3.