JP2020171242A - Stem cell proliferation promoter - Google Patents

Abstract

To discover a new material having an increased proliferation promoting activity on stem cells, and provide a stem cell proliferation promoter having high biological safety.SOLUTION: A stem cell proliferation promoter contains supercritical extract of Ganoderma lucidum spores, or water and/or organic solvent extract of Ganoderma lucidum spores as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a stem cell growth-promoting agent and a stem cell growth-promoting method.

In vertebrate (especially mammal) tissues, when cell / organ damage occurs due to injury or disease, aging, etc., the regeneration system works to recover the cell / organ damage. Stem cells in the tissue play a major role in this action. It has been clarified that stem cells are present in all organs / tissues such as bone marrow, liver, pancreas, skin, fat, and brain, and it has been found that they control the regeneration and homeostasis of each organ / tissue ( See Non-Patent Documents 1 to 4).

In recent years, it has become clear that stem cells existing in organs and tissues are aging (see Non-Patent Document 5). 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 proliferative capacity of neural stem cells and hematopoietic stem cells that produce blood cells existing in the brain decreases significantly with aging (see Non-Patent Documents 6 to 7). In addition, 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 8 to 9).

Based on the above findings, technologies for proliferating 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.

Ganoderma lucidum (scientific name: Ganoderma lucidum) is a mushroom of the family Ganoderma lucidum, and is also called Ganoderma lucidum (Litchi) or Polyporaceae. Ganoderma lucidum (Ganoderma lucidum) contains β-glucan, terpenoids, etc., and has various effects such as immunity improving effect, anticancer effect, blood pressure lowering effect, and blood sugar level lowering effect, and is mainly decocted as a healthy tea. It's being used. It is known that fruiting bodies of Ganoderma lucidum, mycelium, or extracts of cultures thereof have an effect of maintaining undifferentiation of stem cells and an effect of promoting proliferation (Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2008-301726 Japanese Unexamined Patent Publication No. 2011-211956

Goodell M.A. et al., Nat. Med., 1997, Vol. 3, pp. 1337-1345 Zulewski H. et al., Diabetes, 2001, Vol. 50, pp. 521-533 Suzuki A. et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 Zuk P.A. et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Beane O.S. et al., PLoS One, 2014, Vol. 9, 12, e115963 Molofsky A.V. et al., Nature, 2006, Vol. 443, 7110, pp. 448-452 Geiger H. et al., Nat. Rev. Immunol., 2013, Vol. 13, 5, pp. 376-389 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 new substance having higher growth promoting activity on stem cells and to provide a growth promoting agent for stem cells having excellent biosafety.

As a result of diligent research to solve the above problems, the present inventors have found that an extract of Ganoderma lucidum spores has an excellent growth promoting effect on stem cells, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A stem cell growth-promoting agent containing a supercritical extract of Ganoderma spores or a water and / or organic solvent extract of Ganoderma lucidum as an active ingredient.
(2) A method for promoting proliferation of stem cells, which comprises a step of culturing stem cells in a medium containing a supercritical extract of Mannentake spores or a water and / or organic solvent extract of Mannentake spores.
(3) A method for producing stem cells, which comprises a step of culturing stem cells in a medium containing a supercritical extract of Mannentake spores or a water and / or organic solvent extract of Mannentake spores.
(4) A composition for promoting stem cell proliferation, which comprises the stem cell proliferation promoting agent according to (1).

According to the present invention, stem cells can be efficiently proliferated. Therefore, the present invention can make a great contribution in the fields of regenerative medicine, regenerative cosmetology, and anti-aging.

Hereinafter, the present invention will be described in detail.
The stem cell growth promoter according to the present invention is described as a supercritical extract of Ganoderma lucidum spores or a water and / or organic solvent extract of Ganoderma lucidum spores (hereinafter, these are collectively referred to as "Ganoderma lucidum spore extract". In some cases) as an active ingredient.

Ganoderma is a basidiomycete belonging to the genus Ganoderma of the family Ganodermataceae and is used in the crude drug "Ganoderma lucidum". 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. Examples thereof include spores of turf and white turf, but spores of red turf (Ganoderma lucidum) and black turf (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.

The active ingredient of the stem cell growth promoter according to the present invention may be a supercritical extract of Ganoderma lucidum spores (also referred to as Ganoderma lucidum spore oil), or a water and / or organic solvent extract of Ganoderma lucidum spores. ..

In the present invention, the extraction method for obtaining a supercritical extract of Mannentake spores is not particularly limited as long as it is a method of contacting Mannentake spores with a fluid in a supercritical state (supercritical fluid), but it is safe and easy. An extraction method using carbon dioxide (supercritical carbon dioxide) in a supercritical state is preferable in that solvent removal can be performed. 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 (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.

The extraction method for obtaining the water and / or organic solvent extract of Ganoderma lucidum spores 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, squalane, 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.

When Ganoderma spores are extracted with water and / or an organic solvent, the ratio of Ganoderma lucidum spores to the solvent is, for example, 1 to 50% (w / w), preferably 5 to 25% (w / w). The extraction temperature and time vary depending on 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 can be exemplified. For example, an extract of Ganoderma spores can be obtained by adding water to a dried product of Ganoderma lucidum and performing hot water extraction at 95 to 100 ° C. Alternatively, dried mannentake spores with lower alcohols (eg ethanol, etc.), liquid polyhydric alcohols (eg, propylene glycol, 1,3-butylene glycol, etc.) and / or hydrocarbons (hexane, heptane, liquid paraffin, squalane, etc.) Etc.) is added and extraction is performed at room temperature (for example, 15 to 35 ° C.) to obtain an extract of Mannentake spores.

After the above extraction, the obtained solvent phase itself can be used as an extract of Ganoderma lucidum spores. Alternatively, if necessary, the obtained solvent phase is subjected to treatments such as concentration, dilution, filtration and drying, and decolorization and deodorization treatments with activated carbon or the like, and the obtained product is used as an extract of Ganoderma lucidum spores. be able to. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze drying and the like, and the obtained dried product may be used as an extract of Ganoderma lucidum spores.

The extract of Ganoderma lucidum spores thus obtained is used as an active ingredient of the stem cell growth promoter according to the present invention. Since the extract of Mannentake spores has an action of efficiently proliferating stem cells at the biological level or the culture level, the stem cell proliferation promoter according to the present invention is a medium for stem cell culture for efficiently proliferating stem cells. It can be blended and applied to pharmaceuticals, non-pharmaceutical products, cosmetics, foods, etc., including additives, research reagents, medical reagents, and cell transplant agents.

The blending amount of the extract of Ganoderma lucidum spores in the stem cell growth promoter of the present invention is not particularly limited, but is preferably 0.00001 to 10% by weight in terms of a dried product with respect to the total amount of the drug. , 0.0001 to 1% by weight, more preferably. If it is less than 0.00001% by weight, it may be difficult to sufficiently exert the effect.

By applying the stem cell growth-promoting agent according to the present invention to mammalian stem cells including humans, the growth of stem cells can be promoted. The stem cells to which the stem cell growth-promoting agent according to the present invention is applied are not particularly limited as long as they meet the object of the present invention, and are, for example, bone marrow, blood, skin (epidermal, dermal, subcutaneous tissue), fat, and hair follicles. , Somatic stem cells present in brain, nerve, liver, pancreas, kidney, muscle and other tissues; Embryonic stem cells (ES cells); Stem cells artificially produced by gene transfer (artificial pluripotent stem cells) : IPS cells). Examples of ES cells include ES cells established by culturing early embryos before implantation, and ES cells established by culturing early embryos prepared by nuclear transplantation of somatic cell nuclei. And ES cells in which genes on the chromosomes of those ES cells are modified by using genetic engineering techniques. Such ES cells can be produced, for example, by a method known per se, but can be obtained from a predetermined institution, and a commercially available product can also be purchased. Further, these stem cells may be either primary cultured cells, subcultured cells or frozen cells.

Furthermore, the stem cell proliferation promoter according to the present invention can be applied to all mammalian-derived stem cells as long as they have the same characteristics regarding the direction of stem cell differentiation and the process of differentiation. For example, the stem cell proliferation promoter according to the present invention exerts an effect on mammalian stem cells such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs. can do.

The application of the growth-promoting agent for stem cells according to the present invention to stem cells may be in vitro or in vivo, and in either case, the action can be exerted. Therefore, the stem cell growth-promoting agent according to the present invention promotes stem cell growth by culturing the stem cells in a stem cell culture medium to which an effective amount thereof has been added, or by administering the stem cells to mammals including humans. can do.

In the stem cell growth-promoting agent according to the present invention, since the extract of Mannentake spores, which is an active ingredient, has an excellent stem cell growth-promoting effect, in vivo such as skin, osteoblast, cartilage, muscle, nerve, fat, and liver. It is effective in treating, ameliorating, and preventing damage or damage to the tissue or organ by acting on the stem cells of the tissue or organ. In addition, since stem cells decrease or decrease in function with aging, the stem cell proliferation-promoting agent according to the present invention treats diseases related to the decrease or function of stem cells in tissues or organs in the living body. It is effective for improvement and prevention. Here, as diseases related to tissue or organ damage or damage, reduction of stem cells or functional deterioration, for example, in the case of skin, wrinkles, tarmi, stains, dullness, rough skin, thickening of the skin, opening of pores, scars of acne , Wounds, scars, keloids, etc., and also includes scalp and hair damage such as thinning hair and hair loss. In addition, osteoporosis and fractures (spine compression fractures, femoral neck fractures, etc.) are involved in bone-related diseases, and degenerative arthritis, rheumatoid arthritis, and disc hernia are involved in cartilage diseases. Spinal cord injury and facial nerve palsy are involved in nerve-related diseases. , Alzheimer's disease, muscle atrophic lateral sclerosis, Parkinson's disease, age-related memory loss, etc., blood-related, regenerative anemia, leukemia, etc., cardiovascular-related, myocardial infarction, obstructive arteriosclerosis, etc. Related items include periodontal disease and alveolar bone damage due to alveolar pyorrhea, ophthalmology-related items include retinal pigment degeneration, age-related macular degeneration, and glaucoma, and liver / pancreas-related items include hepatitis, liver cirrhosis, and diabetes. Not limited to.

The present invention also relates to a method for producing stem cells and a method for promoting proliferation of stem cells, which comprises a step of culturing stem cells in a medium containing an extract of Mannentake spores.

In the method according to the present invention, the medium for culturing the stem cells and the additive used at the same time are not particularly limited, and a medium and an additive generally used for the growth of stem cells may be used.

Specifically, as the medium for culturing stem cells, a basal medium containing components necessary for the survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), for example, Dulvecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basic Medium Eagle (BME), Dulvecco's Modern Eagle Medium (D-MEM), Dulvecco's Modified Eagle Medium / Examples thereof include Eagle's Medium (Grasgow MEM) and Hank's balanced salt solution (Hanks solution). In addition, basic fibroblast growth factor (bFGF) and / or leukemia inhibitory factor (LIF) may be added to the medium as growth factors. In addition, if desired, the medium is epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -Supplement, N2-supplement, ITS-supplement, antibiotics, etc. may be contained.

In addition to the above, it is preferable that serum is contained in the medium 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.

Commercially available media include Invitrogen's mesenchymal stem cell basal medium, Sanko Pure Chemical's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hanks solution (Hank's balanced salt solution). ) Etc. can be used.

The incubator used for culturing stem cells is not particularly limited as long as it can cultivate stem cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. ..

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 Mannentake spore extract added to the medium used for culturing the stem cells can be appropriately determined according to the blending amount of the Mannentake spore extract in the stem cell growth promoter according to the present invention described above. Concentrations of 1 to 1000 μg / mL, preferably 10 to 400 μg / mL, in terms of dried spores of Mannentake. In addition, an extract of Ganoderma lucidum spores may be added to the medium on a regular basis during the culture period of stem cells.

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.

For example, the number of cells of the stem cells cultured in the presence of the stem cell growth promoter according to the present invention is compared with the stem cells cultured in the absence of the stem cell growth promoter according to the present invention. Can be evaluated by whether or not is significantly increased. The cell number can be measured by using a commercially available cell number measurement kit by, for example, the MTT method or the WST method. As a result of the measurement, the relative ratio between the number of stem cells at the start of culturing and the number of stem cells after culturing for a predetermined time in the presence of the stem cell growth promoter of the present invention is not the stem cell growth promoter of the present invention. When it is larger than the same relative ratio (control) when cultured in the presence, it can be determined that the proliferation of stem cells could be promoted.

The stem cells prepared by the above method according to the present invention can be used as a transplant material (cell transplant agent).

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

When the above-mentioned stem cell proliferation-promoting agent according to the present invention is administered in vivo, it can be administered as it is, but it is a cosmetic or quasi-drug together with an appropriate additive as long as the effect of the present invention is not impaired. , Pharmaceuticals, foods and drinks, etc. can be blended and provided. The pharmaceutical product of the present invention also includes a drug used for animals, that is, a veterinary drug.

When the stem cell growth promoter according to the present invention is blended in cosmetics or non-pharmaceutical products, the dosage form is aqueous solution type, solubilization type, emulsification type, powder type, powder dispersion type, oil liquid type, gel type. , Ointment type, aerosol type, water-oil two-layer system, water-oil-powder three-layer system, or 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 growth promoter of stem cells. It 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 stem cell growth-promoting agent according to the present invention is blended in a pharmaceutical product, it is mixed with a pharmacologically and pharmaceutically acceptable additive and formulated into various preparations in a preparation form suitable for application to an affected area. can do. 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 adjusters, propellants , Colorants, 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, eye drops, sprays, trans Examples include parenteral agents such as skin absorbents, transmucosal absorbents, and patches. 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 stem cell proliferation promoter according to the present invention as a pharmaceutical product for treating, ameliorating, or preventing the skin-related damage or disease is an external preparation, for example, an ointment, a cream, or the like. Examples include gel agents, liquid agents, 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.

The pharmaceutical product of the present invention functions as a preventive agent for suppressing the onset of the above-mentioned diseases and / or as a therapeutic agent for improving a 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 stem cell growth-promoting agent in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but it is converted into a dried product of the extract of Mannentake spores with respect to the total weight of the preparation (composition). It is preferably 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 stem cell proliferation promoter according to the present invention can also be blended in foods and drinks. Further, in the present invention, the food and drink are not only general foods and drinks but also foods other than pharmaceuticals that can be ingested for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or special uses. It is used to include food. 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 Health 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. Here, indications such as specific health effects and functions of nutritional components attached to food and drink are displayed on product containers, packaging, manuals, package inserts, etc., product leaflets and pamphlets, newspapers, magazines, etc. It can be used as an advertisement for a product of.

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, carbonated 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 an extract of Ganoderma lucidum spores 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 extract of Mannentake spores, and a tableting machine It can be manufactured by applying pressure to the product. Further, other materials (e.g., vitamin C, vitamin B _2, vitamin such as vitamin B _6, minerals such as calcium, fiber, etc.) optionally may be added.

The blending amount of the extract of Ganoderma lucidum spores in the food and drink of the present invention may be an amount capable of exerting the growth promoting effect of stem cells, but the general intake amount of the target food and drink, the form of the food and drink, the efficacy and effect, It may be set appropriately in consideration of taste, taste, cost and the like.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Example of production of Ganoderma lucidum extract An extract of Ganoderma lucidum was produced as follows.

(Production Example 1) Production of hot water extract of Ganoderma lucidum spores 1 L of distilled water was added to 100 g of Ganoderma lucidum spores, and the mixture was extracted at 100 ° C. for 2 hours. After filtering the obtained extract, the filtrate was concentrated and freeze-dried to obtain 11 g of a hot water extract of Ganoderma lucidum spores.

(Production Example 2) Production of 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 thereto. The mixture was supplied for about 4.5 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.

(Comparative Production Example 1) Production of Hot Water Extract of Ganoderma lucidum Fruiting Body 1L of distilled water was added to 20 g of crushed Ganoderma lucidum fruiting body, and the mixture was extracted at 100 ° C. for 1 hour. After filtering the obtained extract, the filtrate was concentrated and freeze-dried to obtain 1.4 g of a hot water extract of Ganoderma lucidum fruiting body.

(Comparative Production Example 2) Production of Supercritical Extract of Mannentakeko Body 1 kg of crushed Mannentakeko body is charged into an extraction tank having an internal volume of 5 L, and supercritical carbon dioxide (temperature 60 ° C., pressure 25 MPa, carbon dioxide supply) is charged thereto. A quantity of 15 m ³ ) was supplied for about 4.5 hours, led to a separation tank (temperature 40 ° C., pressure 4 MPa) connected to the extraction tank to separate carbon dioxide and the extract, and 10 supercritical extracts of the mannentake child substance were separated. .1 g was obtained.

[Example 2] Growth promoting effect of Ganoderma lucidum extract on stem cells The following shows experimental examples of the growth promoting effect on stem cells using the Ganoderma lucidum extract produced in Example 1.

(Experimental Example 1) Evaluation of Proliferation-Promoting Effect of Mannentake Extract on Epidermal Stem Cells A method described in JP-A-2017-055721 using commercially available normal human adult epidermal keratinocytes (manufactured by Kurabo) as epidermal-derived cells. Epidermal stem cells were isolated using NGFR (nerve growth factor receptor: GenBank number: Nucleotide NM_002507.3; Protein NP_002498.1) as an index. The above epidermal stem cells maintained in HuMedia-KG2 medium (manufactured by Kurabo Industries) were seeded on a 96-well plate (manufactured by Falcon) so that the number of cells was 5 × 10 ³ . Next, the test substance (each extract of Ganoderma lucidum produced in Example 1) was added so as to have a final concentration of 100 μg / mL, and cultured for 24 hours. After completion of the culture, the cells were washed 3 times with PBS (-) and the number of cells was counted. When the total number of cells when the test substance was not added was used as the control and the control was set to 100 (%), the increase / decrease (%) in the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated.

(Experimental Example 2) Proliferation-promoting effect of Mannentake extract on dermis stem cells Commercially available human skin fibroblasts (manufactured by Toyo Boseki Co., Ltd.) were used as dermis-derived cells, and according to the method described in JP-A-2017-093383. , NGFR (nerve growth factor receptor: GenBank number: Nucleotide NM_002507.3; Protein NP_002498.1) was used as an index to isolate dermal stem cells. The above dermal stem cells maintained in 1% FBS Minimum Essential Media alpha medium (manufactured by Gibco) were seeded on a 96-well plate (manufactured by Falcon) so that the number of cells was 5 × 10 ³ . Next, the test substance (each extract of Ganoderma lucidum produced in Example 1) was added so as to have a final concentration of 100 μg / mL, and cultured for 24 hours. After completion of the culture, the cells were washed 3 times with PBS (-) and the number of cells was counted. When the total number of cells when the test substance was not added was used as the control and the control was set to 100 (%), the increase / decrease (%) in the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated.

(Experimental Example 3) Proliferation-promoting effect of Mannentake extract on hematopoietic stem cells A6 cells (RIKEN BRC Cell Bank) were used as hematopoietic stem cells. The above hematopoietic stem cells maintained in 20% FBS DMEM / F12 (10 μg / mL bovine insulin, 10 μg / mL bovine Transferrin, 10 ng / mL bFGF) medium (manufactured by Gibco) so that the number of cells is 2 × 10 ⁴ It was sown on a 96-well plate (manufactured by Falcon). Next, the test substance (each extract of Ganoderma lucidum produced in Example 1) was added so as to have a final concentration of 100 μg / ml, and the cells were cultured for 36 hours. After completion of the culture, the cells were washed 3 times with PBS (-) and the number of cells was counted. When the total number of cells when the test substance was not added was used as the control and the control was set to 100 (%), the increase / decrease (%) in the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated.

(Experimental Example 4) Proliferation-promoting effect of Ganoderma lucidum extract on neural stem cells MEB5 cells (JCRB cell bank) were used as neural stem cells. Dulbecco's Modified Eagle's Medium-high glucose (5 μg / mL insulin, 10 ng / mL EGF, 50 μg / mL holo-transferrin, 10 ng / mL biotin, 30nM Na-selenite) The cells were sown on 96-well plates (manufactured by Falcon) so that the number was 1 × 10 ⁴ . Next, the test substance (each extract of Ganoderma lucidum produced in Example 1) was added so as to have a final concentration of 100 μg / ml, and the cells were cultured for 24 hours. After completion of the culture, the cells were washed 3 times with PBS (-) and the number of cells was counted. When the total number of cells when the test substance was not added was used as the control and the control was set to 100 (%), the increase / decrease (%) in the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated.

The results obtained in Experimental Examples 1 to 4 are shown in Table 1.

As shown in Table 1, the extracts of Mannentake spores (Production Examples 1 and 2) were found to have an excellent growth promoting effect on stem cells (epidermal stem cells, dermal stem cells, hematopoietic stem cells, neural stem cells). The effect was higher than that of the extract of the mannen bamboo body (Comparative Production Examples 1 and 2), and the effect of the supercritical extract of Mannentake was particularly remarkable (Production Example 2). In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of promoting stem cell proliferation was observed.

Since the stem cell proliferation promoter of the present invention can promote stem cell proliferation, the drug is useful in the fields of tissue regeneration and homeostasis maintenance such as regenerative medicine, regenerative beauty, and anti-aging.

Claims (4)

A stem cell growth-promoting agent containing a supercritical extract of Ganoderma spores or a water and / or organic solvent extract of Ganoderma lucidum as an active ingredient. A method for promoting proliferation of stem cells, which comprises a step of culturing stem cells in a medium containing a supercritical extract of Mannentake spores or a water and / or organic solvent extract of Mannentake spores. A method for producing stem cells, which comprises a step of culturing stem cells in a medium containing a supercritical extract of Mannentake spores or a water and / or organic solvent extract of Mannentake spores. A composition for promoting stem cell proliferation, which comprises the stem cell proliferation promoting agent according to claim 1.