JP2024011649A - Stem cell growth promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide materials with excellent stem cell growth-promoting action.

SOLUTION: In the process of cultivating Ganoderma lucidum, we have found that Ganoderma lucidum obtained by cultivating it in a medium containing a specific amount of bagasse has a significantly superior stem cell growth-promoting action compared to conventional Ganoderma lucidum. The skin preparations, pharmaceuticals, and foods containing the extract of Ganoderma lucidum an active ingredient of the present invention can greatly contribute to the fields of regenerative medicine, regenerative beauty, and the like, and are extremely effective in applications for maintaining tissue homeostasis, repairing and regenerating damaged tissues.

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

TECHNICAL FIELD The present invention relates to a stem cell growth promoter characterized by containing an extract of C. chinensis obtained by a cultivation method using a bagasse-containing medium.

In the tissues of vertebrates (particularly mammals), when cells and organs are damaged due to injury, disease, aging, etc., the regenerative system works to try to recover the damage to the cells and organs. Stem cells in the tissue play a major role in this effect. Stem cells have pluripotency that allows them to differentiate into various cells and organs, and this property is thought to lead to recovery by replacing damaged cells and organs. Expectations are high for regenerative medicine, a next-generation medical treatment that applies such stem cells.

Stem cells have been found to exist in many organs and tissues, such as bone marrow, skin, liver, pancreas, and fat, and have been found to be responsible for the regeneration and homeostasis of each organ and tissue (non-standard). Patent Documents 1 to 4). In addition, stem cells present in each tissue have excellent plasticity, and may be used to regenerate organs and tissues that were previously unable to self-replicate.

On the other hand, it is known that some of these stem cells decrease with age, and research is actively being conducted on technologies to prevent stem cell decrease in order to maintain homeostasis in each tissue ( Non-patent document 5). In addition, in recent years, in order to apply the ability (pluripotency) of stem cells to the regeneration of organs and tissues, stem cells have been isolated from living tissues and cultured in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative beauty). Development of techniques for propagation is underway (Non-Patent Documents 6, 7).

The mantis mushroom is a basidiomycete used in the herbal medicine "Reishi", has a wide range of physiological activities, and is widely used as a traditional Chinese medicine. For example, it is known that the effect of Cinnamon mushroom is to proliferate stem cells (Patent Document 1).

It is known that the mushroom contains active ingredients such as polysaccharides such as β-glucan and triterpenes such as ganoderic acid. β-glucan is known to have anticancer effects, immunostimulatory effects, and the like. Furthermore, ganoderic acid has been reported to have many effects, such as anticancer effects, blood pressure lowering effects, and cholesterol lowering effects. A method of irradiating with red light has been reported as a method for cultivating stone mushrooms containing a large amount of triterpenes (Patent Document 2).

Currently, most of the stone mushrooms on the market are artificially cultivated, and two main methods are used: log cultivation and fungal bed cultivation. Using these methods, various cultivation methods are being studied for the purpose of increasing yield. For example, a method is known in which a chlorella hot water extraction residue or a dry powder of Coccomixa is used as a medium component (Patent Document 3). The mycelium before fruiting body development is used as a cultivated product of Cinnamon mushroom, but the fruiting body and mycelium have completely different components, physiological activities, etc., and are essentially different.

Bagasse is the residue left after extracting the necessary sugar juice during the sugar manufacturing process using sugarcane, and its annual emissions amount to more than 100 million tons worldwide. Bagasse is used for fuel, soil improvement material, livestock feed, paper raw material, construction material, etc.

Japanese Patent Application Publication No. 2008-301726 Japanese Patent Application Publication No. 2006-25765 Japanese Patent Application Publication No. 2014-158441

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 Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of the Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 Zen Kitagawa et al., Journal of the Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18

Although there is a desire for a material that is excellent in promoting stem cell proliferation, the current situation is that a material that is fully satisfactory has not yet been provided.

As a result of intensive research to solve this problem, the inventors of the present invention have found that by cultivating stone mushrooms in a medium containing a specific amount of bagasse, compared to a medium containing no bagasse, The inventors have discovered that an extract of C. chinensis has an excellent stem cell proliferation promoting effect, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A stem cell growth promoter characterized by containing an extract of Cinnamon mushroom grown in a medium containing 10 to 80% by dry weight of bagasse.
(2) The stem cell proliferation promoter according to (1), characterized in that the extract of C. chinensis is an extract in one or more solvents selected from water, lower alcohols, and liquid polyhydric alcohols.
(3) A method for promoting proliferation of stem cells, comprising the step of culturing the stem cells in a medium containing the extract of C. chinensis described in (1) or (2).
(4) An external preparation for skin, characterized by containing the extract of C. chinensis according to (1) or (2) as an active ingredient for stem cell proliferation.
(5) A pharmaceutical product characterized by containing the extract of C. chinensis according to (1) or (2) as an active ingredient for stem cell proliferation.
(6) A food product characterized by containing the extract of Cinnamon mushroom described in (1) or (2) as an active ingredient for stem cell proliferation.

According to the present invention, stem cells can be efficiently proliferated. Therefore, the present invention can greatly contribute to fields such as regenerative medicine and regenerative beauty, and is extremely effective for maintaining tissue homeostasis and repairing and regenerating damaged tissues.

The present invention will be described in detail below.

The strain of Ganoderma used in the present invention is a basidiomycete used in the crude drug "Reishi", and belongs to the Ganodermaceae family and the genus Ganoderma. Regarding mushrooms of the genus Ganoderma, the ancient Chinese pharmacological texts ``Bencao Gangme'' and ``Shennong Bencao Ching'' include red reishi (reishi), black reishi (black reishi), purple reishi (purple reishi), and blue reishi. It is stated that there are (green grass), yellow reishi (huangzhi), and white reishi (white grass). The scientific name of red reishi is (Ganoderma lucidum), and the scientific name of black reishi is (Ganoderma sinense, Ganoderma japonicum, Ganoderma atrum). Ganoderma neojaponicum is known as another mushroom belonging to the genus Ganoderma. These strains may be those that are distributed in the Chinese or Japanese markets, or may be isolated and cultured from the tissues of C. chinensis.

The method for cultivating Cinnamon mushrooms of the present invention can be any commonly used cultivation method, except for using bagasse as one of the medium components. An example is shown below.

The method for cultivating C. monocytogenes of the present invention can be carried out, for example, by fungal bed cultivation, and includes a culture step of inoculating a bacterial bed with a C. monocytogenes strain and then allowing the bacteria to take hold and propagate throughout the body, and a cultivation step to obtain a C. monocytogenes fruiting body. It has a process.

The medium used for the fungal bed in the culture process may be any medium that appropriately contains, in addition to bagasse, a carbon source, a nitrogen source, inorganic substances, and other necessary nutrients necessary for the growth of the mycelia of C. For example, bagasse, known culture medium base materials, nutrients, or mixtures thereof may be used, and additives may also be added.

The bagasse used in the present invention can be the residue of sugarcane squeezed in the sugar production process, or can be processed by washing, drying, pulverizing, etc. Although the shape of the bagasse is not particularly limited, it is preferable to crush or defibrate bagasse forming a fibrous structure into a predetermined size (width, length). Among these, those defibrated to a width of 5 mm or less or lengths of 50 mm or less are more preferred, and those defibrated to a width of 5 mm or less and a length of 50 mm or less are particularly preferred. Furthermore, its length is most preferably 10 to 50 mm. The content of bagasse in the medium is preferably 10 to 80% by dry weight, more preferably 15 to 60% by weight, and most preferably 20 to 30% by weight.

Examples of the medium base material include sawdust (made from hardwoods such as sawtooth oak, Quercus serrata, beech, etc., or softwoods such as pine, fir, hemlock), corn cob, rice hull, beet, cotton hull, buckwheat husk, etc. Sawdust and/or corncob are preferably used. Although the size of the culture medium base material is not particularly limited, it is preferable to use a 10 mm mesh-passed product (passed through a mesh with an opening of 10 mm). The content of the medium base material in the medium depends on other medium compositions, but is preferably 10 to 90% by dry weight, more preferably 30 to 80% by weight, particularly preferably 40 to 70% by weight. , 50-65% by weight is most preferred.

As the nutritional material, wheat bran, rice bran, corn bran, okara, bean hulls, milo, etc. are used, and preferably wheat bran, rice bran, or corn bran is used. Although the size is not particularly limited, it is preferable to use a 5 mm mesh pass product. The content of nutrients in the medium is preferably 1 to 30% by dry weight, more preferably 5 to 10% by weight, although it depends on other medium compositions.

To summarize the above, a medium containing 20 to 30% by dry weight of bagasse, 50 to 65% by dry weight of sawdust, and 5 to 10% by weight of wheat bran is most preferable.

Additives can be added as necessary for purposes such as pH adjustment and growth promotion, and include calcium carbonate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium dihydrogen phosphate, dihydrogen phosphate, etc. Calcium etc. can be used.

The water content in the medium is not particularly limited, but is preferably, for example, 40 to 80% by weight, more preferably 50 to 75% by weight, even more preferably 60 to 70% by weight, based on the total amount of the medium. However, it is preferable to adjust the moisture content as appropriate so that the pores and moisture in the medium are sufficient to allow the growth of the hyphae of C. chinensis and that the medium does not dry out.

The above-mentioned culture medium can be placed in a container such as a commercially available plastic bag or polypot for bacterial bed cultivation, and then subjected to treatments such as sterilization and sterilization using known methods. Concerning the container for storing the culture medium and the method of sterilization and sterilization, any method may be selected as appropriate as long as it achieves the effects of the present invention. In this way, a bacterial bed is obtained.

Inoculate the fungal bed with a strain of Cinnamon mushroom. A known method may be used for inoculation.

The culturing step is preferably carried out in the dark, but is not limited to the dark. The temperature is, for example, preferably 15 to 35°C, more preferably 20 to 30°C. Humidity is not particularly limited, but may be at a level that does not dry the medium. For example, the relative humidity is preferably 40 to 80%, more preferably 50 to 65%. The period of the culturing step is preferably, for example, 30 to 80 days. As the growth of mycelia progresses, the carbon dioxide concentration increases, which may cause poor growth, so ventilate as necessary. The carbon dioxide concentration is preferably less than 1000 ppm. Cultivate under the above conditions and complete the culturing process when the entire medium is covered with white mycelia, but it is not necessary that part of the medium is covered with white mycelia. Culture may be continued later. The cultivation conditions are not limited to the above ranges, and the temperature, relative humidity, period, etc. in the cultivation process may be selected as appropriate as long as the effects of the present invention are achieved.

In the generation step, for example, the temperature is preferably 15 to 35°C, more preferably 25 to 30°C. The relative humidity is preferably 70% or more, more preferably 90% or more. The illumination intensity is preferably 50 to 3000 lux, more preferably 100 to 2000 lux, and most preferably 300 to 1000 lux. In order to promote the growth, stimulation with bacteria scraping, electricity, etc. may be applied. The cultivation conditions in the generation step are not limited to the above-mentioned ranges, and the temperature, relative humidity, and illuminance may be appropriately selected as long as the cultivation conditions exhibit the effects of the present invention. By cultivating in this way, the mantis mushroom of the present invention can be obtained.

The Bamboo shoots used in the present invention are fruiting bodies, and are clearly distinguished from the mycelium before the fruiting bodies develop by their appearance, contained components, and the like.

The mushroom of the present invention can be used raw after harvesting, or can be processed by washing, drying, crushing, extraction, etc.

Extraction solvents include, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, etc.). , 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.). Polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferred, and water, ethanol, 1,3-butylene glycol and propylene glycol are particularly preferred. These solvents may be used alone or in combination of two or more, and for example, a 30 to 70% by weight aqueous ethanol solution may be used. Furthermore, a solvent whose pH has been adjusted by adding an acid or an alkali to the above extraction solvent can also be used.

The extraction method is not particularly limited, and can be performed by methods such as heating extraction, normal temperature extraction, cooling extraction, stirring extraction, pressure extraction, and column extraction. Moreover, the extract and the mantis mushroom itself can also be used together.

The above extract may be used as an extracted solution, but if necessary, it may be subjected to treatments such as concentration, dilution, filtration, decolorization, deodorization, drying, ethanol precipitation, etc. to the extent that the effects of the present invention are achieved. It may be used from Furthermore, the active ingredient may be concentrated or isolated by column purification or the like before use.

The extract of C. chinensis used in the present invention has an effect of efficiently proliferating stem cells, and therefore can be used as a stem cell proliferation promoter. Furthermore, the stem cell proliferation promoter of the present invention can also be used as a cell culture additive and research reagent for efficiently proliferating stem cells.

The proliferation of stem cells can be promoted by applying the extract of the present invention to stem cells of mammals including humans. The stem cells to which the stem cell proliferation promoter of the present invention is applied are not particularly limited as long as they meet the purpose of the present invention; for example, embryonic stem cells (ES cells); bone marrow, blood, skin (epidermis, dermis). , subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidneys, muscles, and other tissues; stem cells artificially created by gene transfer (induced pluripotent stem cells); : iPS cells). Preferably, it is more effective against stem cells derived from bone marrow, blood, skin, or adipose tissue. More preferably, it exhibits the most effect on mesenchymal stem cells.

Furthermore, the stem cell growth-promoting effect of the extract of the present invention can be applied to mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs. It can exert effects on stem cells.

The extract of Cinnamon edulis according to the present invention may be applied to stem cells either in vitro or in vivo, and in either case, it can exhibit its stem cell proliferation promoting effect. Therefore, the extract of C. chinensis according to the present invention can promote the proliferation of stem cells by culturing the stem cells in a stem cell culture medium supplemented with an effective amount thereof or by administering it to mammals including humans. be able to.

When administering the extract of the present invention into a living body, it is possible to administer it as it is, but it can also be used in external preparations for skin use, pharmaceuticals, food and beverages containing appropriate additives within the range that does not impair the effects of the present invention. It can be provided as various compositions such as products. Note that the pharmaceuticals of the present invention include drugs used for animals, that is, veterinary drugs.

Because the extract of the present invention has an excellent effect of promoting proliferation of stem cells, it acts on the stem cells of tissues or organs in the living body such as skin, bone marrow, cartilage, muscle, nerve, fat, liver, etc. or is also effective in treating, ameliorating, and preventing organ disorders or damage. In addition, since stem cells decrease or their function deteriorates with aging, etc., the extract of C. chinensis according to the present invention can treat diseases or aging related to the decrease or function decrease of stem cells in the above-mentioned in-vivo tissues or organs. , amelioration, and prevention. Here, diseases or aging related to tissue or organ disorder or damage, decrease in stem cells or functional decline include, for example, skin-related wrinkles, sagging, spots, dullness, rough skin, thickening of the skin, enlarged pores, Examples include acne scars, wounds, bedsores, burns, scars, keloids, etc., and also include damage to the scalp and hair such as thinning hair and hair loss. In addition, bone-related diseases include osteoporosis and fractures (vertebral compression fractures, femoral neck fractures, etc.), cartilage diseases such as osteoarthritis, rheumatoid arthritis, and herniated discs, and nerve-related diseases such as spinal cord injuries and facial nerve paralysis. , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, age-related memory decline, etc. Blood-related diseases include aplastic anemia, leukemia, etc. Cardiovascular-related diseases include myocardial infarction, arteriosclerosis obliterans, etc., and dental diseases. Related diseases include periodontal disease and alveolar bone damage due to alveolar pyorrhea, ophthalmology related diseases include retinitis pigmentosa, age-related macular degeneration, and glaucoma, and liver/pancreas related diseases such as hepatitis, cirrhosis, and diabetes. but not limited to.

The dosage form of the skin external preparation containing the extract of C. chinensis according to the present invention is an aqueous solution type, solubilized type, emulsion type, powder type, powder dispersion type, oil liquid type, gel type, ointment type, aerosol type, Either a water-oil two-phase system or a water-oil-powder three-phase system may be used. In addition, the skin topical preparation is prepared by selecting various ingredients, additives, bases, etc. that are usually used in skin care compositions according to the type of the skin preparation, as well as the extract of C. chinensis, and blending them as appropriate. It can be manufactured according to the method of The form may be liquid, emulsion, cream, gel, paste, spray, etc. Ingredients include, for example, oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, Squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (myristic acid, etc.), Isopropyl acid, isopropyl palmitate, cetyl octoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, humectants, Examples include ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericidal agents, fragrances, and the like.

Examples of external skin preparations include lotions, emulsions, gels, serums, general creams, sunscreen creams, packs, masks, cleaning agents, cosmetic soaps, foundations, powder, bath additives, body lotions, body shampoos, Examples include hair shampoo, hair conditioner, hair growth agent, etc.

The pharmaceutical product containing the extract of C. chinensis according to the present invention can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various preparations in a dosage form suitable for application to the affected area. can. Pharmacologically and pharmaceutically acceptable additives include pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, and coatings selected as appropriate depending on the dosage form and use. agent, disintegrant or disintegration aid, stabilizer, preservative, preservative, filler, dispersant, wetting agent, buffer, solubilizer or dissolution aid, tonicity agent, pH regulator, propellant , coloring agents, sweeteners, flavoring agents, fragrances, etc. may be added as appropriate, and various formulations that can be administered orally or parenterally systemically or locally may be prepared by various known methods. When the pharmaceutical of the present invention is provided in each of the above forms, it can be manufactured by a manufacturing method commonly used by those skilled in the art, such as the manufacturing method shown in the Japanese Pharmacopoeia's General Preparations [2] Preparation Monograph.

The form of the pharmaceutical of the present invention is not particularly limited, but includes, for example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs. Oral preparations, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, Examples include parenteral preparations such as skin absorption agents, transmucosal absorption agents, and patches. It may also be a dry product that is reconstituted before use, or, in the case of injectable preparations, presented in unit-dose ampoules or multi-dose containers.

Suitable forms for using the extract of the present invention as a pharmaceutical for the treatment, amelioration, and prevention of skin-related injuries and diseases include external preparations, such as ointments, creams, and gels. agents, solutions, patches, etc. Ointment refers to a homogeneous semi-solid preparation for external use, and includes oil-based ointment, emulsion ointment, and water-soluble ointment. A gel is an external preparation in which a hydrated compound as a water-insoluble component is suspended in an aqueous liquid. Liquid preparations refer to liquid preparations for external use, and include lotions, suspensions, emulsions, liniments, and the like.

The pharmaceutical of the present invention functions as a prophylactic agent that suppresses the onset of the above-mentioned diseases and/or as a therapeutic agent that improves the condition to normal. When the pharmaceutical of the present invention is used as a pharmaceutical for treating, ameliorating, and preventing the above-mentioned diseases, it can be administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, and cats. Can be done.

The content of the extract of C. chinensis in the skin preparations, pharmaceuticals, and foods of the present invention is not particularly limited, but the content of the extract of C. chinensis in the total weight of the preparation (composition) is 0. 0001 to 30% by weight is preferred, 0.001 to 10% by weight is more preferred, and 0.02 to 2% by weight is most preferred. If it is less than 0.0001% by weight, the effect is low, and even if it exceeds 30% by weight, it is difficult to see a significant enhancement in the effect. The above-mentioned amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, the general usage amount, efficacy and effects, etc. Furthermore, the method of adding the active ingredient during formulation may be either added in advance or added during production, and may be appropriately selected in consideration of workability.

Furthermore, in the present invention, food refers to general food and drink, as well as foods other than pharmaceuticals and quasi-drugs that can be ingested for the purpose of maintaining or promoting health, such as health foods, functional foods, and foods with health claims. It is also used to include special purpose foods. Health foods include foods provided under the names of nutritional supplements, health supplements, nutritional adjustment foods, supplements, and the like. Foods with health claims are defined by the Food Sanitation Act or the Health Promotion Act, and include foods for specified health uses, foods with nutritional function claims, and foods with functional claims that can claim specific health effects, nutritional functions, disease risk reduction, etc. It will be done.

The form of the food may be any edible form, such as solid, liquid, granule, granule, powder, capsule, cream, or paste. In particular, the shapes of the above health foods include, for example, tablet, round, capsule, powder, granule, fine granule, troche, and liquid (including syrup, milk, and suspension). etc. are preferred.

Types of foods include tea drinks (green tea, oolong tea, black tea, etc.), soft drinks (sports drinks, mineral water, near water, etc.), carbonated drinks, milk drinks, coffee drinks, drinks with fruit and vegetable juices, nutritional drinks, Examples include various drinks such as jelly drinks, powdered soups, confectionery (candies, gummies, gum, tablets, etc.), noodles, breads, dairy products, processed marine and livestock foods, oils and fats, and processed oils and fats. is not limited to.

The food of the present invention may contain commonly used additives as appropriate depending on the type of food. Any additive can be used as long as it is permissible under the Food Sanitation Act; for example, sweeteners such as glucose, sucrose, fructose, high-fructose corn syrup, aspartame, and stevia; citric acid, malic acid, etc. , acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, Examples include suspending agents and preservatives.

When the food of the present invention is a general food or drink, it can be manufactured by including a step of adding an extract of Manchuria mushroom in the normal manufacturing process of the food or drink. In addition, in the case of health foods, the manufacturing method for pharmaceuticals mentioned above may be followed; for example, in the case of tablet-shaped supplements, additives such as excipients are added and mixed with the extract of C. It can be manufactured by applying pressure and molding. Further, other materials (for example, vitamins such as vitamin C, vitamin B2, and vitamin B6, minerals such as calcium, dietary fiber, etc.) may be added as necessary.

The content of the extract of C. chinensis in the food of the present invention may be any amount that can exhibit the effect of promoting the proliferation of stem cells, but it may be necessary to It may be set appropriately in consideration of preference, cost, etc.

The present invention also relates to a method for promoting proliferation of stem cells by culturing the stem cells in a medium containing an extract of Stone Mountain. In other words, the method according to the present invention can be said to be a method for producing stem cells and a method for promoting proliferation of stem cells, which includes a step of culturing stem cells in a medium containing an extract of L. chinensis.

In the method according to the present invention, a culture medium generally used for stem cell proliferation may be used for culturing the stem cells. For example, a basic 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), specifically Dulbecco's Modified Eagle Medium (D-MEM). ), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D- MEM/F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Examples include Hank's balanced salt solution. Furthermore, the medium may contain basic fibroblast growth factor (bFGF) and/or leukocyte migration inhibitory factor (LIF) as growth factors. Furthermore, if necessary, the medium may contain epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -Supplements, N2-supplements, ITS-supplements, antibiotics, etc. may be contained.

In addition to the above, it is preferable that the medium contains serum at a content of 1 to 20% by volume. However, since the components of serum differ depending on the lot and the effectiveness varies, it is preferable to use the serum after performing a lot check.

Commercially available media include mesenchymal stem cell basal medium manufactured by Thermo Fisher Scientific, mesenchymal stem cell basal medium manufactured by Takara Bio, MF medium manufactured by TOYOBO, and Hank's solution manufactured by Sigma. balanced salt solution), etc. can be used.

The incubator used for culturing stem cells is not particularly limited as long as it is capable of culturing stem cells, but examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. .

The culture vessel may be either non-adhesive or adhesive, and is appropriately selected depending on the purpose. The cell-adhesive culture vessel may be one treated with a cell-supporting substrate such as an extracellular matrix for the purpose of improving adhesion with cells. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

The concentration of the extract of C. monocytogenes added to the medium used for stem cell culture can be appropriately determined according to the content of the extract of C. monocytogenes in the above-mentioned stem cell growth promoter according to the present invention, but In terms of conversion, the concentration is, for example, 1 to 10,000 μg/mL, preferably 10 to 1,000 μg/mL, and most preferably 100 to 500 μg/mL. Moreover, during the culture period of stem cells, an extract of C. chinensis may be periodically added to the medium.

The culture conditions for stem cells may follow the usual conditions used for culturing stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is approximately 30 to 40°C, preferably 36 to 37°C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%. Note that the medium is preferably replaced 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 the stem cells can survive and proliferate.

Next, in order to explain the present invention in detail, a method for cultivating C. chinensis used in the present invention, a production example, an experimental example, and a formulation example of an extract of C. chinensis used in the present invention will be given as examples; however, the present invention is not limited to these. isn't it. In addition, unless otherwise specified, % shown in the examples indicates weight %.

(1) Cultivation of stone mushrooms Bagasse is defibrated to a width of 5 mm or less (10 to 50 mm in length), sawdust is crushed Quercus spp. wood that has passed a 10 mm mesh, and wheat bran is a 5 mm mesh. A polypropylene bag (commercially available) with a breathable and moisture-retaining filter to prevent contamination was used as the container for containing the culture medium. In the above polypropylene bag, 125 g of bagasse (25% by weight of the dry weight of the medium), 325 g of sawdust (65% by weight of the dry weight of the medium), 50 g of wheat bran (10% by weight of the dry weight of the medium) and A medium mixed with 1000 g of water was added. Heat sterilization was performed using steam, and the material was left to cool to room temperature in a clean space. Thereafter, 60 g of a commercially available Red Reishi inoculum was inoculated. After inoculation, the bag was closed and cultured in the dark at a temperature of 21 to 27°C (average temperature of 25°C) and a relative humidity of 40 to 75% (average relative humidity of 51%). The culturing process was continued until the entire medium was covered with white mycelia, and the culturing process was completed. The duration of the culture process was 60 days.

Thereafter, the process moved to the generation step, in which the temperature was 18 to 35°C (average temperature 27°C), the relative humidity was 68 to 100% (average relative humidity 97%), and the illuminance was 3000 lux or less. This generation process was carried out until the fruiting body of the mountain mushroom was formed, and then the generation process was completed. The duration of the developmental process was 30 days. In addition, in the culture step and generation step, ventilation was performed as necessary so that the carbon dioxide concentration was less than 1000 ppm.

The method for cultivating the above-mentioned stone mushroom was designated as Cultivation Example 1. In addition, Cultivation Example 2 was a method for cultivating stone bamboo using bagasse less than 10 mm in length instead of the bagasse (10 to 50 mm in length) in Cultivation Example 1. In addition, as shown in Table 1, cultivation methods for cultivating Cinderella mushrooms by changing the solid content of the culture medium were used as Cultivation Examples 3 to 6 and Comparative Cultivation Examples 1 to 3, respectively, and the yields of Clover mushrooms were calculated for each cultivation method. Comparison was made with Comparative Cultivation Example 1. In addition, the dry weight of the harvested amount of the stone mushroom was measured.

(2) Extraction
Production Example 1A Hot water extract
200 mL of purified water was added to 10 g of the dry material of C. chinensis obtained in Cultivation Example 1, extracted at 95 to 100° C. for 2 hours, filtered, and the filtrate was concentrated and freeze-dried to obtain a hot water extract. (Table 2).

Production Example 1B 50% Ethanol Extract 200 mL of 50% ethanol was added to 10 g of the dry material of the Cinderella mushroom obtained in Cultivation Example 1, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain a 50% An ethanol extract was obtained (Table 2).

Production Example 1C Ethanol Extract 200 mL of ethanol was added to 10 g of the dry material of the Cinderella mushroom obtained in Cultivation Example 1, and after extraction at room temperature for 7 days, it was filtered, and the filtrate was concentrated to dryness to obtain an ethanol extract. (Table 2).

The mushrooms of Cultivation Examples 2 to 6 and Comparative Cultivation Examples 1 to 2 were extracted in the same manner as in Production Examples 1A to 1C above to obtain Production Examples 2A to 6C and Comparative Production Examples 1A to 2C (Table 2).

Experimental example 1 Stem cell proliferation promotion test

(Experiment Example 1) Evaluation of proliferation promoting effect on stem cells Human adipose tissue-derived mesenchymal stem cells (manufactured by DS Pharma Biomedical) cultured using human stem cell culture medium (manufactured by TOYOBO) were placed in a 6 cm dish at 3 x 10 ^Five cells were seeded, the test substance was added to the final concentration shown in Table 3, and culture was continued for 3 days.

After culturing for 3 days, the cells were washed 3 times with PBS (-), collected using a rubber policeman, and the number of cells was counted. The total cell number when the test substance was not added was used as a control, and when the control was set as 100 (%), the increase or 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 of these tests are shown in Table 3 below.

As shown in Table 3, the extract of C. chinensis cultivated using the bagasse of the present invention was found to have a significantly higher stem cell proliferation promoting effect than the conventional extract of C. chinensis. In particular, in the hot water extract, the difference between the extract of the present invention and the conventional extract was more remarkable. In addition, when the above-mentioned control value was taken as 100%, the number of human adipose tissue-derived mesenchymal stem cells at the start of culture was 25%. In addition, in addition to the stem cells used in this experimental example, similar tests were conducted on epidermal stem cells, dermal stem cells, and hematopoietic stem cells, and it was found that the extract of C. monocytogenes cultivated using the bagasse of the present invention did not contain the extract of C. monocytogenes grown using the bagasse of the present invention. A significantly higher stem cell growth promoting effect than the extract was observed.

Next, an example of a formulation prepared using the extract of the present invention will be shown. Note that the mixed extract refers to a mixture of equal amounts of each extract.

Prescription example 1 Lotion Prescription Content (%)
1. Extract of Production Example 1A 1.0
2.1,3-butylene glycol 8.0
3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Fragrance: Appropriate amount 11. Make the total amount 100% with purified water. [Manufacturing method] Components 1 to 6 and 11 and components 7 to 10 are each uniformly dissolved, mixed and filtered to obtain a product.

In Formulation Example 1, the extract of Production Example 1A is replaced with the extract of Production Example 2A, the extract of Production Example 3A, the extract of Production Example 4A, the extract of Production Example 5A, and the extract of Production Example 6A. were designated as Prescription Examples 2, 3, 4, 5, and 6, respectively.

Prescription example 7 Cream Prescription Content (%)
1. Mixed extract of Production Examples 1A, 2A, 3A, 4A, 5A and 6A 0.5
2. Squalane 5.5
3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20E.O.) 3.0
8. Behenyl alcohol 1.5
9. Glyceryl monostearate 2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.25
12.1,3-butylene glycol 8.5
13. Make the total amount 100% with purified water. [Manufacturing method] Components 2 to 9 are dissolved and mixed by heating, and kept at 70°C to form an oil phase. Components 1 and 11 to 13 are heated and dissolved, mixed, and kept at 75°C to form an aqueous phase. Add the aqueous phase to the oil phase and emulsify, cool while stirring, add component 10 at 45°C, and further cool to 30°C to obtain a product.

In Formulation Example 7, the mixed extracts of Production Examples 1A, 2A, 3A, 4A, 5A and 6A were replaced with the mixed extracts of Production Examples 1B, 2B, 3B, 4B, 5B and 6B, which was designated as Formulation Example 8. .

Prescription example 9 Emulsion Prescription Content (%)
1. Extract of Production Example 1C 1.0
2. Squalane 5.0
3. Olive oil 5.0
4. Jojoba oil 5.0
5. Setanol 1.5
6. Glyceryl monostearate 2.0
7. Polyoxyethylene cetyl ether (20E.O.) 3.0
8. Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. Make the total amount 100% with purified water. [Manufacturing method] Components 1 to 8 are dissolved and mixed by heating, and kept at 70°C to form an oil phase. Components 10 to 13 are dissolved and mixed by heating, and the mixture is kept at 75°C to form an aqueous phase. Add the aqueous phase to the oil phase and emulsify, cool while stirring, add component 9 at 45°C, and further cool to 30°C to obtain a product.

In Formulation Example 9, the extract of Production Example 1C was replaced with the extract of Production Example 2C, the extract of Production Example 3C, the extract of Production Example 4C, and the extract of Production Example 5C, respectively. 10, 11, 12 and 13.

Prescription example 14 Gel Prescription Content (%)
1. Mixed extract of Production Examples 1C and 2C 0.01
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60E.O.) 0.1
5. Fragrance Appropriate amount 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Make the total amount 100% with purified water. [Manufacturing method] Components 1 to 5 and components 6 to 11 are each uniformly dissolved and mixed to form a product.

Prescription example 15 Pack Prescription Content (%)
1. Extract of Production Example 1A 0.1
2. Mixed extract of Production Examples 1C and 2C 0.1
3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5.1,3-butylene glycol 8.0
6. Methyl paraoxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20E.O.) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Fragrance: Appropriate amount 11. Make the total amount 100% with purified water. [Manufacturing method] Components 1 to 11 are uniformly dissolved to form a product.

Prescription example 16 Foundation Prescription Content (%)
1. Mixed extract of Production Examples 1B, 2B, 3B, 4B, 5B and 6B 1.0
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20E.O.) 1.0
4. Polyoxyethylene cetyl ether (20E.O.) 2.0
5. Setanol 1.0
6. liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Carboxymethyl cellulose sodium 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Fragrance: Appropriate amount 19. Make the total amount 100% with purified water. [Production method] Dissolve components 2 to 8 by heating and keep at 80°C to form an oil phase. Component 9 is sufficiently swollen in component 19, and then components 1 and 10 to 13 are added and mixed uniformly. Add components 14 to 17 that have been ground and mixed using a grinder, stir with a homomixer, and keep at 75°C to form an aqueous phase. Add the aqueous phase to the oil phase while stirring and emulsify. Thereafter, it is cooled, and component 18 is added at 45°C, and the mixture is cooled to 30°C while stirring to form a product.

Prescription example 17 Bath additive Prescription Content (%)
1. Extract of Production Example 1A 5.0
2. Extract of Production Example 2A 1.0
3. Sodium hydrogen carbonate 50.0
4. Yellow No. 202 (1) Appropriate amount 5. Fragrance (appropriate amount) 6. Adjust the total amount to 100% with sodium sulfate. [Manufacturing method] Components 1 to 6 are mixed uniformly to form a product.

Prescription example 18 Ointment Prescription Content (%)
1. Mixed extract of Production Examples 1A, 2A, 3A, 4A, 5A and 6A 0.5
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glyceryl monostearate 10.0
4. Liquid paraffin 5.0
5. Setanol 6.0
6. Methyl paraoxybenzoate 0.1
7. Propylene glycol 10.0
8. Make the total amount 100% with purified water. [Manufacturing method] Components 2 to 5 are dissolved and mixed by heating, and kept at 70°C to form an oil phase. Components 1 and 6 to 8 are heated and dissolved, mixed, and kept at 75°C to form an aqueous phase. Add the aqueous phase to the oil phase, emulsify, and cool to 30°C while stirring to obtain a product.

Prescription example 19 Powder Prescription Content (%)
1. Mixed extract of Production Examples 1B, 2B, 3B, 4B, 5B and 6B 20.0
2. Dried cornstarch 30.0
3. Microcrystalline cellulose 50.0
[Manufacturing method] Components 1 to 3 are mixed to form a powder.

Prescription example 20 Tablet Prescription Content (%)
1. Mixed extract of Production Examples 1C, 2C, 3C, 4C, 5C and 6C 3.0
2. Extract of Production Example 1A 0.1
3. Dried cornstarch 26.9
4. Carboxymethyl cellulose calcium 20.0
5. Microcrystalline cellulose 40.0
6. Polyvinylpyrrolidone 7.0
7. Talc 3.0
[Manufacturing method] Components 1 to 5 are mixed, and then an aqueous solution of component 6 is added as a binder to form granules. Add component 7 to the formed granules and tablet. One tablet weighs 0.52 g.

Prescription example 21 Tablet confectionery Prescription Content (%)
1. Mixed extract of Production Examples 1A, 2A, 3A, 4A, 5A and 6A 0.5
2. Dried cornstarch 50.0
3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance (appropriate amount) 7. Make the total amount 100% with purified water. [Production method] Mix components 1 to 4 and 7 and form into granules. Components 5 and 6 are added to the formed granules and compressed into tablets. One grain is 1.0g.

Prescription example 22 Beverage Prescription Content (%)
1. Mixed extract of Production Examples 1A, 2A, 3A, 4A, 5A and 6A 2.0
2. Fructose glucose liquid sugar 12.5
3. Citric acid 0.1
4. Fragrance 0.05
5. Make the total amount 100% with purified water. [Manufacturing method] Mix ingredients 1 to 5 and make a drink.

Prescription example 23 Powdered drink Prescription Content (%)
1. Mixed extract of Production Examples 1A, 2A, 3A, 4A, 5A and 6A 10.0
2. Powdered sugar 65.0
3. Powdered peach juice 15.0
4. L-ascorbic acid 8.0
5. Crystalline citric acid 1.2
6. Sodium citrate 0.75
7. Aspartame 0.02
8. Powdered peach flavor 0.03
[Production method] Mix ingredients 1 to 8 to make a powdered drink.

From the above, the extract of C. chinensis according to the present invention exhibited a superior stem cell growth promoting effect compared to the extract of C. chinensis cultivated by the conventional method. Therefore, the extract of the present invention is expected to be applied to fields such as regenerative medicine and regenerative beauty, since it has a very excellent effect of promoting stem cell proliferation.

Claims (6)

1. A stem cell growth promoter characterized by containing an extract of Cinnamon mushroom grown in a medium containing 10 to 80% by dry weight of bagasse. 2. The stem cell proliferation promoter according to claim 1, wherein the extract of C. chinensis is an extract using one or more solvents selected from water, lower alcohols, and liquid polyhydric alcohols. A method for promoting the growth of stem cells, comprising the step of culturing the stem cells in a medium containing the extract of C. chinensis according to claim 1 or 2. 3. A skin preparation for external use, characterized in that it contains the extract of C. chinensis according to claim 1 or 2 as an active ingredient for stem cell proliferation. A medicament containing the extract of C. chinensis according to claim 1 or 2 as an active ingredient for stem cell proliferation. A food product characterized by containing the extract of C. chinensis according to claim 1 or 2 as an active ingredient for stem cell proliferation.