Stem cell proliferation promoter

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a stem cell proliferation promoter characterized by comprising an extract of Ganoderma lucidum obtained by a cultivation method using a bagasse-containing medium. [Background Art]

[0002] When cells or organs are damaged in vertebrate (particularly mammalian) tissues due to injury, disease, aging, or other causes, the regenerative system acts to repair the damaged cells and organs. Stem cells present in the relevant tissue play a major role in this process. Stem cells have pluripotency that allows them to differentiate into various cells and organs, and this property is thought to promote recovery by compensating for damaged parts of cells and organs. Expectations are growing for regenerative medicine, a next-generation medical treatment that applies such stem cells.

[0003] It has been revealed that stem cells are present in many organs and tissues such as bone marrow, skin, liver, pancreas, and adipose tissue, and it has been found that they govern the regeneration and homeostasis maintenance of each organ and tissue (Non-Patent Documents 1 to 4). Furthermore, stem cells present in each tissue have excellent plasticity, and may potentially be used for regeneration of organs and tissues for which self-replication was previously considered impossible.

[0004] On the other hand, it is known that some of these stem cells decrease with age, and active research has been conducted on techniques for preventing the decrease of stem cells to maintain homeostasis of each tissue (Non-Patent Document 5). Furthermore, in recent years, in order to apply the capacity (pluripotency) of stem cells to the regeneration of organs and tissues, the development of techniques for isolating stem cells from biological tissues and then culturing and proliferating them has been advanced in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative aesthetics) (Non-Patent Documents 6 and 7).

[0005] Ganoderma lucidum is a basidiomycete used in the crude drug "Reishi", has a wide range of physiological activities, and is widely used as a traditional Chinese medicine. For example, the stem cell proliferative effect is known as an effect possessed by Ganoderma lucidum (Patent Document 1).

[0006] Ganoderma lucidum is known to contain active ingredients such as polysaccharides like β-glucan and triterpenes like ganoderic acid. β-glucan is known to have anticancer and immunostimulatory effects. Ganoderic acid has also been reported to have many effects, including anticancer, blood pressure lowering, and cholesterol-lowering effects. One method for cultivating Ganoderma lucidum, which is rich in triterpenes, is to irradiate it with red light (Patent Document 2).

[0007] Currently, most commercially available Ganoderma lucidum are artificially cultivated, and the two main cultivation methods used are log cultivation and substrate cultivation. Various cultivation methods are being investigated using these methods with the aim of increasing yields. For example, a method using Chlorella hot water extraction residue or Coccomyxa dried powder as culture medium components is known (Patent Document 3). As a cultivated product of Ganoderma lucidum, the mycelium before fruiting body development is used, but the components and physiological activity of the fruiting body and mycelium are completely different, and they are fundamentally different.

[0008] Bagasse is the residue left over after extracting the necessary sugar juice from sugarcane during the sugar production process. The annual amount of bagasse produced worldwide exceeds 100 million tons. Bagasse is used as fuel, soil conditioner, livestock feed, paper raw material, and building material, among other applications. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2008-301726 [Patent Document 2] Japanese Patent Publication No. 2006-25765 [Patent Document 3] Japanese Patent Publication No. 2014-158441 [Non-patent literature]

[0010] [Non-Patent Document 1] Goodell MA et al., Nat. Med., 1997, Vol. 3, pp. 1337-1345. [Non-Patent Document 2] Zulewski H. et al., Diabetes, 2001, Vol. 50, pp. 521-533 [Non-Patent Document 3] Suzuki A. et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 [Non-Patent Document 4] Zuk PA et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 [Non-Patent Document 5] Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 [Non-Patent Document 6] Mabuchi, Y. et al., Journal of the Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268. [Non-Patent Document 7] Kitagawa, Zen et al., Journal of the Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18. [Overview of the project] [Problems that the invention aims to solve]

[0011] While there is a strong demand for materials that promote stem cell proliferation, currently, no materials that fully meet expectations have been provided. [Means for solving the problem]

[0012] The inventors of this invention conducted extensive research to solve this problem and, as a result, discovered that when cultivating Ganoderma lucidum in a culture medium containing a specific amount of bagasse, the resulting Ganoderma lucidum extract has superior stem cell proliferation-promoting activity compared to culture medium without bagasse, thus completing the present invention.

[0013] In other words, the present invention encompasses the following inventions. (1) A stem cell proliferation promoter, characterized by comprising an extract of *Ganoderma lucidum* cultivated 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 *Ganoderma lucidum* is an extract obtained with one or two or more solvents selected from the group consisting of water, lower alcohols and liquid polyhydric alcohols. (3) A method for promoting proliferation of stem cells, comprising the step of culturing stem cells in a medium containing the *Ganoderma lucidum* extract according to (1) or (2). (4) An external preparation for skin, characterized by comprising the *Ganoderma lucidum* extract according to (1) or (2) as an active ingredient for stem cell proliferation. (5) A pharmaceutical product, characterized by comprising the *Ganoderma lucidum* extract according to (1) or (2) as an active ingredient for stem cell proliferation. (6) A food product, characterized by comprising the *Ganoderma lucidum* extract according to (1) or (2) as an active ingredient for stem cell proliferation. Effects of the Invention

[0014] According to the present invention, stem cells can be efficiently proliferated. Accordingly, the present invention can greatly contribute to fields such as regenerative medicine and regenerative aesthetic medicine, and is extremely effective for uses in maintaining tissue homeostasis and repairing / regenerating damaged tissues. Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention is described in detail.

[0016] The Ganoderma strain used in the present invention is a basidiomycete used for the crude drug "Reishi", and belongs to the family Ganodermataceae and the genus Ganoderma. Regarding mushrooms of the genus Ganoderma, ancient Chinese pharmaceutical classics such as "Compendium of Materia Medica" and "Shennong's Classic of Materia Medica" record that there exist red Reishi (Reishi), black Reishi (black Ganoderma), purple Reishi (purple Ganoderma), green Reishi (green Ganoderma), yellow Reishi (yellow Ganoderma) and white Reishi (white Ganoderma). The scientific name of red Reishi is (Ganoderma lucidum), and the scientific names of black Reishi are (Ganoderma sinense, Ganoderma japonicum, Ganoderma atrum). As another mushroom of the genus Ganoderma, Ganoderma neojaponicum is known. These strains may be those distributed in markets such as China and Japan, or may be those prepared by isolation and culture from tissues of Ganoderma or the like.

[0017] In the cultivation method of Ganoderma of the present invention, generally used cultivation methods can be used except that bagasse is used as one of the medium components. One example thereof is shown below.

[0018] The cultivation method of Ganoderma of the present invention can be carried out, for example, by fungal bed cultivation, and comprises a culture step of inoculating a Ganoderma strain into a fungal bed, allowing the fungi to take root and propagate throughout the fungal bed, and a generation step of obtaining Ganoderma fruiting bodies.

[0019] The medium used for the fungal bed in the culture step may be any medium that appropriately contains, in addition to bagasse, carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients required for the growth of Ganoderma mycelia. For example, bagasse and a known medium base material, nutrient material, or a mixture thereof are used, and an additive can be further added.

[0020] The bagasse used in the present invention can be the residue of sugarcane extracted during the sugar production process, or it may be processed by washing, drying, and grinding. The shape of the bagasse is not particularly limited, but it is preferable to use bagasse that forms a fibrous structure, which has been ground or defibrated to a predetermined size (width, length). Among these, it is more preferable that the width is 5 mm or less or the length is 50 mm or less, and it is particularly preferable that the width is 5 mm or less and the length is 50 mm or less. Furthermore, the length is most preferably 10 to 50 mm. The bagasse content in the culture medium is preferably 10 to 80% by weight, more preferably 15 to 60% by weight, and most preferably 20 to 30% by weight, based on dry weight.

[0021] As the culture medium substrate, for example, sawdust (made from broad-leaved trees such as sawtooth oak, Japanese oak, and beech, or from coniferous trees such as pine, fir, and hemlock), corn cob, rice hull, beet, cotton hull, buckwheat hull, etc. are used, with sawdust and / or corn cob being preferred. The size of the culture medium substrate is not particularly limited, but it is preferable to use a 10 mm mesh pass product (one that has passed through a mesh with a 10 mm opening). The content of the culture medium substrate in the culture medium depends on the other culture medium composition, but for example, it is preferably 10 to 90% by weight, more preferably 30 to 80% by weight, particularly preferably 40 to 70% by weight, and most preferably 50 to 65% by weight by dry weight.

[0022] As nutritional materials, wheat bran, rice bran, corn bran, okara, bean hulls, milo, etc., are used, and preferably wheat bran, rice bran, or corn bran are selected and used. The size is not particularly limited, but it is preferable to use a 5 mm mesh-pass grade. The content of nutritional materials in the culture medium depends on the other composition of the culture medium, but it is preferably 1 to 30% by weight by dry weight, and more preferably 5 to 10% by weight.

[0023] In summary, the most preferred culture medium contains 20-30% by dry weight of bagasse, 50-65% by dry weight of sawdust, and 5-10% by dry weight of wheat bran.

[0024] Additives can be added as needed for purposes such as pH adjustment or growth promotion, and examples of additives that can be used include calcium carbonate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium dihydrogen phosphate, and dicalcium hydrogen phosphate.

[0025] The moisture content in the culture medium is not particularly limited, but is preferably 40-80% by weight, more preferably 50-75% by weight, and even more preferably 60-70% by weight relative to the total amount of the medium. However, it is preferable to adjust the moisture content appropriately so that there is sufficient void and moisture for the growth of the Ganoderma lucidum mycelium in the culture medium, and the culture medium does not dry out.

[0026] The culture medium described above can be placed in a commercially available container such as a plastic bag or plastic pot for mushroom cultivation, and subjected to sterilization or other treatments by known methods. The container for the culture medium and the methods for sterilization and other treatments can be appropriately selected as long as they achieve the effects of the present invention. In this way, a mushroom bed is obtained.

[0027] The Ganoderma lucidum strain is inoculated onto the substrate. Any known method of inoculation is acceptable.

[0028] The cultivation process is preferably carried out in the dark, but is not limited to the dark. The temperature is preferably 15-35°C, and more preferably 20-30°C. The humidity is not particularly limited, but it should be sufficient as long as the culture medium does not dry out. For example, the relative humidity is preferably 40-80%, and more preferably 50-65%. The duration of the cultivation process is preferably 30-80 days. As mycelial growth progresses, the carbon dioxide concentration will rise, which may cause poor growth, so ventilation should be performed as needed. The carbon dioxide concentration is preferably less than 1000 ppm. The cultivation process can be completed when the entire culture medium is covered with white mycelium under the above conditions, but it is also acceptable if part of the culture medium is not covered with mycelium, and cultivation can be continued even after it is covered with white mycelium. The cultivation conditions are not limited to the above range, and the temperature, relative humidity, and duration of the cultivation process can be appropriately selected as long as they are within the range that achieves the effects of the present invention.

[0029] For example, the temperature during the growth process is preferably 15-35°C, more preferably 25-30°C. The relative humidity is preferably 70% or higher, more preferably 90% or higher. The illuminance is preferably 50-3000 lux, more preferably 100-2000 lux, and most preferably 300-1000 lux. To promote growth, stimulation such as scraping the fungus or applying electricity may be used. The cultivation conditions during the growth process are not limited to the above ranges, and temperature, relative humidity, and illuminance may be appropriately selected as long as the cultivation conditions produce the effects of the present invention. By cultivating in this manner, the Ganoderma lucidum of the present invention can be obtained.

[0030] The Ganoderma lucidum used in this invention refers to the fruiting body, which is clearly distinguishable from the mycelium before fruiting body development by its appearance, components, etc.

[0031] The Ganoderma lucidum of the present invention can be used raw after harvesting, for example, or it can be processed by washing, drying, crushing, and extracting.

[0032] Examples of extraction solvents include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are used, and particularly preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol are used. These solvents may be used individually or in mixtures of two or more, for example, a 30-70% by weight aqueous solution of ethanol can be used. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0033] The extraction method is not particularly limited and can be performed by methods such as heat extraction, room temperature extraction, cold extraction, stirring extraction, pressurized extraction, and column extraction. Furthermore, the extract can be used in combination with the Ganoderma lucidum mushroom itself.

[0034] The above extract may be used as is, but if necessary, it may be used after being treated with methods such as concentration, dilution, filtration, decolorization, deodorization, drying, or ethanol precipitation, to the extent that the effects of the present invention are achieved. Furthermore, the active ingredients may be concentrated or isolated by methods such as column purification before use.

[0035] The Ganoderma lucidum extract used in this invention has the effect of efficiently promoting the proliferation of stem cells, and therefore can be used as a stem cell proliferation promoter. Furthermore, the stem cell proliferation promoter of this invention can also be used as a cell culture additive and research reagent for efficiently promoting the proliferation of stem cells.

[0036] The extract of Ganoderma lucidum according to the present invention can be applied to stem cells of mammals, including humans, to promote the proliferation of stem cells. The stem cells to which the stem cell proliferation promoter according to the present invention is applied are not particularly limited as long as they are in line with the objectives of the present invention, and include, for example, embryonic stem cells (ES cells); somatic stem cells present in bone marrow, blood, skin (epidermis, dermis, subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidneys, muscles, and other tissues; and stem cells artificially created by gene transfer, etc. (induced pluripotent stem cells: iPS cells). Preferably, it is more effective on stem cells derived from bone marrow, blood, skin, or adipose tissue. More preferably, it is most effective on mesenchymal stem cells.

[0037] Furthermore, the stem cell proliferation-promoting effect of the Ganoderma lucidum extract according to the present invention can be effective against stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats, and pigs.

[0038] The application of the Ganoderma lucidum extract according to the present invention to stem cells may be in vitro or in vivo, and in either case, it can exert its stem cell proliferation-promoting effect. Therefore, the Ganoderma lucidum extract according to the present invention can promote stem cell proliferation by culturing stem cells in a stem cell culture medium to which an effective amount thereof has been added, or by administering it to mammals, including humans.

[0039] When administering the Ganoderma lucidum extract according to the present invention into a living organism, it can be administered as is, but it can also be provided as various compositions such as topical skin preparations, pharmaceuticals, and food and beverages, containing appropriate additives within a range that does not impair the effects of the present invention. Furthermore, the pharmaceuticals of the present invention also include drugs for use in animals, i.e., veterinary drugs.

[0040] The Ganoderma lucidum extract according to the present invention has excellent stem cell proliferation-promoting properties, and is therefore effective in acting on stem cells in tissues or organs within the body, such as skin, bone marrow, cartilage, muscle, nerve, fat, and liver, to treat, improve, and prevent damage or injury to said tissues or organs. Furthermore, since stem cells decrease or their function declines with age, the Ganoderma lucidum extract according to the present invention is effective in treating, improving, and preventing diseases or aging related to the decrease or decline in function of stem cells in the tissues or organs mentioned above. Here, examples of diseases or aging related to tissue or organ damage or injury, and the decrease or decline in function of stem cells, include, for example, skin-related issues such as wrinkles, sagging, age spots, dullness, rough skin, skin thickening, enlarged pores, acne scars, wounds, pressure ulcers, burns, scars, and keloids, as well as damage to the scalp and hair, such as thinning hair and hair loss. Furthermore, bone-related conditions include osteoporosis and fractures (such as vertebral compression fractures and femoral neck fractures); cartilage diseases include osteoarthritis, rheumatoid arthritis, and herniated discs; neurological conditions include spinal cord injury, facial nerve paralysis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and age-related memory loss; blood-related conditions include aplastic anemia and leukemia; cardiovascular conditions include myocardial infarction and obstructive arteriosclerosis; dental conditions include periodontal disease and alveolar bone damage due to pyorrhea; ophthalmological conditions include retinitis pigmentosa, age-related macular degeneration, and glaucoma; and liver and pancreatic conditions include hepatitis, cirrhosis, and diabetes, but are not limited to these.

[0041] The dosage form of the topical skin preparation containing the Ganoderma lucidum extract according to the present invention may be any of the following: aqueous solution, solubilized, emulsified, powder, powder dispersion, oil-liquid, gel, ointment, aerosol, water-oil two-phase system, or water-oil-powder three-phase system. Furthermore, the topical skin preparation may be manufactured by selecting and appropriately blending various components, additives, bases, etc., commonly used in topical skin compositions, together with the Ganoderma lucidum extract, according to the type of component, and following methods known in the art. The form may be any of the following: liquid, emulsion, cream, gel, paste, spray, etc. The 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.), and esters (isopropyl myristate, i) Examples of ingredients include sopropyl, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, humectants, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0042] Examples of topical skin preparations include lotions, emulsions, gels, serums, general creams, sunscreens, packs, masks, cleansers, cosmetic soaps, foundations, face powders, bath additives, body lotions, body washes, hair shampoos, hair conditioners, and hair growth products.

[0043] The pharmaceutical product containing the Ganoderma lucidum extract according to the present invention can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations suitable for application to the affected area. Pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and use, appropriately selected formulation bases or carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances, etc., which can then be prepared into various formulations that can be administered orally or parenterally systemically or topically by various known methods. When providing the pharmaceutical product of the present invention in the above forms, it can be manufactured by methods commonly used by those skilled in the art, such as those indicated in the individual articles of the General Provisions for Formulations of the Japanese Pharmacopoeia [2].

[0044] The form of the pharmaceutical product of the present invention is not particularly limited, but examples include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injectable preparations (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections); drip preparations; suppositories; ointments; lotions; eye drops; sprays; transdermal preparations; transmucosal preparations; and patches. It may also be provided as a dried product that is redissolved before use, and in the case of injectable preparations, it may be provided in the form of unit dose ampoules or multi-dose containers.

[0045] When using the extract of Ganoderma lucidum according to the present invention as a pharmaceutical for the treatment, improvement, and prevention of skin-related injuries and diseases, the most suitable form is a topical preparation, such as an ointment, cream, gel, liquid, or patch. An ointment is a homogeneous, semi-solid topical preparation and includes oily ointments, emulsion ointments, and water-soluble ointments. A gel is a topical preparation in which a water-insoluble water-holding compound is suspended in an aqueous solution. A liquid preparation is a liquid topical preparation and includes lotions, suspensions, emulsions, liniments, and the like.

[0046] The pharmaceutical product of the present invention functions as a preventive agent to suppress the onset of the above-mentioned diseases, and / or as a therapeutic agent to improve them to a normal state. When the pharmaceutical product of the present invention is used as a pharmaceutical product for the treatment, improvement, and prevention of the aforementioned diseases, it can be administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, and cats.

[0047] The content of Ganoderma lucidum extract in the topical skin preparations, pharmaceuticals, and foods of the present invention is not particularly limited, but is preferably 0.0001 to 30% by weight, more preferably 0.001 to 10% by weight, and most preferably 0.02 to 2% by weight, when calculated as the dry solid content of Ganoderma lucidum extract relative to the total weight of the preparation (composition). Below 0.0001% by weight, the effect is low, and above 30% by weight, a significant increase in effect is unlikely to be observed. The above amounts are merely examples, and should be set and adjusted as appropriate considering the type and form of the composition, general usage amount, efficacy, etc. Furthermore, regarding the method of adding the active ingredient in formulation, it may be added in advance or added during manufacturing, and the appropriate method should be selected considering workability.

[0048] Furthermore, in this invention, "food" is used to mean not only general food and beverages, but also foods other than pharmaceuticals and quasi-drugs that can be consumed for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or foods for special dietary uses. Health foods include foods provided under names such as nutritional supplements, health supplements, nutritional adjustment foods, and supplements. Health functional foods are defined by the Food Sanitation Act or the Health Promotion Act and include foods for specified health uses, nutrient function foods, and foods with functional claims that can display specific health effects, the functions of nutritional components, or the reduction of disease risk.

[0049] The food product may take any form suitable for consumption, such as solid, liquid, granular, granular, powder, capsule, cream, or paste. In particular, for the health foods mentioned above, preferred forms include tablets, rounds, capsules, powder, granules, fine granules, lozenges, and liquids (including syrup, milk, and suspension).

[0050] Examples of food products include, but are not limited to, tea beverages (green tea, oolong tea, black tea, etc.), soft drinks (sports drinks, mineral water, near-water, etc.), carbonated drinks, dairy beverages, coffee beverages, fruit and vegetable juice beverages, nutritional drinks, jelly drinks and other beverages, powdered soups, confectionery (candy, gummies, gum, tablets, etc.), noodles, bread, dairy products, processed seafood and livestock products, oils and fats and processed oils and fats.

[0051] The food of the present invention may contain additives that are commonly used depending on its type. Any additive that is permissible under the Food Sanitation Act can be used, but examples include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorings, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0052] If the food of the present invention is a general food or beverage, it can be manufactured by adding a step of adding a Ganoderma lucidum extract to the normal manufacturing process of that food or beverage. If it is a health food, the manufacturing method can be the same as that for pharmaceuticals. For example, in the case of a tablet-shaped supplement, it can be manufactured by adding and mixing an excipient or other additive to a Ganoderma lucidum extract and then molding it under pressure using a tablet press or the like. In addition, other materials (for example, vitamins such as vitamin C, vitamin B2, and vitamin B6, minerals such as calcium, dietary fiber, etc.) can be added as needed.

[0053] The amount of Ganoderma lucidum extract contained in the food of the present invention should be sufficient to exert a stem cell proliferation-promoting effect, but it should be set appropriately considering the general intake amount of the target food, the form of the food, efficacy and effects, taste, palatability, and cost.

[0054] The present invention also relates to a method for promoting the proliferation of stem cells by culturing them in a culture medium containing an extract of Ganoderma lucidum. In other words, the method according to the present invention can be described as a method for producing stem cells and a method for promoting the proliferation of stem cells, which include the step of culturing stem cells in a culture medium containing an extract of Ganoderma lucidum.

[0055] In the method according to the present invention, any culture medium commonly used for stem cell proliferation may be used for culturing 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) can be used. Specifically, examples include Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), and Hank's balanced salt solution. The medium may also contain basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) as growth factors. Furthermore, the culture medium may contain, if necessary, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, ITS supplement, antibiotics, etc.

[0056] In addition to the above, it is preferable that serum be included in the culture medium at a concentration of 1 to 20% by volume. However, since the composition of serum varies depending on the lot and its effects can vary, it is preferable to use it after performing a lot check.

[0057] Commercially available culture media include mesenchymal stem cell basal medium from Thermo Fisher Scientific, mesenchymal stem cell basal medium from Takara Bio, MF medium from TOYOBO, and Hank's balanced salt solution from Sigma.

[0058] The incubators used for culturing stem cells are not particularly limited as long as they are capable of culturing stem cells, but examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc.

[0059] The culture vessel may be either non-adherent or adherent, and can be appropriately selected according to the purpose. Cell-adherent culture vessels may be treated with cell-supporting substrates such as extracellular matrix to improve cell adhesion. Examples of extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0060] The concentration of Ganoderma lucidum extract added to the culture medium used for stem cell culture can be appropriately determined in accordance with the Ganoderma lucidum extract content in the stem cell proliferation promoter according to the present invention described above, but in terms of solid content, for example, concentrations of 1 to 10,000 μg / mL, preferably 10 to 1,000 μg / mL, and most preferably 100 to 500 μg / mL can be used. Furthermore, Ganoderma lucidum extract may be added to the culture medium periodically during the stem cell culture period.

[0061] The culture conditions for stem cells should 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 approximately 30-40°C, preferably 36-37°C. The CO2 gas concentration is, for example, approximately 1-10%, preferably approximately 2-5%. It is preferable to change the culture medium once every 2-3 days, and more preferably daily. The culture conditions can also be appropriately varied within a range in which stem cells can survive and proliferate.

[0062] Next, in order to describe the present invention in detail, examples of cultivation methods for the Ganoderma lucidum used in the present invention, examples of production of Ganoderma lucidum extract, experimental examples, and formulation examples are given, but the present invention is not limited thereto. Unless otherwise specified, the percentages shown in the examples refer to weight percentages. [Examples]

[0063] (1) Cultivation of Ganoderma lucidum Bagasse was defibrated to a width of 5 mm or less (length 10-50 mm), sawdust was made from crushed Quercus serrata wood that passed through a 10 mm mesh, and wheat bran was passed through a 5 mm mesh. The container for the culture medium was a commercially available polypropylene bag with a filter that prevents contamination by bacteria, while also being breathable and moisturizing. A culture medium made by mixing 125 g of bagasse (25% of the dry weight of the culture medium), 325 g of sawdust (65% of the dry weight of the culture medium), 50 g of wheat bran (10% of the dry weight of the culture medium), and 1000 g of water was placed in the polypropylene bag. The mixture was sterilized by steam heating and allowed to cool to room temperature in a clean space. After that, 60 g of commercially available red reishi mushroom spawn was inoculated. After inoculation, the bag was sealed and cultured in the dark at a temperature of 21-27°C (average temperature 25°C) and a relative humidity of 40-75% (average relative humidity 51%). The culture process was continued until the entire culture medium was covered with mycelium, and the culture process was terminated. The duration of the culture process was 60 days.

[0064] Subsequently, the process moved to fruiting, where the mushrooms were grown at a temperature of 18-35°C (average temperature 27°C), relative humidity of 68-100% (average relative humidity 97%), and illumination of 3000 lux or less. This fruiting process was continued until the fruiting bodies of the Ganoderma lucidum were formed, at which point the fruiting process was terminated. The fruiting process lasted 30 days. During both the cultivation and fruiting processes, ventilation was performed as needed to maintain a carbon dioxide concentration of less than 1000 ppm.

[0065] The cultivation method for Ganoderma lucidum described above was designated as Cultivation Example 1. Furthermore, Cultivation Example 2 involved cultivating Ganoderma lucidum using bagasse (less than 10 mm in length) instead of the bagasse (10-50 mm in length) used in Cultivation Example 1. Additionally, as shown in Table 1, cultivation methods using Ganoderma lucidum with altered solid content in the growing medium were designated as Cultivation Examples 3-6 and Comparative Cultivation Examples 1-3, respectively. The yield of Ganoderma lucidum from each method was compared with Comparative Cultivation Example 1. Note that the yield of Ganoderma lucidum was measured as dry weight.

[0066] [Table 1]

[0067] (2) Extraction Production example 1A Hot water extract 10 g of dried Ganoderma lucidum obtained in cultivation example 1 was mixed with 200 mL of purified water, extracted at 95-100°C for 2 hours, filtered, concentrated the filtrate, and freeze-dried to obtain a hot water extract (Table 2).

[0068] Manufacturing Example 1B: 50% Ethanol Extract 10 g of dried Ganoderma lucidum obtained in cultivation example 1 was mixed with 200 mL of 50% ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain a 50% ethanol extract (Table 2).

[0069] Manufacturing Example 1C: Ethanol Extract 10 g of dried Ganoderma lucidum obtained in cultivation example 1 was mixed with 200 mL of ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain an ethanol extract (Table 2).

[0070] Using the Ganoderma lucidum specimens from cultivation examples 2-6 and comparative cultivation examples 1-2, extraction was performed in the same manner as in the above production examples 1A-1C, resulting in production examples 2A-6C and comparative production examples 1A-2C (Table 2).

[0071] [Table 2] [Examples]

[0072] Experimental Example 1: Stem Cell Proliferation Promotion Test

[0073] (Experimental Example 1) Evaluation of the proliferation-promoting effect on stem cells Human adipose tissue-derived mesenchymal stem cells (manufactured by DS Pharma Biomedical Co., Ltd.), cultured using human stem cell culture medium (manufactured by TOYOBO Co., Ltd.), are placed in a 6cm dish in a 3x10 cm arrangement. 5 Individual seeds were sown, and the test substance was added to the final concentration shown in Table 3. Cultivation was continued for 3 days.

[0074] After 3 days of culture, the cells were washed three times with PBS(-), collected using a rubber policeman, and the number of cells in each cell group was counted. The total number of cells without the test substance was used as the control, and the increase or decrease in the number of cells after the addition of the test substance was calculated relative to the control (100%) to evaluate the stem cell proliferation-promoting effect. These test results are shown in Table 3 below.

[0075] [Table 3]

[0076] As shown in Table 3, the extract of Ganoderma lucidum cultivated using the bagasse of the present invention showed a significantly higher stem cell proliferation-promoting effect than conventional Ganoderma lucidum extracts. In particular, the difference between the extract of the present invention and conventional extracts was even more pronounced in the hot water extract. When the control value described above is set to 100%, the number of human adipose tissue-derived mesenchymal stem cells at the start of culture was 25%. In addition to the stem cells used in this experiment, similar tests were conducted on epidermal stem cells, dermal stem cells, and hematopoietic stem cells, and the extract of Ganoderma lucidum cultivated using the bagasse of the present invention showed a significantly higher stem cell proliferation-promoting effect than conventional Ganoderma lucidum extracts.

[0077] Next, we show an example of a formulation prepared using the Ganoderma lucidum extract of the present invention. Note that a mixed extract refers to a mixture of equal amounts of each extract. [Examples]

[0078] Prescription Example 1: Lotion Formula Content (%) 1. Extract from 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 parahydroxybenzoate 0.1 9. Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1 10.Fragrance (appropriate amount) 11. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Components 1-6 and 11 and components 7-10 are uniformly dissolved, mixed together, and filtered to obtain the product.

[0079] In Formulation Example 1, the extracts from Production Example 1A were replaced with the extracts from Production Example 2A, Production Example 3A, Production Example 4A, Production Example 5A, and Production Example 6A, respectively, to obtain Formulation Examples 2, 3, 4, 5, and 6.

[0080] Prescription example 7: Cream Formula Content (%) 1. Manufacturing Example: Mixed extract of 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 (20 E.O.) 3.0 8. Behenyl alcohol 1.5 9. Glyceryl monostearate 2.5 10.Fragrance 0.1 11. Methyl parahydroxybenzoate 0.25 12.1,3-Butylene glycol 8.5 13. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 2-9, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 1 and 11-13, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring, add component 10 at 45°C, and further cool to 30°C to obtain the product.

[0081] In Formulation Example 7, Formulation Example 8 was obtained by replacing the mixed extracts of Production Examples 1A, 2A, 3A, 4A, 5A, and 6A with the mixed extracts of Production Examples 1B, 2B, 3B, 4B, 5B, and 6B.

[0082] Prescription example 9: Emulsion Formula Content (%) 1. Extract from Production Example 1C 1.0 2. Squalane 5.0 3. Olive oil 5.0 4. Jojoba oil 5.0 5. Cetanol 1.5 6. Glyceryl monostearate 2.0 7. Polyoxyethylene cetyl ether (20 E.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 parahydroxybenzoate 0.2 13. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 1-8, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 10-13, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring. At 45°C, add component 9, and further cool to 30°C to obtain the final product.

[0083] In Formulation Example 9, the extract from Manufacturing Example 1C was replaced with the extracts from Manufacturing Example 2C, Manufacturing Example 3C, Manufacturing Example 4C, and Manufacturing Example 5C, respectively, to obtain Formulation Examples 10, 11, 12, and 13.

[0084] Prescription example 14: Gel Formula Content (%) 1. Mixed extract of production example 1C and 2C 0.01 2. Ethanol 5.0 3. Methyl parahydroxybenzoate 0.1 4. Polyoxyethylene hydrogenated castor oil (60 E.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. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve ingredients 1-5 and ingredients 6-11 uniformly, then mix them together to form the product.

[0085] Prescription example 15 packs Formula Content (%) 1. Extract from manufacturing 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 parahydroxybenzoate 0.2 7. Polyoxyethylene hydrogenated castor oil (20 E.O.) 0.5 8. Citric acid 0.1 9. Sodium citrate 0.3 10.Fragrance (appropriate amount) 11. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve ingredients 1-11 uniformly to form the product.

[0086] Prescription Example 16: Foundation Formula Content (%) 1. Manufacturing Example: Mixed extract of 1B, 2B, 3B, 4B, 5B and 6B 1.0 2. Stearic acid 2.4 3. Polyoxyethylene sorbitan monostearate (20 E.O.) 1.0 4. Polyoxyethylene cetyl ether (20 E.O.) 2.0 5. Cetanol 1.0 6. Liquid lanolin 2.0 7. Liquid paraffin 3.0 8. Isopropyl myristate 6.5 9. Sodium carboxymethylcellulose 0.1 10. Bentonite 0.5 11. Propylene glycol 4.0 12. Triethanolamine 1.1 13. Methyl parahydroxybenzoate 0.2 14. Titanium dioxide 8.0 15. Talc 4.0 16. Bengara 1.0 17. Yellow iron oxide 2.0 18.Fragrance (appropriate amount) 19. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 2-8, maintain at 80°C to form the oil phase. Swell component 9 thoroughly in component 19, then add components 1 and 10-13 and mix uniformly. Add components 14-17, which have been crushed and mixed in a pulverizer, and stir with a homomixer, maintaining at 75°C to form the aqueous phase. Add the aqueous phase to this oil phase while stirring and emulsify. Then cool, add component 18 at 45°C, and cool to 30°C while stirring to obtain the product.

[0087] Prescription Example 17: Bath Additive Formula Content (%) 1. Extract from Production Example 1A 5.0 2. Extract from manufacturing example 2A 1.0 3. Sodium bicarbonate 50.0 4. Yellow No. 202 (1) appropriate amount 5.Fragrance (appropriate amount) 6. Add sodium sulfate to bring the total volume to 100. [Manufacturing Method] Mix ingredients 1-6 uniformly to form the product.

[0088] Prescription Example 18: Ointment Formula Content (%) 1. Manufacturing Example: Mixed extract of 1A, 2A, 3A, 4A, 5A, and 6A 0.5 2. Polyoxyethylene cetyl ether (30 E.O.) 2.0 3. Glyceryl monostearate 10.0 4. Liquid paraffin 5.0 5. Cetanol 6.0 6. Methyl parahydroxybenzoate 0.1 7. Propylene glycol 10.0 8. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 2-5, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 1 and 6-8, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool to 30°C while stirring to obtain the final product.

[0089] Prescription Example 19: Powder Formula Content (%) 1. Manufacturing Example: Mixed extract of 1B, 2B, 3B, 4B, 5B, and 6B 20.0 2. Dried corn starch 30.0 3. Microcrystalline cellulose 50.0 [Manufacturing method] Mix ingredients 1-3 and prepare as a powder.

[0090] Prescription example 20: Tablets Formula Content (%) 1. Manufacturing Example: Mixed extract of 1C, 2C, 3C, 4C, 5C, and 6C 3.0 2. Extract from manufacturing example 1A 0.1 3. Dried cornstarch 26.9 4. Carboxymethylcellulose calcium 20.0 5. Microcrystalline cellulose 40.0 6. Polyvinylpyrrolidone 7.0 7. Talc 3.0 [Manufacturing Method] Mix ingredients 1-5, then add an aqueous solution of ingredient 6 as a binder and form into granules. Add ingredient 7 to the formed granules and compress into tablets. Each tablet should weigh 0.52g.

[0091] Prescription example 21: Tablet confectionery Formula Content (%) 1. Manufacturing Example: Mixed extract of 1A, 2A, 3A, 4A, 5A, and 6A 0.5 2. Dried corn starch 50.0 3. Erythritol 40.0 4. Citric acid 5.0 5. Sucrose fatty acid ester 3.0 6.Fragrance (appropriate amount) 7. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Mix ingredients 1-4 and 7 and form into granules. Add ingredients 5 and 6 to the formed granules and compress into tablets. Each tablet should weigh 1.0g.

[0092] Prescription Example 22: Beverages Formula Content (%) 1. Manufacturing Example: Mixed extract of 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. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Mix ingredients 1-5 together to make a beverage.

[0093] Prescription Example 23: Powdered Beverage Formula Content (%) 1. Manufacturing Example: Mixed extract of 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 flavoring 0.03 [Manufacturing Method] Mix ingredients 1-8 to make a powdered beverage. [Industrial applicability]

[0094] Based on the above, the Ganoderma lucidum extract of the present invention showed superior stem cell proliferation-promoting activity compared to extracts of Ganoderma lucidum cultivated by conventional methods. Therefore, because the Ganoderma lucidum extract of the present invention has excellent stem cell proliferation-promoting effects, it is expected to have applications in fields such as regenerative medicine and regenerative beauty.

Claims

1. A stem cell proliferation promoter characterized by containing an extract of the fruiting body of Ganoderma lucidum cultivated in a culture medium containing 10 to 80% by weight of bagasse on a dry weight basis.

2. The stem cell proliferation promoter according to claim 1, characterized in that the extract of the fruiting body of Ganoderma lucidum is an extract obtained by dissolving it in one or more solvents selected from water, lower alcohols, and liquid polyhydric alcohols.

3. A method for promoting the proliferation of stem cells, comprising the step of culturing stem cells in a culture medium containing an extract of the fruiting body of Ganoderma lucidum according to claim 1 or 2.

4. A topical skin preparation characterized by containing an extract of the fruiting body of Ganoderma lucidum according to claim 1 or 2 as an active ingredient for stem cell proliferation.

5. A pharmaceutical product characterized by containing an extract of the fruiting body of Ganoderma lucidum according to claim 1 or 2 as an active ingredient for stem cell proliferation.

6. A food product characterized by containing an extract of the fruiting body of Ganoderma lucidum according to claim 1 or 2 as an active ingredient for stem cell proliferation.

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