Stem cell proliferation promoter

Abstract

The objective is to discover novel substances that have higher proliferation-promoting activity for stem cells, and to provide stem cell proliferation promoters, methods for promoting stem cell proliferation, and cosmetics, pharmaceuticals, quasi-drugs, or food and beverages containing stem cell proliferation promoters. [Solution] We have invented a stem cell proliferation promoter containing an extract of cocoa husk using a polar solvent as an active ingredient. That is, the present invention can be used as a method for preparing stem cells and / or as a stem cell proliferation promoter in regenerative medicine and regenerative beauty.

Description

Technical Field

[0001] The present invention relates to a stem cell growth promoter and a method for promoting stem cell growth.

Background Art

[0002] When a tissue of a vertebrate (particularly a mammal) is damaged in cells or organs due to injury, disease, aging, etc., a regeneration system works to recover the damage in cells or organs. Stem cells present in the tissue play a major role in this action. Stem cells have the pluripotency to differentiate into all cells and organs, and it is considered that this property leads to recovery by supplementing the damaged parts of cells and organs. There is an expectation for regenerative medicine, which is a next-generation medicine applying such stem cells.

[0003] The most advanced tissue in stem cell research in mammals is the bone marrow. Hematopoietic stem cells, which can self-replicate to produce the same cells as themselves and have the ability to differentiate into all blood cell types, exist in the bone marrow. Therefore, since it is possible to reconstruct blood cells by transplanting hematopoietic stem cells, hematopoietic stem cell transplantation is performed as a treatment method for blood diseases and immunodeficiency diseases. However, it has been reported that the proliferative ability and differentiation ability of hematopoietic stem cells decline with aging (Non-Patent Document 1), and there are also situations where blood cell system reconstruction cannot be achieved even after hematopoietic stem cell transplantation.

[0004] Therefore, in order to prevent or improve the decline in QOL caused by incomplete reconstruction of the blood cell system, it is considered important to restore the function of hematopoietic stem cells (such as restoring the proliferative ability and differentiation ability).

[0005] 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 the decrease of stem cells in order to maintain homeostasis in each tissue (Non-Patent Literature 2). In recent years, in order to apply the capabilities (pluripotency) of stem cells to the regeneration of organs and tissues, the development of technologies to culture and proliferate stem cells after isolating them from living tissue is progressing in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative beauty) (Non-Patent Literature 3, 4).

[0006] Cacao (scientific name: Theobroma cacao) is an evergreen tree belonging to the genus Theobroma in the family Malvaceae (or Sterileaceae) of the order Malvales. It has been known that extracts from cacao nibs, which are the endosperm of cacao, have an inhibitory effect on melanin production (Patent Document 1), extracts from cacao seeds (cocoa) have an inhibitory effect on the adhesion of leukocytes to vascular endothelial cells (Patent Document 2), and extracts from cacao husks, which are the outer shell of cacao beans, have collagenase inhibitory activity (Patent Document 3). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2007-15943 [Patent Document 2] Japanese Patent Application Publication No. 10-120587 [Patent Document 3] Japanese Patent Application Publication No. 3-44331 [Non-patent literature]

[0008] [Non-Patent Document 1] Age-associated characteristics of murine hematopoietic stem cells.J.Exp.Med.2000 Nov 6;192(9):1273-80. [Non-Patent Document 2] Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 [Non-Patent Document 3] Mabuchi, Y. et al., Journal of the Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268. [Non-Patent Document 4] 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]

[0009] In view of the above circumstances, the object of the present invention is to discover a novel substance that promotes the proliferation of stem cells, and to provide a stem cell proliferation promoter, a method for promoting the proliferation of stem cells, and a composition for promoting the proliferation of stem cells. [Means for solving the problem]

[0010] The inventors of this invention conducted extensive research to solve the above problems and, as a result, discovered that an extract of cocoa husk using a polar solvent has an excellent promoting effect on stem cell proliferation, thus completing the present invention.

[0011] In other words, the present invention encompasses the following inventions. (1) A stem cell proliferation promoter characterized by containing an extract of cocoa husk in a polar solvent. (2) A stem cell proliferation promoter wherein the polar solvent described in (1) is one or more solvents selected from water, ethanol, 1,3-butylene glycol, and propylene glycol. (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 cocoa husk in a polar solvent. (4) A stem cell proliferation promoting composition containing the stem cell proliferation promoting agent described in (1). [Effects of the Invention]

[0012] According to the present invention, stem cells can be efficiently proliferated. Therefore, the present invention can make a significant contribution to fields such as regenerative medicine and regenerative beauty. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below. The stem cell proliferation promoter according to the present invention contains an extract of cocoa husk in a polar solvent as an active ingredient.

[0014] The cacao plant (scientific name: Theobroma cacao) used in this invention is an evergreen tree belonging to the genus Theobroma in the family Malvaceae (or Sterileaceae) of the order Malvales. Native to the tropical regions of Central and South America, it is cultivated as a raw material for chocolate and cocoa. The tree grows to a height of about 5 to 10 meters, and its leaves are oblong, about 30 centimeters long, pointed at the tip, and arranged alternately. It flowers almost year-round, and the flowers are small, about 1.5 centimeters in diameter, white, and dry flowers that grow in clusters directly on the trunk or large branches. The fruit is oblong, about 15 to 25 centimeters long and 8 to 15 centimeters in diameter, and the pericarp is thick and hard, and comes in red, yellow, or purplish-brown. The fruit is divided into five chambers, and the seeds are arranged in a single row inside, and these seeds become cacao beans. Cacao beans are mainly divided into cacao husk (outer shell) and cacao nibs (endosperm). Cacao husks are generally unused parts of the cacao plant; some are used for compost, but most are discarded. Cacao nibs are used in chocolate, and cacao mass is generally made by roasting cacao nibs and then making a paste from them. Cacao husks have a different chemical composition from cacao nibs and cacao mass, and therefore their physiological activities are also completely different.

[0015] The cocoa husk used in this invention can be obtained from the above-mentioned growing regions or purchased commercially. For extraction, the cocoa husk itself may be extracted as is, or it may be subjected to processing such as heating, drying, grinding, or enzyme treatment.

[0016] The extraction method using a solvent is not particularly limited. For example, it can be carried out by heating extraction (e.g., 40 to 100 °C), normal temperature extraction (e.g., 15 to 25 °C), low temperature extraction (e.g., 0 to 15 °C), stirring extraction, column extraction, or the like. As the extraction solvent, any polar solvent may be used. Examples include water, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), and liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.). Preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol are good. These solvents may be used alone or in combination of two or more. The most preferred extraction solvent is water. In addition, an acid or alkali can be added to the above extraction solvent to use a solvent with adjusted pH.

[0017] Regarding the amount of the solvent used, there is no particular limitation. For example, it may be 5 times or more, preferably 10 times or more, based on the weight of the cacao husk (dry weight). However, for the convenience of the operation when performing concentration or filtration after extraction, it is preferably 100 times or less. Also, the extraction temperature and time can be appropriately selected depending on the type of the solvent used, the pressure during extraction, and the like.

[0018] The above extract may be used as the extracted solution as it is. However, if necessary, within the range where the effects of the present invention are achieved, it may be used after performing treatments such as concentration (concentration by reduced pressure concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon or the like, deodorization, ethanol precipitation, and the like. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0019] The extract of cacao husk using a polar solvent used in the present invention has the effect of efficiently growing stem cells, and thus can be used as a stem cell growth promoter. Furthermore, the stem cell growth promoter of the present invention can also be used as an additive for cell culture or the like for efficiently growing stem cells.

[0020] The stem cell proliferation promoter according to the present invention can be applied to mammalian stem cells, including human stem cells, to promote stem cell proliferation. 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 artificially produced stem cells (induced pluripotent stem cells: iPS cells) by gene transfer, etc. Preferably, the stem cell proliferation promoter according to the present invention is more effective on stem cells derived from bone marrow, blood, skin, or adipose tissue. Examples of ES cells include ES cells established by culturing early embryos before implantation, ES cells established by culturing early embryos produced by nuclear transplantation of somatic cell nuclei, and ES cells in which the genes on the chromosomes of such ES cells have been modified using genetic engineering techniques. Such ES cells can be produced, for example, by methods known to the public, but can also be obtained from designated institutions, or even purchased as commercially available products. Furthermore, these stem cells may be primary cultured cells, subcultured cells, or frozen cells.

[0021] Furthermore, the stem cell proliferation promoter 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.

[0022] The stem cell proliferation promoter according to the present invention may be applied to stem cells either in vitro or in vivo, and its effects can be exerted in either case. Therefore, the stem cell proliferation promoter according to the present invention can promote the proliferation of stem cells 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.

[0023] When administering the stem cell proliferation promoter 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 cosmetics, quasi-drugs, pharmaceuticals, and food and beverages, containing the promoter together with appropriate additives, as long as the effects of the present invention are not impaired. Furthermore, the compositions of the present invention also include drugs for use in animals, i.e., veterinary drugs.

[0024] The stem cell proliferation promoter according to the present invention is effective in treating, improving, and preventing damage to tissues or organs in the body, such as skin, osteoblasts, cartilage, muscle, nerve, fat, and liver, because the extract of cocoa husk, which is the active ingredient, has excellent stem cell proliferation-promoting properties. Furthermore, since stem cells decrease or their function declines with age, the stem cell proliferation promoter according to the present invention is effective in treating, improving, and preventing diseases related to the decrease or decline in function of stem cells in the above-mentioned tissues or organs in the body. Diseases related to tissue or organ damage, and the decrease or decline in function of stem cells, include, for example, skin-related conditions 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.

[0025] The dosage form of cosmetics and quasi-drugs containing the stem cell proliferation promoter 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, these cosmetics and quasi-drugs can be manufactured by selecting and appropriately blending various components, additives, bases, etc., commonly used in topical skin compositions, together with the stem cell proliferation promoter, according to their respective types, 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.

[0026] Examples of cosmetics and quasi-drugs 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.

[0027] A pharmaceutical product containing the stem cell proliferation promoter 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 application, 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 be appropriately added to prepare 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].

[0028] 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.

[0029] When the stem cell proliferation promoter according to the present invention is used as a pharmaceutical for treating, improving, and preventing the aforementioned skin-related injuries and diseases, a 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.

[0030] 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.

[0031] The amount of stem cell proliferation promoter in the compositions of cosmetics, pharmaceuticals, quasi-drugs, etc. 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 5% by weight, based on the total weight of the formulation (composition) and converted to the dry solid content of the extract of cocoa husk in a polar solvent. Below 0.0001% by weight the effect is low, and above 30% by weight no significant enhancement of the effect is likely 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, the method of adding the active ingredient in formulation may be added in advance or added during manufacturing, and the appropriate method should be selected considering workability.

[0032] Furthermore, in this invention, "food and beverages" refers not only to general food and beverages but also to 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.

[0033] The form of food and beverages may be any form suitable for consumption, such as solid, liquid, granular, granular, powder, capsule, cream, or paste. In particular, for the above-mentioned health foods, preferred forms include tablets, rounds, capsules, powder, granules, fine granules, lozenges, and liquids (including syrup, milk, and suspension).

[0034] Examples of food and beverages 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, 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.

[0035] The food and beverages of the present invention may contain additives that are commonly used depending on their type. Any additives that are 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.

[0036] If the food or beverage of the present invention is a general food or beverage, it can be manufactured by including a step of adding an extract of cocoa husk in a polar solvent during the normal manufacturing process of the food or beverage. If it is a health food, the manufacturing method can be the same as that of the pharmaceutical described above. For example, in the case of a tablet-shaped supplement, it can be manufactured by adding and mixing an extract of cocoa husk in a polar solvent with additives such as excipients, 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.

[0037] The amount of cocoa husk extract in the food and beverage 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 food and beverage, the form of the food and beverage, efficacy and effects, taste, palatability, and cost.

[0038] 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 cocoa husk in a polar solvent. 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 cocoa husk in a polar solvent.

[0039] 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.

[0040] In addition to the above, it is preferable that serum be included in the culture medium at a concentration of 1-20%. 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.

[0041] Commercially available culture media include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hank's balanced salt solution.

[0042] 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.

[0043] 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.

[0044] The concentration of the cocoa husk extract in a polar solvent added to the culture medium used for stem cell culture can be appropriately determined in accordance with the content of the cocoa husk extract in a polar solvent in the stem cell proliferation promoter according to the present invention described above. However, when converted to solid content, for example, concentrations of 10 to 100,000 μg / mL, preferably 100 to 10,000 μg / mL, and most preferably 200 to 5,000 μg / mL can be used. Furthermore, the cocoa husk extract in a polar solvent may be added to the culture medium periodically during the stem cell culture period.

[0045] 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. 2 The gas concentration is, for example, about 1-10%, preferably about 2-5%. The culture medium is preferably changed once every 2-3 days, and more preferably daily. The culture conditions can also be adjusted as appropriate within a range that allows stem cells to survive and proliferate.

[0046] Next, in order to describe the present invention in more detail, examples of the production, experimental, and formulation of the extract used in the present invention will be given as examples, but the technical scope of the present invention is not limited to these. In the production examples, % refers to weight percentage, and in the examples, parts refer to parts by weight. [Examples]

[0047] Example of preparation of cocoa husk extract using a polar solvent. Extracts of cocoa husk using polar solvents were prepared as follows. In preparation examples 1 to 4, dried cocoa husk was used as the extraction material.

[0048] (Manufacturing Example 1) Preparation of Hot Water Extract of Cocoa Husk 10 g of ground cocoa husk was added to 200 mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 3.6 g of hot water extract of cocoa husk.

[0049] (Manufacturing Example 2) Preparation of 50% ethanol extract from cocoa husk 10 g of crushed cocoa husk was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the substance. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 1.5 g of 50% ethanol extract of cocoa husk.

[0050] (Manufacturing Example 3) Preparation of ethanol extract from cocoa husk 10 g of crushed cocoa husk was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.37 g of cocoa husk ethanol extract.

[0051] (Manufacturing Example 4) Preparation of 1,3-butylene glycoside extract from cocoa husk 10 g of crushed cocoa husk was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days to extract the substance. The resulting extract was filtered to obtain 190 g of 1,3-butylene glycol extract from cocoa husk. [Examples]

[0052] Prescription Example 1: Lotion Formulation Content (per portion) 1. Hot water extract of cocoa husk (production example 1) 0.1 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.

[0053] Prescription Example 2: Cream Formulation Content (per portion) 1. 50% ethanol extract from cocoa husk (Production Example 2) 0.05 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.

[0054] Prescription example 3: Emulsion Formulation Content (per portion) 1. Ethanol extract from cocoa husk (Production Example 3) 0.05 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.

[0055] Prescription example 4: Gel Formulation Content (per portion) 1. 1,3-butylene glycoside extract from cocoa husk (Production Example 4) 0.1 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 components 2-5 and components 1 and 6-11 uniformly, then mix them together to obtain the product.

[0056] Prescription example 5 pack Formulation Content (per portion) 1. Hot water extract of cocoa husk (Production example 1) 1.0 2. 1,3-butylene glycoside extract from cocoa husk (Production Example 4) 5.0 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.

[0057] Formula example 6: Foundation Formulation Content (per portion) 1. 50% ethanol extract from cocoa husk (Production Example 2) 0.1 2. Stearic acid 2.4 3. Polyoxyethylene sorbitan monostearate (20E.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] Components 2-8 are heated and dissolved, and maintained at 80°C to form the oil phase. Component 9 is thoroughly swollen into component 19, and then components 1 and 10-13 are added and mixed uniformly. Components 14-17, which have been crushed and mixed in a pulverizer, are added to this mixture, and the mixture is stirred with a homomixer and maintained at 75°C to form the aqueous phase. The aqueous phase is added to this oil phase while stirring, then cooled, component 18 is added at 45°C, and the mixture is stirred while cooling to 30°C to obtain the final product.

[0058] Prescription Example 7: Bath Additive Formulation Content (per portion) 1. Hot water extract of cocoa husk (production example 1) 0.1 2. Sodium bicarbonate 50.0 3. Yellow No. 202 (1) appropriate amount 4.Fragrance (appropriate amount) 5. Add sodium sulfate to bring the total volume to 100. [Manufacturing Method] Mix ingredients 1-5 uniformly to form the product.

[0059] Prescription example 8: Ointment Formulation Content (per portion) 1. Hot water extract of cocoa husk (Production example 1) 1.0 2. 1,3-butylene glycoside extract from cocoa husk (Production Example 4) 1.0 3. Polyoxyethylene cetyl ether (30 E.O.) 2.0 4. Glyceryl monostearate 10.0 5. Liquid paraffin 5.0 6. Cetanol 6.0 7. Methyl parahydroxybenzoate 0.1 8. Propylene glycol 10.0 9. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 3-6, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 1, 2 and 7-9, 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.

[0060] Prescription example 9: Powder Formulation Content (per portion) 1. Hot water extract of cocoa husk (Production example 1) 1.0 2. Dried Corn Starch 39.0 3. Microcrystalline Cellulose 60.0 [Manufacturing method] Mix ingredients 1-3 and prepare as a powder.

[0061] Prescription example 10: Tablets Formulation Content (per portion) 1. Ethanol extract of cocoa husk (Production Example 3) 3.0 2. Dried corn starch 27.0 3. Carboxymethylcellulose calcium 20.0 4. Microcrystalline cellulose 40.0 5. Polyvinylpyrrolidone 7.0 6. Talc 3.0 [Manufacturing Method] Mix ingredients 1-4, then add an aqueous solution of ingredient 5 as a binder and form into granules. Add ingredient 6 to the formed granules and compress into tablets. Each tablet should weigh 0.52g.

[0062] Prescription example 11: Tablet confectionery Formulation Content (per portion) 1. Ethanol extract from cocoa husk (Production example 3) 2.0 2. Dried Corn Starch 49.8 3. Erythritol 40.0 4. Citric acid 5.0 5. Sucrose fatty acid ester 3.0 6.Fragrance 0.1 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.

[0063] Prescription Example 12: Beverages Formulation Content (per portion) 1. Hot water extract of cocoa husk (production example 1) 0.05 2. Stevia 0.05 3. Malic acid 5.0 4.Fragrance 0.1 5. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve ingredients 2 and 3 in a small amount of water. Then add ingredients 1, 4 and 5 and mix.

[0064] Next, to explain the effects of the present invention in detail, experimental examples will be given. [Examples]

[0065] Experimental Example 1: Stem Cell Proliferation Promotion Test A6 cells (RIKEN BRC Cell Bank) were used as hematopoietic stem cells. These hematopoietic stem cells, maintained in 20% FBS DMEM / F12 (10 μg / mL bovine insulin, 10 μg / mL bovine transferrin, 10 ng / mL bFGF) medium (Gibco), were divided into groups of 5 × 10⁶ cells. 4 Cells were seeded into 96-well plates (Falcon) to a total concentration of 100 cells. Next, the test substance was added to a final concentration of 625 μg / mL, and the cells were cultured for 24 hours. After the culture period, the cells were washed three times with PBS(-) and the number of cells 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 effect of promoting stem cell proliferation.

[0066] The experimental results are shown in Table 1. The cocoa husk extract in a polar solvent according to the present invention significantly promoted the proliferation of stem cells. On the other hand, when a hexane extract of cocoa husk was used as the test substance and evaluated similarly, no effect on promoting stem cell proliferation was observed.

[0067] [Table 1] [Industrial applicability]

[0068] Based on the above, the extract of cocoa husk using a polar solvent exhibited excellent stem cell proliferation-promoting properties. Therefore, the extract of cocoa husk using a polar solvent can be used in the fields of tissue regeneration and homeostasis maintenance in regenerative medicine and regenerative cosmetics.

Claims

1. A stem cell proliferation promoter characterized by containing an extract of cocoa husk in a polar solvent.

2. A stem cell proliferation promoter, wherein the polar solvent described in claim 1 is one or more solvents selected from water, ethanol, 1,3-butylene glycol, and propylene glycol.

3. A method for promoting stem cell proliferation, comprising the step of culturing stem cells in a culture medium containing an extract of cocoa husk in a polar solvent.

4. A stem cell proliferation promoting composition comprising the stem cell proliferation promoting agent described in claim 1.

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