Beige adipocyte differentiation inducer promoter

Abstract

To provide an agent that can control the differentiation and balance of cells of adipose tissue to increase and activate beige adipocytes, efficiently burning fat.SOLUTION: The present invention provides an agent for promoting the induction of differentiation of adipose tissue-derived stem cells to beige adipocytes, containing Akebia extract as an active ingredient, an agent for promoting the transformation of white adipocytes to beige adipocytes, and a fat combustion promoter.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent for promoting the differentiation induction of adipose-derived stem cells into beige adipocytes, an agent for promoting the conversion of white adipocytes into beige adipocytes, and an agent for promoting fat burning.

Background Art

[0002] Adipose tissue is one of the important organs present in the living body and is roughly classified into two types: white adipose tissue and brown adipose tissue. White adipose tissue is composed of white adipocytes that store energy as neutral lipids inside cells and has the function of storing excess energy in the living body. Also, these white adipocytes decompose the stored neutral lipids into free fatty acids when the living body needs energy, such as during exercise or starvation, and synthesize adenosine triphosphate (ATP) through the electron transport system present in mitochondria, thereby playing a role in supplying energy to the living body.

[0003] On the other hand, brown adipocytes also store neutral lipids inside cells like white adipocytes, but brown adipocytes express uncoupling protein 1 (UCP1), and due to the function of this gene, they convert neutral lipids into heat without going through ATP synthesis in the electron transport system and directly consume energy.

[0004] Therefore, in the living body, white adipocytes store and supply energy when needed, while brown adipocytes consume energy through heat production, thus regulating the energy balance in the living body (Non-Patent Documents 1 to 3).

[0005] Recent research results have revealed that abnormal balance in the roles of these white and brown adipocytes is closely associated with the development of obesity and metabolic syndrome (Non-Patent Documents 4 and 5). In particular, obesity is caused by the storage of excess ingested energy in white adipose tissue, which is considered the main cause of the onset of metabolic syndrome. To address this, it is necessary to efficiently consume energy.

[0006] Therefore, for the prevention and treatment of diseases such as obesity and metabolic syndrome, it is necessary to reduce white adipocytes that excessively accumulate energy in the body and increase brown adipocytes that consume energy, and the challenge is how to control the balance of these cells. In particular, the function of brown adipocytes is important, and the development of technologies to efficiently increase these cells in the body may be directly applicable to the improvement of obesity and metabolic syndrome in the future.

[0007] In recent years, it has been confirmed that white adipocytes can change into cells that express UCP1, similar to brown adipocytes, in response to cold stimulation, hormones induced by β3-adrenergic receptor, or exercise, such as Irisin (Non-Patent Documents 6 and 7). The brown adipocyte-like cells generated from white adipocytes are called "beige adipocytes" or "bright cells," and it is known that they can switch from the role of storing energy in white adipocytes to directly consuming energy, similar to brown adipocytes. Therefore, promoting the browning of white adipocytes that accumulate neutral lipids and inducing their differentiation into beige adipocytes is considered an effective new approach for improving obesity and metabolic syndrome. However, there is a problem that it is difficult to control the balance of such cells in the body. So far, substances having the effect of inducing the differentiation of white adipocytes into beige adipocytes, such as resveratrol polymer compounds (Patent Document 1) and Saposhnikovia divaricata acid extract (Patent Document 2), have been reported, but the search for more active factors is desired.

Prior Art Documents

Patent Document

[0008]

Patent Document 1

Patent Document 2

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0010] In view of the above circumstances, an object of the present invention is to provide a drug that can control the differentiation and balance of cells in adipose tissue, increase and activate beige adipocytes, and efficiently burn fat.

Means for Solving the Problems

[0011] As a result of intensive research to solve the above problems, the present inventors have found that an extract of Akebia quinata has the effects of promoting the induction of differentiation from stem cells in adipose tissue into beige adipocytes and promoting the conversion from white adipocytes to beige adipocytes, and have thus completed the present invention.

[0012] That is, the present invention includes the following inventions. (1) A promoter for promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes, containing an extract of Akebia quinata as an active ingredient. (2) A promoter for promoting the conversion from white adipocytes to beige adipocytes, containing an extract of Akebia quinata as an active ingredient. (3) The agent according to (1) or (2), which enhances the expression of uncoupling protein 1 (UCP1) in beige adipocytes. (4) A fat burning promoter, containing an extract of Akebia quinata as an active ingredient. (5) A composition for promoting the increase in beige adipocytes, containing the agent according to any one of (1) to (4). (6) A method for promoting the induction of differentiation of adipose tissue-derived stem cells into beige adipocytes, including the step of culturing adipose tissue-derived stem cells in a medium containing an extract of Akebia quinata. (7) A method for converting white adipocytes into beige adipocytes, including the step of culturing white adipocytes in a medium containing an extract of Akebia quinata.

Effects of the Invention

[0013] The extract of Akebia quinata, which is the active ingredient of the present invention, has the effects of promoting the induction of differentiation from adipose stem cells to beige adipocytes and promoting the conversion of white adipocytes to beige adipocytes. Therefore, by applying the extract of Akebia quinata to a living body, while suppressing the differentiation of adipose stem cells into white adipocytes, it promotes the induction of differentiation into beige adipocytes, and can also convert white adipocytes that have already accumulated fat into beige adipocytes. As a result, white adipocytes that store energy as fat are digested, and beige adipocytes that convert energy into heat increase, so fat burning in the living body is promoted. Therefore, the agent containing the extract of Akebia quinata of the present invention as an active ingredient is effective in improving and preventing obesity caused by fat accumulation.

Embodiments for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail.

[0015] 1. Agent for promoting the induction of differentiation from adipose tissue-derived stem cells to beige adipocytes, agent for promoting the conversion from white adipocytes to beige adipocytes, and agent for promoting fat burning The agent for promoting the induction of differentiation from adipose tissue-derived stem cells to beige adipocytes, the agent for promoting the conversion from white adipocytes to beige adipocytes, and the agent for promoting fat burning of the present invention (hereinafter referred to as "the agent of the present invention") contain the extract of Akebia quinata as an active ingredient.

[0016] In the present invention, "adipose tissue-derived stem cells" (also simply referred to as "adipose stem cells") are stem cells contained in adipose tissue, and refer to cells that can differentiate into mature adipocytes (white adipocytes, brown adipocytes) having lipid droplets in the cytoplasm. Also, "beige adipocytes" are brownish cells rich in mitochondria, also called bright adipocytes and brown-like adipocytes, highly express uncoupling protein 1 (UCP1), and actively produce heat to consume the accumulated energy.

[0017] In the present invention, "promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes" and "promoting the conversion from white adipocytes into beige adipocytes" mean promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes at the in vivo level or the culture level, promoting the conversion from white adipocytes into beige adipocytes, increasing beige adipocytes, and activating beige adipocytes by enhancing the expression of uncoupling protein 1 (UCP1).

[0018] The Akebia (also known as "Tongcao" or "Mutong", scientific name: Akebia quinata) used in the present invention is a trailing deciduous shrub belonging to the genus Akebia in the family Lardizabalaceae. Examples of Akebia that can be used in the present invention include Akebia quinata (scientific name), as well as its closely related species in the same genus, such as Akebia trifolia (scientific name), Akebia pentaphylla (scientific name), Akebia longeracemosa (scientific name), etc. Among them, Akebia quinata and Akebia trifolia are preferred.

[0019] In the present invention, the extract of Akebia refers to the extract of the whole plant of Akebia, or a part of the plant such as flowers, flower spikes, fruits, pericarp, stems, leaves, branches, branch leaves, roots, seeds, etc., or a mixture thereof. However, the parts used as extraction raw materials in the present invention are preferably fruits, pericarp, and seeds. Also, for extraction, these plants may be used as they are, or may be subjected to treatments such as drying, pulverization, and cutting into small pieces.

[0020] Examples of the solvent used for the extraction of Akebia include water or hot water, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (such as acetone, methyl ethyl ketone, etc.), acetonitrile, esters (such as ethyl acetate, butyl acetate, etc.), hydrocarbons (such as hexane, heptane, liquid paraffin, etc.), ethers (such as ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water or hot water, lower alcohols, and liquid polyhydric alcohols are preferred, and water or hot water, ethanol, and 1,3-butylene glycol are more preferred. These solvents may be used alone or in combination of two or more. For example, an aqueous ethanol solution of 30 to 70 v / v% can be preferably used. In addition, an acid or an alkali can be added to the above extraction solvent to use a solvent with adjusted pH.

[0021] When using Akebia seeds as the extraction material, they can be directly extracted with the above solvent. However, in some cases, after removing fats and oils with hydrocarbons (such as hexane, heptane, etc.) or ethers (such as ethyl ether, tetrahydrofuran, propyl ether, etc.) to defat them, the defatted seeds can be further extracted with the above extraction solvent. For the seeds, the residue after pressing or extraction can also be used. For example, it is preferable to extract the residue after pressing Akebia seeds with hydrocarbons and then extract the residue with an extraction solvent of water, lower alcohols, liquid polyhydric alcohols, ketones, or hydrocarbons (which may be used alone or in combination of two or more). An extraction solvent of water, ethanol, or hexane (which may be used alone or in combination of two or more) is more preferable, and hydrous ethanol (a mixed solvent of water and ethanol) or ethanol is most preferable.

[0022] The amount of solvent used for extraction is generally 2 to 200 parts by weight, preferably 10 to 100 parts by weight, per 1 part by weight of the extraction raw material. If the amount of extraction solvent is less than this range, the extraction solvent may not be evenly distributed throughout the extraction raw material, and the extraction efficiency may decrease. If the amount of extraction solvent exceeds this range, the burden during subsequent removal of the extraction solvent will increase.

[0023] The extraction temperature is a temperature below the boiling point of the solvent used for extraction, and is not particularly limited. For example, it may be extraction by heating or extraction at room temperature.

[0024] The extraction time is 1 to 2 weeks in the case of room temperature extraction, and 30 minutes to 24 hours, preferably 1 to 10 hours, in the case of heating extraction. However, it can be appropriately adjusted according to conditions such as the type of extraction solvent and extraction temperature. The number of extraction operations is not particularly limited, and it may be 1 time, or after the first extraction, fresh extraction solvent may be added again and extraction operations may be carried out for the second and subsequent times. Also, extraction operations may be carried out multiple times using the same extraction solvent.

[0025] The extraction method may be any method commonly used in the art, and is carried out using any device at room temperature or under heating. Specifically, the extraction raw material is put into an extraction treatment tank filled with the extraction solvent, and the soluble components are eluted while stirring from time to time as necessary, and then filtered to remove the residue to obtain the extract.

[0026] The above-mentioned extract may be used as the extracted solution as it is, but if necessary, within the range that does not affect its effect, it may be used after treatment such as concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc. 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.

[0027] The above-mentioned extract of Akebia has the effects of promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes and promoting the conversion from white adipocytes to beige adipocytes at the in vivo level or the culture level. Therefore, it can increase beige adipocytes, activate them through enhanced expression of uncoupling protein 1 (UCP1), and efficiently burn fat. Thus, the extract of Akebia can be used as an active ingredient of an agent for promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes, an agent for promoting the conversion from white adipocytes to beige adipocytes, and an agent for promoting fat burning.

[0028] The content of the extract of Akebia in the agent of the present invention is not particularly limited. For example, it is preferably 0.00001 to 10% by weight in terms of the dried product based on the total amount of the drug, and more preferably 0.0001 to 1% by weight.

[0029] In vitro, the agent of the present invention can be used as an additive for cell culture media, a research reagent, and a medical reagent for promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes and promoting the conversion from white adipocytes to beige adipocytes.

[0030] When the agent of the present invention is used in vivo, it can be used as it is. However, within the range that does not impair the effects of the present invention, it can be formulated with appropriate additives into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, foods, and drinks, and provided as a composition for promoting the increase of beige adipocytes. Note that the pharmaceuticals of the present invention include drugs for animals, that is, veterinary drugs. When the agent of the present invention is used for purposes such as slimming effects or face slimming effects, it is preferably in the form of cosmetics or quasi-drugs, or in the form of foods and drinks such as beauty drinks. When the agent of the present invention is used for purposes such as treating and / or preventing diseases caused by obesity, it is preferably used in the form of pharmaceuticals.

[0031] When the agent of the present invention is incorporated into cosmetics or quasi-drugs, a composition for external use on the skin is preferred, and any dosage form such as an aqueous solution system, solubilized system, emulsified system, powder system, powder dispersion system, oil solution system, gel system, ointment system, aerosol system, water-oil two-layer system, or water-oil-powder three-layer system may be used. Further, the cosmetics and quasi-drugs can be produced according to known methods in the art by selecting and appropriately blending various components, additives, bases, etc. usually used in compositions for external use on the skin, together with the above-mentioned extract of Akebia quinata, according to their types. The form may be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of the components to be incorporated into the composition for external use on the skin include fats and oils (such as olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (such as lanolin, beeswax, carnauba wax, etc.), hydrocarbons (such as liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (such as myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (such as isopropyl myristate, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (such as citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidonecarboxylic acid, etc.), saccharides (such as maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and hydrolysates of proteins, amino acids and their salts, vitamins (such as β-carotene (vitamin A), vitamin C, vitamin E, etc.), plant and animal extract components, various surfactants, humectants, ultraviolet absorbers, pH adjusters, stabilizers, preservatives, bactericides, fragrances, etc.

[0032] Examples of the types of cosmetics and quasi-drugs include, but are not limited to, lotion, emulsion, gel, essence, general cream, sunscreen cream, pack, mask, facial wash, cosmetic soap, foundation, powder, bath agent, body lotion, body shampoo, etc.

[0033] When the agent of the present invention is formulated into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various pharmaceutical preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharmaceutically acceptable additives, depending on the dosage form and use, appropriately selected pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegrant aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizing aids, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances, etc. can be appropriately added, and it can be prepared into various dosage forms that can be administered orally or parenterally, systemically or locally by various known methods. When the pharmaceutical product of the present invention is provided in the above-mentioned various forms, it can be manufactured by manufacturing methods commonly used by those skilled in the art, for example, the manufacturing methods shown in each article of the General Rules of Pharmaceutical Preparations of the Japanese Pharmacopoeia [2].

[0034] For oral dosage forms, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegrant aids such as carboxymethyl cellulose, carboxymethyl cellulose calcium, starch, or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropyl methylcellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat, etc. can be used, but are not limited thereto.

[0035] For parenteral dosage forms, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, vegetable oil, etc.; isotonic agents such as glucose, sodium chloride, D-mannitol, etc.; pH adjusters such as inorganic acids, organic acids, inorganic bases or organic bases, etc. can be used, but are not limited thereto.

[0036] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs, and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, suppositories, transdermal absorption preparations, transmucosal absorption preparations, and patches. In addition, the pharmaceutical may be a dried product that is redissolved when used, and in the case of an injection preparation, it is provided in the form of a unit-dose ampule or a multi-dose container.

[0037] The Akebia extract, which is an active ingredient of the agent of the present invention, has an effect of promoting differentiation induction from adipose tissue-derived stem cells to beige fat cells and an effect of promoting conversion from white fat cells to beige fat cells, and is therefore effective as a medicine for improving and preventing obesity and metabolic syndrome (visceral fat syndrome), or for treating and / or preventing diseases caused by obesity and metabolic syndrome. Here, examples of "diseases caused by obesity and metabolic syndrome" include arteriosclerosis, hyperlipidemia (hypertriglyceridemia, hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, etc.), fatty liver, hypertension, diabetes, ischemic heart disease (angina pectoris, myocardial infarction, etc.), and cerebrovascular disorders (cerebral infarction, cerebral hemorrhage, etc.). The pharmaceutical of the present invention functions as a preventive drug that suppresses the onset of the above-mentioned diseases and / or as a therapeutic drug that improves the condition to a normal state.

[0038] Since the active ingredient of the pharmaceutical of the present invention is derived from a natural product, it is extremely safe and has no side effects. Therefore, when used as a pharmaceutical for the prevention and / or treatment of the above-mentioned diseases, it can be administered orally or parenterally in a wide range of dosages to mammals such as humans, mice, rats, rabbits, dogs, and cats.

[0039] The dosage or administration amount of the cosmetics, quasi-drugs, and pharmaceuticals of the present invention can be appropriately determined according to their types, forms, the age, sex, body weight, degree of symptoms, etc. of the subject of use or administration. For example, when orally administering to adults, as the extract of Akebia quinata, it is in the range of 0.1 to 1000 mg / day, preferably 1 to 500 mg / day, more preferably 5 to 300 mg / day, and is administered once to several times a day respectively. In some cases, an amount less than the above dosage range may be sufficient, and in other cases, it may be necessary to administer beyond the range.

[0040] When the extract of Akebia quinata is formulated into cosmetics, quasi-drugs, and pharmaceuticals, its content is not particularly limited, but in terms of the dry solid content of the extract of Akebia quinata relative to the total weight of the preparation (composition), 0.001 to 30% by weight (w / w) is preferred, and 0.01 to 10% by weight (w / w) is more preferred. If it is less than 0.001% by weight (w / w), the effect is low, and even if it exceeds 30% by weight (w / w), a significant enhancement in the effect is hardly observed. Also, regarding the method of adding the active ingredient in formulation, it may be added in advance or during the manufacturing process, and it may be appropriately selected considering workability.

[0041] In addition, the agent of the present invention can also be formulated into foods and drinks. By providing it in the form of foods and drinks, it becomes easy to ingest the active ingredient of the present invention daily or continuously. In the present invention, foods and drinks include, in addition to general foods and drinks, foods that can be ingested for the purpose of maintaining or promoting health other than pharmaceuticals, for example, health foods, functional foods, health functional foods, or special-purpose foods. Health foods include foods provided under names such as dietary supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Law or the Food Promotion Law, and include foods for specified health uses and nutritional functional foods that can display specific health effects, functions of nutritional components, reduction of disease risks, etc. The form of foods and drinks may be any form suitable for consumption, for example, solid, liquid, granular, pellet, powder, capsule, cream, or paste.

[0042] Examples of food and drink products include, but are not limited to, breads, noodles, confectioneries, dairy products, processed fishery and livestock foods, fats and oils and processed oil and fat products, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, milk drinks, etc.) and concentrated stock solutions and powder for adjustment of such beverages.

[0043] The food and drink products of the present invention may be appropriately blended with additives commonly used according to their types. Any additives that are acceptable in food hygiene can be used. For example, 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, flavors, colorants, buffering agents, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, antiseptics, etc.

[0044] The blending amount of the extract of Akebia quinata in the food and drink products of the present invention may be any amount that can exhibit the effects of promoting the induction of differentiation from adipose tissue-derived stem cells into beige adipocytes, promoting the conversion of white adipocytes into beige adipocytes, and promoting fat burning, but it may be appropriately set in consideration of the general intake amount of the target food and drink products, the form of the food and drink products, efficacy and effects, taste, palatability, and cost.

[0045] 2. Method for promoting the induction of differentiation of adipose tissue-derived stem cells into beige adipocytes and method for converting white adipocytes into beige adipocytes The present invention also relates to a method for promoting the induction of differentiation of adipose tissue-derived stem cells into beige adipocytes, which includes a step of culturing adipose tissue-derived stem cells in a medium containing an extract of Akebia quinata, and a method for converting white adipocytes into beige adipocytes, which includes a step of culturing white adipocytes in a medium containing an extract of Akebia quinata.

[0046] In the method according to the present invention, a medium generally used for the proliferation, differentiation, and maturation of preadipocytes (adipose stem cells, mesenchymal stromal cells, mesenchymal stem cells) or adipocytes may be used. Also, the conditions and operations of the culture method can be carried out according to the conventional conditions and operations in the art. For example, a basal medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), specifically, Dulbecco’s Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Hank’s balanced salt solution, etc. can be mentioned. When inducing differentiation, as adipocyte differentiation inducing factors, one or more of dexamethasone (DEX), isobutylmethylxanthine (IBMX), indomethacin (IDMM), insulin (Ins), and biotin are added. Also, basic fibroblast growth factor (bFGF) and / or leukemia inhibitory factor (LIF) may be added as growth factors. Furthermore, if necessary, the medium may contain epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, antibiotics, etc.

[0047] In addition to the above, it is preferable that serum is contained in the medium at a content rate of 1 to 20%. However, since the components of serum vary depending on the lot and there are variations in its effects, it is preferable to use it after lot checking.

[0048] As commercially available media, media such as the mesenchymal stem cell basal medium manufactured by Invitrogen, the mesenchymal stem cell basal medium manufactured by Sanko Junyaku, the MF medium manufactured by TOYOBO, and the Hank’s balanced salt solution manufactured by Sigma can be used.

[0049] Examples of the culture vessels used for cell culture include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, and the like.

[0050] The culture vessel may be non-cell adhesive or cell adhesive, and is appropriately selected according to the purpose. For the purpose of improving the adhesiveness with cells, a culture vessel with cell adhesiveness may be treated with a cell support substrate such as an extracellular matrix. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, and the like.

[0051] The addition concentration of the extract of Akebia quinata to the medium used for cell culture is, for example, 0.1 to 1000 μg / mL, preferably 1 to 100 μg / mL. Further, during the cell culture period, the extract of Akebia quinata may be periodically added to the medium.

[0052] The cell culture conditions may follow normal conditions and do not require special control. For example, the culture temperature is not particularly limited but is about 30 to 40°C, preferably 36 to 37°C. The CO2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. It is preferable to change the medium once every 2 to 3 days, and more preferably every day. The above culture conditions can also be appropriately varied and set within the range where the cells can survive and proliferate.

[0053] Inducing the differentiation of adipose tissue-derived stem cells into beige adipocytes and converting white adipocytes into beige adipocytes can be confirmed, for example, by determining whether the expression level of uncoupling protein 1 (UCP1), which is a marker of beige adipocytes, is significantly increased at the mRNA level or protein level compared to the expression level at the start of culture in cells cultured in the presence of an extract of Akebia quinata as compared to cells cultured in the absence of the extract of Akebia quinata. At the mRNA level, methods of confirmation include, for example, RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific to the UCP1 gene. Also, at the protein level, immunological methods such as ELISA, flow cytometry, and Western blotting using an antibody specific to the protein encoded by the UCP1 gene can be mentioned.

Example

[0054] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited thereto.

[0055] [Example 1] (Production Example 1) Preparation of hot water extract of Akebia quinata fruit 400 mL of purified water was added to 20 g of the dried product of the fruit of Akebia quinata, and extraction was carried out at 95 to 100 °C for 2 hours, followed by filtration. The filtrate was concentrated and freeze-dried to obtain 9.4 g of a hot water extract of Akebia quinata fruit.

[0056] (Production Example 2) Preparation of 50% ethanol extract of Akebia quinata fruit 400 mL of 50% ethanol was added to 20 g of the dried product of the fruit of Akebia quinata, and extraction was carried out at room temperature for 7 days, followed by filtration. The filtrate was concentrated to dryness to obtain 10.4 g of a 50% ethanol extract of Akebia quinata fruit.

[0057] (Production Example 3) Preparation of ethanol extract of Akebia quinata fruit 20 g of the dried fruit of Akebia quinata was added to 400 mL of ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain 7.1 g of the ethanol extract of Akebia fruit.

[0058] (Production Example 4) Preparation of the hot water extract of Akebia pericarp 20 g of the dried pericarp of Akebia quinata was added to 400 mL of purified water, extracted at 95 - 100 °C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried to obtain 9.7 g of the hot water extract of Akebia pericarp.

[0059] (Production Example 5) Preparation of the 50% ethanol extract of Akebia pericarp 20 g of the dried pericarp of Akebia quinata was added to 400 mL of 50% ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain 9.4 g of the 50% ethanol extract of Akebia pericarp.

[0060] (Production Example 6) Preparation of the ethanol extract of Akebia pericarp 50 g of the dried pericarp of Akebia quinata was added to 1000 mL of ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain 7.6 g of the ethanol extract of Akebia pericarp.

[0061] (Production Example 7) Preparation of the hot water extract of Akebia seed 20 g of the residue after pressing the seeds of Akebia quinata was added to 400 mL of purified water, extracted at 95 - 100 °C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried to obtain 3.4 g of the hot water extract of Akebia seed.

[0062] (Production Example 8) Preparation of the 50% ethanol extract of Akebia seed 20 g of the residue after pressing the seeds of Akebia quinata was added to 400 mL of 50% ethanol, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness to obtain 4.1 g of the 50% ethanol extract of Akebia seed.

[0063] (Production Example 9) Preparation of Ethanol Extract of Akebia Seeds To 50 g of the residue after pressing the seeds of Akebia quinata, 1000 mL of ethanol was added, and after extraction at room temperature for 7 days, it was filtered, and the filtrate was concentrated to dryness to obtain 7.0 g of an ethanol extract of Akebia seeds.

[0064] [Example 2] (Experimental Example 1) Promoting Effect of Akebia Extract on Differentiation Induction from Human Adipose Tissue-Derived Stem Cells into Beige Adipocytes The cell growth medium (Culture Solution 1) was prepared by adding 25 mM HEPES, 3% GLUTAMAX-1 (manufactured by GIBCO), 1% Insulin Transferrin Selenium Ethanolamine Solution (manufactured by GIBCO), 1% fetal bovine serum inactivated by heating at 56°C for 30 minutes (manufactured by SIGMA), 10 ng / mL FGF-basic (manufactured by PEPROTECH), and 0.4 μg / mL hydrocortison (manufactured by WAKO) to 50% αMEM (manufactured by SIGMA) and 50% DMEM (manufactured by SIGMA).

[0065] The white adipocyte differentiation induction medium (Culture Solution 2) was prepared by adding 1 μM dexamethason (manufactured by SIGMA), 0.5 mM 3-isobutyl-1-methylxanthine (manufactured by SIGMA), 0.2 mM indomethacin (manufactured by SIGMA), 10% fetal bovine serum (manufactured by SIGMA), 10 μg / mL insulin (manufactured by SIGMA), and 33 μM biotin (manufactured by SIGMA) to DMEM (manufactured by Nacalai).

[0066] Human adipose-derived stem cells (ASC) (manufactured by DS Pharma) were suspended in Culture Medium 1, seeded in a 24-well plate for tissue culture, and cultured in an incubator until confluent. After confirming the confluent state, the medium was replaced with Culture Medium 2, and after culturing for 3 days, it was further replaced with fresh Culture Medium 2 and cultured for another 3 days. When replacing with Culture Medium 2, each extract of Akebia prepared in Example 1 (Production Examples 1 to 9) was added to the same medium so that the final concentration was 100 μg / mL as a test sample.

[0067] After culturing for 6 days with the addition of the test sample, the cells were collected, and the effect of promoting the induction of differentiation from adipose tissue-derived stem cells to beige adipocytes was evaluated using the gene expression level of UCP1, which is a beige adipocyte marker, as an index.

[0068] Gene expression analysis was performed as follows. The collected cells were washed twice with PBS(-), and then RNA was extracted from the cells using Trizol Reagent (manufactured by Invitrogen). After reverse-transcribing the extracted RNA into cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was performed using the following primer sets with ABI7300 (manufactured by Applied Biosystems) to confirm the expression of UCP1. Other operations were carried out according to the defined methods.

[0069] Primer set for UCP1: 5’-GGCTTCAGCGGCAAATCAG-3’ (SEQ ID NO: 1) 5’-AACTCCTGGACCGTGTCGTA-3’ (SEQ ID NO: 2) Primer set for 18S rRNA (internal standard): 5’-CCGAGCCGCCTGGATAC-3’ (SEQ ID NO: 3) 5’-CAGTTCCGAAAACCAACAAAATAGA-3’ (SEQ ID NO: 4)

[0070] The expression of UCP1 was calculated as the ratio of the expression level of UCP mRNA in cells without the addition of the test sample to the expression level of 18S ribosomal RNA (18S rRNA) as the internal standard (UCP1 gene expression level / 18S rRNA gene expression level), with the value in cells without the test sample set to 1. In contrast, the relative gene expression level of UCP1 in cells cultured with the test sample added was calculated and evaluated. The results of these tests are shown in Table 1 below.

[0071] [Table 1]

[0072] As shown in Table 1, when the extract of Akebia quinata was added to human adipose-derived stem cells (ASC), it was confirmed that the relative expression level of the UCP1 gene was improved and the induction of differentiation into beige adipocytes was promoted. In particular, the effect was high in the 50% ethanol extract of Akebia quinata fruit.

[0073] (Experimental Example 2) Effect of Akebia quinata extract on promoting the conversion of white adipocytes to beige adipocytes Human adipose-derived stem cells (ASC) were differentiated into white adipocytes in the same manner as in Experimental Example 1. Subsequently, the cells were cultured for 3 days after being replaced with a white adipocyte maturation medium (a medium prepared by adding 1% fetal bovine serum (manufactured by SIGMA), 2% BSA (manufactured by Fujifilm Wako Pure Chemical Corporation), and 500 μM oleic acid (manufactured by CAYMAN CHEMICAL) to DMEM medium (manufactured by Nacalai Tesque)). When replacing with the white adipocyte maturation medium, each extract of Akebia quinata prepared in Example 1 (Production Examples 1 to 9) was added to the medium at a final concentration of 100 μg / mL as the test sample.

[0074] After culturing for 3 days with the test sample added, the cells were collected, and the effect of promoting the conversion of white adipocytes to beige adipocytes was evaluated in the same manner as in Experimental Example 1 using the gene expression level of UCP1, a beige adipocyte marker, as an index. The results of these tests are shown in Table 2 below.

[0075] [Table 2]

[0076] As shown in Table 2, when the extract of Akebia quinata was added to white adipocytes, it was confirmed that the relative expression level of the UCP1 gene was improved and the conversion into beige adipocytes was promoted. In particular, the effect was high in the 50% ethanol extract of Akebia quinata fruit.

Industrial Applicability

[0077] The present invention can be used in the field of manufacturing pharmaceuticals, quasi-drugs, cosmetics, functional foods, supplements and other food and drink products for the purpose of improving and preventing obesity.

Claims

**Claim 1** A promoter for promoting the differentiation of adipose tissue-derived stem cells into beige adipocytes, containing as an active ingredient a hot water extract, a 50% ethanol extract, or an ethanol extract of fruits, peels, or seeds of Akebia quinata. **Claim 2** An accelerator for promoting the conversion of white adipocytes into beige adipocytes, containing as an active ingredient a hot water extract, a 50% ethanol extract, or an ethanol extract of fruits, peels, or seeds of Akebia quinata. **Claim 3** The agent according to claim 1 or 2, which enhances the expression of uncoupling protein 1 (UCP1) in beige adipocytes. **Claim 4** A fat burning promoter, containing as an active ingredient a hot water extract, a 50% ethanol extract, or an ethanol extract of fruits, peels, or seeds of Akebia quinata. **Claim 5** A composition for promoting the increase in beige adipocytes, containing the agent according to any one of claims 1 to 4. **Claim 6** A method for promoting the induction of differentiation of adipose tissue-derived stem cells into beige adipocytes, comprising the step of culturing adipose tissue-derived stem cells in a medium containing a hot water extract, a 50% ethanol extract, or an ethanol extract of fruits, peels, or seeds of Akebia quinata. **Claim 7** A method for converting white adipocytes into beige adipocytes, comprising the step of culturing white adipocytes in a medium containing a hot water extract, a 50% ethanol extract, or an ethanol extract of fruits, peels, or seeds of Akebia quinata.

Images