Composition containing specific component

Abstract

To provide a composition that exerts UCP1 expression promoting effect and / or brown adipocyte differentiation promoting effect without side effects and is ingestible continuously for a long period of time.SOLUTION: The above-mentioned purpose is solved by a composition containing at least one kind of component selected from the group consisting of Humulus lupulus, trehalose, aspartame, Hordeum vulgare, ceramide, placenta, alanine, and mint, as well as having UCP1 expression promoting effect and / or brown adipocyte differentiation promoting effect.

Description

[Technical Field]

[0001] The present invention relates to a composition that contains specific ingredients and has the effect of promoting UCP1 expression and / or promoting brown adipocyte differentiation. [Background technology]

[0002] Uncoupling proteins (UCPs) uncouple oxidative phosphorylation reactions in the inner mitochondrial membrane and dissipate energy as heat (see Non-Patent Document 1). In particular, UCP1 is specifically expressed in brown adipocytes, the site of heat production, and plays a role in converting neutral lipids into heat and directly consuming energy without ATP synthesis in the electron transport chain. Furthermore, if differentiation of precursor cells into brown adipocytes can be promoted, the expression level of UCP1 can be increased, making it possible to convert neutral lipids into heat. Therefore, if the expression level of UCP1 or brown adipocyte differentiation can be promoted, obesity may be alleviated through the consumption of neutral lipids. Patent Document 1 describes such brown adipocyte differentiation promoters and UCP1 expression promoters that contain catechins as active ingredients. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-65672 [Non-patent literature]

[0004] [Non-Patent Document 1] Proceedings of the 124th Japanese Medical Association Symposium, pp.62-70 Summary of the Invention [Problem to be solved by the invention]

[0005] The substance capable of promoting the expression level and activity of UCP1 described in Patent Document 1 contains, as an active ingredient, a specific substance isolated and purified from a natural product. However, because such a specific substance has a strong pharmacological effect, caution is required when ingesting it, and excessive intake may cause side effects.

[0006] The present invention has been made in view of the above circumstances, and an object of the invention is to provide a composition that exhibits the effect of promoting UCP1 expression and / or the effect of promoting brown adipocyte differentiation, which is free from side effects and can be taken over a long period of time. [Means for solving the problem]

[0007] The present inventors conducted extensive research to solve the above-mentioned problems and surprisingly found that the inclusion of specific components results in a particularly significant effect of promoting UCP1 expression and / or brown adipocyte differentiation. They then succeeded in creating compositions containing the specific components as compositions for promoting UCP1 expression, metabolism, anti-obesity, body fat reduction, and thermogenesis. The present invention was completed based on these findings and successful examples.

[0008] Therefore, according to the present invention, there is provided a cosmetic composition containing at least one ingredient selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0009] Another aspect of the present invention provides a composition for promoting UCP1 expression, which contains at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0010] Another aspect of the present invention provides a composition for activating brown adipocytes, which comprises at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0011] Another aspect of the present invention provides a metabolism-enhancing composition containing at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0012] Another aspect of the present invention provides an anti-obesity composition containing at least one ingredient selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0013] Another aspect of the present invention provides a composition for reducing body fat, which comprises at least one ingredient selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0014] Another aspect of the present invention provides a composition for promoting thermogenesis, which comprises at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0015] Another aspect of the present invention provides a composition containing at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint. [Effects of the Invention]

[0016] The composition of the present invention has the effect of promoting UCP1 expression and / or brown adipocyte differentiation, and furthermore, in relation to these effects, is expected to exhibit brown adipocyte activation, metabolism promotion, anti-obesity, body fat reduction, thermogenesis promotion, UCP1 expression promotion, energy production promotion, triglyceride lowering, sebum suppression, fat metabolism promotion, etc.

[0017] The specific components used in the composition of the present invention have a proven track record of use in pharmaceuticals, cosmetics, etc., and therefore the composition of the present invention is highly safe. Therefore, by virtue of having the above-mentioned effects, the composition of the present invention is useful as a composition for promoting UCP1 expression, a composition for promoting metabolism, an anti-obesity composition, a composition for reducing body fat, and a composition for promoting thermogenesis, and is expected to be provided in parenteral or oral forms. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below. The composition of the present invention has the effect of promoting UCP1 expression and / or brown adipocyte differentiation, and contains at least any of the following ingredients (hereinafter referred to as the specific ingredient): hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

[0019] The composition of the present invention may have either one of the effects of promoting UCP1 expression and the effect of promoting brown adipocyte differentiation, or both of these effects. Furthermore, the specific component may be any one of the specific components, or may be two or more components.

[0020] In addition to the effects of promoting UCP1 expression and brown adipocyte differentiation, the composition of the present invention can also exhibit brown adipocyte activation, metabolism promotion, anti-obesity, body fat reduction, thermogenesis promotion, energy production promotion, neutral lipid lowering, sebum suppression, and fat metabolism promotion.

[0021] By virtue of the above-mentioned effects, the composition of the present invention can be used in the form of a composition for promoting UCP1 expression, a composition for promoting metabolism, an anti-obesity composition, a composition for reducing body fat, and a composition for promoting thermogenesis.

[0022] Hops are made from the common lupin ( Hops hopsThe plant is not particularly limited as long as it is known as hops, and parts such as flowers (cones, hop cones), seeds, fruits, leaves, stems, and roots can be used, but it is preferable to use hop cones.

[0023] Mint is Mentha piperita ( Mint pepper The mint is not particularly limited as long as it is known as mint, and for example, its flowers, seeds, fruits, leaves, stems, roots, and other parts can be used, but it is preferable to use mint leaves.

[0024] Barley is Hordeum vulgare ( Barley vulgar The plant material is not particularly limited as long as it is known as barley. For example, its flowers, seeds, fruits, leaves, stems, roots, and other parts can be used, but it is preferable to use barley leaves and stems.

[0025] The placenta is not particularly limited as long as it is a commonly known placenta, and examples thereof include animal placenta, which is an extract derived from the placenta of pigs, humans, cows, horses, sheep, etc., or a component thereof; marine placenta, which is an extract derived from the ovarian membrane of fish, etc., or a component thereof; and plant placenta, which is an extract derived from the germ of a plant, etc., or a component thereof. Animal placenta is preferred, and animal placenta derived from pigs is more preferred.

[0026] The ceramide is not particularly limited as long as it is a compound in which sphingosine and a fatty acid are bonded by an amide bond, and examples include ceramides derived from natural products and ceramides obtained by synthesis. Ceramides derived from natural products are preferred, and ceramides derived from rice, corn, konjac, maitake mushroom, maitake mushroom, soybean, wheat, beet, etc. are more preferred, and ceramides derived from rice (glucosylceramide) are even more preferred.

[0027] The trehalose is not particularly limited as long as it is a commonly known trehalose, and examples thereof include trehalose itself and trehalose derivatives such as its hydrates and optical isomers, with D-(+)-trehalose being preferred.

[0028] Aspartame is not particularly limited as long as it is aspartame as is commonly known, and examples thereof include aspartame itself and aspartame derivatives such as its optical isomers, with aspartame having CAS registration number 22839-47-0 being preferred.

[0029] The alanine is not particularly limited as long as it is a commonly known alanine, and examples thereof include alanine itself and derivatives thereof such as optical isomers, and preferably alanine having CAS Registry Number 338-69-2.

[0030] Of the specific components, natural products may be processed products thereof. Examples of processed products include, but are not limited to, dry powders, shredded products and their dry powders, squeezed juice and their dry powders, and extracts and their dry powders. However, from the viewpoints of ease of processing, storage, transportation, etc., and versatility in usage form, dry powders are preferred.

[0031] There are no particular limitations on the method for obtaining dried natural products. For example, barley stem and leaf powder can be produced by the method disclosed in Japanese Patent No. 3277181. Specifically, after harvesting, barley stem and leaf are washed with water to remove dirt and other impurities, drained, and then cut to an appropriate length. If necessary, a blanching treatment such as hot water treatment or steam treatment is then carried out. The temperature and time of this treatment can be determined appropriately depending on the amount of barley stem and leaf to be treated and the pH of the hot water. The blanched barley stem and leaf are immediately cooled and dehydrated by centrifugation or other methods. Next, they are dried to a moisture content of 5 wt% or less. Further steps, such as coarse grinding, heating, and fine grinding, are carried out to obtain barley stem and leaf powder.

[0032] The specific ingredients are natural products, derivatives thereof, and synthetic compounds that have been taken by humans for a long time, and are therefore highly safe. Therefore, the composition of the present invention is highly practical and can be used for a variety of purposes in parenteral or oral forms, either as is or after processing.

[0033] The specific component may be one produced by a method generally known to those skilled in the art, or may be one that is commercially available.

[0034] The specific component exhibits a UCP1 expression-promoting effect or a brown adipocyte differentiation-promoting effect. In this specification, the "expression-promoting effect" refers to, for example, in the case of a UCP1 expression-promoting effect, at least one of promoting the expression level of the UCP1 gene in a subject, increasing the translation level of the UCP1 protein, and activating UCP1. Furthermore, the brown adipocyte activation effect refers to at least one of activating brown adipocytes and increasing the number of brown adipocytes.

[0035] Regarding the UCP1 expression-promoting effect, as described in Non-Patent Document 1, it is known that UCP1 function is reduced in obese animals, that UCP1 levels are increased in animals that do not become obese even when they eat a lot, and that mice in which UCP1 expression has been artificially reduced become obese, while mice with high UCP1 expression become thin. Therefore, an anti-obesity effect can be expected from the UCP1 expression-promoting effect, and substances that actually have the effect of promoting UCP1 expression can promote lipolysis in white adipocytes and simultaneously activate UCP1, converting free fatty acids into heat and ultimately reducing body fat.

[0036] Thermogenesis-promoting effect refers to, for example, the effect of promoting thermogenesis from neutral lipids by brown fat cells. Neutral lipid-lowering effect refers to, for example, the effect of decomposing neutral lipids to reduce the total amount of neutral lipids in the body. Sebum-inhibiting effect refers to, for example, the effect of suppressing sebum production. Fat metabolism-promoting effect refers to, for example, the effect of qualitatively converting fat cells and adipose tissue that cause obesity into small, normal fat cells. Body fat-reducing effect refers to, for example, the effect of reducing body fat by converting free fatty acids into heat. Metabolism-promoting effect and anti-obesity effect refer to, for example, the effect of promoting metabolism so as to reduce body fat and the effect of suppressing obesity as a result of said effect. Energy production-promoting effect refers to, for example, the effect of producing energy through thermogenesis from neutral lipids by brown fat cells. Each effect in this paragraph includes the effect described above, but also includes those achieved by other mechanisms of action.

[0037] The anti-disease effect achieved by an anti-disease composition such as the anti-obesity composition of the present invention refers, for example, to the prevention or delay of the onset of current or future obesity or obesity in a subject, or the improvement of that state. Furthermore, insulin resistance refers to a state in which insulin's main function of promoting glucose absorption, such as in the liver, adipocytes, and skeletal muscle, is weakened. The anti-insulin resistance effect refers to the prevention or delay of a worsening of a value that serves as an indicator of current or future insulin resistance in a subject, as measured by the steady-state plasma glucose (SSPG) method or the like, or the improvement of that value.

[0038] Obesity is known to cause various diseases, including type 2 diabetes, hypertension, and hyperlipidemia. Furthermore, these diseases can lead to stroke and ischemic heart disease. Obesity leads to adipocyte hypertrophy and the secretion of TNF-α and free fatty acids (FFA), which inhibit glucose uptake in insulin target cells such as muscle cells and liver cells. This suppresses the secretion of adiponectin, which promotes insulin action, leading to decreased insulin sensitivity at the cellular level and insulin resistance. The above-mentioned obesity-related diseases are considered to be a series of metabolic abnormalities caused by insulin resistance. Therefore, the use of the composition of the present invention can prevent or ameliorate obesity and obesity-associated diseases such as type 2 diabetes, hypertension, and hyperlipidemia.

[0039] Here, prevention and amelioration of type 2 diabetes refers to suppressing or delaying the onset of a diabetic state or borderline state, or bringing these states closer to a state known as the normal range. Prevention and amelioration of hypertension refers to suppressing or delaying the onset of a hypertensive state or borderline state, or bringing these states closer to a state known as the normal range. Prevention and amelioration of hyperlipidemia refers to suppressing or delaying the onset of a hyperlipidemic state or borderline state, or bringing these states closer to a state known as the normal range.

[0040] The method for producing the composition of the present invention is not particularly limited. For example, a solid composition can be obtained by incorporating a specific component at room temperature or under heating. Alternatively, this solid composition can be dissolved in a solvent such as water to obtain a liquid composition. Furthermore, a liquid composition can be obtained by adding and mixing other solid components to a liquid product of the specific component. The obtained liquid composition may be dried and then powdered. In this case, drying methods include, but are not limited to, spray drying and freeze drying.

[0041] The composition of the present invention can be used as is or mixed with other components depending on the application. In this way, the composition of the present invention can contain various ingredients in addition to the specific components as long as the object of the present invention can be achieved.

[0042] For example, the composition of the present invention may further contain additives used in conventional processing, such as excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavors, flavor oils, etc. The amount of additive used is not particularly limited as long as it does not interfere with the solution of the problems of the present invention, and may be adjusted appropriately.

[0043] The composition of the present invention is not particularly limited as long as it is in a commonly used form, and may take any of various forms, such as a liquid, lotion, mousse, gel, emulsion, suspension, cream, ointment, sheet, aerosol, spray, stick, powder, granules, granules, tablet, rod, plate, block, solid, paste, capsule, or caplet.

[0044] The composition of the present invention can be used as a parenteral composition, for example, in a form suitable for cosmetics. That is, one embodiment of the composition of the present invention is a cosmetic composition. For example, the composition of the present invention can be processed into various forms such as lotions, emulsions, gels, creams, and ointments, either as is or mixed with additives commonly used in cosmetic processing. Specifically, the composition can be used as a lotion, cosmetic cream, emulsion, cream, pack, hair tonic, hair cream, shampoo, hair rinse, treatment, facial cleanser, foundation, hair growth agent, aqueous ointment, spray, etc.

[0045] The content of the specific component contained in the composition of the present invention is not particularly limited as long as it is an amount that is confirmed to promote UCP1 expression and / or brown adipocyte differentiation, but for oral compositions, it is, for example, 0.0001 wt% or more, preferably 0.001 wt% or more, relative to the total composition, and for parenteral compositions such as cosmetics, it is, for example, 0.00001 wt% or more, preferably 0.0001 wt% or more.

[0046] A specific embodiment of the composition of the present invention is, for example, a composition containing any one of the specific components in an amount of 0.001 to 100 wt% relative to the total composition, and among these, a composition containing the specific component in an amount of 0.01 to 80 wt% is more preferred.

[0047] Another specific embodiment of the composition of the present invention is a composition containing any two of the specific components in a total amount of 10 to 100 wt % relative to the entire composition, including a composition containing one of the two components in an amount of 10 to 90 wt % and the other in an amount of 90 to 10 wt %.

[0048] Another specific embodiment of the composition of the present invention is a composition containing any three specific components in a total amount of 10 to 100 wt% relative to the entire composition, including a composition containing 10 to 50 wt% of the first component, 30 to 40 wt% of the second component, and 60 to 10 wt% of the third component.

[0049] Below, formulation examples according to specific embodiments of the composition of the present invention are shown, but the present invention is not limited to these formulation examples, and the present invention can take various forms as long as the object of the present invention can be solved.

[0050] (Example 1: Lotion) The composition of the present invention in the form of a lotion was prepared by mixing 0.01 parts by mass of trehalose, 0.01 parts by mass of ginger extract, 0.01 parts by mass of Cassia japonica, 10 parts by mass of glycerin, 3 parts by mass of diglycerin, 12 parts by mass of 1,3-butylene glycol, 3 parts by mass of pentylene glycol, 0.1 parts by mass of sodium hyaluronate, 0.01 parts by mass of citric acid, 0.02 parts by mass of sodium citrate, 0.1 parts by mass of xanthan gum, 0.15 parts by mass of methylparaben, 0.2 parts by mass of carbomer, 0.03 parts by mass of sodium hydroxide, and the remainder being water, making the total 100 parts by mass.

[0051] (Example 2: Shampoo) A composition of the present invention in the form of a shampoo was prepared by mixing 0.02 parts by mass of hop extract, 0.01 parts by mass of caffeine, 7.5 parts by mass of sodium laureth sulfate, 4.2 parts by mass of cocamidopropyl betaine, 3 parts by mass of cocamide DEA, 0.1 parts by mass of 1,3-butylene glycol, 0.225 parts by mass of polyquaternium-10, 0.15 parts by mass of citric acid, 0.05 parts by mass of sodium citrate, 0.9 parts by mass of phenoxyethanol, and the remainder, water, to a total of 100 parts by mass.

[0052] (Example 3: Soap) The composition of the present invention in the form of soap was prepared by mixing and solidifying 0.5 parts by mass of aspartame, 0.2 parts by mass of melilot extract, 0.1 parts by mass of elastin, 2 parts by mass of glycerin, 1 part by mass of olive oil, 0.1 parts by mass of EDTA-tetrasodium, 0.2 parts by mass of tetrasodium etidronate, and the remainder of the soap base, with the total amount being 100 parts by mass.

[0053] (Formulation example 4: Emulsion) The composition of the present invention was prepared in the form of an emulsion by mixing 0.1 parts by mass of ceramide, 0.1 parts by mass of alanine, 0.1 parts by mass of arginine, 3 parts by mass of sucrose fatty acid ester, 12 parts by mass of glycerin, 6 parts by mass of squalane, 24 parts by mass of dimethyl silicone oil, 1 part by mass of polypropylene glycol, 0.06 parts by mass of thickener, 0.2 parts by mass of phenoxyethanol, 5 parts by mass of ethanol, 0.01 parts by mass of sodium hydroxide, and the remainder being purified water, taking the total to 100 parts by mass.

[0054] (Example 5: Cosmetic cream) Based on 100 parts by mass of the total, the ingredients are: alanine 0.1 part by mass, fructooligosaccharide 0.1 part by weight, squalane 15.0 parts by mass, octyldodecyl myristate 4.0 parts by mass, hydrogenated soybean phospholipid 0.2 parts by mass, butyl alcohol 2.4 parts by mass, hydrogenated oil 1.5 parts by mass, stearic acid 1.5 parts by mass, lipophilic glycerin monostearate 1.5 parts by mass, polyglyceryl monostearate 0.5 parts by mass, behenyl alcohol 0.8 parts by mass, polyglyceryl monomyristate 0.7 parts by mass, white beeswax 0.3 parts by mass, d-δ-tocopherol 0.1 part by mass, methylparaben 0.3 parts by mass, C10-30 alkyl-modified carboxyvinyl polymer 0.2 parts by mass, carboxyvinyl polymer 0.1 part by mass, 1,3-butanediol 18.0 parts by mass, sodium hydroxide 0.1 parts by mass of the above-mentioned powder and the remainder of the powder were mixed together to prepare a cosmetic cream composition of the present invention.

[0055] (Example 6: Pack) The composition of the present invention was prepared in the form of a pack by mixing 0.1 part by mass of placenta, 0.01 part by mass of gallic acid, 20.0 parts by mass of polyvinyl alcohol, 5.0 parts by mass of glycerin, 20.0 parts by mass of ethanol, 6.0 parts by mass of kaolin, 0.2 parts by mass of preservative, 0.1 part by mass of fragrance, and the remainder being purified water, making the total 100 parts by mass.

[0056] (Composition Example 7: Tablets) The composition of the present invention was prepared in the form of a tablet by mixing and compressing 10 parts by mass of barley leaf powder, 8 parts by mass of ginger extract, 5 parts by mass of caffeine, 5 parts by mass of melilot, 20 parts by mass of crystalline cellulose, 50 parts by mass of lactose, 4 parts by mass of magnesium stearate, and the remainder of cornstarch, with the total amount being 100 parts by mass.

[0057] (Composition Example 8: Tablets) The composition of the present invention was prepared in the form of a tablet by mixing and compressing 0.5 parts by mass of alanine, 8 parts by mass of Cassia powder, 5 parts by mass of melilot, 0.5 parts by mass of fructooligosaccharide, 20 parts by mass of crystalline cellulose, 50 parts by mass of lactose, 4 parts by mass of magnesium stearate, and the remainder of cornstarch, with the total amount being 100 parts by mass.

[0058] (Formulation Example 9: Granules) The composition of the present invention was prepared in the form of granules by mixing and granulating 15 parts by mass of hop powder, 5 parts by mass of Cassia japonica extract, 10 parts by mass of lactose, 1 part by mass of calcium stearate, and the remainder of the crystalline cellulose, with the total amount being 100 parts by mass.

[0059] (Formulation Example 10: Capsules) The composition of the present invention was prepared in the form of a capsule by mixing 10 parts by mass of mint extract, 20 parts by mass of ginger extract, 5 parts by mass of elastin, 8 parts by mass of lecithin, and the remainder of olive oil, with the total weight being 100 parts by mass, and encapsulating this mixture in a capsule shell to form the content liquid.

[0060] (Example 11: Liquid) The composition of the present invention was prepared in the form of a liquid by mixing 0.84 parts by mass of young barley leaf extract, 1 part by mass of arginine, 1 part by mass of alanine, 10 parts by mass of high fructose corn syrup, 1 part by mass of citric acid, 0.02 parts by mass of sodium benzoate, 2 parts by mass of a flavoring preparation, 0.05 parts by mass of sucralose, 0.03 parts by mass of acesulfame potassium, and the remainder being purified water, making a total of 100 parts by mass.

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved. [Example]

[0062] It was demonstrated as follows that the specific ingredients trehalose, aspartame, barley, ceramide, placenta, alanine, mint and hops have a particularly significant UCP1 expression promoting effect.

[0063] (1) Test substance Of the specific components, trehalose, aspartame, ceramide, placenta, alanine, mint and hops were commercially available.

[0064] The barley used was dried barley stem and leaf powder prepared as follows. Specifically, barley stems and leaves harvested at a height of approximately 30 cm were washed with water to remove any adhering mud, and then pretreated by cutting into pieces approximately 5 to 10 cm in size. The pretreated stem and leaf were blanched once in hot water at 90 to 100°C for 90 to 120 seconds, and then cooled in cold water. The resulting stem and leaf were then dried in a dryer for 20 to 180 minutes with hot air at 80 to 130°C until the moisture content was 5% by mass or less. The dried stem and leaf were coarsely pulverized to a size of approximately 1 mm using a mixer. The resulting barley stem and leaf were then finely pulverized using a pulverizer so that 90% or more passed through a 200-mesh screen, yielding a dried barley stem and leaf powder.

[0065] (2) Preparation of collagenase solution A collagenase solution was prepared by shaking DMEM medium, which had been adjusted to contain 0.2% (W / V) collagenase and 1% (W / V) BSA, at room temperature for 1 hour.

[0066] (3) Preparation of brown adipocyte suspension ICR mice (Kyudo) were euthanized with diethyl ether, and brown adipose tissue was removed from the back. The resulting brown adipose tissue was then removed with tweezers from the surrounding adipose tissue, including adipose tissue, blood vessels, and muscle. The brown adipose tissue was then washed three times in DMEM. The brown adipose tissue was then immersed in collagenase solution and finely chopped with scissors. The chopped brown adipose tissue was incubated in the collagenase solution at 37°C for 1 hour to obtain a cell dispersion. The resulting cell dispersion was filtered through a 100 μm pore cell strainer and then centrifuged (800 rpm, 5 min, 20°C). The precipitated cells were then suspended in DMEM to obtain a cell suspension. The resulting cell suspension was then filtered through a 25 μm filter sterilized by autoclaving. The filtered supernatant was centrifuged (800 rpm, 5 min, 20°C), and the precipitated cells were suspended in DMEM containing 10 vol% FBS to prepare a brown adipocyte solution.

[0067] (4) Preparation of culture medium DMEM containing 10 vol% FBS was used as the growth medium.

[0068] A differentiation-inducing medium containing 2.5 μM dexamethasone, 10 μg / ml insulin, and 0.5 mM IBMX in a growth medium was prepared.

[0069] As a maintenance medium, a growth medium containing 10 μg / ml insulin was prepared using the insulin solution described above.

[0070] Test substance-containing media were prepared as follows: Each test substance was dissolved in DMSO, diluted with maintenance medium to a final DMSO concentration of 0.5 vol%, and filter-sterilized. After sterilization, the test substances (hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint) were diluted to a predetermined concentration of 20 μg / ml using 0.5 vol% DMSO-containing maintenance medium.

[0071] (5)Cell culture 75cm2 5 ml of the collagen coating solution was placed in the flask, and the flask was left to stand at room temperature for about 1 hour. The collagen coating solution was then removed by suction, and the flask was washed twice with 5 ml of PBS to obtain a collagen-coated flask.

[0072] The brown adipocyte solution was added to the collagen-coated flask, and the brown adipocytes were cultured for 24 hours in a 37°C, 5% CO2 incubator. After the culture, the brown adipocytes were washed once with growth medium, replaced with fresh growth medium, and cultured until they became subconfluent.

[0073] After culturing, the brown adipocytes were trypsinized and suspended. The resulting suspended cells were seeded into each well of a collagen-coated 24-well plate (collagen coating solution 350 μL / well, PBS washing 350 μL / well twice).

[0074] Brown adipocytes were cultured in a growth medium until confluent. After removing the medium from each well, 500 μl of differentiation-inducing medium was added to each well and cultured for 48 hours.

[0075] After removing the differentiation-inducing medium, 500 μl of medium containing 20 μg / ml of the test substance was added and cultured for 6 days. The medium was changed every other day. As a control, 500 μl of medium containing no test substance was added and cultured for 6 days. The medium was changed every other day.

[0076] After culturing for 8 days, the supernatant was collected, RNA was collected using an RNeasy Mini Kit (Qiagen), and cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen).

[0077] Using the resulting cDNA as a template and primers for the UCP1 gene (Qiagen), quantitative real-time PCR was performed with the Rotor-Gene SYBR Green PCR Kit (Qiagen) to measure the expression level of UCP1 mRNA. As an endogenous control, GAPDH mRNA expression level was measured using GAPDH primers (Qiagen).

[0078] (6) Evaluation Table 1 shows the results of measuring the UCP1 mRNA expression level (relative expression level when the control mRNA expression level was set to 1.00) when each of the test substances, hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint, was administered. [Table 1]

[0079] As shown in Table 1, when hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint were used as test substances, the amount of UCP1 expression was high. [Industrial Applicability]

[0080] The compositions of the present invention contain specific components that can be administered either parenterally or orally, and are particularly useful for subjects suffering from cellulite, obesity, and associated diseases, contributing to the health and well-being of such subjects.

Claims

[Claim 1] A metabolism-promoting composition containing crushed barley stalks and leaves as an active ingredient (excluding herbal medicines containing aspergillus oryzae, germ and young barley leaves).

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