JP7617656B2 - Composition containing specific ingredient - Google Patents

Description

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

Uncoupling proteins (UCPs) have the function of uncoupling oxidative phosphorylation reactions in the inner mitochondrial membrane and dissipating energy as heat (see Non-Patent Document 1). In particular, UCP1 is specifically expressed in brown fat cells, which are the site of heat production, and plays a role in converting neutral lipids into heat and directly consuming energy without going through ATP synthesis in the electron transport system. In addition, if the differentiation of precursor cells into brown fat cells 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 fat cell differentiation can be promoted, it may be possible to eliminate obesity through the consumption of neutral lipids. Patent Document 1 describes such brown fat cell differentiation promoters and UCP1 expression promoters that contain catechins as active ingredients.

JP 2014-65672 A

Proceedings of the 124th Symposium of the Japanese Medical Association, pp. 62-70

The substance capable of promoting the expression level and activation of UCP1 described in Patent Document 1 contains a specific substance isolated and purified from a natural product as an active ingredient. However, because such a specific substance has a strong pharmacological effect, care must be taken when ingesting it, and excessive intake may cause side effects.

The present invention has been made in consideration of these circumstances, and the problem that the invention aims to solve 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 of side effects and can be taken for a long period of time and sustainedly.

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

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.

According to another aspect of the present invention, there is provided a composition for promoting UCP1 expression, comprising at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint.

According to another aspect of the present invention, there is provided a composition for activating brown adipocytes, which contains at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

According to another aspect of the present invention, there is provided a composition for promoting metabolism, comprising at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint.

According to another aspect of the present invention, there is provided an anti-obesity composition containing at least one ingredient selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint.

According to another aspect of the present invention, there is provided a composition for reducing body fat, which contains at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint.

According to another aspect of the present invention, there is provided a composition for promoting thermogenesis, which contains at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

According to another aspect of the present invention, there is provided a composition containing at least one component selected from the group consisting of hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint.

The composition of the present invention has a UCP1 expression promoting effect and/or a brown fat cell differentiation promoting effect, and furthermore, in relation to these effects, it is expected to exhibit brown fat cell activation effect, metabolism promoting effect, anti-obesity effect, body fat reducing effect, heat production promoting effect, UCP1 expression promoting effect, energy production promoting effect, neutral lipid lowering effect, sebum suppressing effect, fat metabolism promoting effect, etc.

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 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 a parenteral or oral form.

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 one of the following ingredients (hereinafter referred to as specific ingredients): hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint.

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 may have both of these effects. Furthermore, the specific component may be any one of the specific components, or may be two or more components.

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

By having 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.

The hops are not particularly limited as long as they are known as the common lupulus ( Humulus lupulus ), and parts such as the flowers (cones, seeds, fruits, leaves, stems, and roots can be used, but it is preferable to use hop cones.

The mint is not particularly limited as long as it is known as Mentha piperita , and for example, its flowers, seeds, fruits, leaves, stems, roots, etc. can be used, but it is preferable to use mint leaves.

The barley is not particularly limited as long as it is known as barley ( Hordeum vulgare ), and 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.

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.

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, Pleurotus venus, soybean, wheat, beet, etc. are more preferred, and ceramides derived from rice (glucosylceramide) are even more preferred.

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

Aspartame is not particularly limited as long as it is aspartame as is commonly known, but examples include aspartame itself and aspartame derivatives such as its optical isomers, and preferably aspartame with CAS registration number 22839-47-0.

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

Among the specific components, natural products may be processed products. Examples of processed products include, but are not limited to, dried powders, shredded products and their dried powders, squeezed juice and their dried powders, and extracts and their dried powders. However, from the standpoint of ease of processing, storage, transportation, etc., and versatility of use, it is preferable to use dried powders.

The method for obtaining dried natural products is not particularly limited, but for example, barley stalk and leaf powder can be produced by the method disclosed in Patent Publication No. 3277181. That is, after harvesting barley stalks and leaves, they are washed with water to remove dirt and the like, 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 appropriately determined depending on the amount of barley stalks and leaves to be treated and the pH of the hot water. The blanched barley stalks and leaves are immediately cooled and dehydrated by centrifugation or the like. They are then dried so that the moisture content is 5 wt% or less. Barley stalk and leaf powder can be obtained by further passing through a coarse grinding process, a heating process, a fine grinding process, etc.

The specific ingredients are natural products or their derivatives or synthetic compounds that have been ingested by humans for a long time and are therefore highly safe, so 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.

The specific ingredient may be one produced by a method generally known to those skilled in the art, or may be one that is available on the market.

The specific component exhibits a UCP1 expression promoting effect or a brown fat cell 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 the effects of promoting the expression level of the UCP1 gene in a subject, increasing the translation level of the UCP1 protein, and activating UCP1. In addition, the brown fat cell activation effect refers to at least one of the effects of activating brown fat cells and increasing the number of brown fat cells.

As described in Non-Patent Document 1, it is known that UCP1 function is reduced in obese animals, that animals that do not become obese even when they eat a lot have increased UCP1 levels, 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 UPC1 expression can promote fat breakdown in white fat cells while at the same time activating UCP1, converting liberated fatty acids into heat, and ultimately reducing body fat.

Thermogenesis promoting action refers to, for example, the action of promoting heat production from neutral lipids by brown fat cells. The neutral lipid lowering action refers to, for example, the action of decomposing neutral lipids and lowering the total amount of neutral lipids in the body. The sebum suppressing action refers to, for example, the action of suppressing the generation of sebum. The fat metabolism promoting action refers to, for example, the action of qualitatively converting fat cells and fat tissues that cause obesity into small normal fat cells. The body fat reducing action refers to, for example, the action of reducing body fat by converting free fatty acids into heat. The metabolism promoting action and anti-obesity action refer to, for example, the action of promoting metabolism so as to reduce body fat and the action of suppressing obesity through said action. The energy production promoting action refers to, for example, the action of producing energy through thermogenesis from neutral lipids by brown fat cells. Each action in this paragraph includes the action described above, but also includes those realized by other action mechanisms.

The anti-disease effect of an anti-disease composition such as the anti-obesity composition of the present invention means, for example, in the case of an anti-obesity effect of an anti-obesity composition, suppressing or delaying the onset of a current or future state considered to be obesity or obesity in a subject, or improving that state. Insulin resistance means a state in which the main function of insulin, the promotion of sugar absorption, is weakened in the liver, fat cells, skeletal muscles, etc. An anti-insulin resistance effect means suppressing or delaying the worsening of a value that is an index of current or future insulin resistance in a subject, as measured by the SSPG (steady-state plasma glucose) method, or improving that value.

Obesity is known to cause various diseases, such as type 2 diabetes, hypertension, and hyperlipidemia. Furthermore, these diseases may lead to stroke and ischemic heart disease. When an individual is obese, fat cells become enlarged, and TNF-α and free fatty acids (FFA) are secreted. These inhibit the uptake of sugar in insulin target cells such as muscle cells and liver cells, and suppress the secretion of adiponectin, which promotes insulin action, causing a decrease in insulin sensitivity at the cellular level, resulting in insulin resistance. The above-mentioned diseases caused by obesity are considered to be a series of metabolic abnormalities based on insulin resistance. Therefore, by using the composition of the present invention, it is possible to prevent or improve obesity and diseases associated with obesity, such as type 2 diabetes, hypertension, and hyperlipidemia.

Here, prevention and improvement of type 2 diabetes refers to suppressing or delaying the onset of a diabetic state or borderline state, or moving from these states closer to a state known as the normal range. Prevention and improvement of hypertension refers to suppressing or delaying the onset of a state known as hypertension or borderline state, or moving from these states closer to a state known as the normal range. Prevention and improvement of hyperlipidemia refers to suppressing or delaying the onset of a hyperlipidemic state or borderline state, or moving from these states closer to a state known as the normal range.

The method for producing the composition of the present invention is not particularly limited, and 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 a specific component. The obtained liquid composition may be powdered through a drying process. In this case, the drying process can be performed by spray drying, freeze drying, etc., but is not limited to these.

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

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

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

The composition of the present invention can be used as a non-oral composition, for example, in a form suitable for cosmetics. That is, one aspect 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 that are usually used in the processing of cosmetics. Specifically, it can be used as a lotion, cosmetic cream, emulsion, cream, pack, hair tonic, hair cream, shampoo, hair rinse, treatment, face wash, foundation, hair growth agent, aqueous ointment, spray, etc.

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 recognized 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 entire composition, and for non-oral compositions such as cosmetics, it is, for example, 0.00001 wt% or more, preferably 0.0001 wt% or more.

Specific embodiments of the composition of the present invention include, for example, a composition containing 0.001 to 100 wt % of any one of the specific components relative to the entire composition. Of these, a composition containing 0.01 to 80 wt % of the specific component is more preferred.

Another specific embodiment of the composition of the present invention is a composition that contains any two of the specific components in a total amount of 10 to 100 wt % relative to the entire composition. Among these, there is a composition that contains one of the two components in a ratio of 10 to 90 wt % and the other component in a ratio of 90 to 10 wt %.

Another specific embodiment of the composition of the present invention is a composition that contains three specific components in a total amount of 10 to 100 wt% relative to the entire composition. Among these, there is a composition that contains 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.

The following are examples of formulations according to specific embodiments of the composition of the present invention, but the present invention is not limited to these formulations, and the present invention can take various forms as long as the object of the present invention can be solved.

(Formulation 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 of the mixture, making the total 100 parts by mass.

(Formulation example 2: Shampoo)
The 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, assuming a total of 100 parts by mass.

(Formulation Example 3: Soap)
The composition of the present invention in the form of a soap was prepared by mixing and solidifying 0.5 parts by weight of aspartame, 0.2 parts by weight of melilot extract, 0.1 parts by weight of elastin, 2 parts by weight of glycerin, 1 part by weight of olive oil, 0.1 parts by weight of EDTA-4 sodium, 0.2 parts by weight of tetrasodium etidronate, and the remainder of the soap base, making the total amount 100 parts by weight.

(Formulation Example 4: Milk Lotion)
The composition of the present invention was prepared in the form of an emulsion by mixing 0.1 part by mass of ceramide, 0.1 part by mass of alanine, 0.1 part 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 part by mass of thickener, 0.2 part by mass of phenoxyethanol, 5 parts by mass of ethanol, 0.01 part by mass of sodium hydroxide, and the remainder, purified water, making the total 100 parts by mass.

(Formulation Example 5: Cosmetic cream)
Based on a total of 100 parts by mass, the following ingredients are added: alanine 0.1 parts by mass, fructooligosaccharide 0.1 parts 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, hardened 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 parts by mass, methylparaben 0.3 parts by mass, C10-30 alkyl-modified carboxyvinyl polymer 0.2 parts by mass, carboxyvinyl polymer 0.1 parts by mass, 1,3-butanediol 18.0 parts by mass, sodium hydroxide 0.1 parts by mass of each of the above components and the remainder purified water were mixed together to prepare a composition of the present invention in the form of a cosmetic cream.

(Formulation Example 6: Pack Agent)
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, purified water, making the total 100 parts by mass.

(Formulation Example 7: Tablets)
The composition of the present invention in the form of tablets was prepared by mixing and tableting 10 parts by weight of barley leaf powder, 8 parts by weight of ginger extract, 5 parts by weight of caffeine, 5 parts by weight of melilot, 20 parts by weight of crystalline cellulose, 50 parts by weight of lactose, 4 parts by weight of magnesium stearate, and the remainder of corn starch, making a total of 100 parts by weight.

(Formulation Example 8: Tablets)
The composition of the present invention in the form of tablets was prepared by mixing and tableting 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 corn starch, making a total of 100 parts by mass.

(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, making the total amount 100 parts by mass.

Formulation Example 10: Capsules
The composition of the present invention was prepared in the form of a capsule by mixing 10 parts by weight of mint extract, 20 parts by weight of ginger extract, 5 parts by weight of elastin, 8 parts by weight of lecithin, and the remainder of olive oil (total of 100 parts by weight). The resulting mixture was used as the content liquid, and this was encapsulated in a capsule shell.

(Formulation 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 fructose glucose liquid sugar, 1 part by mass of citric acid, 0.02 parts by mass of sodium benzoate, 2 parts by mass of a flavor preparation, 0.05 parts by mass of sucralose, 0.03 parts by mass of acesulfame potassium, and the remainder, making a total of 100 parts by mass.

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 solved.

It has been demonstrated that the specific ingredients trehalose, aspartame, barley, ceramide, placenta, alanine, mint and hops have a particularly significant effect of promoting UCP1 expression, as shown below.

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

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

(2) Preparation of Collagenase Solution A collagenase solution was prepared by shaking a DMEM medium containing 0.2% (w/v) collagenase and 1% (w/v) BSA at room temperature for 1 hour.

(3) Preparation of brown fat cell solution ICR mice (Kyudo) were euthanized with diethyl ether, and brown fat tissue was extracted from the back. After removing fat tissue, blood vessels, muscles, etc. attached around the brown fat from the obtained brown fat tissue with tweezers, the brown fat was washed three times in DMEM. Next, the brown fat was immersed in a collagenase solution and subjected to a shredding process in which the brown fat was finely chopped with scissors in the solution. The shredded brown fat was incubated in the collagenase solution at 37°C for 1 hour to obtain a cell dispersion. The obtained cell dispersion was filtered with a cell strainer with a pore size of 100 μm, and then subjected to centrifugation (800 rpm, 5 min, 20°C). The precipitated cells were then suspended in DMEM to obtain a cell suspension. The obtained cell suspension was then filtered using a 25 μm filter sterilized by autoclave. The filtered supernatant was centrifuged (800 rpm, 5 min, 20° C.), and the precipitated cells were suspended in DMEM containing 10 vol % FBS to obtain a brown adipocyte solution.

(4) Preparation of medium As a growth medium, DMEM containing 10 vol % FBS was used.

A differentiation-inducing medium was prepared by adding 2.5 μM dexamethasone, 10 μg/ml insulin, and 0.5 mM IBMX to the proliferation medium.

The above insulin solution was used to prepare a maintenance medium containing 10 μg/ml insulin in growth medium.

The test substance-containing medium was prepared as follows: each test substance was dissolved in DMSO, then diluted with maintenance medium to adjust the final DMSO concentration to 0.5 vol%, and filter sterilized. After sterilization, the test substances were diluted to the prescribed concentration, i.e., hops, trehalose, aspartame, barley, ceramide, placenta, alanine, and mint, to 20 μg/ml using 0.5 vol% DMSO-containing maintenance medium.

(5) 5 ml of the collagen coating solution was placed in a 75 ^cm2 cell culture 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.

The brown adipocyte liquid was added to the collagen-coated flask, and the brown adipocytes were cultured for 24 hours in a 5% _CO2 incubator at 37°C. The cultured brown adipocytes were then washed once with growth medium, which was then replaced with fresh growth medium, and cultured until they became subconfluent.

After the culture, the brown adipocytes were subjected to trypsin treatment 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 x 2 times).

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

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 not containing the test substance was added and cultured for 6 days. The medium was changed every other day.

After 8 days of culture, the supernatant was collected, RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen).

Using the obtained cDNA as a template and a primer 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, the expression level of GAPDH mRNA was measured using a GAPDH primer (Qiagen).

(6) Evaluation The results of measuring the expression level of UCP1 mRNA when each of the test substances, namely hops, trehalose, aspartame, barley, ceramide, placenta, alanine and mint, was administered (relative expression level when the mRNA expression level of the control was set at 1.00) are shown in Table 1.

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.

The composition of the present invention contains specific components that can be administered either parenterally or orally, and is particularly useful for subjects suffering from cellulite, obesity, and associated diseases, contributing to the health and well-being of such subjects.

Claims (2)

A composition for promoting energy consumption that contains crushed barley stalks and leaves as an active ingredient (excluding herbal medicines that contain aspergillus oryzae, germ, and young barley leaves). A composition for promoting thermogenesis, characterized by containing crushed barley stalks and leaves as an active ingredient (excluding herbal medicines containing aspergillus oryzae, germ and young barley leaves).