JP2022529570A - Composition for promoting adipocyte differentiation containing theaflavin - Google Patents

Abstract

The present specification relates to a composition for promoting adipocyte differentiation, which comprises theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient. The composition has the effect of promoting the differentiation of adipocytes to improve skin elasticity, wrinkle improvement, skin volume increase, skin regeneration, and subcutaneous fat reduction. [Selection diagram] Fig. 1

Description

The present specification describes a composition for promoting adipocyte differentiation containing theaflavin.

(Cross-reference to related applications)
This application has priority based on Korean Patent Application No. 10-2019-0048189 filed on April 25, 2019 and Korean Patent Application No. 10-2020-0029539 filed on March 10, 2020. It is alleged and the entire content of the application is incorporated into this application as a reference.

Subcutaneous fat is one of the main skin organs that support the structure of the skin, and unlike visceral fat, it is known that it gradually loses its efficacy as it gets older and loses its function as a structure. ing. For this reason, we tried to realize anti-aging by various methods in order to suppress and complement the skin dent phenomenon of the facial part, except for the treatment method approached from esthetics as a method to realize anti-aging by subcutaneous fat. Until now, there is no clear method that can promote the fat differentiation of subcutaneous fat.

Korean Published Patent No. 10-2018-0091991 Korean Published Patent No. 10-2008-0052675 International Publication No. 02/39956 International Publication No. 2006/078600 Chinese Patent Application Publication No. 106256357

PARK, P. J. et al., Theaflavin-Enriched Fraction Stimulates Adipogensis in Human Subcutaneous Fat Cells, International journal of molecular sciences, 15 April 2019, vol.20, thesis no .: 2034, page 9.

From one point of view, the problem to be solved by the present invention is that when the subcutaneous fat derived from human is treated with theaflavin, which is easily changed to another substance among various polyphenols derived from green tea and whose function could not be assured. It is to provide the effect of specifically accumulating fat in subcutaneous fat.

In one aspect, the present invention provides a composition for promoting adipocyte differentiation, which comprises theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient.

In one aspect, the present invention provides the effects of promoting adipocyte differentiation to improve skin elasticity, wrinkle improvement, skin volume increase, skin regeneration, and subcutaneous fat reduction.

The analysis results of the HPLC components for each extract are: (A) standard chemicals (1; theaflavin (TF), 2; theaflavin-3-gallate (TF3G), 3; theaflavin- 3'-gallate (theaflavin-3'-gallate, TF3'G), and 4; theaflavin-3,3'-jigallate (theaflavin-3, 3'-digallate, TFDG)), (B) non-fermented green tea extract (GT), (C) Theaflavin concentrated raw fermented extract (CoF-GT), (D) Green tea fermented extract (F-GT). The figure showing the cell proliferation efficacy of theaflavin concentrated raw fermented extract (CoF-GT), green tea fermented extract (F-GT), theaflavin (TF), theaflavin-3,3'-digalate (TFDG). be. Analysis results of cell viability (72 hours) efficacy of theaflavin concentrated raw fermented extract (CoF-GT), green tea fermented extract (F-GT), theaflavin (TF), theaflavin-3,3'-digalate (TFDG) It is a figure which showed. It is a figure which confirmed the subcutaneous fat differentiation promotion effect of theaflavin (TF) and theaflavin-3,3'-digalate (TFDG). It is a figure which confirmed the subcutaneous fat differentiation promotion effect of theaflavin concentrated raw fermented extract (CoF-GT) and green tea fermented extract (F-GT). Theaflavin (TF), theaflavin-3,3'-digalate (TFDG), theaflavin concentrated raw fermented extract (CoF-GT), and green tea fermented extract (F-GT) are treated respectively before producing subcutaneous fat. It is a figure which showed the result of having measured the quantity. Analysis of gene expression after treatment of theaflavin (TF), theaflavin-3,3'-digalate (TFDG), theaflavin concentrated raw fermented extract (CoF-GT), and green tea fermented extract (F-GT), respectively. It is a figure which showed the result. Adiponectin after treatment of theaflavin (TF), theaflavin-3,3'-digalate (TFDG), theaflavin concentrated raw fermented extract (CoF-GT), and green tea fermented extract (F-GT), respectively. It is a figure which showed the measurement result of the production amount. It is a figure which showed the analysis result of cell proliferation effect (24 hours) and cell viability (72 hours) effect by concentration of theaflavin (TF). It is a figure which showed the analysis result of the differentiation promotion effect of the adipocyte according to the concentration of theaflavin (TF).

Hereinafter, examples of the present application will be described in more detail with reference to the attached drawings. However, the techniques disclosed in this application are not limited to the examples described herein and may be embodied in other forms. It should be noted that the examples presented herein are provided so that the disclosed content can be thorough and complete, and that the ideas of the present application can be fully communicated to those skilled in the art. In order to clearly express each component in the drawings, the sizes such as the width and thickness of the components are shown in a slightly enlarged size. Also, for convenience of explanation, only a part of the component is illustrated, but a person skilled in the art will be able to easily grasp the remaining part of the component. In addition, a person having ordinary knowledge in the art may be able to embody the ideas of the present application in various other forms without departing from the technical ideas of the present application.

In one embodiment, the invention can provide a composition comprising theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient.

As used herein, the term "isomer" refers specifically to optical isomers (eg, essentially pure isomers), essentially pure pure diastereomers, or mixtures thereof. ), As well as conformation isomers (ie, isomers that differ only in their angle of one or more chemical bonds), position isomers (particularly tautomers), Alternatively, it comprises geometric isomers (eg, cis-trans isomers).

As used herein, "essentially pure" means, for example, a specific compound such as an enantiomer or a diastereomer when used in connection with an enantiomer or a diastereomer. It is present in an amount of about 90% or more, preferably about 95% or more, more preferably about 97% or more, or about 98% or more, still more preferably about 99% or more, and most preferably about 99.5% or more (w / w). Means that.

As used herein, "pharmaceutically acceptable" means to be used in animals, more specifically in humans, by avoiding significant toxic effects when used at conventional pharmaceutical doses. Can be approved by the government or an equivalent regulatory body, or can be approved, listed in the general pharmacopoeia, or certified as listed in other general pharmacopoeias. means.

As used herein, "pharmaceutically acceptable salt" means a salt according to one aspect of the invention that is pharmaceutically acceptable and has the preferred pharmacological activity of the parent compound. Is the salt formed from (1) inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or acetic acid, propionic acid, hexane acid, cyclopentenepropionic acid, glycolic acid, pyruvate, Lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamon acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-Ethan-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2 , 2] -oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucon It may include an acid addition salt formed from an organic acid such as an acid; or (2) a salt formed when the acidic protons present in the parent compound are substituted.

As used herein, "hydrate" means a compound to which water is bound, and is a broad concept including inclusion compounds having no chemical binding force between water and a mixture.

As used herein, the term "solvate" means a higher-order compound formed between a molecule or ion of a solute and a molecule or ion of a solvent.

Another embodiment provides the use of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof for use in the production of a composition for promoting adipocyte differentiation. can do. Another embodiment is a fat comprising administering theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in an effective amount to a subject in need thereof. A method for promoting cell differentiation can be provided. Another embodiment provides the teaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient for use in a composition for promoting adipocyte differentiation. can do. Another embodiment can provide theaflavins for use in promoting adipocyte differentiation, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof or solvates thereof. Further, as an active ingredient for promoting adipocyte differentiation, non-therapeutic use of the theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof can be provided. ..

The present invention comprises the theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient, whereby a human-derived, for example, subcutaneous adipocyte of a facial site is contained. When treated on cells, the following effects can be exhibited. Specifically, theaflavin is water-soluble and easily soluble in water, so that it is easily mixed with other dosage forms, and as a result of treatment with human-derived subcutaneous adipocytes, cell proliferation and toxicity do not change regardless of the concentration. Theaflavin, which is the active ingredient of the present invention, promotes the differentiation of subcutaneous fat in a concentration-dependent manner, whereas other theaflavins exhibit an existing anti-obesity effect and an inhibitory effect on adipose differentiation. As an example, the composition may be used for increasing skin volume by promoting the differentiation of adipocytes. As an example, the composition may be for suppressing subcutaneous fat loss by promoting the differentiation of adipocytes.

In one example, the content of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof contained in the composition is not limited, but for example, the total composition. It may be 0.01 to 100% by weight, specifically 0.01 to 50% by weight, based on the weight. More specifically, theflavin, its isomers, its pharmaceutically acceptable salts, its hydrates or its solvates are 0.01% by weight or more, 0. 1% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 15% by weight or more, 20% by weight or more, 25% by weight or more, 30% by weight or more, 35% by weight or more, 40% by weight or more, 45% by weight or more, 50% by weight or more, 55% by weight or more, 60% by weight or more, 65% by weight or more, 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, Alternatively, it may be 95% by weight or more. More specifically, the content of theflavin, its isomers, its pharmaceutically acceptable salts, its hydrates or its solvates is 100% by weight or less, 95% by weight, based on the total weight of the composition. % Or less, 90% by weight or less, 85% by weight or less, 80% by weight or less, 75% by weight or less, 70% by weight or less, 65% by weight or less, 60% by weight or less, 55% by weight or less, 50% by weight or less, 45% by weight % Or less, 40% by weight or less, 35% by weight or less, 30% by weight or less, 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, 1% by weight or less, or 0 It may be 1% by weight or less.

In one example, the concentration in the composition of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof was 0.01 to 10,000 mg / kg. Well, this can vary depending on the route of administration of the composition. In one example, the dose of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof may be 0.01 to 10,000 mg / kg / day. , This may vary depending on the route of administration of the composition. More specifically, the concentration or daily dose of the theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate is 0.01 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2 mg / kg or more, 3 mg / kg or more, 4 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 15 mg / kg or more, 20 mg / kg or more, 25 mg / kg or more, 30 mg / kg or more, 35 mg / kg or more, 40 mg / kg or more, 45 mg / kg or more, 50 mg / kg or more, 55 mg / kg or more, 60 mg / kg or more, 65 mg / kg or more, 70 mg / kg or more, 75 mg / kg or more, 80 mg / kg or more, 85 mg / kg or more, 90 mg / kg or more, 95 mg / kg or more, 100 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, 500 mg / kg or more, 600 mg / kg or more, 700 mg / kg or more, 800 mg / kg or more, 900 mg / kg or more, 1,000 mg / kg or more, 2,000 mg / kg or more, 3,000 mg / kg or more, 4,000 mg / kg or more, 5, It may be 000 mg / kg or more, 6,000 mg / kg or more, 7,000 mg / kg or more, 8,000 mg / kg or more, and 9,000 mg / kg or more. More specifically, the concentration of the theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate is 10,000 mg / kg or less, 9,000 mg / kg or less, 8, 000 mg / kg or less, 7,000 mg / kg or less, 6,000 mg / kg or less, 5,000 mg / kg or less, 4,000 mg / kg or less, 3,000 mg / kg or less, 2,000 mg / kg or less, 1,000 mg / Kg or less, 900 mg / kg or less, 800 mg / kg or less, 700 mg / kg or less, 600 mg / kg or less, 500 mg / kg or less, 400 mg / kg or less, 300 mg / kg or less, 200 mg / kg or less, 100 mg / kg or less, 95 mg / Kg or less, 90 mg / kg or less, 85 mg / kg or less, 80 mg / kg or less, 75 mg / kg or less, 70 mg / kg or less, 65 mg / kg or less, 60 mg / kg or less, 55 mg / kg or less, 50 mg / kg or less, 45 mg / Kg or less, 40 mg / kg or less, 35 mg / kg or less, 30 mg / kg or less, 25 mg / kg or less, 20 mg / kg or less, 15 mg / kg or less, 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0 It may be 5.5 mg / kg or less, or 0.1 mg / kg or less.

Specifically, the concentration or daily dose of theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate at the time of oral administration is 0.01 to 1,000 mg /. It may be kg, but is not limited to this. The concentration or daily dose of theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate at the time of skin application was 0.01 to 3,000 mg / kg. Good, but not limited to this. The concentration or daily dose of theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate at the time of direct subcutaneous administration is 0.01 to 100 mg / kg. However, it is not limited to this.

In one embodiment, the theaflavin may be contained in a fermented green tea extract. That is, the composition may contain a fermented green tea extract containing theaflavin.

Tea is made by processing the buds and leaves of a tea tree (Camellia sinensis O, Kuntze) belonging to Theaceae and Camellia, and the fresh leaves of tea have 75-80% water content. It contains 20 to 25% solids, which contain organic substances such as catechin, caffeine, amino acids, fibrinos, and pectin, as well as various components such as lipids, resins, essential oils, vitamins, and chlorophyll. Has been done. The teas are usually unfermented tea (green tea) having a degree of fermentation of 0%, semi-fermented tea (white tea, flower tea, baozhong tea and oolong tea) having a degree of fermentation of 20 to 60%, and 80% or more, depending on the degree of fermentation. It is roughly divided into fermented tea (black tea). Traditional fermented tea is called enzyme fermented tea because it uses fermentation with enzymes contained in tea leaves. Black tea, which is an enzyme-fermented tea, undergoes a wilting process to soften the leaves and induces a biochemical reaction necessary for the production of aroma, and then undergoes a kneading process to combine oxidative enzymes and polyphenols. By promoting the reaction and promoting the oxidative polymerization of catechins in the fermentation step, theaflavins are produced, and aroma and taste are produced with browning. Enzyme-fermented tea produces theaflavins (theaflavins) that are reddish-yellow due to the oxidative polymerization reaction of catechins such as epicatechin and epigallocateingalate due to oxidative enzymes as the fermentation progresses, and mainly theaflavins, 4 species such as theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate Has been reported. These theaflavins are known to have effects such as antioxidant activity and anti-mutation, and an oxidative polymer is produced by the action of an oxidase, which increases the redness of tea and makes it aroma and taste. Differences occur, and there are also differences in the biophysiological activity of the product.

In the present specification, "fermented green tea" means green tea in a fermented state, and includes all differences depending on the degree of fermentation.

The fermented green tea extract according to an embodiment of the present invention contains theaflavins containing theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-jigallate, and at this time, the present invention. Theaflavin, which is the active ingredient of the present invention, may be contained in an amount of 30 to 50% by weight based on the total weight of the theaflavins.

As an example, the fermented green tea extract is (a) a step of preparing a mixture of green tea and spring chrysanthemum; (b) a step of fermenting and drying the mixture under reduced pressure; and (c) the fermentation. And may be produced by a method comprising the step of extracting the dried mixture.

As an example of the method for producing fermented green tea according to the present invention, the step (a) is a step of preparing a mixture obtained by mixing green tea and chrysanthemum.

In the present invention, green tea may contain one or more species selected from the group consisting of green tea leaves, stems of green tea trees and roots of green tea trees, and may include, for example, green tea leaves. Further, as the green tea, a growing green tea may be used to promote the enzymatic reaction.

In one embodiment of the present invention, the chrysanthemum may be used as a fermentation additive composition that promotes fermentation of green tea. The chrysanthemum contains polyphenol oxidase and peroxidase, which can promote the fermentation of green tea. In one embodiment of the present invention, the chrysanthemum may be used in the form of powdered chrysanthemum grounds or juice. The pulverized spring chrysanthemum pulverized product is not particularly limited as long as it is known in the art, but the powder primary pulverized by a crusher is re-pulverized into fine particles of about 1 to 500 μm by a crusher. It may be produced as a fine powder by a ball mill, a hammer mill, or the like without primary pulverization. The juice is not particularly limited as long as it is known in the art.

As an example, the green tea and chrysanthemum may be mixed in a weight ratio of 1: 1 to 99: 1, specifically, may be mixed in a weight ratio of 20: 1 to 5: 1, and more specifically. The mixture may be mixed at a weight ratio of 10: 1. If the amount of spring chrysanthemum is more than the weight of green tea, green tea fermentation can be carried out in a short time, but the oxidation rate may be increased and theaflavins may be reduced, and green tea and spring chrysanthemum may be 99: 1. When the weight ratio is exceeded, the content of theaflavin among theaflavins is not increased due to the control of the enzyme contained in green tea.

As an example, the mixture of green tea and spring chrysanthemum may further contain distilled water, ionized water, clean water, groundwater containing minerals, and less than 30% alcohol as a solvent. As an example, the solvent may be contained in an amount of 50 to 200 parts by weight, specifically 80 to 150 parts by weight, based on 100 parts by weight of green tea.

In one embodiment, step (b) is a step of fermenting and drying the mixture prepared in step (a) under reduced pressure. As used herein, "fermentation" means that it occurs by the action of naturally occurring oxidases in green tea. The reduced pressure state refers to a state in which contact with oxygen is blocked, and in one embodiment, fermentation and drying may be performed in a reduced pressure state of 10 to 50 mmHg. Specifically, the fermentation under the reduced pressure state may be carried out at a temperature of 20 to 50 ° C. for 1 to 10 hours, and more specifically, may be carried out for 2 to 5 hours. If the fermentation temperature is less than 20 ° C., the fermentation time may be prolonged, and if it exceeds 50 ° C., the deactivation rate of the enzyme may increase and incomplete fermentation may occur. In addition, the step (b) may dry the fermented mixture, and the drying may be freeze-drying. The method of freeze-drying is not particularly limited, and may be performed by a method known in the art.

In one embodiment, step (c) is the step of extracting the mixture fermented and dried in step (b). The extraction is carried out using an extraction solvent, and the extraction solvent contains one or more selected from the group consisting of water and alcohol having 1 to 5 carbon atoms, and specifically, is selected from the group consisting of water and ethanol. May include one or more of the following species. The extraction temperature and the extraction time are not particularly limited, but for example, the extraction temperature is normal temperature to 80 ° C., and the extraction time is 1 to 24 hours. In the step (c), the fermentation and dry mixture may be extracted by various methods, for example, a method using a hot water extract or a lower alcohol extract having 1 to 5 carbon atoms.

For example, as a first embodiment, water of about 10 to 30 times the weight of the fermented and dried mixture is added, primary extraction is performed at 100 ° C. for 4 hours, secondary extraction is performed for 4 hours by the same method, and then secondary extraction is performed. After the primary and secondary extracts are combined and filtered, the filtrate is concentrated with a concentrator. The concentrated extract is dried using a spray dryer or a vacuum freezing freeze dryer. As a second embodiment, the method for producing a 70% ethanol extract is as follows: after adding 70% ethanol, which is about 10 to 30 times the weight of the fermented and dried mixture, and extracting at 50 to 80 ° C. for 30 minutes to 3 hours. , Filter and concentrate the filtrate with a concentrator. The concentrated extract is dried using a spray dryer or a vacuum freezing freeze dryer. As a third embodiment, in the method for producing a 95% ethanol extract, 95% ethanol, which is about 10 to 30 times the weight of the fermented and dried mixture, is added and left at room temperature for 1 to 3 days, every 6 hours. After repeating the shaking operation to extract, the extract is obtained by repeating the process twice in the same manner. After the primary and secondary extracts are combined and filtered, the filtrate is concentrated with a concentrator. The concentrated extract is dried using a spray dryer or a vacuum freezing freeze dryer.

Generally, in fermented green tea extract, as the fermentation progresses, oxidative polymerization reaction of catechins such as epicatechin and epigalocatechingalate occurs by oxidative enzyme to produce theaflavins (theaflavins) having a reddish yellow tinge, and the theaflavins are produced. Theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-jigalate (theaflavin-3,3'- digitalate) is included.

The fermented green tea of the present invention according to the above-mentioned example contains theaflavin among theaflavins in a high content with respect to other theaflavins, thereby promoting the differentiation of human-derived subcutaneous adipocytes and inducing high adipocyte production. Show efficacy. Specifically, the fermented green tea extract may contain theaflavin in an amount of 30 to 50% by weight based on the total weight of theaflavins.

As an example, the content of the fermented green tea extract contained in the composition according to the present invention is not particularly limited, but as an example, 0.01 to 99.9% by weight, specifically, 0.01 to 99.9% by weight based on the total weight of the composition. , 0.1 to 50% by weight may be contained. When it is contained in the above content, it is not only suitable for exhibiting the intended effect of the present invention, but also can satisfy both the stability and safety of the composition, and is also preferable in terms of cost effectiveness.

In one embodiment, the composition according to the present invention may be a cosmetic composition. The cosmetic composition may be formulated containing a cosmetically or dermatologically acceptable medium or base. This is any dosage form suitable for topical application and may be for transdermal administration. For example, solutions, gels, solids, anhydrous products, emulsions obtained by dispersing the oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, fine granulocytes or ionic (lipolips) and nonionic forms. It may be provided in the form of a vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or concealed stick. It may also be used in the form of foam or in the form of an aerosol composition further containing a compressed propellant. Other examples may be, but are not limited to, injection forms such as fillers, suppositories, patches and the like. These compositions may be produced by conventional methods in the art.

As an example, the composition according to the present invention may contain other ingredients that can give a synergistic effect to the main effect, preferably, as long as the main effect is not impaired together with the active ingredient. In addition to the active ingredient of the present invention, other ingredients may be appropriately selected and blended by those skilled in the art according to the dosage form or purpose of use of other cosmetic compositions. Further, as an example, the cosmetic composition according to the present invention may contain other components to be blended with a normal cosmetic composition, if necessary, together with the above components. For example, moisturizers, emollients, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, colds. Examples include sensitive agents, antiseptic agents, purified water and the like. The other compounding components that may be contained in the cosmetic composition according to the present invention are not limited to these, and the compounding amount of the components may be within a range that does not impair the object and effect of the present invention.

The composition according to the embodiment of the present invention may be a food composition containing the active ingredient. For example, it may be processed as a functional food such as fermented milk containing the active ingredient, cheese, yogurt, juice, live bacterial preparation, health food, etc., and may be used in various other food additive forms. As an example, the composition may be a composition for health foods. As an example, the composition for health food can be formulated into pills, capsules, tablets, granules, caramel agents, drinks and the like. As another embodiment, it may be processed into a liquid, powder, granule, tablet or tea bag. The composition may be administered by various methods such as simple drinking, injection administration, spray method or squeeze method. The composition may contain other components that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, additives such as fragrances, pigments, bactericides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers, and synthetic polymer substances may be further contained for improving physical properties. In addition, auxiliary components such as water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, and seaweed extracts may be further contained. The above-mentioned components may be appropriately selected and blended by those skilled in the art according to the dosage form or the purpose of use, and the addition amount thereof may be selected within a range that does not impair the purpose and effect of the present invention. For example, the amount of the component added may be 0.0001% by weight to 99.9% by weight based on the total weight of the composition.

The composition according to the embodiment of the present invention may be a pharmaceutical composition containing the active ingredient. The pharmaceutical compositions are preservatives, stabilizers, wettable powders or emulsion promoters, pharmaceutical aids such as salts and / or buffers for osmoregulation, and other therapeutically useful. It may further contain a substance.

As an example, the pharmaceutical composition may be an oral administration, wherein the oral administration is, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups. , Powders, powders, syrups, granules, pellets, etc. These dosage forms include active ingredients, surfactants, diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (eg silica, talc, stearic acid and magnesium thereof). Alternatively, it may contain a calcium salt and polyethylene glycol). The tablets may further contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidin, optionally starch, agar, starch or sodium salts thereof. It may contain pharmaceutical additives such as agents, absorbents, colorants, flavors, and sweeteners. The tablets may be produced by conventional mixing, granulation or coating methods.

As an example, the pharmaceutical composition may be a parenteral administration agent, and the parenteral administration agent may be a rectal, topical, subcutaneous, dermal-administered dosage form. For example, it may be in the form of an injection such as a filler, a drip, an ointment, a skin, a lotion, a gel, a cream, a spray, a suspension, an emulsion, a suppository, a patch, etc., but is limited thereto. It is not something that will be done.

As an example, the dose of the pharmaceutical composition is the age, sex, body weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration and the prescribing person. It can vary depending on the judgment of. Dosage determination based on such factors is within the standards of those of skill in the art. For example, the dose may be 0.01 to 10,000 mg divided into 1 to 3 times a day, but the dose does not limit the scope of the present specification by any method. do not have.

Hereinafter, the present invention will be described in detail with reference to Examples, Comparative Examples, and Test Examples. It will be obvious to those skilled in the art that these are merely examples for more specific explanation of the present invention, and the scope of the present invention is not limited by these Examples, Comparative Examples and Test Examples. ..

[Example 1] Production of CoF-GT (fresh green tea / co-fermentation) fraction with enhanced theaflavin content Fresh green tea and chrysanthemum leaves were washed twice with deionized water and excessive water was tapped to remove them. .. These leaves were immersed in liquid nitrogen, crushed and stored at −80 ° C. CoF-GT was manufactured as follows. CoF-GT fermented a mixture of fresh green tea powder (100 g) and frozen raw chrysanthemum (50 mg). After the reaction for 3 hours, the mixture was extracted with 70% ethyl alcohol (v / v) for 2 hours. The solids and residues were passed through a 90 mesh sieve and then filtered through a 0.22 μm filter (Dow Corning, Corning, ny, USA). The filtered sample was evaporated in a Hei-VAP rotary concentrated evaporator (Heidolph Instruments, Schwabach, Germany) and then pulverized using a FreeZone lyophilizer (Labconco, Kansas City, MO, USA).

[Comparative Example 1] Production of a normally fermented F-GT (green tea general fermentation) fraction In F-GT, the leaves of fresh green tea were washed twice with deionized water and excessive water was tapped to remove them. .. The leaves were soaked in liquid nitrogen, crushed and stored at −80 ° C. It was produced by fermenting fresh green tea powder (100 g) frozen in a heat jacket z-blade mixer (IKA, Staufen im Breisgau, Germany) at 37.5 ° C. After the reaction for 3 hours, the mixture was extracted with 70% ethyl alcohol (v / v) for 2 hours. The solids and residues were passed through a 90 mesh sieve and then filtered through a 0.22 μm filter (Dow Corning, Corning, NY, USA). The filtered sample was evaporated in a Hei-VAP rotary concentrated evaporator (Heidolph Instruments, Schwabach, Germany) and then pulverized using a FreeZone lyophilizer (Labconco, Kansas City, MO, USA).

[Test Example 1] HPLC analysis of CoF-GT and F-GT fractions Each sample (8 g) is dissolved in a mixture of 100 ml DMSO / methanol / ethanol (5:45:50, v / v) after ultrasonic treatment for 30 minutes. After centrifuging, the mixture was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) injector filter (Pall Corp., Port Washington, NY, USA). An 82 ml sample was filled in a 50 ml sample roof and injected into an AKTA purifier 10 (GE Healthcare, Stockholm, Sweden) with a photodiode array detector and analyzed at 275 and 365 nm. Purification and separation were performed on an AQ-HG octadecyclyl column (120 Å, 10 μm, 20 × 250 mm, mercury volume = 78.5 ml) (YMC Co., Kyoto, Japan). Gradient elution was performed with pure water (solvent A) and acetonitrile (solvent B). 8.2 ml of the sample solution was injected at one time, and the flow rate of the mobile phase was 10 ml / min. All solvents were filtered, degassed and used under appropriate pressure. Fractions (110 ml) were collected after injection of the sample. Four fractions were obtained for each cycle and collected in separate bottles over 10 cycles. The fourth fraction was collected in one each time, the solvent was removed with a Hei-VAP rotary evaporator (Heidolph Instruments), lyophilized and powdered, and then stored at −20 ° C. until analysis.

Theaflavin (TF), theaflavin-3-gallate (theaflavin, TF) of the fermented green tea extracts produced in Examples 1 and 1 using HPLC (High performance liquid chromatografy) for the detection of theaflavin components in the fermented tea. theaflavin-3-gallate, TF-3G), theaflavin-3'-gallate (theaflavin-3'-gallate, TF-3'G), and theaflavin-3,3'-jigallate (theaflavin-3,3'-digalate). , TF-3,3'DG) content analysis was performed. The theaflavin standard was purchased from Wako, and the sample was dissolved in a 50% ethanol solvent, ultrasonically extracted, filtered through a 0.45 μm PVDF filter, and injected into the instrument. Analysis was performed using HPLC (Waters Alliance 2695 system, Waters, USA) with a detection wavelength in the 270 nm region. The column was analyzed using Optimapak C18 (150 x 4.6 mm, 5 μm) in a gradient mode using 0.05% trifluoroacetic acid (TFA) and acetonitrile (Actonirile, ACN) solvent, and the concentration of the solvent. The composition ratios are shown in Table 1 below. All analytical solvents were HPLC grade reagents and data processing was performed using the Waters Emper 3 program.

As a result, as shown in FIG. 1, in the case of the green tea fermented extract (F-GT) to which the existing green tea fermentation technique (Comparative Example 1) is applied, the four main polyphenol components are uniformly increased. However, the theaflavin raw fermented extract (CoF-GT, Example 1) to which the co-fermentation technique was applied was produced in a large amount with priority given to the content of theaflavin (1).

[Test Example 2] Cell culture and differentiation and analysis of cell viability
Cell culture and differentiation
Human-derived subcutaneous adipocytes (human Subcutaneous Fat cells, hSCF) and undifferentiated subcutaneous adipocyte culture medium were purchased from ZenBio (Research Triangle Park, NC, USA). The hSCF was cultured in a 5% CO ₂ incubator during the culture period. 10 μg / ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, 1 μg troglitazone (both Sigma-Aldrich) to induce differentiation 21 days in Dulbecco's modified Eagle's medium (Lonza, Walsville, MD, MD, USA) containing 10% fetal serum (PAA, Walkersville, MD, USA) with (purchased from USA). It was cultured. The medium was changed every other day.

Cell viability analysis The viability of hSCF was measured with the EZ-Cytox test kit (DAEIL LAB SERVICE, Seoul, Republic of Korea) according to the manufacturer's guidelines. Simply, for cells cultured for 7 days, theaflavin-enriched raw fermented extract (CoF) at various concentrations of 0.1, 0.5, 1, 5 and 10 μg / ml over 24 and 72 hours after drug treatment, respectively. -GT), fermented green tea extract (F-GT), theaflavin (TF) and theaflavin-3,3'-digalate (TFDG) were treated, respectively. EZ-Cytox solution (10 μl) was added to each well and cultured at 37 ° C. for 2 hours. Absorbance at 450 nm was measured with a spectrophotometer (Syrgenergy H2; BioTek, Winosk, VT, USA). The experiment was repeated 3 times and the data expressed the absorbance value as a comparative activity ratio (%) to the control group.

As a result of confirming the concentration showing an influence on hSCFs of each of the treated substances, all the treated substances had the total concentration of the growth effect experiment (FIG. 2) of the 24-hour treatment and the toxicity confirmation experiment (FIG. 3) of the 72-hour treatment. It was confirmed that it is a safe substance that does not show any toxic effect except for a slight cell proliferation effect in the section.

[Test Example 3] Triglyceride (TG) content analysis by ORO (Oil Red O) staining The results of subcutaneous adipocyte differentiation confirmed adipogenesis qualitatively and quantitatively by Oil Red O staining.

The hSCF was washed twice with cold phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde (Sigma-Aldrich) for 1 hour. The immobilized cells were washed with 60% propylene glycol (propylene glycol, Sigma-Aldrich) for 30 minutes and then stained with a solution of Oil Red O (0.7% ORO stock was prepared by diluting with 60% propylene glycol). .. Again, the cells were washed 3 times with 85% propylene glycol and then rinsed with running tap water. Lipid droplets (LD) stained with ORO were observed and photographed with an IX71 microscope (Olympus, Tokyo, Japan). To quantify LD, cells stained with 70% propylene glycol (Sigma-Aldrich) were washed and a solution of isopropyl alcohol (isopropanol alcohol) mixed with 4% Nonidet P-40 (Sigma-Aldrich) was applied to the cells. Only ORO was extracted from the cells stained with ORO for 20 minutes. The absorbance of the extract was measured at 490 nm with a spectrophotometer.

It takes about 21 days for hSCFs to differentiate into mature lipids that produce lipid droplets. Therefore, theaflavin (TF) and theaflavin-3,3'-digalate (TFDG) 0.5, 2 μg / ml, respectively, theaflavin concentrated raw fermented extract (CoF-GT) and green tea fermented extract (F-GT). They were cultured for 21 days in media treated at 1, 5 and 10 μg / ml, respectively. As a result, in the case of theaflavin (TF) and theaflavin concentrated raw fermented extract (CoF-GT), which are examples of the present invention, it was confirmed by the Oil Red O staining method that the fat production was increased in a concentration-dependent manner. In contrast, when TFDG and F-GT were treated, it was confirmed that adipogenesis, which is an anti-obesity effect of existing green tea, occurred (Fig. 4). In order to quantify the amount of stained fat in FIGS. 3 and 4, the amount of triglyceride (TG) was measured and represented graphically in FIG.

[Test Example 4] Real-time quantitative PCR (Realtime Quantitative Polymerase chain reaction; RT-qPCR)
Total RNA is extracted using TRIzol reagents (TRIzol, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, and complementary DNA (cDNA) is reverse transcription kit, Promega, Mad. It was synthesized using WI, USA). The cDNA was used as a template for RT-qPCR amplification in a 7500 high-speed real-time PCR system (Fast7500 Real-time PCR, Life Technologies) using a TaqMan® probe (Taqman probe), and was used as a template for RT-qPCR amplification. Hs01115513_m1), adiponectin (ADIPOQ; # Hs00605917_m1) and glyceraldehyde (glyceraldehyde 3-phosphate dehydrogenase, GAPDH; # 4352239E) were used as control genes. Data are obtained in three independently repeated experiments, and sample values are presented in varying numbers relative to GAPDH levels.

As a result of measuring the amounts of PPARγ and adiponectin (Adiponectin), which are genes related to adipose differentiation, as shown in FIG. 7, the staining results of Test Example 3 and the results similar to the theaflavin amount were confirmed.

[Test Example 5] Analysis of adiponectin eluted in the medium Adiponectin, which is one of the adipocyte-specific hormones (adipocyte specific hormones) eluted in the medium to confirm the condition after the differentiation of subcutaneous fat. The increase in the amount was confirmed by the ELISA (Enzyme-linked immunoglobulin assay) measurement method.

The hSCF is treated with various concentrations of theaflavin (TF), theaflavin-3,3'-digalate (TFDG), theaflavin concentrated raw fermented extract (CoF-GT), and green tea fermented extract (F-GT), respectively. Differentiated for 21 days. The culture medium was removed, and the foreign matter was removed after centrifugation at 13,000 rpm for 15 minutes. ELISA (Enzo Life Sciences, Farmingdale, NY, USA) measured the level of adiponectin eluted using according to the manufacturer's guidelines. As a result, as shown in FIG. 8, adiponectin, which is one of the adipocyte-derived hormones (adipokine) eluted in the medium after fat differentiation, is theaflavin-3,3'-digalate (TFDG) and green tea fermentation extraction. While it decreases when the substance (F-GT) is treated, its concentration increases when the theaflavin (TF) and theaflavin concentrated raw fermented extract (CoF-GT), which are examples of the present invention, are treated. As a result, the amount of adiponectin also increased. This is in agreement with the experimental results of Test Examples 3 and 4 above.

[Test Example 6] Cell culture and differentiation and cell viability analysis by concentration of theaflavin (TF) 0.01 to 100 μg / ml theaflavin (TF) exerts an influence on hSCFs by the same method as in Test Example 2 above. The concentration was confirmed and this is shown in FIG. As a result, it was found that both the 24-hour treatment proliferation effect experiment and the 72-hour treatment toxicity confirmation experiment were safe substances showing no toxic effect except for a slight cell proliferation effect at all the treated concentrations. confirmed.

[Test Example 7] Analysis of Triglyceride (TG) Increased Efficacy by Concentration of Theaflavin (TF) By the same method as in Test Example 3, the theaflavin (TF) of 0 to 100 μg / ml was subjected to subcutaneous fat cells by concentration. The differentiation efficacy was analyzed.

As a result, as shown in FIG. 10, theaflavin (TF) promoted fat differentiation in a concentration-dependent manner up to 50 μg / ml, and at 100 μg / ml, the fat differentiation ability was slightly reduced from 50 μg / ml.

Hereinafter, a dosage form example of the composition according to one embodiment of the present specification will be described, but application to various other dosage forms is also possible, and this is not merely specific to the present specification. It is for the purpose of explaining.

[Dosage form example 1] Soft lotion (skin lotion)
A soft lotion was produced according to a usual method with the compositions shown in the table below.

[Dosage form example 2] Nutritional lotion (milk lotion)
A nutritional lotion was produced according to a usual method with the composition shown in the table below.

[Dosage Form Example 3] Massage Cream A massage cream was produced according to a usual method with the compositions shown in the table below.

[Dosage Form Example 4] Tablets Theaflavin (100 mg), lactose (400 mg), corn starch (400 mg) and magnesium stearate (2 mg) were mixed and then tableted according to a usual method for producing tablets to produce tablets.

[Dosage Form Example 5] Capsule After mixing 100 mg of teaflavin, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate, the capsule was filled into a gelatin capsule according to a usual method for producing a capsule to produce a capsule.

[Dosage Form Example 6] Granules Theaflavin 50 mg, anhydrous crystalline glucose 250 mg and starch 550 mg were mixed, formed into granules using a fluidized bed granulator, and then packed in sachets.

[Dosage Form Example 7] Drinking agent After mixing 50 mg of teaflavin, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide, 300 ml of purified water is added, and 200 ml of each bottle is filled. After filling the bottle, it was sterilized at 130 ° C. for 4 to 5 seconds to produce a drink.

[Dosage Form Example 8] Caramel form Theaflavin 50 mg, corn syrup 1.8 g, defatted milk 0.5 g, soybean lecithin 0.5 g, butter 0.6 g, hydrogenated vegetable oil 0.4 g, sugar 1. 4 g, 0.58 g of margarine, and 20 mg of salt were mixed and caramelized.

[Dosage form example 9] Health food

The composition ratio of the vitamin and the inorganic mixture is a mixture of ingredients that are relatively suitable for health foods as an example, but the composition ratio may be arbitrarily modified and according to a normal method for producing health foods. After mixing the above components, granules may be produced and used in the production of health food compositions according to conventional methods.

[Dosage Form Example 10] Health drink

As shown in the table above, the remaining amount of purified water is added so as to have a total volume of 900 ml, the components are mixed according to a normal method for producing a healthy beverage, and then the mixture is stirred and heated at 85 ° C. for about 1 hour to prepare the mixture. The filtrate obtained by filtering the resulting solution may be placed in a sterilized 2 liter container, sealed and sterilized, and then stored in a refrigerator for use in the production of a health beverage composition.

[Dosage Form Example 11] Injection An injection was produced according to a usual method with the compositions shown in the table below.

The present invention can provide the following embodiments as an example.

The first embodiment can provide a composition for promoting adipocyte differentiation, which comprises theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient. ..

A second embodiment, in the first embodiment, can provide a composition in which the composition comprises a fermented green tea extract containing theaflavin.

The third embodiment is one or more of the first embodiment and the second embodiment, wherein the fermented green tea extract is theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'. -It is possible to provide a composition containing theaflavins including digarates and containing theaflavins in an amount of 30 to 50% by weight based on the total weight of the theaflavins.

The fourth embodiment is one or more of the first to third embodiments, wherein the fermented green tea extract is used.
(A) Steps to prepare a mixture by mixing green tea and chrysanthemum;
Compositions can be provided that are produced by a method comprising (b) a step of fermenting and drying the mixture under reduced pressure; and (c) a step of extracting the fermented and dried mixture.

A fifth embodiment is in the composition of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in one or more of the first to fourth embodiments. Compositions can be provided that have a concentration of 0.01 to 10,000 mg / kg.

In the sixth embodiment, in one or more of the first to fifth embodiments, the dose of theaflavin, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate is , 0.01 to 10,000 mg / kg / day, can be provided.

The seventh embodiment is one or more of the first to sixth embodiments, and the composition can provide a composition for increasing the skin volume by promoting the differentiation of adipocytes.

The eighth embodiment is one or more of the first to seventh embodiments, and the composition can provide a composition for suppressing subcutaneous fat loss by promoting the differentiation of adipocytes.

A ninth embodiment can provide a composition for transdermal administration in one or more of the first to eighth embodiments.

The tenth embodiment is one or more of the first to ninth embodiments, and the composition can provide a composition which is a cosmetic composition.

The eleventh embodiment is one or more of the first to tenth embodiments, and the composition can provide a composition which is a pharmaceutical composition.

The twelfth embodiment is one or more of the first to eleventh embodiments, and the composition can provide a composition which is a food composition.

The embodiments are disclosed for the purpose of explaining the present invention, and the above description does not limit the scope of the present invention. Accordingly, various modifications, modifications, and replacements may arise from ordinary technicians in the art, as long as they do not deviate from the meaning and scope of the invention.

Claims (12)

A composition for promoting adipocyte differentiation, which comprises theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient. The composition according to claim 1, wherein the composition contains a fermented green tea extract containing theaflavin. The fermented green tea extract contains theaflavins, including theaflavins, theaflavins-3-gallate, theaflavin-3'-gallate, and theaflavins-3,3'-digalate, wherein theaflavins are 30 relative to the total weight of the theaflavins. The composition according to claim 2, which comprises ~ 50% by weight. The fermented green tea extract is
(A) Steps to prepare a mixture by mixing green tea and chrysanthemum;
The composition according to claim 2, wherein (b) is produced by a method comprising a step of fermenting and drying the mixture under reduced pressure; and (c) a step of extracting the fermented and dried mixture. The concentration of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in the composition is 0.01 to 10,000 mg / kg, according to claim 1. The composition described. The first aspect of claim 1, wherein the dose of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof is 0.01 to 10,000 mg / kg / day. Composition. The composition according to claim 1, wherein the composition is for increasing the skin volume by promoting the differentiation of adipocytes. The composition according to claim 1, wherein the composition is for suppressing the decrease in subcutaneous fat by promoting the differentiation of adipocytes. The composition according to any one of claims 1 to 8, wherein the composition is for transdermal administration. The composition according to any one of claims 1 to 8, wherein the composition is a cosmetic composition. The composition according to any one of claims 1 to 8, wherein the composition is a pharmaceutical composition. The composition according to any one of claims 1 to 8, wherein the composition is a food composition.

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