KR102359446B1 - Composition comprising theaflavin for promoting differentiation of adipocyte - Google Patents

Abstract

The present specification relates to a composition for promoting adipocyte differentiation comprising theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient. The composition promotes the differentiation of adipocytes to provide effects of improving skin elasticity, wrinkle improvement, skin volume increase, skin regeneration, and subcutaneous fat reduction.

Description

A composition comprising theaflavin for promoting differentiation of adipocytes

The present specification describes a composition for promoting differentiation of adipocytes comprising theaflavin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Korean Patent Application No. 10-2019-0048189, filed on April 25, 2019, the entire contents of which are incorporated herein by reference.

Subcutaneous fat is one of the main skin organs that support the structure of the skin, and unlike visceral fat, it is well known that it gradually loses its effectiveness with age and loses its function as a structure. Therefore, it was attempted to implement anti-aging in various ways in order to suppress and compensate for the skin off phenomenon in the face area, but until now, except for the treatment method approaching esthetic, subcutaneous fat as a method of realizing anti-aging through subcutaneous fat. There was no clear method to promote fat differentiation.

Republic of Korea Patent Registration No. 10-0856556 Republic of Korea Patent Registration No. 10-0916196

From one point of view, the problem to be solved by the present invention is to accumulate fat specifically in the subcutaneous fat when theaflavin, which is easily changed into other substances among various green tea-derived polyphenols and its function is not certain, to human-derived subcutaneous fat. to provide possible effects.

In one aspect, the present invention provides a composition for promoting adipocyte differentiation comprising 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 effects of improving skin elasticity, wrinkle improvement, skin volume increase, skin regeneration, and subcutaneous fat reduction by promoting adipocyte differentiation.

1 is a result of HPLC component analysis for each extract: (A) Standard chemical (1; theaflavin (TF), 2; theaflavin-3-gallate (TF3G), 3) ; theaflavin-3'-gallate (TF3'G), and 4; theaflavin-3, 3'-digallate (TFDG)) , (B) unfermented green tea extract (GT), (C) theaflavin concentrated live fermented extract (CoF-GT), (D) green tea fermented extract (F-GT).
2 shows the cell proliferation efficacy of theaflavin-concentrated live ferment extract (CoF-GT), green tea ferment extract (F-GT), theaflavin (TF), and theaflavin-3,3'-digallate (TFDG) It is a diagram showing the analysis result.
Figure 3 shows the cell viability of theaflavin-concentrated live ferment extract (CoF-GT), green tea ferment extract (F-GT), theaflavin (TF), and theaflavin-3,3'-digallate (TFDG). (72 hours) is a diagram showing the results of the efficacy analysis.
4 is a diagram confirming the differentiation promoting effect of theaflavin (TF) and theaflavin-3,3'-digalate (TFDG) in subcutaneous fat.
5 is a diagram confirming the subcutaneous fat differentiation promoting effect of the aflavin-enriched live fermented extract (CoF-GT) and green tea fermented extract (F-GT).
6 is the aflavin (TF), theaflavin-3,3'-digallate (TFDG), theaflavin concentrated live fermented extract (CoF-GT) and green tea fermented extract (F-GT), respectively It is a diagram showing the results of measuring the amount of fat production in the post-subcutaneous fat.
7 is the aflavin (TF), theaflavin-3,3'-digallate (TFDG), theaflavin concentrated live fermented extract (CoF-GT) and green tea fermented extract (F-GT) treated respectively A diagram showing the results of post-gene expression analysis.
8 is the aflavin (TF), theaflavin-3,3'-digallate (TFDG), theaflavin concentrated live fermented extract (CoF-GT) and green tea fermented extract (F-GT) treated respectively It is a diagram showing the measurement result of adiponectin (Adiponectin) production after.
9 is a diagram showing the results of analysis of cell proliferation efficacy (24 hours) and cell viability (72 hours) efficacy by concentration of theaflavin (TF).
10 is a diagram showing the results of analysis of adipocyte differentiation promoting efficacy according to concentrations of theaflavin (TF).

Hereinafter, embodiments of the present application will be described in more detail with reference to the accompanying drawings. However, the technology disclosed in the present application is not limited to the embodiments described herein and may be embodied in other forms. However, the embodiments introduced herein are provided so that the disclosed content may be thorough and complete, and the spirit of the present application may be sufficiently conveyed to those skilled in the art. In order to clearly express each component in the drawing, the size of the component, such as width or thickness, is slightly enlarged. In addition, although only some of the components are illustrated for convenience of description, those skilled in the art will be able to easily understand the remaining parts of the components. In addition, those of ordinary skill in the relevant field will be able to implement the idea of the present application in various other forms without departing from the technical spirit of the present application.

In one embodiment, the present invention may 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, "isomers" are in particular optical isomers (e.g. essentially pure enantiomers, essentially pure diastereomers or mixtures thereof), as well as conformational isomers ( includes conformation isomers (i.e. isomers that differ only in the angle of one or more chemical bonds), position isomers (especially tautomers) or geometric isomers (e.g. cis-trans isomers) do.

As used herein, "essentially pure", for example, when used in reference to an enantiomer or diastereomer, contains at least about 90%, preferably at least about 95% of a specific compound exemplified by enantiomer or diastereomer. , more preferably at least about 97% or at least about 98%, even more preferably at least about 99%, even more preferably at least about 99.5% (w/w).

As used herein, "pharmaceutically acceptable" means avoiding significant toxic effects when used in conventional medicinal dosages, thereby obtaining approval from the government or equivalent regulatory agencies that it can be used in animals, more specifically humans. available, approved, or recognized as being listed in a pharmacopoeia or other general pharmacopoeia.

As used herein, "pharmaceutically acceptable salt" refers to a salt according to an aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salts are formed by (1) inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-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-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert -acid addition salts formed with organic acids such as butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (2) salts formed when an acidic proton present in the parent compound is substituted.

As used herein, the term “hydrate” refers to a compound to which water is bonded, and is a broad concept including an encapsulated compound that does not have a chemical bonding force between water and the compound.

As used herein, the term “solvate” refers to a higher-order compound formed between a molecule or ion of a solute and a molecule or ion of a solvent.

Another embodiment may provide the use of the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof for use in the preparation of a composition for promoting adipocyte differentiation. Another embodiment provides a method for promoting adipocyte differentiation, comprising administering the 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. have. Another embodiment may provide the theaflavin, 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 differentiation of adipocytes comprising. In addition, non-therapeutic use of the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient for promoting adipocyte differentiation may be provided.

The present invention includes the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient. , can exhibit the following effects. Specifically, theaflavin is water-soluble, easily dissolved in water, and easily mixed with other formulations, and as a result of treatment on human-derived subcutaneous fat cells, it does not cause changes in cell proliferation and toxicity regardless of concentration. Whereas other theaflavins exhibit the anti-obesity inhibitory effect known as an anti-obesity effect, the active ingredient theaflavin of the present invention promotes the differentiation of subcutaneous fat in a concentration-dependent manner. As an embodiment, the composition may be for increasing skin volume by promoting differentiation of adipocytes. As an embodiment, the composition may be for inhibiting the reduction of subcutaneous fat by promoting differentiation of adipocytes.

In one embodiment, the content of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof included in the composition is not limited, but for example, 0.01 to about the total weight of the composition. It may be included in an amount of 100% by weight, specifically, it may be included in an amount of 0.01 to 50% by weight. More specifically, theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, based on the total weight of the composition, 0.01% by weight or more, 0.1% by weight or more, 1% by weight or more, 5 % or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55 It may be included in an amount of at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, or at least 95 wt%. More specifically, theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof is 100% by weight or less, 95% by weight or less, 90% by weight or less, 85% by weight or less, based on the total weight of the composition % or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35 Weight % or less, 30 wt% or less, 25 wt% or less, 20 wt% or less, 15 wt% or less, 10 wt% or less, 5 wt% or less, 1 wt% or less, or 0.1 wt% or less may be included.

In one embodiment, the concentration of the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in the composition may be 0.01 to 10,000 mg/kg, which may vary depending on the administration route of the composition. can In one embodiment, the dosage 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, which may vary depending on the route of administration of the composition can More specifically, the concentration or daily dose of the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof 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, 5,000 mg/kg or more, 6,000 mg/kg or more, 7,000 mg/kg or more, 8,000 mg/kg or more, 9,000 mg/kg or more. More specifically, the concentration of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof is 10,000 mg/kg or less, 9,000 mg/kg or less, 8,000 mg/kg or less, 7,000 mg/kg 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 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, 0.5 mg/kg or less, or 0.1 mg/kg or less.

Specifically, the concentration or daily dose of the aflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof upon oral administration may be 0.01 to 1,000 mg/kg, but is not limited thereto. . When applied to the skin, the concentration or daily dose of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof may be 0.01 to 3,000 mg/kg, but is not limited thereto. When administered directly subcutaneously, the concentration or daily dose of theaflavin, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof may be 0.01 to 100 mg/kg, but is not limited thereto.

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

Tea is a processed bud or leaf of the tea tree ( Camellia sinensis O, Kuntze) belonging to the Camellia family (Theaceae) and Camellia (Camellia). , the solids contain organic substances such as catechins, caffeine, amino acids, fiber, and pectin, as well as various components such as lipids, resins, essential oils, vitamins, and chlorophyll. Depending on the degree of fermentation, tea is generally classified into unfermented tea (green tea) with a degree of fermentation of 0%, semi-fermented tea (white tea, hwacha, pojong tea, and oolong tea) with 20 to 60%, and fermented tea (black tea) with more than 80% fermentation. are doing Traditional fermented tea is called enzyme fermented tea because it uses the fermentation by enzymes contained in tea leaves. Black tea, which is an enzyme fermented tea, softens the leaves through a forgery process after harvesting the tea leaves, induces biochemical reactions necessary for fragrance generation, and a mindful process It promotes the reaction of oxidases and polyphenols through the fermentation process, and promotes the oxidative polymerization of catechins through the fermentation process to produce theaflavins and browning, resulting in flavor and taste. As fermentation proceeds, oxidative polymerization of catechins such as epicatechin and epigallocatechin gallate occurs by oxidative enzymes to produce red-yellow theaflavins, mainly theaflavin. , theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (theaflavin-3) ,3'-digallate) and 4 other species have been reported. These theaflavins are known to have effects such as antioxidant activity and anti-mutation, and oxidized polymers are produced according to the action of oxidase, which increases the redness of tea and causes differences in flavor and taste. There is also a difference in the physiological activity of the product in the body.

As used herein, "fermented green tea" refers to green tea in a fermented state, and includes all differences depending on the degree of fermentation.

The fermented green tea extract according to an exemplary embodiment of the present invention contains The aflavins are included, and in this case, the theaflavin, which is the active ingredient of the present invention, may be included in an amount of 30 to 50% by weight based on the total weight of the theaflavins.

As an embodiment, the fermented green tea extract is prepared by mixing (a) green tea and mugwort gatton to prepare a mixture; (b) fermenting and drying the mixture under reduced pressure; And (c) may be prepared by a method comprising the step of extracting the fermentation and dried mixture.

As an embodiment of the method for producing fermented green tea of the present invention, step (a) is a step of preparing a mixture by mixing green tea and sagebrush.

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

In an embodiment of the present invention, the mugwort gat can be used as a fermentation additive composition that promotes the fermentation of green tea. The mugwort herb contains polyphenol oxidase and peroxidase, which can promote the fermentation of green tea. In one embodiment of the present invention, the mugwort gatton may be used in the form of powdered wormwood garnish or juice. The pulverization is not particularly limited as long as it is known in the art, but the powder first pulverized in a crusher is re- pulverized into fine particles of about 1 to 500 μm in a grinder, or It may be prepared as a fine powder in a ball mill or a hammer mill without pulverization. The juice is not particularly limited as long as it is known in the art.

As an embodiment, the green tea and mugwort 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 10:1 It can be mixed by weight ratio. If the mugwort gatton contains more than the weight of green tea, green tea fermentation can be performed in a short time, but the oxidation rate is accelerated and the aflavins may decrease. It is governed by the included enzyme, so the content of theaflavins among theaflavins does not increase.

In one embodiment, the mixture mixed by mixing green tea and mugwort herb may further include distilled water, ionized water, tap water, mineral-containing groundwater and less than 30% alcohol as a solvent. As an embodiment, the solvent may be included in an amount of 50 to 200 parts by weight based on 100 parts by weight of green tea, and specifically, it may be included in an amount of 80 to 150 parts by weight.

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 occurring by the action of an oxidase naturally present in green tea. The reduced pressure may be a state in which contact with oxygen is blocked, and in an embodiment, fermentation and drying may be performed under a reduced pressure of 10 to 50 mmHg. Specifically, the fermentation under the reduced pressure may be performed at a temperature of 20 to 50° C. for 1 to 10 hours, and more specifically, for 2 to 5 hours. If the fermentation temperature is less than 20 ℃, the fermentation time may be long, and if it exceeds 50 ℃, the deactivation rate of the enzyme may be increased and incomplete fermentation may proceed. In addition, the step (b) dries the fermented mixture, the drying may be freeze-drying. The freeze-drying method is not particularly limited and may be performed by a method known in the art.

In one embodiment, step (c) is a step of extracting the mixture fermented and dried in step (b). The extraction is performed using an extraction solvent, and the extraction solvent includes at least one selected from the group consisting of water and alcohols having 1 to 5 carbon atoms, specifically, at least one selected from the group consisting of water and ethanol. may include Although the extraction temperature and extraction time are not particularly limited, for example, the extraction temperature is room temperature to 80° C., and the extraction time is 1 to 24 hours. In step (c), the fermentation and dry mixture may be extracted through various methods, for example, a method using a hot water extract or a lower alcohol extract having 1 to 5 carbon atoms.

For example, in the first embodiment, by adding water 10 to 30 times the weight of the fermented and dried mixture, primary extraction is performed at 100° C. for 4 hours, and secondary extraction is performed for 4 hours in the same manner, After the first and second extracts are combined and filtered, the filtrate is concentrated with a concentrator. The concentrated extract is dried using a spray dryer or vacuum freeze-drying machine. In the second embodiment, in the method for preparing a 70% ethanol extract, 70% ethanol in an amount of 10 to 30 times the weight of the fermented and dried mixture is added, extracted at 50 to 80° C. for 30 minutes to 3 hours, and then filtered and the filtrate is concentrated with a concentrator. The concentrated extract is dried using a spray dryer or vacuum freeze-drying machine. In the third embodiment, the 95% ethanol extract preparation method is performed by adding 95% ethanol in an amount of 10 to 30 times the weight of the fermented and dried mixture and leaving it at room temperature for 1 to 3 days (shaking every 6 hours) After repeating extraction, repeat the same process twice to obtain an extract.The first and second extracts are combined and filtered, and then the filtrate is concentrated with a concentrator.The concentrated extract is spray dried or vacuum-frozen. Dry using a freeze dryer.

In general, in fermented green tea extract, as fermentation proceeds, oxidative polymerization of catechins such as epicatechin and epigallocatechin gallate occurs by oxidase to produce red-yellow theaflavins. Ravins include theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3, 3'-digallate (theaflavin-3,3'-digallate).

The fermented green tea of the present invention according to the above embodiments contains theaflavin in a high content compared to other theaflavins, which promotes the differentiation of human-derived subcutaneous fat cells and induces high adipocyte production. shows efficacy. Specifically, the fermented green tea extract may contain 30 to 50% by weight of theaflavins based on the total weight of theaflavins.

As an embodiment, the content of the fermented green tea extract included in the composition according to the present invention is not particularly limited, but as an example, it may be included in an amount of 0.01 to 99.9% by weight, specifically 0.1 to 50% by weight, based on the total weight of the composition. . In the case of the above content, it is not only suitable to exhibit the intended effect of the present invention, but also the stability and safety of the composition can be satisfied, and it is 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. These are any formulations suitable for topical application, and may be for transdermal administration. For example, in the form of solutions, gels, solids, kneaded dry products, emulsions obtained by dispersing the oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and nonionic vesicular dispersants, Alternatively, it may be provided in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. As another example, it may be in the form of injections such as fillers, suppositories, patches, etc., but is not limited thereto. These compositions can be prepared according to conventional methods in the art.

As an embodiment, the composition according to the present invention may contain other ingredients capable of giving a synergistic effect to the main effect, preferably within a range that does not impair the main effect, along with the active ingredient, and In addition to the ingredients, a person skilled in the art can appropriately select and mix other ingredients according to the formulation or purpose of use of the cosmetic composition. In addition, as an embodiment, the cosmetic composition of the present invention may include other components that are usually formulated in the cosmetic composition, if necessary, in addition to the above components. For example, moisturizing agents, emollients, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, blood circulation promoters, cooling agents, limiting agents, Purified water etc. are mentioned. Other compounding components that may be included in the cosmetic composition of the present invention are not limited thereto, and the compounding amount of the components is possible within a range that does not impair the purpose and effect of the present invention.

The composition according to embodiments of the present invention may be a food composition including the active ingredient. For example, it may be processed into functional foods such as fermented milk, cheese, yogurt, juice, probiotics and health food containing the active ingredient, and may be used in the form of other various food additives. In one embodiment, the composition may be a composition for health food. As an embodiment, the composition for health food may be formulated in pills, capsules, tablets, granules, caramel, or drinks. As another embodiment, it may be processed in the form of 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 ingredients capable of giving a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, in order to improve physical properties, additives such as fragrances, pigments, bactericides, antioxidants, preservatives, humectants, thickeners, inorganic salts, emulsifiers, and synthetic polymers may be further included. In addition, auxiliary components such as water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular weight polysaccharides and seaweed extract may be further included. The above ingredients can be appropriately selected by those skilled in the art according to the formulation or purpose of use, and the amount added can be selected within a range that does not impair the purpose and effect of the present invention. For example, the amount of the components added may be 0.0001 wt% to 99.9 wt%, based on the total weight of the composition.

The composition according to the embodiments of the present invention may be a pharmaceutical composition including the active ingredient. The pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents or emulsifying agents, salts and/or buffers for regulating osmotic pressure, and other therapeutically useful substances.

In one embodiment, the pharmaceutical composition may be an oral dosage form, and the oral dosage form is, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine granules, There are granules, pellets, and the like. These formulations contain, in addition to the active ingredient, surfactants, diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt It may contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavoring agents, and sweetening agents, such as. The tablet may be prepared by a conventional mixing, granulating or coating method.

As an embodiment, the pharmaceutical composition may be a parenteral administration agent, and the parenteral administration agent may be a rectal, topical, subcutaneous, or transdermal dosage form. For example, injections such as fillers, drops, ointments, skins, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches, etc. may be formulations, but is not limited thereto. .

In one embodiment, the dosage of the pharmaceutical composition will vary depending on the age, sex, and 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 judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art. For example, the dosage may be administered in divided doses of 0.01 to 10,000 mg 1 to 3 times a day, but the dosage is not intended to limit the scope of the present specification in any way.

Hereinafter, the present invention will be described in detail with reference to Examples, Comparative Examples and Test Examples. These are only presented by way of example to describe the present invention in more detail, and it is for those of ordinary skill in the art that the scope of the present invention is not limited by these Examples, Comparative Examples and Test Examples. it will be self-evident

[Example 1] Production of CoF-GT (Co-Fermented Green Tea) Fraction with Enhanced Theaflavin Content

Fresh green tea and mugwort leaves were washed twice with deionized water, and excess water was lightly tapped to remove. The leaves were immersed in liquid nitrogen and then crushed and stored at -80°C. CoF-GT was prepared as follows. CoF-GT is a fermented mixture of raw green tea powder (100g) and frozen fresh mustard green tea (50mg). After 3 hours 　 reaction, the mixture was extracted with 70% ethyl alcohol (v/v) for 2 hours. The solid content and the residue were passed through a 90 mesh sieve and then filtered through a 0.22 μm filter (Dow Corning, Corning, NY, USA). The filtered samples were evaporated with a Hei-VAP rotary evaporator (Heidolph Instruments, Schwabach, Germany) and then pulverized using a FreeZone freeze dryer (Labconco, Kansas City, MO, USA).

[Comparative Example 1] Production of conventionally fermented F-GT (general fermentation of green tea) fraction

The F-GT was removed by washing the fresh green tea leaves twice with deionized water and tapping the excess water lightly. The leaves were immersed in liquid nitrogen and then crushed and stored at -80°C. It was prepared by fermenting frozen green tea powder (100 g) in a heat jacketed z-blade mixer (IKA, Staufen im Breisgau, Germany) at 37.5°C. After 3 hours 　 reaction, the mixture was extracted with 70% ethyl alcohol (v/v) for 2 hours. The solid content and the residue were passed through a 90 mesh sieve and then filtered through a 0.22 μm filter (Dow Corning, Corning, NY, USA). The filtered samples were evaporated with a Hei-VAP rotary evaporator (Heidolph Instruments, Schwabach, Germany) and then pulverized using a FreeZone freeze dryer (Labconco, Kansas City, MO, USA).

[Test Example 1] HPLC analysis of CoF-GT and F-GT fractions

KTApurifier 10 (GE Healthcare, Stockholm, Sweden)에 주입하여 275와 365 nm에서 분석했다. 정제 분리는 AQ-HG octadecylsilyl 컬럼 (120Å, 10um, 20x250mm, column volume = 78.5ml) (YMC Co., Kyoto, Japan)으로 수행하였다. 순수한 물 (용매 A) 및 아세토 니트릴 (용매 B)로 기울기 용출을 수행하였다. 1회에 8.2 ml의 샘플용액을 주입하고 이동상의 유속은 10 ml/min이었다. 모든 용매는 여과하고, 탈기시키고, 적절한 압력하에 사용되였다. 샘플 주입 후 분획 (110 ml)을 수집하였다. 각 사이클마다 4 개의 분획물을 얻고 10번 사이클에 걸쳐 개별 병에 수집하였다. 매 4 번째 분획을 하나로 모아　Hei-VAP 회전식 증발기 (Heidolph Instruments)에서 용매를 제거하고, 동결 건조하여 분말화한 후, 분석 할 때까지 -20℃에서 저장하였다.Each sample (8 g) was dissolved in a mixture of 100 ml DMSO/methanol/ethanol (5:45:50, v/v) after 30 min sonication and centrifuged to 0.45 μm polytetrafluoroethylene (PTFE) Filtered with a syringe filter (Pall Corp., Port Washington, NY, USA). 82 ml of sample was filled in a 50 ml sample loop and a photodiode array detector was installed.

It was injected into KTApurifier 10 (GE Healthcare, Stockholm, Sweden) and analyzed at 275 and 365 nm. Purification separation was performed with an AQ-HG octadecylsilyl column (120 Å, 10 μm, 20×250 mm, column 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 a 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 sample injection. Four fractions were obtained for each cycle and collected into individual bottles over ten cycles. Every fourth fraction was collected and the solvent was removed in a Hei-VAP rotary evaporator (Heidolph Instruments), lyophilized to powder, and stored at -20°C until analysis.

Theaflavin (TF), theaflavin-3 of the fermented green tea extracts prepared in Example 1 and Comparative Example 1 using high performance liquid chromatography (HPLC) to detect the aflavin component of the fermented tea -Gallate (theaflavin-3-gallate, TF-3G), theaflavin-3'-gallate (TF-3'G) and theaflavin-3,3'-digallate (theaflavin-3,3'-digallate, TF-3,3'DG) content analysis was performed. Theaflavin standard was purchased from Wako, and the sample was dissolved in 50% ethanol solvent, extracted with ultrasonic waves, filtered through a 0.45 μm PVDF filter, and injected into the instrument. HPLC (Waters Alliance 2695 system, Waters, USA) was used as the instrument, and the detection wavelength was analyzed in the 270 nm region. The column was analyzed by a gradient solvent composition method using 0.05% trifluoroacetic acid (TFA) and acetonitrile (ACN) solvent using Optimapak C18 (150 x 4.6 mm, 5 μm). The concentration composition ratio is shown in Table 1 below. HPLC grade reagents were used for all analysis solvents, and Empower 3 program from Waters was used for data processing.

As a result, as shown in FIG. 1 , in the case of green tea fermented extract (F-GT) to which the existing green tea fermentation technique (Comparative Example 1) was applied, four major polyphenol components were evenly increased, but the aflavin production to which the co-fermentation technique was applied. In the ferment extract (CoF-GT, Example 1), the content of theaflavin (1) was preferentially produced.

[Test Example 2] Cell culture and differentiation and cell viability analysis

Cell Culture and Differentiation

Human subcutaneous fat cells (hSCF) and undifferentiated subcutaneous fat cell culture medium were purchased from ZenBio (Research Triangle Park, NC, USA). hSCF was cultured in a 5% CO ₂ incubator during the incubation period. To induce differentiation, 10 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, 1 μg troglitazone (all Sigma-Aldrich (USA) ) and 10% fetal serum (PAA, Walkersville, MD, USA) containing Dulbecco's modified Eagle's medium (Lonza, Walsvill, MD, MD, USA) for 21 days. The medium was changed every other day.

Cell viability assay

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

As a result of confirming the concentration showing an influence on hSCFs of each treatment material, all of the treatment materials showed a slight cell proliferation in the pre-concentration period of the 24-hour treatment experiment (FIG. 2) and the 72-hour treatment toxicity confirmation experiment (FIG. 3) It was confirmed that it was a safe substance without any toxic effects except for the effect.

[Test Example 3] Triglyceride (TG) content analysis by ORO (Oil Red O) staining

As for the differentiation result of subcutaneous adipocytes, the adipogenesis was confirmed qualitatively and quantitatively through Oil Red O staining.

hSCF was washed twice with cold phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde (Sigma-Aldrich) for 1 hour. The fixed 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 diluted with 60% propylene glycol). Again, cells were washed three times with 85% propylene glycol and rinsed with running tap water. ORO-stained lipid droplets (LD) 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 cells were treated with a solution of isopropyl alcohol mixed with 4% Nonidet P-40 (Sigma-Aldrich) 20 Only ORO was extracted from cells stained with ORO for minutes. The absorbance of the extract was measured at 490 nm in a spectrophotometer.

It takes about 21 days for hSCFs to differentiate into mature adipocytes that produce lipid droplets. Therefore, theaflavin (TF) and theaflavin-3,3'-digallate (TFDG) were respectively 0.5 and 2 μg/ml, theaflavin concentrated live ferment extract (CoF-GT) and green tea ferment extract (F-GT) ) were incubated for 21 days in media treated with 1, 5, and 10 μg/ml, respectively. As a result, it was confirmed through Oil Red O staining that the theaflavin (TF) and theaflavin concentrated live ferment extract (CoF-GT), which are an embodiment of the present invention, increased fat production in a concentration-dependent manner, in contrast to this When TFDG and F-GT were treated with , it was confirmed that lipolysis, which is the anti-obesity effect of existing green tea, occurred (FIG. 4). In order to quantify the amount of the stained fat of FIGS. 3 and 4, the amount of triglyceride (TG) was measured and expressed as a graph in FIG. 6 .

[Test Example 4] Realtime quantitative polymerization chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (TRIzol, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, and complementary DNA (cDNA) was prepared using the Reverse Transcription Kit (Reverse Transcription Kit, Promega, Madison, WI, USA) was used for synthesis. cDNA was used as a template for RT-qPCR amplification in a 7500 Fast real-time PCR system (Fast7500 Real-time PCR, Life Technologies) using a Taqman probe, and PPARγ (Peroxisome Proliferator Activated Receptor Gamma #Hs01115513_m1), Adiponectin (ADIPOQ; #Hs00605917_m1) and glyceraldehyde 3-phosphate dehydrogenase, GAPDH; #4352339E) were used as control genes. Data were obtained from three independently repeated experiments, and sample values are presented as relative values for GAPDH levels.

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

[Test Example 5] Analysis of adiponectin eluted with the medium

To check the condition after subcutaneous fat differentiation, an increase in the amount of adiponectin, one of adipocyte cytokines, adipokine eluted into the medium, was confirmed by ELISA (Enzyme-linked immunosorbent assay).

Various concentrations of theaflavin (TF), theaflavin-3,3'-digallate (TFDG), theaflavin concentrated live ferment extract (CoF-GT) and green tea ferment extract (F-GT) were added to hSCF, respectively. treated and differentiated for 21 days. The culture medium was collected and centrifuged at 13,000 rpm for 15 minutes to remove foreign substances. ELISA (Enzo Life Sciences, Farmingdale, NY, USA) was used according to the manufacturer's instructions to measure the level of eluted adiponectin. As a result, as shown in FIG. 8 , adiponectin, one of adipokines eluted into the medium after adipogenesis, is the aflavin-3,3'-digalate (TFDG) and green tea ferment extract (F- GT) was decreased when treated, whereas the adiponectin amount increased as the concentration increased during treatment with theaflavin (TF) and theaflavin concentrated live fermented extract (CoF-GT), which are an embodiment of the present invention. This is also consistent 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)

According to the same method as in Test Example 2, a concentration of 0.01 to 100 μg/ml of theaflavin (TF) having an effect on hSCFs was confirmed, and this is shown in FIG. 9 . As a result, it was confirmed that both the proliferative effect test of the 24-hour treatment and the toxicity confirmation test of the 72-hour treatment were safe substances without any toxic effects except for a slight cell proliferation effect at all the treated concentrations.

[Test Example 7] Efficacy analysis of increasing triglyceride (TG) by concentration of theaflavin (TF)

In the same manner as in Test Example 3, the differentiation efficacy of subcutaneous adipose cells was analyzed for each concentration of 0 to 100 μg/ml of theaflavin (TF).

As a result, as shown in FIG. 10 , theaflavin (TF) promoted adipogenesis in a concentration-dependent manner up to 50 µg/ml, and at 100 µg/ml, the ability to differentiate into adipocytes was slightly decreased than that of 50 µg/ml.

Formulation examples of the composition according to an embodiment of the present specification will be described below, but other various formulations are also applicable, which is not intended to limit the present specification, but merely to describe it in detail.

[Formulation Example 1] Softening lotion (skin lotion)

Softening lotion was prepared in a conventional manner according to the composition described in the table below.

compounding ingredients content (wt%) theaflavin 10 glycerin 3.0 butylene glycol 2.0 propylene glycol 2.0 Carboxyvinyl Polymer 0.1 PEG-12 Nonylphenyl Ether 0.2 Polysorbate 80 0.4 ethanol 10.0 triethanolamine 0.1 Preservatives, Colors, Flavors appropriate amount Purified water remaining amount

[Formulation Example 2] Nutrient lotion (milk lotion)

Nutrient lotion was prepared in a conventional manner according to the composition described in the table below.

compounding ingredients content (wt%) theaflavin 10 glycerin 3.0 butylene glycol 3.0 propylene glycol 3.0 Carboxyvinyl Polymer 0.1 beeswax 4.0 Polysorbate 60 1.5 Caprylic/Capric Triglycerides 5.0 squalane 5.0 sorbitasesquioleate 1.5 liquid paraffin 0.5 cetearyl alcohol 1.0 triethanolamine 0.2 Preservatives, Colors, Flavors appropriate amount Purified water remaining amount

[Formulation Example 3] Massage Cream

A massage cream was prepared in a conventional manner according to the composition described in the table below.

compounding ingredients content (wt%) theaflavin 10 glycerin 8.0 butylene glycol 4.0 liquid paraffin 45.0 beta glucan 7.0 Carbomer 0.1 Caprylic/Capric Triglycerides 3.0 beeswax 4.0 cetearyl glucoside 1.5 Sesquioleic Acid Sorbitan 0.9 Vaseline 3.0 paraffin 1.5 Preservatives, Colors, Flavors appropriate amount Purified water remaining amount

[Formulation Example 4] Tablet

100 mg of theaflavin, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed, and then tablets were prepared by tableting according to a conventional manufacturing method.

[Formulation Example 5] Capsules

100 mg of theaflavin, 400 mg of lactose, 400 mg of corn starch, and 2 mg of magnesium stearate were mixed, and then filled into gelatin capsules according to a conventional capsule preparation method to prepare capsules.

[Formulation Example 6] Granules

50 mg of theaflavin, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed, formed into granules using a fluidized bed granulator, and then filled in a bag.

[Formulation Example 7] Drink formulation

After mixing 50 mg of theaflavin, 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 prepare a drink.

[Formulation Example 8] Caramel formulation

Theaflavin 50mg, corn syrup 1.8g, skim milk 0.5g, soybean lecithin 0.5g, butter 0.6g, hydrogenated vegetable oil 0.4g, sugar 1.4g, margarine 0.58g, and salt 20mg were mixed and caramelized. .

[Formulation Example 9] Health food

ingredient content theaflavin 100 mg vitamin mixture vitamin A acetate 70 μg vitamin E 1.0 mg vitamin B1 0.13 mg vitamin B2 0.15 mg vitamin B6 0.5 mg vitamin B12 0.2 μg vitamin C 10 mg biotin 10 μg Nicotinamide 1.7 mg folic acid 50 μg Calcium Pantothenate 0.5 mg mineral mixture ferrous sulfate 1.75 mg zinc oxide 0.82 mg magnesium carbonate 25.3 mg monobasic potassium phosphate 15 mg dicalcium phosphate 55 mg potassium citrate 90 mg calcium carbonate 100 mg magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is a composition that is relatively suitable for health food, but the mixing ratio may be arbitrarily modified. And it can be used in the manufacture of a health food composition according to a conventional method.

[Formulation Example 10] Health drink

ingredient content theaflavin 10 mg citric acid 1000 mg oligosaccharide 100 g Plum Concentrate 2 g Taurine 1 g Purified water remaining amount total volume 900 ml

As shown in the table above, the remaining amount of purified water is added to make a total volume of 900 ml, and the above ingredients are mixed according to a conventional health drink manufacturing method, and then stirred and heated at 85° C. for about 1 hour, and then the resulting solution is filtered and sterilized. It can be obtained in a 2L container, sealed and sterilized, and then stored in a refrigerator for use in the manufacture of health beverage compositions.

[Formulation Example 11] Injection

Injections were prepared in a conventional manner according to the composition described in the table below.

compounding ingredients content theaflavin 10-50 mg Sterile distilled water for injection appropriate amount pH adjuster appropriate amount

The above embodiments have been disclosed for the purpose of explanation of the present invention, and the above description is not intended to limit the scope of the present invention. Accordingly, various modifications, variations, and substitutions may occur to those skilled in the art without departing from the spirit and scope of the present invention.

Claims (15)

A cosmetic composition for inhibiting subcutaneous fat reduction by promoting adipocyte differentiation, comprising theaflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient. The composition of claim 1, wherein the composition comprises a fermented green tea extract containing theaflavin. 3. The tea according to claim 2, wherein the fermented green tea extract contains theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digalate. A composition comprising rabines, wherein the theaflavin is included in an amount of 30 to 50% by weight based on the total weight of the theaflavins. The method of claim 2, wherein the fermented green tea extract
(a) preparing a mixture by mixing green tea and mugwort radish;
(b) fermenting and drying the mixture under reduced pressure; and
(c) the composition, which is prepared by a method comprising the step of extracting the fermentation and dried mixture. The composition according to claim 1, wherein the concentration of the aflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in the composition is 0.01 to 10,000 mg/kg. The composition of claim 1, wherein the dosage of the aflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof is 0.01 to 10,000 mg/kg/day. The composition of claim 1, wherein the composition is for increasing skin volume by promoting adipocyte differentiation. The composition according to any one of claims 1 to 7, wherein the composition is for transdermal administration. A food composition for inhibiting subcutaneous fat reduction by promoting adipocyte differentiation, comprising theaflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient. The composition of claim 9, wherein the composition comprises a fermented green tea extract containing theaflavin. 11. The method of claim 10, wherein the fermented green tea extract contains theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digalate. A composition comprising rabines, wherein the theaflavin is included in an amount of 30 to 50% by weight based on the total weight of the theaflavins. 11. The method of claim 10, wherein the fermented green tea extract is
(a) preparing a mixture by mixing green tea and mugwort radish;
(b) fermenting and drying the mixture under reduced pressure; and
(c) the composition, which is prepared by a method comprising the step of extracting the fermentation and dried mixture. The composition according to claim 9, wherein the concentration of theaflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof in the composition is 0.01 to 10,000 mg/kg. The composition according to claim 9, wherein the dosage of theaflavin, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof is 0.01 to 10,000 mg/kg/day. The composition of claim 9, wherein the composition is for increasing skin volume by promoting adipocyte differentiation.

Images