KR102273583B1 - Antiobesity composition comprising epigallocatechin gallate nano-emulsion as effective gradient and method for preparing the same - Google Patents

Abstract

The present invention is epigallocatechin gallate (EGCG) in which an oil-layer emulsifier and a water-layer emulsifier are added, and nanoparticulate; and an anti-browning agent; the oil layer emulsifier is any one selected from lecithin, polycaprolactone, polypropylene glycol and olive oil, and the water layer emulsifier is pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside , and any one selected from carrageenan, and the anti-browning agent relates to an anti-obesity composition comprising as an active ingredient nanoized epigallocatechin gallate, which is any one selected from vitamin C and gallic acid. Thereby, when applied as a drinking material of epigallocatechin gallate (EGCG), it overcomes insolubility, which is a disadvantage, and increases solubility to nano particles so that it can be applied to transparent beverages or foods, as well as reduced bitterness and anti-obesity activity Therefore, it can be variously applied to diet food, beverage, or medicine.

Description

Antiobesity composition comprising epigallocatechin gallate nano-emulsion as an effective gradient and method for preparing the same

The present invention relates to an anti-obesity composition comprising epigallocatechin gallate nanoemulsion as an active ingredient and a method for manufacturing the same, and more particularly, epigallocatechin gallate nanoemulsion and bitterness reduction applied to various foods, etc. It relates to a possible anti-obesity composition and a method for preparing the same.

In recent years, in industries such as food, pharmaceuticals, and daily products, the development of natural ingredients with excellent biocompatibility and biosafety as well as excellent bioactivity for the human body for the discovery of bioactive materials has become more important. Catechins, which give green tea astringent taste, are components of what are called tannins or polyphenols, which are oxidized to give them a reddish-brown color. Catechin accounts for the largest portion of tea, and has many hydroxyl groups -OH in its chemical structure, so it can be easily combined with various substances. These properties act as antioxidants to remove heavy metals or free radicals from the body. It is known that an appropriate amount of active oxygen has a positive effect on the sterilization of phagocytes related to the immune function in vivo, but an excessive amount of active oxygen disrupts the balance between oxidation and antioxidant, resulting in protein denaturation, in vivo lipid oxidation, DNA Green tea catechins are known to have excellent ability to remove these free radicals.

In particular, catechins present in green tea include EC (Epicatechin), EGC (Epigallocatechin), ECG (Epicatechin gallate), EGCG (Epigallocatechin gallate), and optical isomers thereof. In general, these four are called green tea catechins, and among them, EGCG accounts for the largest amount and is known to have the highest physiological activity such as showing the strongest antioxidant ability. It is widely known that high concentration of EGCG in 3T3-L1 adipocyte line is in lipolysis, which affects the expression of Resistin mRNA and regulates fat metabolism, that is, increases body fat by promoting triglyceride decomposition and fatty acidization. Result However, EGCG has been known to have many restrictions on its use as an active ingredient in foods and beverages because it has a property of poor solubility in which only a very small amount is dissolved in water.

Several methods have been devised to increase the stability of green tea-derived EGCG. For example, a method using a derivative of EGCG or a method using a lipophilic microcapsule has been proposed, but these methods have a limitation in that they can ensure the stability only in very limited cases. In addition, a method for stabilizing EGCG in aqueous phase using a cationic polymer or anionic polymer, an oxidation stabilizer, etc. has been proposed, but when other polar substances are added to the stabilization formulation, it can be mixed with a cationic polymer or an anionic polymer, an antioxidant, etc. If there is a very strong interaction, there is a disadvantage in that the degree of stabilization is reduced. In addition, a method for stabilizing EGCG in an aqueous phase by confining it in a water-insoluble capsule using an anode or anode polymer, an oxidation stabilizer, a water-insoluble polymer, etc. has been proposed. There is a problem with the use of transparent beverages, etc. that require transparent dissolution conditions in the fields of cosmetics, pharmaceuticals, food and household products, such as having a suspended or suspended appearance.

On the other hand, nano-emulsion has a particle size of about 100 nm and is thermodynamically known as an equilibrium system. Ordinary emulsions have large particle diameters and appear cloudy because light is scattered, whereas nanoemulsions have small particle diameters and appear transparent because light is not scattered. The homogeneity of the particles is good and the storage stability is very high. In addition, since EGCG has a unique bitter taste of tannins, which is inconvenient when consumed, there is a need for an EGCG processing method that can be applied to various diet foods or medicines by reducing bitterness and improving storage stability.

Korean Patent Publication No. 2006-28916

It is an object of the present invention to solve the problem of insolubility and bitter taste, which are disadvantages when applying epigallocatechin gallate (EGCG) as a drinking material. Epigallocatechin gallate nanoemulsion with improved anti-obesity effect is effective by making gallocatechin gallate into nanoparticles to increase solubility and improve storage stability while reducing bitterness to make it easier to apply to foods or drugs such as transparent beverages. To provide an anti-obesity composition comprising as a component.

According to one aspect of the present invention,

oil layer emulsifier, water layer emulsifier and anti-browning agent, and nanoparticulate epigallocatechin gallate (EGCG);

The oil layer emulsifier is any one selected from lecithin, polycaprolactone, polypropylene glycol and olive oil,

The aqueous layer emulsifier is any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside and carrageenan,

The anti-browning agent is provided with an anti-obesity composition comprising as an active ingredient any one selected from vitamin C and gallic acid, epigallocatechin gallate nanoemulsion.

The nanoparticulated epigallocatechin gallate may be nanoparticulated by spraying by ultrasonic treatment.

The nanoparticulated epigallocatechin gallate was ultrasonically sprayed three times by sequentially adding the oil-layer emulsifier, water-layer emulsifier and anti-browning agent to a mixture of solvent and epigallocatechin powder. can

The nanoparticulated epigallocatechin gallate may have an average particle diameter of 100 to 200 nm.

The nanoparticulated epigallocatechin gallate may have a uniformity (PDI) of 0.1 to 0.2.

The anti-obesity composition may have an adipogenesis inhibitory activity and a lipolytic activity.

The oil layer emulsifier may be lecithin.

The aqueous emulsifier may be pectin or cyclodextrin.

Lecithin as the oil emulsifier and pectin or cyclodextrin as the water emulsifier may be used simultaneously.

The anti-browning agent may be gallic acid.

The anti-obesity composition may include 5 to 20 parts by weight of an oil emulsifier, 0.05 to 2 parts by weight of an aqueous emulsifier, and 0.05 to 2 parts by weight of an anti-browning agent based on 100 parts by weight of epigallocatechin gallate.

According to another aspect of the present invention,

There is provided a food or beverage composition for improving or preventing obesity comprising the anti-obesity composition.

The food or beverage composition may be included in the food or beverage having a transparent or translucent property.

According to another aspect of the present invention,

There is provided a pharmaceutical composition for treating or preventing obesity comprising the anti-obesity composition.

According to another aspect of the present invention,

(a) preparing a primary emulsion containing primary epigallocatechin gallate nanoparticles by spraying water to which epigallocatechin gallate and an oil emulsifier are added by ultrasonic treatment;

(b) preparing a secondary emulsion containing secondary epigallocatechin gallate nanoparticles by adding a water layer emulsifier to the primary emulsion and spraying by ultrasonication; and

(c) preparing a tertiary emulsion containing tertiary epigallocatechin gallate nanoparticles by adding an anti-browning agent to the secondary emulsion and spraying by sonication;

The oil layer emulsifier is any one selected from lecithin, polycaprolactone, polypropylene glycol, and olive oil,

The aqueous emulsifier is any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside, and carrageenan,

The anti-browning agent provides a method for preparing an anti-obesity composition comprising as an active ingredient any one selected from vitamin C and gallic acid, epigallocatechin gallate nanoemulsion.

The epigallocatechin gallate may be used in a solution of 10 to 30% (w/v).

The oil layer emulsifier may be mixed in an amount of 1 to 30 parts by weight based on 100 parts by weight of the epigallocatechin gallate.

The oil layer emulsifier may be lecithin.

The aqueous emulsifier may be pectin or cyclodextrin.

Lecithin as the oil emulsifier and pectin or cyclodextrin as the water emulsifier may be used simultaneously.

According to another aspect of the present invention,

There is provided a method for preparing a food or beverage composition for improving or preventing obesity, including the method for preparing the anti-obesity composition.

The food or beverage composition may be included in the food or beverage having a transparent or translucent property.

According to another aspect of the present invention,

There is provided a method for preparing a pharmaceutical composition for treating or preventing obesity, including the method for preparing the anti-obesity composition.

The anti-obesity composition comprising the epigallocatechin gallate nanoemulsion of the present invention as an active ingredient contains epigallocatechin gallate nanoparticles by treatment with an oil layer emulsifier, a water layer emulsifier and an antibrowning agent, and emulsion preparation by ultrasonic spraying. By making it easier to apply to foods or medicines such as transparent beverages by reducing bitterness while increasing solubility and improving storage stability, it is also possible to improve the anti-obesity effect.

1 is a photograph showing the state of the emulsion according to 10 types of oil emulsifiers of Experimental Example 1 and their concentrations.
2 is a photograph showing the state of the emulsion according to the aqueous layer emulsifier of Experimental Example 1 and its concentration;
3 is an EGCG nano-optimization test result according to the type of oil-layer emulsifier and water-layer emulsifier of Experimental Example 2.
4 is a photograph showing the results of EGCG nano-optimization experiments according to the types of oil layer emulsifier and water layer emulsifier of Experimental Example 2.
5 is an EGCG nano-optimization experiment result according to the type of the aqueous emulsifier of Experimental Example 2.
6 is an EGCG nano-optimization experiment result according to the lecithin mixing ratio of Experimental Example 2.
7 is an EGCG nano-optimization experiment result according to the EGCG concentration of Experimental Example 2.
8 is a FE-TEM photograph of the EGCG emulsion of Experimental Example 3.
9 is an EGCG average particle size, dispersion state (A), and zeta potential (B) measurement results according to Experimental Example 3.
10 is an FTIR analysis result of the EGCG emulsion according to Experimental Example 4.
11 is a photograph of staining of 3T3-L1 differentiated cells of Experimental Example 6.
12 is a measurement result of the adipogenesis inhibitory ability of Experimental Example 6.
13 is a result of measuring lipolysis in Experimental Example 6.
14 schematically shows a method for analyzing the bitter taste reduction effect of Experimental Example 7.
15 is a measurement result of bitter taste according to the electrophysiological technique of Experimental Example 7.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, element, or combination thereof described in the specification is present, and includes one or more other features or numbers, It should be understood that it does not preclude the possibility of the presence or addition of steps, components, or combinations thereof.

Hereinafter, an anti-obesity composition comprising epigallocatechin gallate of the present invention as an active ingredient will be described.

Epigallocatechin gallate used as the material of the present invention is catechin extracted from green tea, purified water, glycerin, butylene glycol, hexanediol, methylpropanediol, propanediol, propylene glycol, ethanol, methanol, ethyl It may be extracted with an extraction solvent such as acetate, dichloromethane, ethyl ether, hexane, and more preferably, it may be extracted with purified water, methylpropanediol, or hexanediol, but the scope of the present invention is not limited thereto. , various extraction solvents that can be used for extraction of plant material can be used.

The anti-obesity composition comprising epigallocatechin gallate as an active ingredient of the present invention includes an oil-layer emulsifier, a water-layer emulsifier and an anti-browning agent, and comprises nanoparticulated epigallocatechin gallate (EGCG). do.

An emulsifying agent refers to a third substance that converts two immiscible liquids into a stable emulsion (emulsion). In the present invention, the oil layer emulsifier is defined as an emulsifier having a Hydrophile Lipophile Balance (HLB) value in the range of 3 to 7. A water layer emulsifier is also defined as an emulsifier having an HLB value in the range of 8 to 18.

The oil layer emulsifier may be any one selected from lecithin, polycaprolactone, polypropylene glycol, and olive oil, and preferably lecithin.

The aqueous emulsifier may be any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside, and carrageenan, preferably pectin or cyclodextrin, more preferably pectin.

The anti-browning agent may be any one selected from vitamin C and gallic acid, and more preferably gallic acid.

It is preferable to use lecithin as the oil emulsifier and pectin or cyclodextrin as the water emulsifier, more preferably lecithin as the oil emulsifier, and pectin as the water emulsifier. Even more preferably, lecithin as the oil emulsifier, pectin as the water emulsifier, and gallic acid as the anti-browning agent can be used simultaneously.

The nanoparticulated epigallocatechin gallate may be nanoparticulated by spraying by ultrasonic treatment.

Specifically, the nanoparticulated epigallocatechin gallate is ultrasonically sprayed three times by sequentially adding the oil-layer emulsifier, water-layer emulsifier and anti-browning agent to a mixture in which a solvent and epigallocatechin powder are mixed. It is preferably treated.

The nanoparticulated epigallocatechin gallate may have an average particle diameter of preferably 100 to 200 nm, more preferably 100 to 180 nm, and still more preferably 100 to 150 nm. If the nano is smaller than 100 nm, it may harm the human body when drinking, and if it is larger than 200 nm, the particle stability may be lowered and the transparent property may be deteriorated, or the bitter taste reducing effect and anti-obesity effect may be reduced.

The nanoparticulated epigallocatechin gallate may have a uniformity (PDI) of 0.1 to 0.2, more preferably 0.15 to 0.17, more preferably 0.155 to 0.165.

The anti-obesity composition may have an adipogenesis inhibitory activity and a lipolytic activity.

The anti-obesity composition of the present invention may be used in an amount of 5 to 20 parts by weight of an oil emulsifier, 0.05 to 2 parts by weight of an aqueous emulsifier, and 0.05 to 2 parts by weight of an anti-browning agent based on 100 parts by weight of epigallocatechin gallate, more preferably It is possible to use 7 to 15 parts by weight of an oil emulsifier, 0.1 to 1.5 parts by weight of an aqueous emulsifier, and 0.1 to 1.5 parts by weight of an anti-browning agent, and even more preferably 8 to 12 parts by weight of an oil emulsifier, 0.2 to 1.2 parts by weight of an aqueous emulsifier, 0.2 to 1.2 parts by weight of a browning agent may be used. Most preferably, based on 100 parts by weight of epigallocatechin gallate, 9 to 11 parts by weight of lecithin as an oil layer emulsifier, 0.3 to 1 parts by weight of pectin as an aqueous layer emulsifier, and 0.3 to 1 parts by weight of gallic acid as an anti-browning agent may be used. At such a content ratio, the anti-obesity properties are high, the nanoparticles of the emulsion particles are easy to be nanosized, and the particles can be stably maintained.

Hereinafter, a method for preparing an anti-obesity composition comprising the epigallocatechin gallate nanoemulsion of the present invention as an active ingredient will be described.

first, Epigallocatechin gallate and oil layer Water with emulsifier added is sprayed by ultrasonic treatment to epigallocatechin gallate Primary containing nanoparticles emulsion prepared (step a).

The epigallocatechin gallate may be used in the form of a solution of 10 to 30% (w/v) in which the powder is mixed with a solvent. The solution may be an alcohol solution or an aqueous solution.

The oil layer emulsifier may be any one selected from lecithin, polycaprolactone, polypropylene glycol, and olive oil, and preferably lecithin.

Next, the first in emulsion water layer Secondary by adding emulsifier and spraying by sonication epigallocatechin gallate secondary containing nanoparticles emulsion prepared (step b).

The aqueous emulsifier may be any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside, and carrageenan, preferably pectin or cyclodextrin, more preferably pectin.

It is preferable to use lecithin as the oil emulsifier and pectin or cyclodextrin as the water emulsifier, more preferably lecithin as the oil emulsifier, and pectin as the water emulsifier.

After that, the second in emulsion anti-browning drug add (step c).

The anti-browning agent may be at least one selected from vitamin C and gallic acid, preferably gallic acid.

Even more preferably, lecithin as an oil emulsifier, pectin as an aqueous emulsifier, and gallic acid as an anti-browning agent can be used simultaneously.

The oil emulsifier is preferably mixed in an amount of 1 to 30 parts by weight based on 100 parts by weight of the epigallocatechin gallate, more preferably 5 to 20 parts by weight, even more preferably 8 to 15 parts by weight, most preferably Preferably, it can be mixed in an amount of about 10 parts by weight.

The present invention provides a food or beverage composition for improving or preventing obesity, comprising an anti-obesity composition comprising the epigallocatechin gallate nanoemulsion as an active ingredient.

The food or beverage composition may be included in food or beverage having a transparent or translucent property.

The food or beverage composition according to the present invention may be used as a functional food or beverage, or may be added to various foods or beverages. Foods or beverages to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, bread noodles, other noodles, gums, ice creams, vitamin complexes, There are health supplements, etc.

The food composition of the present invention may include not only nano-sized epigallocatechin gallate as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and Contains flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink or beverage, citric acid, fructose liquid, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, etc. may be additionally included in addition to the nanoized epigallocatechin gallate. can

The present invention provides a health functional food or health functional beverage comprising a food or beverage composition for improving or preventing obesity comprising the anti-obesity composition.

Health functional food or beverage is a food prepared by adding nanoized epigallocatechin gallate to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, or suspension, etc. It means that it has a specific effect on health, but unlike general drugs, it has the advantage of not having side effects that may occur when taking a drug for a long period of time using food as a raw material. The health functional food or beverage of the present invention obtained in this way is very useful because it can be ingested on a daily basis. The amount of nano-enhanced epigallocatechin gallate added in such a health functional food or beverage may vary depending on the type of health functional food or beverage and cannot be uniformly defined, but within a range that does not impair the original taste of the food. It may be added, and it is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, based on the target food. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in an amount of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of obesity comprising the anti-obesity composition.

The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and lubricants. Or a flavoring agent or the like may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be solutions, syrups, juices, suspensions, emulsions, drops, or the like. For example, if necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The pharmaceutical composition of the present invention is preferably administered orally.

[ Example ]

Materials and reagents

EGCG (Epigallocatechin gallate) used in this test was extracted from green nemulcha with 92% purity, and then lyophilized and powdered for use. Standards such as lecithin, tocopherol, oleic acid, β-cyclodextrin, alginate, pectin, stevioside, vitamin C, and gallic acid are supplied by Sigama Chemical Co., Ltd. (St Louis, MO, USA) solvents such as methanol and ethyl acetate were prepared by Mallincrodt Baker Inc. for HPLC. (Phillipsburg, NJ, USA) product was used. Polypheonol oxidase (PPO) and catechin substrates for anti-browning effect were purchased from Sigma-Aldrich (St. Luis, MO, USA). As reagents for measuring antioxidant activity, ABTS and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were manufactured by Sigma Chemical Co., Ltd. product was used. All analytical reagents were used with a purity of 90% or more. In addition, insulin (insulin, dexamethasone) and IBMX used for differentiation of 3T3-L1 preadipocytes and Oil Red O used to confirm triglyceride production in adipocytes were obtained from Sigma-Aldrich (St. Luis, MO, USA).

Example One: EGCG emulsion Produce

In order to solubilize the epigallocatechin gallate (EGCG) alcohol mixture (10%~30%, w/v), 1% (w/v) of an oil emulsifier based on EGCG (10%, w/v) was first added. In other words, 10% of the EGCG weight of the oil layer emulsifier was mixed, followed by ultrasonication in 10 times distilled water for 5 minutes and sprayed slowly. Second, an aqueous layer emulsifier (0.1%, w/v) as an EGCG solubilization accelerator in the aqueous layer was slowly sprayed with sonication for 5 minutes in the first solution. Third, an anti-browning agent (0.1%, w/v) was slowly sprayed into the secondary solution by ultrasonication for 5 minutes.

Lecithin was used as an oil emulsifier.

As the aqueous emulsifier, β-cyclodextrin, alginate, pectin, and stevioside were used.

As an anti-browning agent, vitamin C and gallic acid were used.

Ultrasonic treatment conditions were emulsified at 30% power (700W 100%), 3 sec on /1 sec off 5 min (50 cycles), and the equipment used was John Morris Ultrasonic processor 750 watts, 20khz with CV334 converter & 630-0220 ( 1/2'') 13mm probe was used.

[ Experimental example ]

Experimental example One: oil layer emulsifiers and water layer Choosing an emulsifier

A mixed solution of EGCG 5% (w/v) ethanol was used, each emulsifier was dissolved in distilled water at a content of 0.1 to 1% (w/v), and the mixed solution was ultrasonically dispersed to observe turbidity and properties.

1 is a photograph showing the state of the emulsion according to 10 types of oil emulsifiers and their concentrations, and Table 1 below summarizes the turbidity accordingly.

turn Types of oil emulsifiers EGCG 5% + Emulsifier Concentration (%) 0.1% 0.5% One% 5% 10% One Polycaprolactone 0.12 1.15 1.28 2.07 2.00 2 Glyceryl monostearate 0.09 0.41 0.55 1.89 2.41 3 Hydroxy Propyl Methlycellulose 0.12 0.14 0.23 0.61 0.15 4 black pepper oil 0.11 0.21 0.69 2.07 1.90 5 Polypropylene glycol 0.12 0.20 0.33 2.08 2.04 6 olive oil 0.18 025 0.54 1.90 2.10 7 Tween 80 0.21 1.19 1.80 2.62 2.18 8 Oleic acid 0.12 0.43 1.16 2.12 2.70 9 Chitosan 0.17 0.20 0.20 0.18 0.42 10 Lecithin 0.12 0.20 0.33 0.51 1.90

Accordingly, it was determined that four types of oil emulsifiers including polycaprolactone, polypropylene glycol, olive oil, and lecithin could be applied.

On the other hand, Figure 2 is a photograph showing the state of the emulsion according to the eight kinds of water-layer emulsifiers and their concentrations, and Table 2 below summarizes the turbidity accordingly. Here, sodium alginate, pectin and carrageenan were added only 0.1 times the indicated concentrations due to solubility limitations.

turn Types of water layer emulsifiers EGCG 5% + Emulsifier Concentration (%) 0.1% 0.5% One% 5% 10% One Hydroxy Propyl Methylcellulose 0.12 0.14 0.23 0.61 0.15 2 Whey Protein Concentrate Isolates (WCI) 0.23 0.47 0.95 2.32 2.84 3 Sodium caseinate 0.11 0.32 0.45 2.22 2.55 4 Cyclodextrin 0.11 0.12 0.10 0.12 0.13 5 Sodium alginate 0.09 0.11 0.12 0.11 0.13 6 Pectin 0.10 0.09 0.10 0.13 0.14 7 Steaviosides 0.10 0.09 0.18 0.13 0.13 8 Carrageenan 0.11 0.12 0.23 0.33 0.13

Accordingly, it was determined that it was appropriate to use six types of aqueous emulsifiers including hydroxypropyl methylcellulose, cyclodextrin, sodium alginate, pectin, stevioside, and carrageenan.

Experimental example 2: EGCG nanoization Optimization and Nanoparticle Sizing

(One) oil layer emulsifiers and water layer According to the type of emulsifier EGCG nanoization optimization

First, the results of adding 4 types of oil emulsifiers at a concentration of 1% (w/v) by concentration of EGCG (5 to 90% (w/v)) are shown in FIG. 3 . According to this, it was found that lecithin solubilizes EGCG up to 50% (w/v) EGCG concentration among the four types of oil emulsifiers.

Based on these results, an emulsion was prepared by dispersing water under the conditions of 50% (w/v) of EGCG + 1% (w/v) of each type of emulsifier + 0.1% (w/v) of each type of emulsifier, and a photograph of the emulsion was taken. 4 is shown. In addition, the results of measuring the average particle diameter (nm) of EGCG nanoparticles nanosized in the emulsion prepared in this way are shown in Table 3, and the results of measuring the particle distribution index (PDI) of the EGCG nanoparticles are shown in Table 4.

According to this, it was confirmed that the combination of pectin, an emulsifier in the water layer, and lecithin, an emulsifier in the oil layer, formed the most efficiently small-sized EGCG nanoparticles, showing a low particle distribution and uniform particle distribution. In addition, the combination of cyclodextrin and lecithin was also shown to efficiently produce nano-EGCG.

EGCG (50%) Insect Emulsifier (0.1%) Control Methyl
cellulose Whey Protein Isolate Cyclo
dextrin Alginate Pectin Carra
geenan oil emulsifier
(One %) 732.4 757.6 826.6 543.3 471.6 493.8 492.1 492.1 711.8 623.9 600.3 522.2 492.1 338.5 471.6 471.6 643.9 522.2 543.6 471.6 493.8 367.8 492.1 492.1 522.2 341.5 338.5 345.9 342.0 250.3 493.8 493.8 342.1 363.5 471.6 247.4 317.8 198.2 471.6 471.6

EGCG (50%) water emulsifier (0.1%) Control Methyl
cellulose Whey Protein Isolate Cyclo
dextrin Alginate Pectin Carra
geenan oil emulsifier
(One %) 0.48 0.52 0.43 0.43 0.33 0.31 0.52 0.52 0.43 0.35 0.35 0.36 0.35 0.29 0.46 0.46 0.35 0.42 0.37 0.25 0.25 0.23 0.46 0.46 0.31 0.26 0.55 0.21 0.20 0.17 0.37 0.37 0.27 0.27 0.42 0.17 0.21 0.15 0.23 0.23

On the other hand, an emulsion was prepared by dispersing water under the conditions of EGCG 30% (w/v) + 1% (w/v) of lecithin as an oil emulsifier + 0.1% (w/v) of 4 types of water-layer emulsifier. 5, the average particle diameter and uniformity (PDI) are summarized and shown in Table 5.

EGCG content fat soluble emulsifier water-soluble emulsifier
(0.1%) size
(d.nm) uniformity
(PDI) 30% additive-free water 711.8 0.385 b-cyclodextrin 496.1 0.372 Alginate 427.0 0.327 Pectin 282.9 0.296 Stevioside 349.6 0.292 Lecithin 1% water 302.3 0.26 b-cyclodextran 187.4 0.18 Alginate 242.5 0.23 Pectin 132.2 0.15 Stevioside 162.3 0.16

According to this, after nanoization by adding 30% (w/v) of EGCG and 1% (w/v) of lecithin, the second water-soluble emulsifiers cyclodextrin, alginate, pectin and stevioside were treated with 0.1% (w/v) each. The degree of promoting EGCG solubilization was investigated. In particular, pectin has the ability to solubilize EGCG by itself, but in this case, the uniformity of the nano-sized particles is lowered and the stability of the generated particles is lowered, so that they are released immediately after 3 days. However, after the first emulsification with lecithin and the second addition of a water-soluble solubilizer, cyclodextrin, stevioside, and pectin were all stable, uniform, and sized at 187.4, 162.3, and 132.2 nm, respectively, to form 100 nm level nanoparticles. Among the oil emulsifiers, pectin had the best particle size and uniformity, and in particular, pectin and lecithin were used together, thereby showing a synergistic effect in dissolving and nanoizing EGCG.

(2) According to the lecithin mixing ratio EGCG nanoization optimization

Based on EGCG (10%, w/v), 0.1%, 0.5%, 1%, 2%, 3%, 4% (w/v) of lecithin was added for each concentration by DLS irradiation for size and uniformity measurement. The results are shown in FIG. 6 and Table 6. According to this, the uniformity is high when 0.5% to 1% (w/v) is added, and in particular, when 1% is added, that is, when 10% lecithin is added based on EGCG, solubilization is the highest and nanoization is good.

EGCG
(%, w/v) lecithin
(%, w/v) size
(d.nm) uniformity
(PDI) Zeta potential
(mV) turbidity
(OD600) 10 0.1 302.1 0.279 -46.0 0.04 0.5 152.1 0.186 -52.6 0.05 One 135.8 0.178 -64.1 0.09 2 167.7 0.198 -55.3 0.14 3 187.1 0.219 -49.9 0.15 4 210.7 0.346 -47.0 0.16

(3) EGCG according to concentration EGCG nanoization optimization

On the other hand, considering that nanoization is most efficient when 10% lecithin is added in the EGCG standard as described above, based on the 10% lecithin addition amount in the EGCG standard, EGCG concentration (10 to 80%, w/v), lecithin is 1 After adding ~8%%, DLS irradiation was performed to analyze the physical properties, and the results are shown in FIG. 7 and Table 7. According to this, when 10% lecithin is added in the EGCG standard, it was found that using EGCG at a concentration of 10% to 30% (w/v) has a high degree of solubilization and the best EGCG nanoization.

EGCG
(%, w/v) lecithin
(%, w/v) size
(d.nm) uniformity
(PDI) Zeta potential
(mV) turbidity
(OD600) 10 One 133.3 0.172 -67.2 0.08 20 2 142.8 0.178 -58.6 0.10 30 3 148.5 0.160 -52.7 0.15 40 4 174.2 0.292 -50.7 0.24 50 5 187.5 0.296 -48.1 0.48 60 6 194.2 0.380 -41.1 0.67 70 7 215.4 0.327 -44.6 0.73 80 8 281.9 0.385 -46.2 0.83

Experimental example 3: EGCG emulsion Physical Characterization

Field emission transmission electron microscope Tecnai20 (FEI Co., The Netherlands) was used for FE-TEM images, and EGCG nanoparticles in liquid were dried by dropping a drop (1 μl) on a 200 mesh carbon-coated copper grid and dried at different magnifications. was irradiated at 100 kV voltage. An EGCG emulsion was prepared under the conditions of Example 1 using lecithin as an emulsifier in an oil layer, pectin as an emulsifier in an aqueous layer, and gallic acid as an anti-browning agent in an EGCG 30% (w/v) alcohol mixture. The thus-prepared EGCG emulsion, that is, the EGCG + lecithin + pectin + gallic acid treatment group (ELPG) and the control group in which only EGCG was dispersed (E), EGCG + lecithin treatment group (EL), EGCG + lecithin + pectin treatment group ( ELP) and FE-TEM photographs are shown in FIG. 8 for comparison. According to this, in the ELPG of Example 1, the EGCG nanoparticles showed the most stable form.

Meanwhile, to examine the physical characteristics of the EGCG emulsion, average particle diameter (Diameter, nm), uniformity (PDI), zeta potential (mV), and trapping ability (%) were investigated. The average particle diameter, uniformity (PDI), and zeta potential of EGCG nanoparticles were measured using dynamic light scattering (DLS) and Zetasizer Nano (Malvern Instruments Ltd., UK). The sample was diluted 25-fold before measurement, and the nano was measured immediately on the same day. On the other hand, the encapsulation efficiency (EE) of EGCG nanoparticles was calculated according to the following equation based on the UV-Vis spectroscopy results, based on the 280-290 nm value for green tea EGCG. Nanosized EGCG was centrifuged at 10,000 rpm for 10 minutes and used in the experiment. (Micro Centrifuge 5415C, Eppendorf, US) (Davidov-Pardo et al., 2015; Luo, Zhang, Whent, Yu, & Wang, 2011)

[expression]

Capture capacity (EE, %) = ((Total EGCG content- Nanoized EGCG content))/ Total EGCG content) *100

On the other hand, the chromaticity of the EGCG emulsion was placed in a transparent plastic cylindrical container (35×10 mm) and measured using a spectrophotometer (Minolta, CM-2500D, Tokyo, Japan) to measure L*, a*, b*, the CIE system, of 3 each. Each was prepared and measured three times, and it was expressed as mean and standard deviation (SD).

The EGCG + lecithin + pectin + gallic acid treatment group (ELPG) of Example 1 and the control group in which only EGCG was dispersed (E), EGCG + lecithin treatment group (EL), EGCG + lecithin + pectin treatment group (ELP) measured in this way ), the average particle diameter, dispersion state (A), and zeta potential (B) measurement results are shown in FIG. 9 as a graph, and the average particle diameter, uniformity (determined to be uniform if within 0.2), and zeta potential (the larger the absolute value, the more stable particles generation), and the collecting ability are summarized and shown in Table 8 below.

experimental group average particle diameter
(nm) uniformity
(PDI) Zeta potential
(mV) trapping ability
(%) E 702.1 0.369 -35.73 - EL 207.5 0.134 -63.48 69.4 ELP 251.0 0.176 -66.96 74.7 ELPG 148.5 0.160 -69.59 88.9

According to this, the average particle diameter was decreased from 702 nm before nanoization to 207.5 nm for the treated sample and 169.3 nm for ELPG after the addition of lecithin. The uniformity was 0.2 or less when lecithin was added, which was the lowest among the samples, and thus the uniformity was high. In Example 1, the ELPG treated group showed a zeta potential of -69.59 mV to generate the most stable particles. The capture ability was 69.4% when lecithin was treated, and 88.9% of the final nanonized EGCG treatment group, indicating that most of the EGCG (30%) was nanosized.

Experimental example 4: EGCG emulsion chemical characterization

For FTIR analysis, changes in functional groups in chemical EGCG emulsion were analyzed using FTIR spectrophotometer (Frontier-89063; PerkinElmer, Inc., Waltham, USA) and MIR TGS detector, and the measurement wavelength was 4000-450 cm ^-1 resolution, experimental data. was collected and analyzed using Origin 8.0 software (OriginLab Corporation, Northampton, USA), and the results are shown in FIG. 10 .

According to this, EGCG and nano-products are composed of three peak ranges in the FTIR spectrum: 1) 3500-2900 cm ^-1 , 2) 1700-1300 cm ^-1 , 3) 1200-900 cm ^-1 For each range, 1) means the generation of OH groups, mainly water or ethanol characteristics, 2) mainly means the presence, increase or generation of phenolic compounds, and represents the main characteristics such as flavonoids and phenols, 3) is It indicates the expansion or increase of the carbonyl group, and the increase of the curve height means the increase of the C=O bond.

EGCG is a flavonoid, and most of the peaks were concentrated at ^{1700-1300 cm -1 indicating the presence of phenolic compounds.} On the other hand lecithin exhibits a characteristic phospholipid in combination with glycerol ^{1) 3500 ~ 2900 cm -1,} 2) 1700 ~ 1300 cm -1, 3) are evenly made up of 1200 ~ 900 cm ^-1, particularly 1 to gauge the OH groups present ) The peak was clear at ^{3500-2900 cm -1 .} In the case of gallic acid, since it has a simple phenolic structure, the size of the curve is smaller than that of EGCG, but peaks were spread at ^{2) 1700-1300 cm -1} and 3) 1200-900 cm ^{-1 .} As for the binding pattern of EGCG and the substances constituting the nano-somes, as lecithin, pectin, and gallic acid were added, the curve pattern was similar, but the curvature of the curve was larger. This seems to be due to the increase of aromatic rings through the formation of OH groups as a whole through the addition of lecithin, pectin, and gallic acid, and the bonding of phenolic structures.

Experimental example 5: EGCG emulsion Stability analysis

The EGCG emulsion treated in the same manner as in the experimental group of Experimental Example 3 was stored at 50° C. for 3 days to investigate changes in the physical properties of the EGCG emulsion, and the results are shown in Table 9 below. Turbidity was measured at 600 nm using a microplate reader.

experimental group Turbidity (600 nm) Chromaticity (L value) 0D 3D 0D 3D E 0.34 0.32 46.91 42.66 EL 1.72 1.89 39.66 38.13 ELP 1.68 2.13 40.37 36.64 ELPG 1.71 2.00 39.88 38.61

According to this, it was confirmed that the changes in turbidity (solubility) and chromaticity of substances were maintained the highest in the EGCG sample of Example 1, which is presumed to be a protective action of gallic acid used as an anti-browning agent.

Experimental example 6: anti-obesity Effect analysis

The 3T3-L1 preadipocytes used in the experiment were purchased from the American Type Culture Collection (Manassas, VA, USA), and were prepared with 10% bovine calf serum (BCS) and 1% penicillin and streptomycin (Gibco BRL, Grand Island, NY, USA). ) containing Dulbecco's Modified EaDCRT Media (DMEM, Gibco BRL) was used to incubate at _{37 °C, 5% CO 2 conditions.}

3T3-L1 is a cell line mainly used for in vitro differentiation and function studies of adipocytes, and has the same properties as WAT (white adipocytes) when differentiated from pre-adipocytes into adipocytes. In order to differentiate from pre-adipocytes into adipocytes, when a medium containing high glucose DMEM 10% FBS and 1% penicillin is treated with MDI solution, the cells become spherical, adipocytes are formed, and gene expression occurs. Adipocyte research can be divided into liposynthesis (measuring inhibition during synthesis) and lipolysis (measuring breakdown after fat is synthesized). For cell research, after seeding, when confluent, MDI solution and sample processing in FBS media on day 0, insulin and sample processing in FBS media on day 3, and sample processing in FBS media on day 6 Ended on day one. For 3T3-L1 differentiation of adipocytes, intracellular toxicity tests of EGCG and nanosized EGCG emulsion samples were performed through MTT assay. As a result, it was confirmed that the EGCG sample had no intracellular toxicity at a concentration of 100 to 300 μM. did.

On the other hand, Oil Red O staining was performed to confirm the formation of small fat particles in cells. The prepared 3T3-L1 cells were washed with PBS, fixed with 3.7% formalin for 10 minutes, washed with 60% isopropanol, treated with Oil Red O solution, and stained for 20 minutes at room temperature. After staining, the Oil Red O solution was removed, washed with distilled water 4 times, and the stained cells were observed using a phase contrast microscope. In addition, for quantitative analysis, after extracting fat using 100% isopropanol, 200 μl of each was transferred to a 96 well plate and absorbance was measured at 500 nm with an ELISA reader, and it was expressed as a percentage of the absorbance value of the control group. Triglycerides are broken down into free fatty acids and glycerol. In this experiment, the content of glycerol eluted into the medium during fat accumulation was measured at 540 nm. (Lin et al., 2005; Wu et al., 2005)

EGCG of 3T3-L1 differentiated cells was treated with 100 μM or 300 μM of each concentration of the nano-staged samples of EGCG (the same as in the experimental group of Experimental Example 3), and then the generated triglycerides were stained with Oil red-O staining method. Oil-red O reagent This is a staining agent that specifically stains fat cells. It shows a difference in the degree of cell staining of pre-adipocytes and dipocytes. The more fat cells, the more red. A photograph for this is shown in FIG. 11 . As a result, it was found that the EGCG emulsion sample of Example 1 had a low degree of red staining due to less accumulation of fat globules.

Meanwhile, as a quantitative analysis result, the measurement result of the adipogenesis inhibitory ability is shown in FIG. According to this, the EGCG emulsion sample of Example 1 showed the highest efficacy in inhibiting fat accumulation in 300 μM treatment. In addition, according to the adipogenesis inhibition, when the untreated control group is 100%, when the sample is treated with 300 μM, EGCG is 85%, EGCG + Lecithin treatment group is 78%, EGCG emulsion of Example 1 In the sample, it was confirmed that the fat production was very low at 67%.

In addition, as a result of quantitative analysis, the measurement result of fat resolution is shown in FIG. 13 . According to this, it was found that the amount of glycerol eluted to the outside increased, and when the sample was treated with 300 μM, EGCG was 120 when compared with the untreated control group as 100% of the fat accumulation results for each treatment according to the promotion of lipolysis. %, the EGCG + Lecithin treatment group was inhibited by 138%, and the EGCG emulsion sample of Example 1 was inhibited by 180%, confirming that the fat resolution was also very excellent.

Experimental example 7: bitter taste reduction Effect analysis

TAS2R + GIRK1/4, a receptor that produces about 5-20 ng of bitter taste after treatment with collagenase after surgically harvesting mature and healthy stage 5 or 6 Xenopus oocytes (frog eggs) from the ovaries. of RNA was microinjected into oocytes using a microinjector. The injected oocytes were treated in ND96 culture medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 5 mM HEPES, 1.8 mM CaCl ₂ , 2.5 mM Na-pyruvate, 50 μg/ml, gentamycin, pH 7.4) in 2- Cultured for 3 days. At this time, the processed high-concentration K ⁺ solution binds bitter taste receptors to the bitter taste receptor (EGCG), opens the ion channel, and K+ ions flow in. The degree of membrane potential difference of the oocytes generated at this time is measured by the TEVC (Two Electrode Voltage Clamp) method. It was measured and a schematic diagram showing the measurement method is shown in FIG. 14 . The stronger the bitter taste, the greater the membrane potential difference of oocytes appears, and the degree of bitterness can be quantitatively compared.

15 is a result of comparing the degree of bitterness of substances before and after EGCG nano-ization by EGCG concentration through an electrophysiological technique. According to this, when frog eggs in which bitter taste receptors were expressed were treated with EGCG0 (untreated group) at a concentration of 100 μM and EGCG2 (Example 1), the final nanomaterial, as a result of TEVC measurement, the current was EGCG0 = 12 nA. , EGCG2 was reduced by 50% to 6 nA.

All experimental results were measured three times and expressed as the mean and standard deviation, and the analysis of variance (ANOVA) was performed to test the significance between the mean values for each treatment using the SAS program (Package relwase 8.01). For items with significant differences between the means, a significance test was performed at p<0.05 level using Duncan's multiple range test.

In the above, although embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or add components within the scope that does not depart from the spirit of the present invention described in the claims. The present invention may be variously modified and changed by, etc., and this will also be included within the scope of the present invention.

Claims (22)

oil layer emulsifier, water layer emulsifier and anti-browning agent, and nanoparticulate epigallocatechin gallate (EGCG);
The oil layer emulsifier is any one selected from lecithin, polycaprolactone, polypropylene glycol and olive oil,
The aqueous layer emulsifier is any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside and carrageenan,
The anti-browning agent is any one selected from vitamin C and gallic acid, an anti-obesity composition comprising an epigallocatechin gallate nanoemulsion as an active ingredient. According to claim 1,
The nanoparticulated epigallocatechin gallate anti-obesity composition, characterized in that the nanoparticles by spraying by ultrasonic treatment. 3. The method of claim 2,
The nanoparticulated epigallocatechin gallate was ultrasonically sprayed three times by sequentially adding the oil layer emulsifier, water layer emulsifier and anti-browning agent to a mixture in which a solvent and epigallocatechin powder were mixed. Anti-obesity composition, characterized in that. According to claim 1,
The nanoparticulated epigallocatechin gallate anti-obesity composition, characterized in that the average particle diameter is 100 to 200 nm. According to claim 1,
The nanoparticulated epigallocatechin gallate anti-obesity composition, characterized in that the uniformity (PDI) of 0.1 to 0.2. According to claim 1,
The anti-obesity composition is an anti-obesity composition, characterized in that it has an adipogenesis inhibitory activity and a lipolytic activity. According to claim 1,
The anti-obesity composition, characterized in that the oil emulsifier is lecithin According to claim 1,
The anti-obesity composition, characterized in that the water-layer emulsifier is pectin or cyclodextrin. According to claim 1,
Anti-obesity composition, characterized in that the simultaneous use of lecithin as the oil emulsifier and pectin or cyclodextrin as the water emulsifier. According to claim 1,
The anti-obesity composition comprises 5 to 20 parts by weight of an oil emulsifier, 0.05 to 2 parts by weight of an aqueous emulsifier, and 0.05 to 2 parts by weight of an anti-browning agent based on 100 parts by weight of epigallocatechin gallate. . A food or beverage composition for improving or preventing obesity comprising the anti-obesity composition of any one of claims 1 to 10. 12. The method of claim 11,
The food or beverage composition is a food or beverage composition for improving or preventing obesity, characterized in that it is included in a food or beverage having a transparent or translucent property. A pharmaceutical composition for treating or preventing obesity comprising the anti-obesity composition of any one of claims 1 to 10. (a) preparing a primary emulsion containing primary epigallocatechin gallate nanoparticles by spraying water to which epigallocatechin gallate and an oil emulsifier are added by ultrasonic treatment;
(b) preparing a secondary emulsion containing secondary epigallocatechin gallate nanoparticles by adding a water layer emulsifier to the primary emulsion and spraying by ultrasonication; and
(c) preparing a tertiary emulsion containing tertiary epigallocatechin gallate nanoparticles by adding an anti-browning agent to the secondary emulsion and spraying by sonication;
The oil layer emulsifier is any one selected from lecithin, polycaprolactone, polypropylene glycol, and olive oil,
The aqueous emulsifier is any one selected from pectin, hydroxypropyl methylcellulose, cyclodextrin, alginate, stevioside, and carrageenan,
The anti-browning agent is any one selected from vitamin C and gallic acid, a method for producing an anti-obesity composition comprising an epigallocatechin gallate nanoemulsion as an active ingredient. 15. The method of claim 14,
The method for producing an anti-obesity composition, characterized in that the epigallocatechin gallate is used in a solution of 10 to 30% (w/v). 15. The method of claim 14,
The method for producing an anti-obesity composition, characterized in that the oil layer emulsifier is mixed in an amount of 1 to 30 parts by weight based on 100 parts by weight of the epigallocatechin gallate. 15. The method of claim 14,
The method for producing an anti-obesity composition, characterized in that the oil emulsifier is lecithin. 15. The method of claim 14,
The method for producing an anti-obesity composition, characterized in that the water-layer emulsifier is pectin or cyclodextrin. 15. The method of claim 14,
Lecithin as the oil layer emulsifier, and pectin or cyclodextrin as the water layer emulsifier. 20. A method of manufacturing a food or beverage composition for improving or preventing obesity, comprising the method for preparing an anti-obesity composition of any one of claims 14 to 19. 21. The method of claim 20,
The food or beverage composition is a method of manufacturing a food or beverage composition for improving or preventing obesity, characterized in that it is included in a food or beverage having a transparent or translucent property. 20. A method of preparing a pharmaceutical composition for treating or preventing obesity, comprising the method for preparing an anti-obesity composition of any one of claims 14 to 19.

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