KR20100109691A - Anti-obese composition comprising the extracts of erythronium japonicum - Google Patents

Abstract

PURPOSE: A composition for suppressing obesity containing Erythronium japonicum extract or organic solvent fraction thereof is provided to effectively suppress differentiation of adipocyte. CONSTITUTION: A composition for suppressing obesity contains Erythronium japonicum extract or organic solvent fraction thereof as an active ingredient. The Erythronium japonicum extract is ethanol or spirit extract. The organic solvent fraction is chloroform or ethylacetate fraction. The Erythronium japonicum extract is 80% ethanol extract. A composition for suppressing obesity further contains Heloniopsis orientalis extract and acetate fraction thereof.

Description

Anti-obese composition comprising the extracts of Erythronium japonicum}

The present invention relates to a composition for inhibiting obesity, and more particularly, to a composition for inhibiting obesity, which can effectively inhibit the differentiation of adipocytes and can be usefully used for the production of functional foods or pharmaceuticals with excellent anti-obesity effect. .

Recently, the obesity population is increasing due to the change of eating habits according to the development of industrial society and the improvement of living standard. Obesity is also known as a risk factor for cardiovascular diseases such as heart disease and stroke, liver disease, diabetes, cancer, and high blood pressure. Such obesity is currently affecting all age groups, especially the incidence of older people.

Obesity represents a state in which the body has an excessive accumulation of fat, and the cause of the accumulation of fat in the body is due to excessive intake of carbohydrate (carbohydrate) or excessive intake of fat. The mechanism of reaching obesity is that by over-ingesting the sugar, the sugar in the food is digested and becomes monosaccharide and absorbed into the body through the small intestine. It takes the cells and converts them into fat.

As a result, studies on various anti-obesity agents have been conducted based on the idea of causing anti-obesity by inhibiting a part of those pathways leading to obesity, but there are usually unpleasant or excessive doses It is also a chemical compound, and since it is mostly a chemical compound, it may cause a problem for the safety of the human body when administered regularly.

The present invention has been made to solve the problems of the prior art as described above, the purpose of which can be safely administered as a natural extract, can effectively inhibit the differentiation of fat cells, functional food with excellent anti-obesity effect Or to provide a composition for inhibiting obesity that can be usefully used in the manufacture of medicines.

The technical problem of the present invention as described above is achieved by the following means.

(1) A composition for inhibiting obesity, comprising an extract or an organic solvent fraction thereof as an active ingredient.

(2) The composition for inhibiting obesity according to item 1, wherein the extract is ethanol or alcohol extract.

(3) The composition for inhibiting obesity according to item 1, wherein the organic solvent fraction is a chloroform or ethyl acetate fraction.

(4) The composition for inhibiting obesity according to item 2, wherein the composition is 80% ethanol extract.

(5) The composition for inhibiting obesity according to item 1, further comprising a virgin skirt extract.

(6) The composition for inhibiting obesity according to item 1, further comprising an acetate fraction of virgin skirting extract.

The composition for inhibiting obesity according to the present invention can effectively inhibit the differentiation of adipocytes and can be usefully used for the production of functional foods or pharmaceuticals excellent in anti-obesity effect.

Hereinafter, the content of the present invention will be described in more detail.

The present invention includes an anti-obesity composition containing an extract or an organic solvent fraction thereof as an active ingredient.

Eji extract according to the present invention is an extract using water extract, ethanol extract or alcohol. Preferably it is 80-100% ethanol extract, More preferably, it is about 80% ethanol extract.

The algae extract according to the present invention has its own antioxidant activity, radical scavenging activity, and was confirmed to have differentiation inhibitory ability against adipocytes.

In the present invention, the organic solvent fraction of the Alzheimer extract may be included in the composition for inhibiting obesity. The organic solvent usable may include chloroform or ethyl acetate, and these may be used alone or as a mixed solvent. When used as a mixed solvent, the compounding ratio of chloroform to ethyl acetate may be used in the range of 10 to 90 to 90 to 10 by weight. The fraction of the organic solvent of such an extract has better obesity inhibitory activity than the extract itself.

In the present invention, the extract or fraction may be used by itself, or may be used by mixing the extract with the fraction. At this time, the mixing ratio is not particularly limited. For example, the mixing ratio may be used in the range of 10 to 90 to 90 to 10 by weight.

In addition, the composition for inhibiting obesity according to the present invention may be further added to the virgin skirt extract or / and fractions in addition to the Erle extract or / and fractions. Virgin skirt extract or fraction according to the present invention was found to be excellent in antioxidant activity, radical scavenging activity and adipocyte differentiation as in the case of Alge. The addition ratio of virgin skirting extract and / or fractions is not particularly limited, and may be added, for example, in the range of 0.1 to 50% by weight based on the whole composition. According to the present invention, while the extract or / and fraction of the extract is confirmed to induce the death of adipocytes at a low concentration, in the case of virgin agaric extract or / and fractions to inhibit the differentiation ability of the adipocytes even at a small concentration As a result, the synergistic effect is exerted in that it is possible to continuously maintain the differentiation inhibitory ability while controlling the death rate of fat cells which may be caused by containing a large amount of extract.

The composition for inhibiting obesity, including the extract or / and fraction of the present invention can be used in a variety of drugs, food and beverages. Foods to which the extract of the present invention may be added include various foods, for example, beverages, gums, teas, vitamin complexes, health supplements, and the like, pills, powders, granules, acupuncture tablets, capsules or beverages. Can be used in the form of phosphorus. At this time, the amount of the extract in the food or beverage can be added to 0.05 to 30% by weight of the total food weight in the case of the health food composition of the present invention, 0.05 to 30 g based on 100 ml for the health beverage composition can be added. have.

Food additives that can be used herein include food additives customary in the art, such as flavorings, flavors, colorants, fillers, stabilizers, and the like. The health beverage composition according to the present invention is an essential ingredient in the proportions indicated, and there is no particular limitation on the ingredients to be added to the extract other than the above extract, and it may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin and the like; Common sugars such as and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tautin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used. . The proportion of such natural carbohydrates is generally about 0.5 to 30 g per 100 ml of the composition of the present invention.

In addition to the above, the composition for inhibiting obesity of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents such as natural flavoring agents, colorants and fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.

In addition, the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not particularly limited but is generally selected from 0 to about 10 parts by weight based on 100 parts by weight of the composition of the present invention.

The composition for inhibiting obesity containing the extract or / and fraction of the present invention as an active ingredient can be parenterally or orally administered as desired, and the amount of 0.001 to 5 g per 1 kg of body weight per day is 1 to several times. It can be administered separately. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, mode of administration, absorption, severity of the disease, and the like.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Obtaining Extract

The crushed or shredded scallions and virgin skirts were immersed in 80% ethanol and extracted for 24 hours, and then filtered to obtain a liquid component, which was concentrated and dried to obtain a powder obtained as an extract.

Example 2 Obtaining Sequential Solvent Fraction

The extract obtained in Example 1 was sequentially treated with an organic solvent to obtain a fraction. The process of obtaining the chloroform and ethyl acetate fractions from the extracts of Algebra and virgin skirts was as follows.

The same amount of hexane was added to the samples of the ethanol extract evaporated 80% ethanol extract and the virgin skirt, and shaken from side to side and left to separate layers. The hexane fraction was separated into an hexane fraction and an aqueous layer. The hexane fraction was transferred to a water phase and the same amount of chloroform was added to the remaining aqueous layer. The two solutions were shaken from side to side to react well and left to separate layers. The chloroform fraction and the aqueous layer were separated. The chloroform fraction was transferred to a water phase and the same amount of ethyl acetate was added to the remaining aqueous layer. The two solutions were shaken from side to side to react well and left to separate layers. The ethyl acetate fraction was separated from the aqueous layer, and the ethyl acetate fraction was transferred to a water phase and the same amount of butanol was added to the remaining aqueous layer. The two solutions were shaken from side to side to react well and left to separate layers. The butanol fraction and the aqueous layer were separated and the butanol fraction was transferred to a water phase and the remaining water layer was also transferred to the water phase. Hexane, chloroform, ethyl acetate, butanol, and dH ₂ O collected in water were evaporated and dissolved in DMSO.

Experimental Example 1 Evaluation of Antioxidant Activity of Extracts and Fractions

The total polyphenol of each solvent fraction was homogeneous by adding 2 ml of 2% sodium carbonate to 100ul of sample, and adding 100ul of 2N polyline solution for 30 minutes at room temperature, and absorbance was measured at 750 nm. -Converted to catechin.

The total polyphenol contents of the extracts and fractions of sage and virgin skirt were analyzed to show high polyphenol contents in ethyl acetate and chloroform fractions in the aging and in most of the extracts and fractions. The polyphenol content of the ethanol extract was high (FIG. 1).

Experimental Example 2 ABTS Radical Scavenging Activity

2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma Chemical Co., USA) 2.5 mM and 2,2'-azobis (2-amidino-propane) die Hydrochloride (AAPH) was dissolved in 100 mM potassium phosphate buffer (pH 7.4) solution each containing 150 mM NaCl, mixed 1: 1, and left in a constant temperature bath at 68 ° C. to form ABTS ⁺ cation for absorbance at 734 nm. The value was adjusted to 0.650 ± 0.02. After a certain amount of the extract diluted with the concentrations of the large and the virgin skirt extract was added to the solution, the reaction was measured for 10 minutes at 37 ° C., and then the change in absorbance was measured.

The ABTS radical scavenging activity of the extracts and fractions of the sperm and virgin skirts was measured. 2, Table 1).

TABLE 1

IC50 (ug) water Ethyl acetate Butanol chloroform Hexane 80% ethanol E. japonicum 344.59 41.30 620.35 41.01 378.76 588.24 H. orientalis 674.76 34.21 63.73 63.73 85.65 40.21

<Experimental Example 3> and dogtooth violet skirt in virgin vitro Anti-obesity efficacy evaluation

end. Induction of 3T3-L1 Culture and Differentiation

Mouse fetal 3T3-L1 cell line cells purchased from the American Type Culture Collection (ATCC) at concentrations of 2.5 × 10 ⁵ cells / ml in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% bovine serum and 1% penicillin / streptomycin Incubated for 72 hours and suspended in a 37 ℃ CO ₂ incubator to be confluent. Differentiation medium (10% FBS, 1% penicillin / streptomycin, 5 μg / ml insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine) was incubated for 48 hours. At this time, the extract was treated with 0.05 mg / ml and 0.1 mg / ml. In order to promote differentiation into adipocytes, the cells were incubated for 48 hours in 10% FBS DMEM medium to which 5 μg / ml insulin was added. Thereafter, medium was exchanged twice with 48% intervals in 10% FBS DMEM to induce differentiation into adipoocytes.

I. Oil red O dye

For staining of the lipid droplets of differentiated adiposite, staining was carried out with Oil red O, which reacts specifically with the fat. After removing the medium, 1 ml of 10% formalin was treated once for 5 minutes and then fixed by treating with 1 ml of fresh 10% formalin for 1 hour. Each well was completely dried after washing with 60% isopropanol. Oil red O dye (6 parts Oil red O stock, 4 parts dH ₂ O) was treated for 10 minutes and washed four times with distilled water. By observing the differentiated cells under the microscope, it was confirmed that the differentiation was inhibited in the cells treated with the ulge and virgin skirt. The wells were completely dried after discarding dH ₂ O. For elution of fat-associated oil red O, the solution was treated with 100% isopropanol for 10 minutes and the absorbance was measured at 490 nm.

As a result of measuring the differentiation of adipocyte differentiation of 80% ethanol extracts of the larvae and virgin aprons, the large suppressed adipocyte differentiation in a concentration-dependent manner (FIG. 3). In addition, it was confirmed that adipocyte differentiation was inhibited depending on the treatment concentration even in the results of evaluating the effect of adipocyte differentiation of the sequential solvent fractions of Alge (FIG. 4).

In the case of virgin skirt, cell viability decreased rapidly even at the low concentration of 24 hours treated with 0.05 mg / ml, and as a result, adipocyte differentiation was also rapidly suppressed from the low concentration (FIG. 5). In addition, it was confirmed that adipocyte differentiation was inhibited depending on the treatment concentration even in the results of evaluating the effect of adipocyte differentiation of the sequential solvent fractions of virgin skirts (FIG. 6).

Intracellular triglyceride concentration was measured using Serum Triglyceride Determination Kit (SIGMA, TR0100-1KT). 160 μl of Free Glycerol Reagent was added to each well in a 96 well plate, and 2 μl of the standard and the sample were mixed well, followed by reaction at 37 ° C. for 5 minutes. Then, free glycerol reagent was measured at 540 nm. Then, 40 μl of triglyceride reagent was added to the wells, mixed well, and reacted at 37 ° C. for 5 minutes, and then measured triglyceride concentration at 540 nm.

In order to measure the inhibitory efficacy of intracellular triglyceride production, the concentration of intracellular triglycerides was measured after 48 hours of treatment with the extract of Algebra and virgin skirts. Both the Algebra and virgin skirts inhibited the intracellular triglyceride production (FIG. 7).

In cells Tris pH 8.0, 150 mM Tris NaCl, 0.02% sodium azide, 0.2% SDS, PMSF (phenylmethylsulfonylfluoride) 100 ug / ml, aprotinin 50 ug / ml, NP-40 1%, NaF 100 mM , 200 μl of RIPA buffer composed of 0.5% sodium deoxycholate, EDTA 0.5 mM, and EGTA 0.1 mM was homogenized and centrifuged at 13,000 rpm for 20 minutes to separate the supernatant. The supernatant was transferred to a new tube, the protein was quantified, electrophoresed with SDS-polyacrylamide gel at a constant concentration of 20 μg, and then Western blotting. The electrophoresis gel was equilibrated with about 50 ml of transfer solution for 30 minutes. PDVF membrane was also cut to a suitable size and soaked in buffer as well as gel and equilibrated for 30 minutes. The western blot cassette was placed in a container containing a transfer buffer solution, and two deckron sponges and two Atman 3MM filter papers soaked in the transfer buffer solution were placed thereon. The gel equilibrated with transfer buffer solution was carefully placed on Whatman 3MM filter paper to prevent bubbles. The membrane equilibrated with the transfer buffer solution on the gel was carefully placed to avoid bubbles, and two filter papers and a decron sponge were placed on it. The cassette was securely fixed to prevent bubbles and the clips were tightened to prevent both cassettes from falling out. After transferring the cassette to the blotting chamber, insert the transfer buffer solution to the marked part so that the membrane in the cassette is sufficiently submerged, install the electrode so that the membrane side is the anode and the gel side is the cathode, and transfer at 4 ° C at 100V and 350 mA for 2 hours. It was. The protein-transferred membrane was immersed in a TBST solution containing 1% bovine serum albumin and left at 4 ° C. for 12 hours to block the non-protein-bound portion of the membrane. The buffer used for blocking was removed and the membrane washed three times for 10 minutes with TBST solution. Primary antibodies (SREBP-1, C / EBPa, PPARγ, FAS, AMPK, β-actin) diluted to appropriate concentrations using TBST buffer solution were added to the membrane so as to sufficiently immerse the reaction at 4 ° C for 12 hours. The membrane was washed three times for 10 minutes with TBST solution. Anti-rabbit-HRP, appropriately diluted with TBST solution, was reacted for 2 hours at room temperature. The membrane was washed three times for 10 minutes with TBST solution. ECL reaction in the dark room to develop a film to check the band.

Western blot analysis was conducted to investigate the effect of oleic and virgin skirt extract on the expression of adipocyte differentiation-related proteins. Expression of FAS was also inhibited. Inhibition of adipocyte differentiation of virgin albino and algae is thought to be expressed through the AMPK pathway (FIG. 8).

After 48 hours of treatment with oleic and virgin aerosol extracts and fractions on mature adipocytes, DAPI staining evaluated the apoptosis induction effect of adipocytes. 9).

Hereinafter, the preparation of extracts obtained by extracting with 80% ethanol using the stems of the larvae and virgin aprons, in which the anti-obesity activity was confirmed by the experimental example of the present invention, and the preparation examples of various formulations using fractions thereof as raw materials will be described below. do.

Production Example 1 Tablet

Tablet 1 :

The tablets were prepared after blending the following ingredients and tableting according to the conventional methods for preparing tablets.

TABLE 1

ingredient content 80% Ethanol Extract of the Stalk of Example 1 300mg Lactose 100mg Starch 100mg Stearic acid Quantity

Tablet 2 :

The tablets were prepared after blending the following ingredients and tableting according to the conventional methods for preparing tablets.

TABLE 2

ingredient content 80% Ethanol Extract of the Stalk of Example 1 150mg 80% Ethanol Extract of Virgin Skirt Stem of Example 1 150mg Lactose 100mg Starch 100mg Stearic acid Quantity

Tablet 3 :

The tablets were prepared after blending the following ingredients and tableting according to the conventional methods for preparing tablets.

TABLE 3

ingredient content Chloroform Fraction of Alzali Extract of Example 1 150mg 80% Ethanol Extract of Virgin Skirt Stem of Example 1 150mg Lactose 100mg Starch 100mg Stearic acid Quantity

Tablet 4 :

The tablets were prepared after blending the following ingredients and tableting according to the conventional methods for preparing tablets.

TABLE 4

ingredient content Ethyl Acetate Fraction of Alzali Extract of Example 1 150mg Ethyl Acetate Fraction of Virgin Skirt Extract of Example 1 150mg Lactose 100mg Starch 100mg Stearic acid Quantity

Preparation Example 2 Capsule

Capsule 1 :

The capsules were prepared by combining the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method.

TABLE 5

ingredient content 80% Ethanol Extract of the Stalk of Example 1 200 mg Lactose 50 mg Starch 50 mg Talc 2mg Magnesium Stearate Quantity

Capsule 2 :

The capsules were prepared by combining the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method.

TABLE 6

ingredient content Chloroform fraction of Alzheimer extract of Example 1 200 mg Lactose 50 mg Starch 50 mg Talc 2mg Magnesium Stearate Quantity

Capsule 3 :

The capsules were prepared by combining the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method.

<Table 7>

ingredient content Ethyl Acetate Fraction of Alge Extract of Example 1 200 mg Lactose 50 mg Starch 50 mg Talc 2mg Magnesium Stearate Quantity

Capsule 4 :

The capsules were prepared by combining the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method.

<Table 8>

ingredient content 80% Ethanol Extract of the Stalk of Example 1 100mg 80% Ethanol Extract of Virgin Skirt Stem of Example 1 100mg Lactose 50 mg Starch 50 mg Talc 2mg Magnesium Stearate Quantity

Preparation Example 3 Liquid (Health Supplement)

Liquid 1 :

The following ingredients were combined and sterilized by filling into a brown bottle to prepare a liquid.

<Table 9>

ingredient content 80% Ethanol Extract of the Stalk of Example 1 200 mg Sugar 10g Iseong Hwadang 15 g Apple flavor 1 g Purified water Adjust the total volume of liquid to 1ℓ

Liquid 2 :

The following ingredients were combined and sterilized by filling into a brown bottle to prepare a liquid.

<Table 10>

ingredient content 80% Ethanol Extract of the Stalk of Example 1 100mg 80% Ethanol Extract of Virgin Skirt Stem of Example 1 100mg Sugar 10g Iseong Hwadang 15 g Apple flavor 1 g Purified water Adjust the total volume of liquid to 1ℓ

Liquid 3 :

The following ingredients were combined and sterilized by filling into a brown bottle to prepare a liquid.

<Table 11>

ingredient content Chloroform fraction of Alzheimer extract of Example 1 100mg 80% Ethanol Extract of Virgin Skirt Stem of Example 1 100mg Sugar 10g Iseong Hwadang 15 g Apple flavor 1 g Purified water Adjust the total volume of liquid to 1ℓ

Liquid 4 :

The following ingredients were combined and sterilized by filling into a brown bottle to prepare a liquid.

TABLE 12

ingredient content Ethyl Acetate Fraction of Alge Extract of Example 1 100mg Ethyl Acetate Fraction of Virgin Skirt Extract of Example 1 100mg Sugar 10g Iseong Hwadang 15 g Apple flavor 1 g Purified water Adjust the total volume of liquid to 1ℓ

As described above, it will be described with reference to preferred embodiments of the present invention, but those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the present invention described in the claims below. It will be appreciated that it can be changed.

1 is a measurement result of the polyphenol content carried out on the extract and its fractions.

Figure 2 shows the results of the antioxidant activity carried out on the extract and virgin skirt extract and fractions thereof [left: Alejandro, right: virgin skirt].

Figure 3 is the result of measuring the inhibition of adipocyte differentiation carried out on 80% ethanol extract of Alejandro.

Figure 4 is an evaluation result of the inhibition of adipocyte differentiation carried out on the sequential solvent fraction of the Alge.

Figure 5 is an evaluation result of the inhibition of adipocyte differentiation carried out on 80% ethanol extract of virgin skirt.

Figure 6 is an evaluation result of the inhibition of adipocyte differentiation carried out on the sequential solvent fraction of virgin skirt.

Figure 7 shows the results of measurement of intracellular triglyceride concentration after 48 hours treatment of the ulge and virgin skirt extract [left: ulge, right: virgin skirt].

8 is a result of Western blot conducted on the extract of Alejandro virgin.

9 is an evaluation result of the apoptosis induction effect of the adipocytes carried out in the extract and fractions

Claims (6)

Composition for inhibiting obesity, which contains oleace extract or organic solvent fractions thereof as an active ingredient The composition for inhibiting obesity according to claim 1, wherein the extract is ethanol or alcohol extract. The composition for inhibiting obesity according to claim 1, wherein the organic solvent fraction is a chloroform or ethyl acetate fraction. The composition for inhibiting obesity according to claim 2, wherein the composition is 80% ethanol extract. The composition for inhibiting obesity according to claim 1, further comprising a virgin skirt extract. The composition for inhibiting obesity according to claim 1, further comprising an acetate fraction of virgin skirting extract.

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