KR101412987B1 - Composition for anti-obesity comprising resveratrol derivative - Google Patents

Abstract

The present invention relates to an anti-obesity composition comprising resveratrol derivatives as an active ingredient, and more particularly to an anti-obesity composition comprising the following formula (1).
The composition according to the present invention has an anti-obesity effect because it contains a low concentration of resveratrol derivative and also has an inhibitory effect on the differentiation of 3TS-L1 cells and an inhibitory effect on FAS expression, which is superior to that of resveratrol.
[Chemical Formula 1]

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to an anti-obesity composition comprising a resveratrol derivative,

The present invention relates to an anti-obesity composition comprising a resveratrol derivative as an active ingredient.

Obesity is a major cause of heart disease, cancer, hypertension, diabetes and degenerative arthritis. According to the World Obesity Federation, one-third of the world's population is obese (body mass index 30 kg / m < ² >). Currently, the world 's anti - obesity material market is over $ 200 billion, and it is expected to grow rapidly every year due to the increase in obesity population. However, currently used anti - obesity drugs such as orlistat, sibutramine, (Sertraline) 's side effects have been reported, and the research and development of anti - obesity substance is going to replace it fiercely.

Fat is composed of fatty acids and glycerol. The enzyme acetyl-CoA carboxylase (ACC), which plays an essential role in the early stage of fatty acid synthesis by cells, induces carboxylation of acetyl-CoA and promotes production of malonyl-CoA. Fatty acid synthesis is carried out using malonyl-CoA as a raw material by an enzyme having a molecular weight of 250 kDa called fatty acid synthase (FAS). Therefore, FAS and ACC inhibitors can be expected to reduce obesity by reducing fat production.

On the other hand, resveratrol ( trans -3,5,4'-trihydroxystilbene) is a kind of naturally occurring stilbenoids, and it is phytoalexin that is naturally biosynthesized against the attack of pathogens such as bacteria and fungi in some plants. The physiological activity of resveratrol is known to be antioxidant, anticancer and cardioprotective effect, and the antiobesity effect is also excellent (Korean Patent No. 10-0681439, Korean Patent Publication No. 10-2012-0003693, etc.) .

Thus, the present inventors have found that resveratrol derivatives exhibit an excellent 3T3-L1 cell differentiation inhibitory effect and an inhibitory effect on FAS expression level, as compared with resveratrol, and thus completed the present invention.

It is an object of the present invention to provide an anti-obesity composition containing resveratrol derivatives by confirming the 3TS-L1 cell differentiation inhibitory effect and FAS expression inhibiting effect of resveratrol derivatives.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating and preventing obesity, which comprises a resveratrol derivative represented by the following formula 1: The pharmaceutical composition may be characterized by inhibiting the differentiation of 3T3-L1, a lipogenic precursor cell, into adipocytes, and may be characterized by inhibiting the expression of a fatty acid synthase (FAS) 1 < / RTI > of resveratrol derivative in an amount of 10 to 100 mg / kg.

In one embodiment, there is provided a health functional food for improving and preventing obesity comprising a resveratrol derivative represented by the following formula (1). The health functional food may be characterized by inhibiting the differentiation of 3T3-L1, a lipogenic precursor cell, into adipocytes, and may be characterized by inhibiting the expression of a fatty acid synthase (FAS) The resveratrol derivative of formula (I) is contained at 10 to 100 mg / kg.

The formula of the resveratrol derivative of the present invention (4- [2- (3,5-Dimethoxyphenyl) vinyl] pyridine) is shown below.

[Chemical Formula 1]

The formula of resveratrol ( trans -3,5,4'-trihydroxystilbene) of the present invention is shown in the following formula (2).

(2)

The composition containing the substance according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral liquid preparations such as suspensions, emulsions, syrups and aerosols, external preparations, pre-filled syringe solution, or lyophilized form. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.2 to 200 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Examples of the foods to which the composition of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in powder, granule, tablet, have.

The composition of the present invention contains a resveratrol derivative having a low concentration, and thus the 3TS-L1 cell differentiation inhibitory effect and FAS expression inhibitory effect are superior to resveratrol and can have an anti-obesity effect.

Brief Description of the Drawings Figure 1 shows the effect of the present derivatives on fat accumulation in 3T3-L1 cells.
Fig. 2 is a graph showing the effect (Oil-Red O staining result) of the present invention on fat accumulation in 3T3-L1 cells.
3 is a graph showing the effect of the derivatives of the present invention on FAS expression in 3T3-L1 cells.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples

Example

Example  One. Resveratrol  The derivative (4- [2- (3,5- Dimethoxyphenyl ) vinyl ] 피리딘 ) Synthesis of

3,5-Dimethoxybenzyl bromide was dissolved in anhydrous benzene and PPh ₃ was added. The mixture was stirred under reflux for 2 hours and then cooled to give a precipitate, which was filtered to obtain 3,5-dimethoxybenzyltriphenylphosphonium bromide. The obtained 3,5-dimethoxybenzyltriphenylphosphonium bromide and 4-pyridinecarboxaldehyde were dissolved in dichloromethane together with tetrabutylammonium bromide as a catalyst, followed by slow addition of 50% aqueous sodium hydroxide solution. After stirring for 4 h, brine was added and extracted with dichloromethane. The residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography and refluxed with iodine to obtain a resveratrol derivative (4- [2- (3,5-Dimethoxyphenyl) vinyl] pyridine).

Example  2. Cell culture and adipocyte differentiation

3T3-L1 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) at 37 ° C and 5% CO ₂ . Differentiation of cells was induced by differentiation into adipocytes using a hormone mixture (10 mg / mL insulin, 0.5 mM dexamethasone and 0.5 mM isobutyl xanthine).

Example  3. Anti-obesity  Effect experiment

3-1. oil Red  Dyeing ( Oil - Red  O staining ) result

In order to confirm whether resveratrol derivatives exhibit anti-obesity effect, 3T3-L1 cells induced lipid differentiation were treated with resveratrol derivatives for 8 days, and the degree of inhibition of adipocyte differentiation was measured by oil red staining.

For oil red staining (Havel 2000), the cells were gently washed twice with PBS, fixed with 3.7% fresh formaldehyde at room temperature for 1 hour in PBS, filtered with an oil red oozing solution (60% isopropanol and 40% Water) for at least 1 hour. After lipid droplets were stained red, the oil red ocher solution was removed and the plates were washed with water and dried. Lipid droplet images of 3T3-L1 adipocytes were collected using an Olympus microscope (Tokyo, Japan).

Finally, the dye retained in the cells was separated into isopropanol and quantified by measuring the optical absorption capacity at 510 nm using a microplate reader (Tecan Austria GmbH, Austria).

As a result, resveratrol derivative (4- [2- (3,5-Dimethoxyphenyl) vinyl] pyridine) showed a concentration-dependent inhibition of adipocyte differentiation, and especially at a low concentration of 12.5 μM, (Fig. 1). The morphological changes of 3T3-L1 cells when treated with resveratrol and resveratrol derivatives at a concentration of 12.5 μM are shown in FIG.

3-2. FAS  Measurement of expression level

FAS, a major enzyme involved in the fatty acid biosynthesis pathway, is known to be an important factor controlling obesity. It was determined whether the resveratrol derivative of Example 1 inhibited the increase of FAS expression in the lipogenesis-induced 3T3-L1 cells of Example 2.

Cells were washed with PBS and then lysed in lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, Sodium orthovanadate, 1 mM NaF, and 0.2% protease inhibitor cocktail, pH 7.2). Cell proteins were collected and centrifuged at 14,000 rpm for 5 minutes. The resulting supernatant was quantitated using Bradford. 30 μg of the protein was electrophoresed using 10% SDS-PAGE, transferred to nitrocellulose membrane, and detected using an antibody (Acetyl-CoA carboxylase 1: 500, Fatty acid synthase 1: 1000, beta actin 1: 4000 dilutions) .

As a result, the expression level of FAS was decreased, and the inhibitory effect was remarkably higher than that of resveratrol (see FIG. 3), especially at a low concentration of 12.5 μM. These results are in agreement with the results of the inhibitory effect of adipocyte differentiation inhibition at low concentrations. Therefore, the inhibitory effect of 4- [2- (3,5-Dimethoxyphenyl) vinyl] pyridine on adipocyte differentiation is due to a decrease in the expression level of FAS .

As a result of the in vitro test in the present invention, the in vivo test results show that the effect data at a concentration of 50 μM resveratrol is similar to that at a concentration of 12.5 μM resveratrol, and that the effect of anti-obesity is shown when the dose is 40-400 mg / kg The dose of resveratrol derivative was estimated to be 10 to 100 mg / kg in the preparation of a pharmaceutical composition containing resveratrol derivatives.

Examples of formulations for the composition of the present invention are illustrated below. However, the following formulation examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following formulation examples.

Formulation example  One: Sanje  Produce

100 mg of the resveratrol derivative of Example 1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2: Preparation of tablets

50 mg of the resveratrol derivative of Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3: Preparation of capsules

50 mg of the resveratrol derivative of Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4: Preparation of injection

50 mg of the resveratrol derivative of Example 1

Sterile sterilized water for injection

pH adjuster

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5: Liquid  Produce

100 mg of the resveratrol derivative of Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6: Manufacture of health food

1000 mg of the resveratrol derivative of Example 1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7: Manufacture of health drinks

1000 mg of the resveratrol derivative of Example 1

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 liter container, ≪ / RTI >

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (8)

A pharmaceutical composition for the treatment and prevention of obesity comprising a resveratrol derivative represented by the following formula (1).
[Chemical Formula 1]

The method according to claim 1,
3T3-L1, which is a lipogenic precursor cell, is suppressed in adipocyte differentiation. The method according to claim 1,
A pharmaceutical composition for the treatment and prevention of obesity characterized by inhibiting the expression of a fatty acid synthase (FAS). The method according to claim 1,
A pharmaceutical composition for treating and preventing obesity, which comprises 10 to 100 mg / kg of a resveratrol derivative of formula (1). A health functional food for improving and preventing obesity comprising a resveratrol derivative represented by the following formula (1).
[Chemical Formula 1]

6. The method of claim 5,
3T3-L1, which is a lipogenic precursor cell, is suppressed in adipocyte differentiation. 6. The method of claim 5,
Wherein the fatty acid synthase (FAS) is inhibited in the expression of the fatty acid synthase (FAS). 6. The method of claim 5,
Wherein the resveratrol derivative of formula (1) is contained in an amount of 10 to 100 mg / kg.

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