KR20140123263A - A composition comprising extract from Trifolium repens L for obesity or metabolic syndrome related diseases - Google Patents

Abstract

The present invention relates to a composition comprising an extract from trifolium repens L as an effective ingredient for preventing and alleviating obesity-related or metabolic syndrome-related diseases, and, more specifically, to a composition comprising an extract from trifolium repens L as an effective ingredient for preventing and alleviating obesity-related or metabolic syndrome-related diseases, wherein the extract has activities inhibiting the differentiation of adipocytes progenitor cells to adipocytes, fat accumulation, and the expression of PPARγ which is a transcription factor centrally involved in the differentiation of the adipocytes. According to the present invention, the extract from the trifolium repens L inhibits the differentiation of the adipocytes and thereby serves as a food composition with an anti-obesity function or anti-metabolic syndrome function for overweight or obese people, thereby contributing national health.

Description

[0001] The present invention relates to a composition for preventing and improving obesity or metabolic syndrome related diseases,

The present invention relates to an extract of Trifolium repens L) as an active ingredient for preventing or ameliorating obesity or metabolic syndrome related diseases. More particularly, clover extract (Trifolium repens L) inhibits the expression of PPAR gamma which plays a pivotal role in the differentiation of adipocytes, thereby being useful as a preventive and remedy for obesity or metabolic syndrome related diseases.

According to the World Health Organization (WHO) in 2011, more than 1.5 billion adults worldwide are overweight and at least 500 million of them are obese. Obesity can be defined as a disease caused by excessive fat cell differentiation and an overpowering supply of energy. Obesity increases blood pressure and blood sugar, increases blood triglyceride levels and decreases HDL cholesterol, which in turn increases the risk of metabolic syndrome and ultimately increases the risk of cardiovascular disease. The metabolic syndrome refers to the simultaneous occurrence of three or more of abdominal obesity, diabetes, dyslipidemia (elevated triglycerides, high-density cholesterol) and hypertension in one person.

Currently, there are methods to correct lifestyle such as diet, exercise, and behavior therapy, and drug therapy and surgical treatment for obesity treatment. However, drugs such as appetite suppressant and lipolysis inhibitor have various side effects, There is a drawback. In recent years, however, the importance of vegetable compounds, which are useful ingredients in plants, has been recognized, and they have been found to be useful as anti-obesity functional foods, which have beneficial effects on overall health and weight control, And the mechanism of action. Background Art [0002] Prior Art 1 (KR 10-2010-0095987 B1) relates to a composition for preventing and treating obesity or metabolic syndrome comprising an extract of Aspergillus oryzae as an active ingredient, (KR 10-2008-0100037 A) relates to a composition for prevention and treatment of obesity, which comprises an extract of Leucophora japonica as an active ingredient. ≪ / RTI >

Anti-obesity effect and action mechanism using natural materials are concentrated on antioxidant activity, anti-platelet inhibition effect, and inhibition of differentiation of body fat tissue. (C / EBPα, C / EBPβ and C / EBPδ), and ADDA / ADPDA (C / EBP), which are differentiated from adipose precursor cells. It is known that transcription factors called SREBP1c (adipocyte determination differentiation factor 1 / sterol regulatory element binding protein 1c) play a pivotal role. These transcription factors induce expression at different time points in the differentiation process of adipocytes and regulate the expression of various adipocyte-specific genes through interaction with each other, gradually inducing the activation of adipocyte metabolism and adipocyte differentiation. Therefore, those having an activity of inhibiting the differentiation into adipocytes of fat precursor cells and inhibiting fat accumulation in adipocytes, or having an activity of inhibiting the expression of PPARγ, C / EBPα, and ADDA / SREBP1c may be used for prevention and prevention of obesity or metabolic syndrome- It can be utilized as an improving agent.

The shamrock ( Trifolium repens L) is native to Europe, and the planting of the plant grows wild and has become a naturalized plant. The shamrock is also called " Clover ". The height is 20 ~ 30cm, and there are no hairs on the whole abdomen. Leaves are leaves with 3 small leaves. The petiole is 5 ~ 15cm long and long. There are three small leaves but some have four, with inverted heart shape, length of 15 ~ 25mm, width of 10 ~ 25mm. The outposts include, but are not limited to, oranges such as Orthazonan, Triacanthanol, Linamarin, Pinitol, Rotastristin, Cummersterol, Mellic Acid, Tiperphen Saponin (Agarcon is Soyasapogenol A, B and C) Folol, bequiol, umbeleferon, adenine, xanthine, hypoxanthin, 180 mg% ascorbic acid, and 1.4 to 6.7 mg% carotene. The leaves may be selected from the group consisting of quercetin-3-glucoside, formononetin, 7,4-dihydroxyflavone, 7,3,4-trihydroxyflavone, Pauline, there is oil. There are female hormones and amino acid components on the ground. However, the mechanism of antiobesity to shamrock ( Trifolium repens L) is not yet known.

Thus, the present inventors have searched for inhibitors that inhibit the expression of PPARγ, which is a transcription factor that plays a pivotal role in the differentiation of adipocytes, from various plant extracts. As a result, it was found that the shamroat extracts inhibited the differentiation- Inhibiting activity of PPAR [gamma], which is a transcription factor that plays a pivotal role in the differentiation of adipocytes, and thus can be useful as a composition for preventing or ameliorating obesity or metabolic syndrome related diseases Thereby completing the invention.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

KR 10-2010-0095987 B1 KR 10-2010-0114510 A KR 10-2008-0100037E

While studying the anti-obesity or anti-metabolic syndrome action from various plant extracts, the present inventors have found that Trifolium repens L) extract has an activity of inhibiting the expression of PPARγ, which is a transcription factor that plays a pivotal role in the differentiation of adipocytes. As a result, it has been found that the anti-obesity Or anti-metabolic syndrome, and completed the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing and ameliorating obesity or metabolic syndrome-related diseases containing the extract of Trifolium repens ( Trifolium repens L) as an active ingredient.

The present invention relates to a shamroat repens L) extract as an active ingredient for the prevention and treatment of obesity or metabolic syndrome related diseases.

The shamrock ( Trifolium repens L) extract can be obtained by mixing water, a lower alcohol having 1 to 4 carbon atoms, glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, isopropyl alcohol, benzene, hexane, acetone, diethyl ether, dichloromethane, And extracts obtained by extracting with a solvent selected from a mixed solvent.

The shamrock ( Trifolium repens L) extract is characterized by comprising an extract obtained by an extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a selected preparation or purified product thereof.

The shamrock ( Trifolium repens L) extract is extracted using any one extraction method selected from hot water extraction, cold extraction, reflux cooling extraction and ultrasonic extraction.

The shamrock ( Trifolium repens L) extract has an activity of inhibiting the expression of PPARγ (Peroxisome proliferator-activated receptor gamma), a transcription factor that plays a pivotal role in the differentiation of adipocytes.

The composition is characterized by being a food composition.

The composition is characterized in that the preparation is selected from soft capsules, tablets, granules, drinks, caramel formulations and diet bars.

Pratense of the present invention (Trifolium repens L) inhibits the proliferation of lipid precursor cells to adipocytes and inhibits the accumulation of fat and inhibits the expression of PPARγ, a transcription factor that plays a pivotal role in the differentiation of adipocytes. Thus, the expression of obesity or metabolic syndrome Can be usefully used as a preventive and remedy for diseases, and such compositions can be commercialized as functional foods and the like.

FIG. 1 shows the results of Trifolium repens L) extract has a fat accumulation-inhibiting activity in adipocytes in a concentration-dependent manner.
Fig. 2 is a graph showing the results of Trifolium repens L) extract has an activity of inhibiting the expression of PPARγ at a gene level in a concentration-dependent manner.

The present invention relates to a shamroat repens L) extract as an active ingredient for preventing or ameliorating obesity or metabolic syndrome related diseases.

While studying the anti-obesity or anti-metabolic syndrome action from various plant extracts, the present inventors have found that Trifolium repens L) extract has an activity of inhibiting the expression of PPARγ, which is a transcription factor that plays a pivotal role in the differentiation of adipocytes. As a result, it has been found that the anti-obesity Or anti-metabolic syndrome, and completed the present invention.

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

In the present invention, the shamrock (hereinafter referred to as "clover") extract is obtained by mixing water, a lower alcohol having 1 to 4 carbon atoms, glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, isopropyl alcohol, , A solvent selected from the group consisting of acetone, diethyl ether, dichloromethane and a mixed solvent thereof, preferably water, methanol, ethanol, ethyl acetate, isopropyl alcohol, acetone or hexane. In addition, in the present invention, the above-mentioned extract may contain any one of the extract obtained by the extraction treatment, the diluted solution or the concentrate of the extract, the dried product obtained by drying the extract, and the adjusted product or the purified product.

An example of a method for obtaining the shamrock extract in the present invention is as follows. The dried shamrock pulverized product is extracted using an eluting solvent. At this time, the blending ratio of dried shamrock pulverized product to elution solvent is preferably 1: 2 to 1:20 w / v, more preferably 1: 3 to 1: 5 w / v, but is not limited thereto. The dissolution solvent may be any of the solvents described above, and a mixed solvent having a mixing ratio of 1: 0.1 to 1: 10 v / v when the water and the lower alcohol is a mixed solvent may be used as an elution solvent. The extraction temperature is preferably 20 to 100 ° C, and the extraction time is preferably 3 hours to 4 days. The extraction method is preferably performed using any one of extraction methods such as hot water extraction, cold extraction, reflux cooling extraction and ultrasonic extraction, more preferably extraction with hot water extraction, filtration under reduced pressure, The extract is concentrated under reduced pressure at 20 to 100 ° C with a vacuum rotary condenser or the like to obtain a crude extract of Shamrock.

As shown in FIG. 1, the shamrock extract of the present invention has an effect of inhibiting the differentiation of adipocyte precursor cells into adipocytes, and also has an activity of inhibiting fat accumulation. In addition, as shown in Fig. 2, it showed an activity of inhibiting the expression of PPARγ, which plays a pivotal role in the differentiation of adipocytes.

(C / EBPα, C / EBPβ and C / EBPδ), and ADDA / ADPDA (C / EBP), which are differentiated from adipose precursor cells. It is known that transcription factors called SREBP1c (adipocyte determination differentiation factor 1 / sterol regulatory element binding protein 1c) play a pivotal role. These transcription factors induce expression at different time points in the differentiation process of adipocytes and regulate the expression of various adipocyte-specific genes through interaction with each other, gradually inducing the activation of adipocyte metabolism and adipocyte differentiation. Therefore, those having the activity of inhibiting the differentiation into adipocytes of fat precursor cells and inhibiting fat accumulation in adipocytes or having the activity of inhibiting the expression of PPARγ, C / EBPα, and ADDA / SREBP1c are used as preventive and therapeutic agents for obesity or metabolic diseases .

In this regard, the shamrock extract of the present invention has excellent activity of inhibiting the differentiation of adipose precursor cells into adipocytes, inhibiting the accumulation of adipocytes and inhibiting the expression of PPARγ, which plays a pivotal role in the differentiation of adipocytes, Obesity or metabolic syndrome.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example  1. Shamrock ( Trifolium repens  L) Preparation of extract

The shamrock (hereinafter referred to as "clover") was thoroughly washed with water, dried in the shade, and pulverized using a blender (Waring, USA). 2 L of water was added to 200 g of the powdered shamrock, After filtration with Filter Papers (Whatman, US), the filtrate was filtered through a vacuum rotary evaporator at 25 ° C to obtain a crude extract of Shamrock as an extracted residue.

Experimental Example  1. Inhibition of differentiation of adipocytes into adipocytes

3T3-L1 (ATCC CL-173), a mouse precursor adipocyte, was cultured in a 10% BCS and DMEM medium at 37 ° C and 5% CO ₂ . 3T3-L1 precursor adipocytes were plated at a density of 5 × 10 ⁴ cells / well in a 24-well plate and maintained at 100% confluency for 2 days. The precursor adipocytes were induced for 2 days in 10% FBS DMEM medium containing MDI (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM Dexamethasone and 10 μg / ml Insulin) mu] g / ml Insulin in DMEM medium supplemented with 10% FBS for 2 days. After that. For 2 days with 10% FBS DMEM medium. During the induction of adipocyte differentiation, shamrock extracts were treated with 25, 50, and 100 μg / ml of each culture medium, and adipocyte differentiation was observed on the 8th day when the differentiation was completed. The degree of adipocyte differentiation was firstly confirmed by microscopy through Oil Red O staining, and the degree of adipocyte staining was measured at 510 nm absorbance.

As a result, as shown in Fig. 1, the shamrock extract had an effect of inhibiting the differentiation of precursor adipocytes into adipocytes in a concentration-dependent manner. Specifically, the inhibition rate of adipocyte differentiation was high even at a low concentration of 25 μg / ml. In addition, it was confirmed that quercetin, which is known to interfere with lipid synthesis and induce atrophy of adipocytes and increase the destruction of mature adipocytes, has an equal or greater effect than quercetin.

Experimental Example  2. Transcription factors involved in adipocyte differentiation PPAR gamma < / RTI >

The expression of PPARγ (Peroxisome proliferator-activated receptor gamma), which plays a pivotal role in the differentiation of adipocytes, was evaluated. At the stage of differentiation into adipocytes, the expression of PPARgamma increases. In the same manner as in Experimental Example 1, the shamrock extract was treated at 25, 50, and 100 μg / ml for 8 days to differentiate adipocytes and the degree of PPARγ expression was confirmed by PCR. After 3 days of 3T3-L1 cells were washed twice with PBS, cell pellets were collected and RNA was isolated with total RNA extract kit (iNtRON). CDNA was synthesized using 0.5 μg of the extracted RNA and PCR was performed using the primers shown in Table 1 below. Β-Actin was used as a control gene.

Table 1 below shows the nucleotide sequences of the primers used in the RT-PCR assays.

Gene Primer sequence  (5 ' to  3 ') PPARγ-Forward ATT CTG GCC CAC CAA CTT CGG PPARγ-Reverse TGG AAG CCT GAT GCT TTA TCC CCA β-Actin-Forward TGG AAT CCT GTG GCA TCC ATG AAA β-Actin-Reverse TAA AAC GCA GCT CAG TAA CAG TCC

 For PCR, 1 μl of cDNA synthesized with 0.5 μg of RNA was used, 1 μl of each 5 pmole primer, 2 μl of 10 × PCR buffer and 10 mM dNTPs (2.5 mM / each). Using 0.2 μl of DNA polymerase (5 U / μl) and 11.8 μl of distilled water, the total volume was adjusted to 20 μl. The PCR cycle consisted of initial denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 20 sec, annealing at 57 ° C for 30 sec, extension at 72 ° C for 30 sec and final extension at 72 ° C for 2 min. Respectively.

As a result, as shown in Fig. 2, the shamroat extract showed an inhibitory effect on the expression of PPARγ, which plays a pivotal role in the differentiation of adipocytes in a concentration-dependent manner. In particular, when the extract was treated at a concentration of 100 μg / ml, the efficacy was similar to that of quercetin.

Hereinafter, formulation examples of the composition will be described, but the present invention is not intended to be limited thereto but specifically described.

Hereinafter, the dried powder of the shamrock extract is powder obtained by adding dextrin to the crude extract obtained after extraction and concentration.

Formulation example  One. Soft capsule

Shamrock extract Dry powder 80 mg

Soybean oil 180 mg

Palm oil 2 mg

Vegetable hardening oil 8 mg

Yellow apple 4 mg

Lecithin 6 mg

The mixture was mixed with 400 mg per capsule according to a conventional method to prepare a soft capsule.

Formulation example  2. Refining

Shamrock extract Dry powder 74 mg

L-carnitine 120 mg

200 mg of galacto-oligosaccharide

Lactose 60 mg

Maltose 140 mg

The mixture was granulated using a fluidized bed drier, and then 6 mg of sugar ester was added thereto, and the mixture was tableted.

Formulation example  3. Granules

Shamrock extract Dry powder 80 mg

L-carnitine 120 mg

Anhydrous crystalline glucose 250 mg

Starch 550 mg

The granules were mixed and granulated into a granule using a fluidized bed granulator, and then filled into a capsule. The final weight of the contents is 1 g.

Formulation example  4. Drink

Shamrock extract Dry powder 80 mg

L-carnitine 120 mg

Glucose 10 g

Citric acid 0.6g

Liquid oligosaccharide 25g

After mixing the above, 300 ml of purified water is added, and 200 ml of the solution is filled into each bottle. And then sterilized at 130 DEG C for 4 to 5 seconds to prepare a beverage.

Formulation example  5. Caramel  Formulation

Shamrock extract Dry powder 80 mg

L-carnitine 120 mg

Corn syrup 1.8 g

Skim milk 0.5g

Soybean lecithin 0.5g

Butter 0.6g

Vegetable hardened oil 0.4g

Sugar 1.4g

Margarine 0.58 g

Salt 20 mg

The caramel was molded by mixing the above. The final weight of the contents is 6g.

Formulation example  6. Diet bars ( bar ) Formulation

Shamrock extract Dry powder 80 mg

L-carnitine 120 mg

Starch 20 g

Flour 9 g

11 g of starch syrup

11.6 g of maltose

Margarine 6 g

Salt 30 mg

Citric acid 30 mg

BSA 140 mg

2 g of sugar ester

Diet bars were formed by mixing the above. The final weight of the contents is 60g.

Claims (7)

Trifolium repens L) extract as an effective ingredient for preventing or ameliorating obesity or metabolic syndrome related diseases.
The method according to claim 1,
The shamrock ( Trifolium repens L) extract can be obtained by mixing water, a lower alcohol having 1 to 4 carbon atoms, glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, isopropyl alcohol, benzene, hexane, acetone, diethyl ether, dichloromethane, Wherein the extract is an extract obtained by extracting with a solvent selected from a mixed solvent.
The method according to claim 1,
The shamrock ( Trifolium repens L) extract comprises any one selected from the group consisting of an extract obtained by extraction, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a controlled preparation or purified product thereof. / RTI >
The method according to claim 1,
The shamrock ( Trifolium repens L) extract is extracted using any one of extraction method selected from hot water extraction, cold extraction, reflux cooling extraction and ultrasonic extraction.
The method according to claim 1,
The shamrock ( Trifolium repens L) extract has an activity of inhibiting the expression of PPARγ (Peroxisome proliferator-activated receptor gamma), which is a transcription factor that plays a pivotal role in differentiation of adipocytes, for the prevention and improvement of obesity or metabolic syndrome related diseases Composition.
The method according to claim 1,
The composition for preventing and improving obesity or metabolic syndrome related diseases, wherein the composition is a food composition.
7. The method according to any one of claims 1 to 6,
Wherein the composition is prepared from a formulation selected from the group consisting of soft capsules, tablets, granules, a drink, a caramel formulation and a diet bar.

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