KR101099718B1 - Composition for prevention and treatment of obesity comprising Punica granatum Extracts - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of obesity, using the pomegranate extract as an active ingredient. The present invention showed a high inhibitory result of the cell proliferation by MTT assay using cold water, ethanol and hot water extracts of pomegranate which inhibit fat accumulation in mature fat cell lines, and mature fat by oil red O staining. Searching for fat accumulation inhibitory activity in the cell line showed a high inhibitory activity. As a result of confirming the expression rate of genes involved in adipose differentiation using real-time PCR, it also showed high inhibitory ability. Because of this, it can be usefully used in functional foods and medicines for the prevention and treatment of obesity.

Obesity, 3T3-L1 mature adipocytes, fat accumulation inhibitor

Description

Composition for prevention and treatment of obesity comprising Punica granatum Extracts}

The present invention relates to a composition for the prevention and treatment of obesity containing pomegranate extract, to search for physiological pharmacological activity of edible biological resources, and to isolate the highly active substances, functional foods, general food additives, pharmaceuticals, etc. It belongs to the field of biological materials industry technology, which is materialized into intermediate raw materials.

     Recently, the obesity population is rapidly increasing due to excessive calorie intake, lack of exercise, and stress due to abundant diet. . Accordingly, various efforts have been made to relieve obesity, but there are no satisfactory results, and obesity has emerged as a social problem in personal matters. Accordingly, the national level is preparing measures and strategies for the two areas of healthy living, nutrition and exercise, to prevent obesity. (Oh, Dae-Gyu, Obesity Prevention and Management Measures at the National Level, Journal of the Korean Society of Obesity, 11 (3), 327, 2002)

     Obesity is a lifestyle disease caused by the imbalance of energy accumulation and energy consumption, which is accompanied by complications, and can be said to be a disease that requires extreme prevention. For this reason, modern people have increased their interest in preventing obesity and healing obesity as a food material, and accordingly, many studies on food materials and natural materials have been reported. (Effects of Caffeine on the Differentiation of Adipocyte Line 3T3-L1: Kim, Ji-Ja et al .: A Nutrigenomic Approach, Journal of Food and Nutrition, 38 (8), 649-655, 2005) Adipocytes synthesize energy in the form of triglyceride (TG) Plays a central role in storage, storage and disassembly. In other words, the amount of heat stored in the form of TG in the case of excess calories is decomposed when the calories are consumed to be used as energy. This fat cell balance requires a number of different enzymes and regulators involved in lipogenesis and lipolysis. When 3T3-L1 cells grow and become 100% confluency, growth stops from this time and the differentiation into adipocytes is gradually progressed. The differentiation of 3T3-L1 cells into adipocytes is caused by the differentiation-inducing factors in serum added to the culture medium. Therefore, differentiation-promoting factors (insulin, Dexamethasone, 3-isobutyl-1-methylxanthine) can be further promoted in the culture medium. Differentiation can also be suppressed by adding. Finally, the differentiated 3T3-L1 cells are expressed as mature fat cells, such as the accumulation of intracellular fat, and activation of enzymes that induce gene expression and fat accumulation peculiar to adipocytes. Representative fat cell differentiation promoters known to date include insulin and insulin-like growth factor-I (IGF-I). (Effect of Alginic Acid on Differentiation of 3T3-L1 Cells, Journal of the Korean Fisheries Society, 33 (6), 541-545, 2000)

Pomegranate ( Punica granatum ) is one of two species of the genus Punica, P. granatum and P. protopunica . The fruit is the seed, juice, bark, and flower part, and the tree is divided into bark, root, leaf, etc., each with different pharmacological activity. For example, juice and fruit peels are known to relieve menopausal disorders in women by having strong antioxidant properties and seed oils exhibiting weak anti-estrogen activity. They also exhibit anticancer activity as interfering factors such as cancer cell proliferation, cell cycle, infiltration and angiogenesis. Anti-inflammatory agents of vegetable origin, of course, play a major underlying role in connection with the protective and therapeutic effects of cancer. In connection with the alleviation of disorders in postmenopausal women, prevention of breast and prostate cancer, and strong arteriosclerosis lesions, the development of functional ingredients has been widely suggested in medicines, functional foods, and functional cosmetics. In addition, the biologically active ingredients and chemical components are contained in the seeds, juice and skin of a pomegranate, which has been regarded as a power fruit or a functional fruit.

     Under these backgrounds, the present inventors completed the present invention by confirming that the pomegranate's hot water and ethanol extract contained a substance showing a fat accumulation inhibitory effect in mature fat cells while studying a micromaterial having various physiological activities from pomegranate. It became.

In order to solve the above problems, the present inventors have studied the micromaterials having various physiological activities in natural biological resources such as spiced vegetables, spices, and edible algae, and inhibit the proliferation of all-cell cells and fat accumulation in mature fat cells. The present invention was completed by selecting pomegranates having the inhibitory effect on the mRNA levels of PPAR-γ and CEBP-α, which are known as major factors for adipocyte differentiation from cold water, ethanol and hot water extracts of pomegranate.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of obesity, containing the pomegranate extract as an active ingredient.

In addition, the present invention has another object to provide a functional food for preventing and improving obesity containing pomegranate extract as an active ingredient.

The present invention as a means for solving the above problems,
A cold water extract obtained by subjecting the pomegranate to heat treatment, pulverizing and extracting cold water at room temperature;
Ethanol extract obtained by ethanol extraction of the residue remaining after the cold water extraction; And
It provides a pharmaceutical composition for the prevention and treatment of obesity containing as an active ingredient at least one pomegranate extract selected from the group consisting of hydrothermal extract obtained by hydrothermal extraction of the residue remaining after the ethanol extraction.
In addition, the present invention
A cold water extract obtained by subjecting the pomegranate to heat treatment, pulverizing and extracting cold water at room temperature;
Ethanol extract obtained by ethanol extraction of the residue remaining after the cold water extraction; And
It provides a functional food for the prevention and improvement of obesity containing one or more pomegranate extract selected from the group consisting of hydrothermal extracts obtained by hydrothermally extracting the residue remaining after the ethanol extraction as an active ingredient.

In the present invention, the pomegranate was heat-treated, pulverized, and extracted in the order of cold water, ethanol and hot water. -2-yl) -2,5-diphenyltetrazolium bromide] assay and Oil-Red O assay, respectively. Inhibition of PPAR-γ and CEBP-α mRNA expression, major transcription factors in adipocyte differentiation, was confirmed by real-time PCR. Could. Accordingly, it can be used as an intermediate raw material of functional food, food medicine, medicine for preventing and alleviating fat accumulation type obesity in white adipose tissue.

Example 1: Confirmation of the ability to inhibit the growth of fat cells and the accumulation of fat in the process of differentiation from pomegranate

     While searching for extracts extracted from various edible biological sources, it was confirmed that pomegranate extracts had a proliferation inhibitory effect on 3T3-L1 cells. The pomegranate was heat treated (blanching) at 100 ° C. for 5 minutes to inactivate and pulverize enzymes in living cells, followed by extraction with cold water, 100% ethanol, and hot water at room temperature. After drying to obtain an extract (Fig. 1).

     The MTT assay system was used to examine the proliferation inhibitory activity of all-cell cells for each extract of pomegranate obtained by the above method. After dispensing 3T3-L1 cells in a 96-well plate to 1x10cells / well and incubating for 24 hours, replacing with fresh medium, each extract of pomegranate was treated to 1, 0.1, 0.01 mg / ml, 48 After incubation for 10 hours, the medium was removed, and 10 uL of MTT solution (5 mg / ml) and serum-free medium were treated in each well. After incubation for 4 hours in an incubator (37 ℃), formazan reduced by dehydrogenase of viable cells was dissolved in DMSO and absorbance was measured at 570 nm with a microplate reader.

     As a result of measuring the growth rate of whole cell according to the extract as described above, it was confirmed that the growth rate is significantly inhibited compared to the control based on 100% control in cold water, ethanol and hot water extract (Fig. 2).

Example 2: Confirmation of fat accumulation inhibitory ability of the extract extracted from pomegranate

To examine the fat accumulation inhibitory activity in mature adipocytes for each extract, cell line cell 3T3-L1 (mouse embryo fibroblast cell line) was cultured on 6 well-plates at a concentration of 2x105 cells / well, and 100% confluency. After 2 more days of incubation, the original medium was removed, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 uM dexamethasone (DEX), and 1 ug / ml insulin (INS), 10% Fetal bovine Samples were added to DMEM with serum (FBS) at a concentration of 1, 0.1 and 0.01 mg / ml and incubated in 37 ° C. and CO ₂ incubator for 48 hours. After removing the medium for 48 hours, 1 ug / ml INS and 10% FBS were added to DMEM at a concentration of 0.1 mg / ml, and then incubated for 48 hours at 37 ° C. in a CO 2 incubator. After removing the medium again, the sample was added to DMEM to which 10% FBS was added at a concentration of 0.1 mg / ml and incubated in 37 ° C. and CO ₂ incubator for 96 hours. The control was kept in the same state in the incubator without the addition of the sample. After 8 days of culture, the cultured cells were measured as follows using the Oil red O method. After complete removal of the culture, it was washed twice with PBS and left for 1 hour to fix cells by adding 10% formalin solution at 400 ul / well. Thereafter, the cells were washed with PBS and oil red O working solution was added per 400 ul / well to stain fat in differentiated adipocytes for 2 hours. After this, remove the oil red O working solution and completely remove the oil red O working solution on the wall of the well by using the distilled water. After addition, the absorbance was measured at 490 nm using a micro plate reader (Model 680 microplate reader, Bio-Rad, USA). The fat accumulation ability of the sample was calculated by the following equation.

[Equation 1]

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     As a result of measuring the fat accumulation ability according to the fraction as described above, it was confirmed that the fat accumulation ability in cold water, ethanol and hot water extract was significantly inhibited compared to the control, based on 100% of the control (Fig. 3).

Example 3: Inhibitory effect of adipocyte differentiation of pomegranate (PPARγ, C / EBPa gene inhibition)

     As described above, the US National Center for Biotechnology Infiemation (NCBI) database targets PPARγ and C / EBPα, which are key transcription factors involved in fat accumulation by treating extracts and components of the present invention and extracting RNA during adipose differentiation. from. Based on the searched gene sequence, forward and reverse primers were designed, and the designed primers were synthesized by Bioneer. Real-time PCR was performed according to the conventional method and compared the degree of expression (Fig. 4, 5).

        1 shows a process for preparing cold water, ethanol, hot water extract from pomegranate.

        Figure 2 is a graph showing the cell-cell proliferation inhibitory activity of pomegranate extract [PP1: cold water extract, PP2: ethanol extract, PP4: hot water extract].

        3 is a graph showing the adipocyte accumulation inhibitory activity of pomegranate extract [PP1: cold water extract, PP2: ethanol extract, PP4: hot water extract].

        Figure 4 is a graph showing the C / EBPa mRNA expression inhibitory activity of pomegranate extract [PP1: cold water extract, PP2: ethanol extract, PP4: hot water extract].

        5 is a graph showing the PPARγ mRNA expression inhibitory activity of the pomegranate extract [PP1: cold water extract, PP2: ethanol extract, PP4: hot water extract].

Claims (6)

A cold water extract obtained by subjecting the pomegranate to heat treatment, pulverizing and extracting cold water at room temperature; Ethanol extract obtained by ethanol extraction of the residue remaining after the cold water extraction; And A pharmaceutical composition for preventing and treating obesity, comprising as an active ingredient one or more pomegranate extracts selected from the group consisting of hydrothermal extracts obtained by hydrothermally extracting the residues remaining after the ethanol extraction. delete delete A cold water extract obtained by subjecting the pomegranate to heat treatment, pulverizing and extracting cold water at room temperature; Ethanol extract obtained by ethanol extraction of the residue remaining after the cold water extraction; And Functional food for the prevention and improvement of obesity containing one or more pomegranate extract selected from the group consisting of hot water extract obtained by hot water extracting the residue left after the ethanol extraction as an active ingredient. The functional food for preventing and improving obesity according to claim 4, wherein the pomegranate extract has a concentration of 0.01 to 1 mg / ml. The pharmaceutical composition for preventing and treating obesity according to claim 1, wherein the pomegranate extract has a concentration of 0.01 to 1 mg / ml.

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