ANTI-OBESITY COMPOSITION INCLUDING GEUMHWAGYU EXTRACT AS ACTIVE INGREDIENT

Abstract

Proposed is a Geumhwagyu extract as an active ingredient. The Geumhwagyu extract of the present disclosure inhibits the differentiation of pre-adipocytes by inducing the degradation of CEBP-α and inhibits the expression of CEBP-α, PPARγ, Perilipin-1, Adiponectin, FABP4, etc., to suppress the accumulation of lipids in adipocytes. It can be easily used as a composition for preventing or managing obesity by inhibiting it and lowering the content of TG (Triacylglycerol) to show anti-obesity activity.

Claims

1. An anti-obesity composition comprising Geumhwagyu (Hibiscus manihot L.) extract or a fraction of the Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient.

2. The composition of claim 1, wherein the Geumhwagyu extract is obtained by performing solvent extraction on parts selected from flowers, stems, leaves, or roots of Geumhwagyu (Hibiscus manihot L.).

3. The composition of claim 2, wherein the solvent is water, hot water, C1 to C4 alcohol, or a mixed solution thereof.

4. A healthy functional food for anti-obesity, the food comprising Geumhwagyu (Hibiscus manihot L.) extract or a fraction of the Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient.

5. The healthy functional food of claim 4, wherein the Geumhwagyu extract is an extract obtained by performing solvent extraction on parts selected from flowers, stems, leaves, or roots of Geumhwagyu.

6. The healthy functional food of claim 5, wherein the solvent is water, hot water, C1 to C4 alcohol, or a mixed solution thereof.

7. A method of preventing or treating obesity, comprising: administering a pharmaceutical composition comprising Geumhwagyu (Hibiscus manihot L.) extract or a fraction of the Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient to a subject.

8. The method of claim 7, wherein the Geumhwagyu extract is obtained by performing solvent extraction on parts selected from flowers, stems, leaves, or roots of Geumhwagyu (Hibiscus manihot L.).

9. The method of claim 8, wherein the solvent is water, hot water, C1 to C4 alcohol, or a mixed solution thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a graph showing a cell image for lipid accumulation according to an Oil Red O experiment of a Geumhwagyu flower extract (HMF), and an absorbance result of a cell eluate, and a TG synthesis result according to TG measurement;

[0025] FIG. 2 is a graph showing a cell image for lipid accumulation according to the Oil Red O experiment of a Geumhwagyu leaf extract (HMF), and an absorbance result of the cell eluate, and a TG synthesis result according to TG measurement;

[0026] FIG. 3 is a graph showing a cell image for lipid accumulation according to the Oil Red O experiment of Geumhwagyu stem extract (HMF), and an absorbance result of the cell eluate, and a TG synthesis result according to TG measurement;

[0027] FIG. 4 is a graph showing a cell image for lipid accumulation according to the Oil Red O experiment of Geumhwagyu root extract (HMF), and an absorbance result of the cell eluate, and a TG synthesis result according to TG measurement;

[0028] FIG. 5 is a Western blot resulting image showing inhibition of expression of PPARγ, CEBPα, Perilipin-1, Adiponectin, FABP4, etc., which are proteins related to lipid accumulation of Geumhwagyu flowers (HMF), leaves (HMF), stems (HMF), and roots (HMF) extracts.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Hereinafter, preferred embodiments of the present disclosure will be described in detail.

[0030] However, the present disclosure is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete and will fully convey the spirit of the disclosure to those skilled in the art.

Example 1: Preparation of Geumhwagyu Extract

[0031] Geumhwagyu flower, leaf, stem, and root extracts were prepared through the following process. First, the flowers, leaves, stems, and roots of Geumhwagyu grown by E-Farm Co., Ltd., an agricultural company located in Punggi, Gyeongsangbuk-do, Korea, were washed thoroughly with distilled water and dried. Water equivalent to 20 times the volume of the dry pulverized product was added to 10 g of dry pulverized product and stirred at room temperature of 25° C. for 3 days to obtain each of the Geumhwagyu flower, leaf, stem, and root extract. Thereafter, the extract was filtered and then freeze-dried to obtain the final extracts of Geumhwagyu flowers (HMF), leaves (HMF), stems (HMF), and roots (HMF).

Example 2: Cytotoxicity Assay

[0032] In order to check whether the Geumhwagyu extract of the present disclosure obtained in Example 1 is toxic to cells, cytotoxicity was analyzed in vitro.

[0033] To this end, 3T3-L1, a pre-adipocyte, was purchased from KCLB and used. After the cells were aliquoted in a cell culture flask, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml) were added to the DMEM medium and cultured and used in an incubator maintained at 37° C., 5% CO.sub.2, and 95% humidity conditions. For cytotoxicity evaluation, the MTT assay was used, in which the method uses a principle that dehydrogenases in mitochondria of cells with intact metabolic processes reduce yellow water-soluble tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltrazolium bromide] (MTT) to non-soluble dark purple MTT formazan crystals. The crystal was evaluated for cytotoxicity by measuring absorbance at an appropriate wavelength (mainly 500-600 nm).

[0034] First, 3T3-L1 cells were aliquoted into 96 wells at a 1×105 cells/well concentration, and then cultured for 24 hours. Then, each part of the extract of Geumhwagyu prepared in Example 1 was treated at a concentration of 0, 50, 100, and 200 μg/mL, respectively, and after incubation for 24 hours, MTT at a concentration of 1 mg/mL was added and incubated at 37° C. for 2 hours. After the reaction, DMSO (dimethyl sulfoxide) was added, and absorbance was measured at 570 nm using a microplate reader.

[0035] As a result of the analysis, it was confirmed that all Geumhwagyu extracts had no cytotoxicity, and then the following experiment was performed (graph not attached).

Example 3: Activity of Inhibiting Lipid Accumulation and Triglyceride (TG) Production of Geumhwagyu Extract

[0036] 3T3-L1 cells, which are pre-adipocytes, were aliquoted into 96 wells at a 1×105 cells/well concentration and cultured for 3 days and then differentiated with the DMI medium (adipogenic medium). As the MDI medium, 10% FBS/DMEM treated with 0.5 mM of IBMX, 1 μM dexamethasone, and 1 μg/ml insulin was used. This time point was regarded as day 0 of differentiation, and after 2 days of differentiation, 1 μg/ml of insulin medium was replaced, and after 2 days of culture, 10% FBS medium was replaced and maintained for up to 8 days. Geumhwagyu extract was added on day 0 of differentiation and treated for 8 days. After 8 days of differentiation, the degree of differentiation of adipocytes and the degree of inhibition of differentiation by Geumhwagyu extract were analyzed through Oil Red O staining. After adipocyte differentiation and Geumhwagyu extract treatment were completed, cells were washed with 1× phosphate-buffered saline (PBS), and then 10% (w/v) formalin was added and fixed at room temperature for 1 hour. After removing the formalin and washing each well in which the cells were cultured with 60% (v/v) isopropanol, the isopropanol was completely blown away from the hood. Oil red O solution was added to the dried cell culture wells, left for 10 minutes, washed with distilled water, and adipocyte differentiation was confirmed by microscopic imaging. The stained cell culture wells were dissolved in 100% isopropanol, and absorbance was measured at 500 nm. The triglyceride (TG) amount in adipocytes was measured using a commercially available TG assay kit (Triglyceride Assay Kit Quantification, ab65336). The results of each experiment are shown in FIGS. 1 to 4.

[0037] The results of the Geumhwagyu flower extract (HMF) are described in FIG. 1, the Geumhwagyu leaf extract (HML) in FIG. 2, the Geumhwagyu stem extract (HMS) in FIG. 3, and the Geumhwagyu root extract (HMR) in FIG. 4.

[0038] Referring to FIGS. 1 to 4, it can be confirmed that the lipid accumulation is suppressed through the cell image and the absorbance result of the cell eluate, which are the results of the Oil Red O experiment, of the extracts for each part of Geumhwagyu, and the content of triglyceride (TG) produced and accumulated in cells is also significantly reduced.

Example 3: Inhibitory Activity of Lipid Accumulation-Related Protein Expression of Geumhwagyu Extract

[0039] 3T3-L1 cells, which are pre-adipocytes, were aliquoted in a 24-well culture at a 1×105 cells/well concentration and cultured for 3 days and then differentiated with MDI medium. This time point was regarded as day 0 of differentiation, and after 2 days of differentiation, 1 μg/ml of insulin medium was replaced, and after 2 days of culture, 10% FBS medium was replaced and maintained for up to 8 days. Geumhwagyu extract was added on day 0 of differentiation and treated for 8 days. After 8 days of differentiation, cells were harvested, and the expression of lipid accumulation-related proteins was investigated through Western blot analysis.

[0040] FIG. 5 is a Western blot image showing lipid accumulation-related protein expression regulation of Geumhwagyu flower (HMF), leaf (HML), stem (HMS), and root (HMR) extracts. It was shown to inhibit the expression of adipocyte lipids accumulation-related proteins, such as PPARγ, CEBPα, Perilipin-1, Adiponectin, and FABP4.

[0041] Through these results (FIGS. 1 to 5), it can be verified that the extracts of Geumhwagyu flowers, leaves, stems, and roots have anti-obesity activity by inhibiting the expression of lipid accumulation-related proteins in adipocytes.

Formulation Example 1. Pharmaceutical Preparations

[0042] 200 g of the extract for each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

Formulation Example 2. Food Manufacturing

Formulation Example 2-1. Preparation of Cooking Condiments

[0043] Cooking condiments for health promotion were prepared by adding the extract of each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure to the cooking condiments at 1% by weight.

Formulation Example 2-2. Flour Food Manufacturing

[0044] The extract for each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure is added to wheat flour in an amount of 0.1% by weight, and the mixture is used to prepare bread, cake, cookies, crackers, and noodles to produce health-promoting food.

Formulation Example 2-3. Preparation of Soup and Gravies

[0045] The extract of each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure was added to soup and gravies in an amount of 0.1% by weight to prepare health-promoting soup and gravies.

Formulation Example 2-4. Manufacture of Dairy Products

[0046] The extract of each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable Juice Production

[0047] Vegetable juice for health promotion was prepared by adding 0.5 g of the extract of each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit Juice Manufacturing

[0048] Fruit juice for health promotion was prepared by adding 0.1 g of the extract for each part of the flowers, stems, leaves, or roots of Geumhwagyu of the present disclosure to 1,000 ml of apple juice or grape juice.