COMPOSITION COMPRISING LOW TEMPERATURE WATER EXTRACT OF HIBISCUS MANIHOT FOR ANTI-OBESITY

Abstract

Provides is an anti-obesity composition, including a low-temperature Geumhwagyu extract as an active ingredient. The extract was obtained by adding Geumhwagyu to low-temperature water at 4 to 50° C. and extracting for 1 to 20 hours. The extract increases energy consumption through an increase in expression of adipose triglyceride lipase (ATGL) and hormone-sensitivity lipase (HSL), lipolysis through inhibition of expression of perilipin-1, and thermal generation through an increase in expression of uncoupling protein (UCP). In addition, the extract improves energy metabolism by increasing the expression of phospho-AMP-activated protein kinase (AMPK), inhibits lipid formation and accumulation of adipocytes by inhibiting cell growth of mature adipocytes, and shows anti-obesity activity, thereby being easily used as a pharmaceutical composition and healthy functional food for obesity.

Claims

1. An anti-obesity pharmaceutical composition comprising a low-temperature Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient.

2. The anti-obesity pharmaceutical composition of claim 1, wherein the low-temperature extract is an anti-obesity pharmaceutical composition in which the low-temperature extract is extracted using low-temperature water of 4 to 50° C. as a solvent.

3. The anti-obesity pharmaceutical composition of claim 1, wherein the extract has the efficacies of: inhibiting the cell growth of adipocytes; increasing the expression of adipose triglyceride lipase (ATGL); increasing the expression of Hormone-sensitive lipase (HSL); suppressing the expression of perilipin-1; increasing the expression of uncoupling protein (UCP); increasing the expression of phosphorylated AMPK (AMP-activated protein kinase); or, increasing the expression of phosphorylated AMPK (phospho-AMP-activated protein kinase).

4. The anti-obesity pharmaceutical composition of claim 1, wherein the Geumhwagyu extract is an extract of flowers, stems, leaves, or roots of Geumhwagyu.

5. A healthy functional food for anti-obesity comprises a low-temperature Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient.

6. The healthy functional food of claim 5, wherein the Geumhwagyu extract is an extract of flowers, stems, leaves, or roots of Geumhwagyu.

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

8. The method of claim 7, wherein the extract has the efficacies of: inhibiting the cell growth of adipocytes; increasing the expression of adipose triglyceride lipase (ATGL); increasing the expression of Hormone-sensitive lipase (HSL); inhibiting the expression of perilipin-1; increasing the expression of uncoupling protein (UCP); or, increasing the expression of phosphorylated AMPK (phospho-AMP-activated protein kinase).

9. The method of claim 7, wherein the Geumhwagyu extract is an extract of flowers, stems, leaves, or roots of Geumhwagyu.

10. A method of preventing or treating obesity, comprising: administering a healthy functional food comprising a low-temperature Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient to a subject.

11. The method of claim 10, wherein the Geumhwagyu extract is an extract of flowers, stems, leaves, or roots of Geumhwagyu.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a cell image of lipid accumulation according to the Oil Red O experiment using a low-temperature extract of Geumhwagyu leaf (HML), the absorbance result of the cell eluate, and an image of Western blot results showing increased expression of lipolysis-related proteins ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase), inhibition of Perilipin-1 expression, increased expression of UCP (uncoupling protein), increased expression of phosphorylated phospho-AMP-activated protein kinase (AMPK), which is a thermogenic protein.

[0023] FIG. 2 is an image of Western blot results showing that a low-temperature extract of Geumhwagyu leaf (HML) inhibits the cell growth of mature adipocytes and suppresses the expression of β-catenin and cyclin D1, which are cell growth-related proteins.

[0024] FIG. 3 is a graph showing the results of cell images and absorbance of cell eluate for lipid accumulation according to the Oil Red O experiment of Geumhwagyu leaf extract (HML) according to extraction temperature and extraction time conditions.

[0025] FIG. 4 is a graph showing the results of cell images and absorbance of the cell eluate for lipid accumulation according to the Oil Red O experiment of Geumhwagyu leaf extract (HML) extracted for 24 hours for each extraction temperature condition.

[0026] FIG. 5 is a graph showing the results of the cell image and the absorbance of the cell eluate for the lipid accumulation according to the Oil Red O experiment of the Geumhwagyu leaf extract (HML) extracted at 40° C. for each extraction time condition.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Hereinafter, preferred embodiments of the present disclosure will be described in detail.

[0028] 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

[0029] Geumhwagyu leaf extract was prepared through the following process. First, each of the leaves was washed and dried with distilled water, and then 20 times the volume of water was added to 10 g of dry pulverized material, and then immersed according to conditions with a temperature difference of 1 to 20 hours at 4 to 100° C. to obtain each leaf extract, respectively. Thereafter, the extract was filtered and then lyophilized to obtain a final Geumhwagyu leaf extract (HML).

Example 2: Cytotoxicity Assay

[0030] 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.

[0031] 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% CO2, 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-diphenyltetrazolium 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).

[0032] 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.

[0033] 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: Lipid Formation and Accumulation Inhibitory Activity of Geumhwagyu Extract

[0034] 313-L1 cells, which are pre-adipocytes, were aliquoted into 24 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 0 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).

[0035] Each experimental result is described in FIGS. 1 to 7. FIG. 1 shows an image of Western blot results showing the increased expression of Adipose triglyceride lipase (ATGL) and Hormone-sensitive lipase (HSL) and expression suppression of Perilipin-1, which are lipolysis-related proteins of an extract of Geumhwagyu leaf (HML) obtained by extraction at 40° C. for 20 hours, increased expression of UCP (uncoupling protein), a thermogenic protein, and increased expression of phosphorylated AMP-activated protein kinase (p-AMPK) related to energy metabolism enhancement. Referring to FIG. 1, it may be confirmed that the Geumhwagyu leaf extract has the most excellent lipid accumulation inhibitory effect when extracted at a low temperature.

[0036] Next, the effects of the Geumhwagyu leaf extract were compared once again with the root, stem, and flower extracts. As a result, as shown in Table 1 below, it can be seen that, like the leaf extract, the extracts for different parts also have very excellent lipid accumulation inhibitory effects.

TABLE-US-00001 TABLE 1 Lipid accumulation (fold) Extract Extract Extract treatment treatment treatment concen- concen- concen- Extract (40° C. tration tration tration water, 20 hours) 0 μg/ml 10 μg/ml 30 μg/ml Fat differentiation induction + 1.00 0.29 0.19 Geumhwagyu root extract Fat differentiation induction + 1.00 0.26 0.21 Geumhwagyu Stem Extract Fat differentiation induction + 1.00 0.21 0.14 Geumhwagyu flower extract Fat differentiation induction + 1.00 0.25 0.17 Geumhwagyu leaf extract

[0037] In FIG. 2, a low-temperature extract of Geumhwagyu leaves (HML) obtained by extraction at 40° C. for 20 hours suggests inhibiting the cell growth of mature adipocytes and suppressing the expression of cell growth-related proteins β-catenin and cyclin D1.

[0038] In FIG. 3, it can be confirmed the lipid accumulation inhibitory effect of the Geumhwagyu leaf extract (HML) according to extraction temperature and extraction time conditions. In FIG. 4, each extraction temperature condition, and in FIG. 5, each extraction time is confirmed in more detail. From these results, based on the cells induced to differentiate into adipocytes, Geumhwagyu leaf extract (HML) had an effect of inhibiting lipid accumulation by up to 88% at 30 μg/ml.

[0039] In addition, FIGS. 6 and 7 show the difference in efficacy between the extract (HMF) extracted with low-temperature water at 40° C. for 20 hours and the ethanol aqueous solution extract and methanol aqueous solution extract.

[0040] As described above, referring to FIGS. 1 to 5, it can be confirmed that each Geumhwagyu leaf extract inhibits lipid accumulation through the cell image, which is the result of the Oil Red O experiment, and the absorbance result of the cell eluate.

[0041] On the other hand, the ethanol aqueous solution extract and the methanol aqueous solution extract of the Geumhwagyu leaves were compared with the water extract of the Geumhwagyu leaves, and the experimental results of Oil Red O were compared in Tables 2 and 3. The ethanol aqueous solution extract and the methanol aqueous solution extract were prepared by extracting using ethanol and methanol as an extraction solvent instead of water, followed by distillation under reduced pressure.

TABLE-US-00002 TABLE 2 Lipid accumulation (fold) Extract treatment Extract (40° C.) concentration 30 μg/ml Induction of fat differentiation 1.00 Fat differentiation induction + 0.19 HML-water extract Fat differentiation induction + 0.95 HML-30 (v/v) % ethanol aqueous solution extract Fat differentiation induction + 0.95 HML-50 (v/v) % ethanol aqueous solution extract Fat differentiation induction + 0.93 HML-70 (v/v) % ethanol aqueous solution extract

TABLE-US-00003 TABLE 3 Lipid accumulation (fold) Extract treatment Extract (40° C.) concentration 30 μg/ml Induction of fat differentiation 1.00 Fat differentiation induction + 0.17 HML-water extract Fat differentiation induction + 0.92 HML-30% (v/v) methanol aqueous solution extract Fat differentiation induction + 0.93 HML-50 (v/v) methanol aqueous solution extract Fat differentiation induction + 0.96 HML-70 (v/v) methanol aqueous solution extract

[0042] Referring to Tables 2 and 3 above, the lipid accumulation effect is very excellent in the extract 30 μg/ml extracted with low-temperature water at 40° C. for 20 hours, but the lipid accumulation effect is very insignificant in the extract extracted with ethanol aqueous solution or methanol aqueous solution.

[0043] Through these results, it can be verified that the Geumhwagyu leaf extract has anti-obesity activity by increasing the energy consumption of adipocytes and enhancing energy metabolism.

Formulation Example 1. Pharmaceutical Preparations

[0044] 200 g of the extract for each part 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

[0045] Cooking condiments for health promotion were prepared by adding the extract of each part of Geumhwagyu of the present disclosure to the cooking condiments at 1% by weight.

Formulation Example 2-2. Flour Food Manufacturing

[0046] The extract for each part 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

[0047] The extract of each part 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

[0048] The extract of each part 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

[0049] Vegetable juice for health promotion was prepared by adding 0.5 g of the extract of each part of Geumhwagyu of the present disclosure to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit Juice Manufacturing

[0050] Fruit juice for health promotion was prepared by adding 0.1 g of the extract for each part of Geumhwagyu of the present disclosure to 1,000 ml of apple juice or grape juice.