COMPOSITION FOR FAT FORMATION INHIBITION AND BODY FAT REDUCTION, CONTAINING HYDRANGENOL AS ACTIVE INGREDIENT

Abstract

Provided is a composition for inhibiting fat formation and reducing body fat, the composition including hydrangenol as an active ingredient. The composition of the present disclosure reduces fat accumulation in adipocytes, reduces phosphorylation of mammalian target of rapamycin (mTOR), and increases phosphorylation of forkhead box O1 (FoxO1), and finally, leading to reduction of an expression level of peroxisome proliferator-activated receptor gamma y (PPAR), and as a result, the composition inhibits formation of triglyceride in adipocytes to exhibit an anti-obesity effect. Accordingly, the composition including hydrangenol disclosed herein as an active ingredient may be usefully applied to the fields of health functional foods or cosmetics for inhibiting fat formation.

Claims

1. A health functional food composition for preventing or improving obesity, the health functional food composition comprising hydrangenol or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The health functional food composition of claim 1, wherein the hydrangenol is represented by the following Formula 1: ##STR00002##

3. The health functional food composition of claim 1, wherein the hydrangenol or the pharmaceutically acceptable salt thereof inhibits fat formation or reduces body fat.

4. The health functional food composition of claim 1, wherein the hydrangenol is isolated from a hydrangea extract.

5. A pharmaceutical composition for preventing or treating obesity, the pharmaceutical composition comprising hydrangenol or a pharmaceutically acceptable salt thereof as an active ingredient.

6. The pharmaceutical composition of claim 5, wherein the hydrangenol or the pharmaceutically acceptable salt thereof inhibits fat formation or reduces body fat.

7. The pharmaceutical composition of claim 5, wherein the hydrangenol is isolated from a hydrangea extract

8. A health functional food composition for preventing or improving obesity, the health functional food composition comprising a hydrangenol-comprising hydrangea extract as an active ingredient.

9. The health functional food composition of claim 8, wherein the hydrangea extract is extracted with water, C1 to C4 alcohol, or a mixed solvent thereof.

10. The health functional food composition of claim 8, wherein the hydrangea extract is a hot water extract.

11. A pharmaceutical composition for preventing or treating obesity, the pharmaceutical composition comprising a hydrangenol-comprising hydrangea extract as an active ingredient.

12-21. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0065] FIG. 1 shows results of HPLC analysis of hydrangenol included in a hot water extract of hydrangea leaves and a hydrangea extract (Hydrangea serrata);

[0066] FIG. 2 shows images and graphs showing results of Oil Red-O staining and quantification to examine changes in triglyceride accumulation in adipocytes treated with hydrangenol or the hydrangea extract;

[0067] FIG. 3 shows results of Western blotting to examine expression of triglyceride-regulating proteins in adipocytes treated with hydrangenol or the hydrangea extract;

[0068] FIG. 4 shows graphs showing changes in the body weight when a hot water extract of hydrangea leaves was administered to mice at the same time with induction of obesity;

[0069] FIG. 5 shows graphs showing changes in the body weight when the hot water extract of hydrangea leaves was administered to mice after induction of obesity;

[0070] FIG. 6 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when the hot water extract of hydrangea leaves was administered to mice at the same time with induction of obesity in order to examine body fat-reducing effects of the hot water extract of hydrangea leaves;

[0071] FIG. 7A shows a graph showing a body fat content when the hot water extract of hydrangea leaves was administered to mice at the same time with induction of obesity; and FIG. 7B shows a graph showing a fat weight when the hot water extract of hydrangea leaves was administered to mice at the same time with induction of obesity;

[0072] FIG. 8 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when the hot water extract of hydrangea leaves was administered to mice after induction of obesity in order to examine body fat-reducing effects of the hot water extract of hydrangea leaves;

[0073] FIG. 9A shows a graph showing a body fat content when the hot water extract of hydrangea leaves was administered to mice after induction of obesity; and FIG. 9B shows a graph showing a fat weight when the hot water extract of hydrangea leaves was administered to mice after induction of obesity;

[0074] FIG. 10 shows microscopic images of adipocytes to examine the effect of reducing the size of lipid droplets by the hot water extract of hydrangea leaves;

[0075] FIG. 11A shows a graph showing cholesterol levels when the hot water extract of hydrangea leaves was administered; and FIG. 11B shows a graph showing low density lipoprotein (LDL) levels when the hot water extract of hydrangea leaves was administered;

[0076] FIG. 12A shows results of examining expression of p-AMPK protein in the adipose tissue when the hot water extract of hydrangea leaves was administered; and FIG. 12B shows results of examining expression of p-AMPK protein in the liver when the hot water extract of hydrangea leaves was administered;

[0077] FIG. 13 shows a graph showing changes in the body weight when hydrangenol was administered to mice in order to examine body weight-reducing effects of hydrangenol;

[0078] FIG. 14 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when hydrangenol was administered to mice in order to examine body fat-reducing effects of hydrangenol;

[0079] FIG. 15A shows a graph showing a body fat content when hydrangenol was administered; and FIG. 15B shows a graph showing a fat weight when hydrangenol was administered;

[0080] FIG. 16 shows microscopic images of adipocytes to examine the effect of reducing the size of lipid droplets by hydrangenol;

[0081] FIG. 17A shows a graph showing cholesterol levels when hydrangenol was administered; and FIG. 17B shows a graph showing LDL levels when hydrangenol was administered; and

[0082] FIG. 18A shows results of examining expression of p-AMPK protein in the adipose tissue when hydrangenol was administered; and FIG. 18B shows results of examining expression of p-AMPK protein in the liver when hydrangenol was administered.

MODE OF DISCLOSURE

[0083] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not be construed as being limited to these exemplary embodiments.

EXAMPLE 1

Preparation of Hydrangenol-Containing Hydrangea Extract

[0084] A hydrangea extract in a composition of the present disclosure was prepared by the following procedure. First, 20 kg of dried hydrangea (Hydrangea serrata) raw material and 300 kg of purified water were put in an extraction tank, and then subjected to reflux extraction at 100° C. for 5 hours. The extracted sample was filtered using a cartridge filter (10 μm), and then concentrated under reduced pressure, and water-soluble powder was obtained by spray-drying.

EXAMPLE 2

Preparation of Hydrangea Extract-Derived Hydrangenol

[0085] The extract powder obtained in Example 1 was subjected to gel filtration using Diaion HP-20. As a developing solvent, solvent fractionation was performed using each 2 L of a mixed solution of 30%, 50%, 70%, 100% methanol and CH.sub.2Cl.sub.2—MeOH (1:1, v/v), and divided into 5 subfractions (392-70EDia1˜5). The subfraction 392-70EDia4 was divided into 7 subfractions (392-70EDia4a˜4g) using Sephadex LH-20 and methanol as a developing solvent. Among them, the 392-70EDia4d fraction was recrystallized in methanol to obtain an amorphous compound 1 (hydrangenol) as a single material.

[0086] The extract powder obtained in Example 1 and hydrangenol obtained in Example 2 of the present disclosure were analyzed using high-performance liquid chromatography (HPLC) and a UV photometric detector (UV/Vis detector). HPLC instrument was Waters e2695 Series system, Waters 24489 UV/Vis detector (Worcester, Mass., USA), and Luna C18(2)(5 μm, 250 X 4.6 mm, Phenomenex, Torrance, Calif., USA) column was used, and all solvents used in the analysis were HPLC grade solvents purchased from J. T. Baker (Phillipsburg, N.J., USA). During the analysis, a temperature of the column was set at 30° C., an injection volume was set at 20 μl, and a measurement wavelength was set at 210 nm. Acetonitrile (ACN) and tertiary distilled water (D.W) were used as a mobile phase, and an ACN-D.W (2:8-10:0, v/v) mixed solution was analyzed for 50 minutes at a rate of 1 ml/min. As an analysis sample, 100 mg of the extract powder obtained in Example 1 was precisely weighed, 10 ml of methanol was added thereto, and then the powder was dissolved in an ultrasonic shaker for 20 minutes, allowed to cool at room temperature, and the supernatant was obtained and then filtered through a 0.45 pm membrane filter for use. 10 mg of the hydrangenol obtained in Example 2 was precisely weighed and 40 ml of methanol was added thereto, and the hydrangenol was dissolved in an ultrasonic shaker for 20 minutes, allowed to cool at room temperature, and methanol was added thereto, and filtered through a 0.45 μm membrane filter for use. For each analysis sample, a chromatogram was extracted at 210 nm, and a peak of hot water extract of hydrangea leaves and a peak of hydrangenol were compared and analyzed (FIG. 1).

[0087] The structure of Example 2 was first identified by ESI MS (positive-ion mode), and as a result, m/z=257[M+H]+ was observed. In 1H-NMR, it was found that the methine proton (H-3) at δH 5.50 at a high magnetic field and the methylene proton (H-4) at δH 3.30 and 3.06 showed vicinal coupling to each other, and the protons were the chemical shift values and were attributable to the C ring. H-2′, 3′ and H-6′, 5′ attributable to the p-substituted benzene ring of the B-ring were ortho-coupled to each other and appeared as doublets (J=8.4 Hz), and peaks of H-2′ and H-6′ and peaks of H-3′ and H-5′ were also ortho-coupled to each other and appeared as doublets, indicating that they had a symmetric structure around the hydroxyl group. In 1,2,3-trisubstituted benzene of A-ring, H-5 and H-7 hydrogens were coupled with H-6 hydrogen, respectively, and H-5 and H-7 hydrogens were ortho-coupled and appeared as doublet, and H-6 proton was ortho- and meta-coupled and appeared as a double of doublets, and all peaks were found to correspond to one hydrogen.

[0088] 13C-NMR showed a total of 15 peaks including para-substituents. The quaternary carbon at δC 172 was a peak attributable to a carbonyl group which is carbon 1 of the compound, and δC 116.9(C-3′, 5′) and 129.6(C-2′, 6′) are attributable to para substituents of an aromatic ring. Peaks at δC 36.1 and 83.1 were expected to be attributable to an aliphatic carbon and an oxygenated carbon, respectively. In addition, in DEPT NMR, 7 protonated carbons were identified, and a peak of δC 36.1 was found to be a methylene group attributable to C-4. 2D NMR was analyzed to analyze their exact structures. The exact positions of the peaks were identified from HSQC, and the position at which the substituent was bound was identified from HMBC. That is, the peak of δH 7.26 (2H, d, J=8.4 Hz, H-2′, 6′) shows a correlation with C-4 of δC 36.1, and the peaks of δH 3.06 and 3.30 attributable to H-4 show a correlation with the peaks of 83.1 (C-3), 119.8 (C-5), 110.0 (C-9), and 142.2 (C-10). Taken together, hydrangenol was identified.

[0089] FIG. 1 shows results of HPLC analysis of hydrangenol included in the hot water extract of hydrangea leaves and the hydrangea extract (Hydrangea serrata).

EXPERIMENTAL EXAMPLE 1

Evaluation of Fat Accumulation Inhibition by Oil Red O Staining

[0090] In this experiment, to induce adipocyte differentiation, 3T3-L1 cells were dispensed onto a plate and cultured in a 10% BS medium until the cell density reached 100%. At the cell differentiation stage, hydrangenol-containing hydrangea (Hydrangea serrata) (25 ug/ml), hydrangenol (2.5 ug/ml) or a positive control pioglatazone (10 uM) was added to a 10% FBS differentiation medium (5 μg/ml of insulin, 1 μM dexametasone, 0.5 mM 3-isobutyl-1-methylxanthine), respectively. After 10 days of treatment oil Red-O staining and quantitative analysis were performed to determine how much fat accumulation was inhibited. For visual evaluation, images were taken after staining, and then the stained cells were completely dried, dissolved in dimethyl sulfoxide (DMSO), transferred to a 96-well plate, and absorbance at 450 nm was measured.

[0091] FIG. 2 shows images and graphs showing the results of Oil Red-O staining and quantification to examine changes in triglyceride accumulation in adipocytes treated with hydrangenol or the hydrangea extract.

EXPERIMENTAL EXAMPLE 2

[0092] Analysis of expression of adipocyte differentiation-related proteins during hydrangenol treatment

[0093] The mechanism of reducing triglyceride in adipocytes was examined for the hydrangenol-containing hydrangea (Hydrangea serrata) and hydrangenol. A pre-adipocyte 3T3-L1 was differentiated for 10 days and treated with hydrangea (Hydrangea serrata) (25 ug/ml) and hydrangenol (2.5 ug/ml) for 24 hours, respectively. Thereafter, the cells were lysed using a modified LIPA buffer, and each 20 ug thereof was used for analysis. p-mTOR(ab109268, Abcam), p-FOXO1(9461S, Cell Signaling), PPARγ(sc-7273, Santa Cruz), and β-actin(A5316, Sigma) primary antibodies were used for analysis, respectively.

[0094] As shown in FIG. 3, hydrangenol-containing hydrangea (Hydrangea serrata) and hydrangenol were found to reduce phosphorylation of mammalian target of rapamycin (mTOR) and to increase phosphorylation of forkhead box 01 (FoxO1), and finally, leading to reduction of an expression level of peroxisome proliferator-activated receptor gamma y (PPAR), and as a result, triglyceride production in adipocytes was suppressed.

[0095] FIG. 3 shows results of Western blotting to examine expression of triglyceride-regulating proteins in adipocytes treated with hydrangenol or the hydrangea extract.

EXPERIMENTAL EXAMPLE 3

Analysis of In Vivo Effect of Hot Water Extract of Hydrangea Leaves (WHS)

[0096] 3-1. Mouse and Experiment Design

[0097] To analyze in vivo anti-obesity efficacy of WHS, animal models of obesity were first prepared. 8-week-old male C57BL/6N mice (specific-pathogen-free (SPF) grade, 20±2 g, Orient Bio) were set into 7 groups as follows, and 10 mice per each group were tested: as normal control groups, normal mice (con) with no high fat diet and no administration, and obese mice (HFD) with induction of obesity by 30% high fat diet and no administration, and as a positive control group, obese mice with oral administration of orlistat which is an anti-obesity agent. As experimental groups, obese mice with oral administration of WHS of 75 mg/kg, 150 mg/kg, or 300 mg/kg, and normal mice with oral administration of WHS of 300 mg/kg were used. Further, experiments were performed by dividing mice into those which were administered with WHS for 12 weeks at the same time with induction of obesity, and those which were administered with WHS after induction of obesity for 10 weeks. WHS was orally administered for 5 days per week during the administration period. A dark: light cycle was maintained at intervals of 12 hours: 12 hours, and mice were allowed to free access to water.

[0098] 3-2. Analysis of Body Weight- and Fat-Reducing Effects of WHS

[0099] An experiment was performed to analyze whether the body weight and fat of mice decreased when WHS was administered. As a result, when changes in the body weight of the mice according to each experimental group and time were examined, the body weight-reducing effects were observed in the positive control group and the WHS-administered group (FIGS. 4 and 5).

[0100] In addition, images of a fat distribution and body fat contents of mice of each experimental group were measured by dual-energy X-ray absorptiometry in the last week of the animal test. Each mice was sacrificed, and a visceral adipose tissue including epididymal fat was separated and a fat was weighed. The experimental results were tested for a significant difference between groups using a t-test in the Sigma plot statistical program (p#<0.05 vs normal control, p*<0.05, p**<0.01, P***<0.001 vs obese group). As a result, it was confirmed that body fat was decreased in the positive control group and the WHS-administered group (FIGS. 6, 7, 8, and 9).

[0101] FIG. 4 shows graphs showing changes in the body weight when WHS was administered to mice at the same time with induction of obesity.

[0102] FIG. 5 shows graphs showing changes in the body weight when WHS was administered to mice after induction of obesity.

[0103] FIG. 6 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when WHS was administered to mice at the same time with induction of obesity, in order to examine body fat-reducing effects of WHS.

[0104] FIG. 7A shows a graph showing a body fat content when WHS was administered to mice at the same time with induction of obesity; and FIG. 7B shows a graph showing a fat weight when WHS was administered to mice at the same time with induction of obesity.

[0105] FIG. 8 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when WHS was administered to mice after induction of obesity, in order to examine body fat-reducing effects of WHS.

[0106] FIG. 9A shows a graph showing a body fat content when WHS was administered to mice after induction of obesity; and FIG. 9B shows a graph showing a fat weight when WHS was administered to mice after induction of obesity.

[0107] 3-3. Analysis of Adipocyte Size-Reducing Effects of WHS

[0108] For histological analysis, the epididymal fat tissues of mice were fixed in 4% paraformalin. Dehydration was performed several times through graded alcohol series and washing, and each tissue was embedded in paraffin. Each tissue section was cut at a thickness of 4 μm, and stained with hematoxylin and eosin. In order to examine the size of white adipocytes, the adipocyte area of each section was measured with cellSence software (Olympus Co., USA). As a result, it was confirmed that the adipocyte size was reduced in the group administered with WHS at the same time with induction of obesity (FIG. 10).

[0109] FIG. 10 shows microscopic images of adipocytes to examine the effect of reducing the size of lipid droplets by WHS.

[0110] 3-4. Analysis of Effects of WHS on Liver and Kidney

[0111] To examine whether WHS induced liver and kidney damage in mice, glutamic oxalacetic transaminase (GOT), glutamic pyruvate transaminase (GPT), and blood urea nitrogen (BUN) of the WHS-administered group with induction of obesity were measured by serum analysis using a biochemical analyzer (AU480 Chemistry Analyzer, Beckman coulter, Calif., USA). As a result, as shown in Table 1, there was no significant difference between 7 groups. These results suggest that WHS does not cause liver and kidney damage.

TABLE-US-00001 TABLE 1 HFD + HFD + HFD + WHS WHS WHS WHS HFD + 75 150 300 300 CON HFD Orlistat mg/kg mg/kg mg/kg mg/kg GOT 60.80 ± 64.83 ± 63.00 ± 62.71 ± 60.50 ± 57.78 ± 58.38 + μL) 15.93 11.32 18.56 17.99 12.82 8.36 15.11 GPT 20.43 ± 25.58 ± 17.60 ± 17.40 ± 18.50 ± 16.71 ± 18.89 ± (μL) 2.44 4.91 2.17 2.32 2.73 2.93 5.04 BUN 22.04 ± 18.75 ± 19.89 ± 18.79 ± 19.47 ± 19.00 ± 25.58 ± (mg/dL) 2.58 2.66 3.23 1.12 1.86 2.11 2.65

[0112] 3-5. Analysis of Changes of Blood Triglyceride and Blood cholesterol by WHS

[0113] To analyze the effects of WHS on blood triglyceride and blood cholesterol, a hematological-biochemical test of the WHS-administered group with induction of obesity was performed using a biochemical analyzer (AU480 Chemistry Analyzer, Beckman coulter, Calif., USA). As a result, as shown in Table 2 and FIG. 11, it was confirmed that the WHS-administered group with induction of obesity showed reduction in the total cholesterol, triglyceride, and LDL levels, but no significant difference in the HDL level. These results indicate that WHS has a prophylactic effect on obesity by reducing total cholesterol, LDL and triglyceride without affecting HDL level.

[0114] FIG. 11A shows a graph showing cholesterol levels when WHS was administered; and FIG. 11B shows a graph showing low density lipoprotein (LDL) levels when WHS was administered.

TABLE-US-00002 TABLE 2 HFD + HFD + WHS HFD + WHS HFD + WHS WHS CON HFD Orlistat 75 mg/kg 150 mg/kg 300 mg/kg 300 mg/kg CHOL 93.00 ± 129.75 ± 115.63 ± 129.11 ± 124.22 ± 117.33 ± 93.60 ± (mg/dl) 3.80 5.93 11.87 5.49 9.31 15.29 11.78 LDL 7.63 ± 9.00 ± 8.60 ± 8.88 ± 8.44 ± 8.20 ± 6.89 ± (mg/dl) 0.92 1.00 0.84 0.64 1.33 0.92 0.93 HDL 69.82 ± 84.73 ± 83.70 ± 85.20 ± 82.44 ± 75.40 ± 72.11 ± (mg/dl) 8.89 4.65 5.96 6.12 8.43 12.20 6.64 TG 46.09 ± 57.27 ± 76.40 ± 44.70 ± 76.11 ± 62.70 ± 64.00 ± (mg/dl) 7.08 9.55 9.47 6.55 14.49 16.87 14.74

[0115] 3-6. Analysis of Expression of Energy Metabolism-Related Proteins by WHS

[0116] AMP-activated protein kinase (AMPK) is activated when energy in hepatocytes decreases to maintain energy homeostasis in the liver, thereby inhibiting synthesis of fat and cholesterol, and conversely, promoting fatty acid oxidation. Therefore, to confirm whether administration of WHS increased AMPK expression, the protein expression level of the WHS-administered group with induction of obesity was analyzed.

[0117] Specifically, adipose and liver tissues were mixed with a protein extraction solution, homogenized using a tissue homogenizer, and then centrifuged at 4° C., 15,000 rpm for 30 minutes to obtain a supernatant, and then a standard curve was created using the Bradford method to quantify protein. 6 X sample buffer was added to 30 pg of the protein, followed by heating in a water bath for 5 minutes. Electrophoresis was performed using a 10% SDS-PAGE gel, and immunoblotting was performed on a PVDF membrane for 1 hour and 20 minutes. After blocking for 1 hour with a Tris-buffered saline-Tween 20 (TBST) buffer solution containing 5% (w/v) skim milk, anti-p-AMPK antibody was diluted to 1:1000 and reacted at 4° C. for 18 hours. After washing three times with the TBST buffer solution for 10 minutes, the membrane was reacted with a peroxidase-conjugated secondary antibody for 2 hours at room temperature. After washing three times for 10 minutes with the TBST buffer solution, a hyper film was color-developed and developed using an enhanced chemiluminescence kit (Amersham Life Sciences, Amersham, U.K.) to examine the change of AMPK phosphorylation in each control group and experimental group by Western blotting. As a result, when WHS was administered, the amount of phosphorylated AMPK protein increased (FIG. 12).

[0118] These results indicate that WHS has the AMPK phosphorylation-inducing effect which is critical in the anti-obesity effect.

[0119] FIG. 12A shows results of examining expression of p-AMPK protein in the adipose tissue when WHS was administered; and FIG. 12B shows results of examining expression of p-AMPK protein in the liver when WHS was administered.

EXPERIMENTAL EXAMPLE 4

Analysis of In Vivo Effect of Hydrangenol (HG)

[0120] 4-1 Mouse and Experiment Design

[0121] To analyze in vivo anti-obesity efficacy of HG, animal models of obesity were prepared. 8-week-old male C57BL/6N mice (SPF grade, 20±2 g, Orient Bio) were set into 7 groups as follows, and 10 mice per each group were tested: as normal control groups, normal mice (con) with no high fat diet and no administration, and obese mice (HFD) with induction of obesity by 30% high fat diet and no administration, and as a positive control group, obese mice with oral administration of orlistat which is an anti-obesity agent. As experimental groups, obese mice with oral administration of HG of 20 mg/kg, 40 mg/kg, or 80 mg/kg, and normal mice with oral administration of HG of 80 mg/kg were used. Mice were administered with HG at the same time with induction of obesity, and HG was administered for 5 days per week for 12 weeks. A dark: light cycle was maintained at intervals of 12 hours: 12 hours, and mice were allowed to free access to water.

[0122] 4-2 Analysis of Body Weight- and Fat-Reducing Effects of HG

[0123] An experiment was performed to analyze whether the body weight and fat of mice decreased when HG was administered. As a result, when changes in the body weight of the mice according to each experimental group and time were examined, the body weight-reducing effects were observed in the positive control group and the HG-administered group (FIG. 13).

[0124] In addition, images of a fat distribution and body fat contents of mice of each experimental group were measured by dual-energy X-ray absorptiometry in the last week of the animal test. Each mice was sacrificed, and a visceral adipose tissue including epididymal fat was separated and a fat was weighed. The experimental results were tested for a significant difference between groups using a t-test in the Sigma plot statistical program (p#<0.05 vs normal control, p*<0.05, r<0.01, P***<0.001 vs obese group). As a result, it was confirmed that body fat was decreased in the positive control group and the HG-administered group (FIGS. 14 and 15).

[0125] FIG. 13 shows a graph showing changes in the body weight when HG was administered to mice in order to examine body weight-reducing effects of HG.

[0126] FIG. 14 shows images of a fat distribution and a body fat content which were measured by dual-energy X-ray absorptiometry, when HG was administered to mice in order to examine body fat-reducing effects of HG.

[0127] FIG. 15A shows a graph showing a body fat content when HG was administered; and FIG. 15B shows a graph showing a fat weight when HG was administered.

[0128] 4-3 Analysis of Adipocyte Size-Reducing Effects of HG

[0129] For histological analysis, the epididymal fat tissues of mice were fixed in 4% paraformalin. Dehydration was performed several times through graded alcohol series and washing, and each tissue was embedded in paraffin. Each tissue section was cut at a thickness of 4 μm, and stained with hematoxylin and eosin. In order to examine the size of white adipocytes, the adipocyte area of each section was measured with cellSence software (Olympus Co., USA). As a result, it was confirmed that the adipocyte size was reduced when HG was administered (FIG. 16).

[0130] FIG. 16 shows microscopic images of adipocytes to examine the effect of reducing the size of lipid droplets by HG.

[0131] 4-4 Analysis of Effects of HG on Liver and Kidney

[0132] To examine whether HG induced liver and kidney damage, glutamic oxalacetic transaminase (GOT), glutamic pyruvate transaminase (GPT), and blood urea nitrogen (BUN) were measured by serum analysis using a biochemical analyzer (AU480 Chemistry Analyzer, Beckman coulter, Calif., USA). As a result, as shown in Table 3, there was no significant difference between 7 groups. These results suggest that HG does not cause liver and kidney damage.

TABLE-US-00003 TABLE 3 HFD + HFD + HFD + HFD + HG 20 HG 40 HG 80 HG 80 CON HFD Orlistat mg/kg mg/kg mg/kg mg/kg GOT 60.80 ± 64.83 ± 63.00 ± 54.88 ± 54.14 ± 53.63 ± 60.14 ± (μL) 15.93 11.32 18.56 11.66 10.29 9.91 8.73 GPT 20.43 ± 25.58 ± 17.60 ± 16.00 ± 15.71 ± 15.00 ± 16.89 ± (μL) 2.44 4.91 2.17 5.03 1.60 2.98 2.62 BUN 22.04 ± 18.75 ± 19.89 ± 21.91 ± 22.93 ± 25.39 ± 27.79 ± (mg/dl) 2.58 2.66 3.23 3.33 2.74 5.02 4.85

[0133] 4-5 Analysis of Changes of Blood Triglyceride and Blood Cholesterol by HG

[0134] To analyze the effects of HG on blood triglyceride and blood cholesterol, a hematological-biochemical test was performed using a biochemical analyzer (AU480 Chemistry Analyzer, Beckman coulter, Calif., USA).

[0135] As a result, as shown in Table 4 and FIG. 17, it was confirmed that the HG-administered group showed reduction in the total cholesterol and LDL, but no significant difference in the triglyceride and HDL levels. These results indicate that HG has the effects of improving bad blood lipid levels caused by obesity by reducing total cholesterol and LDL while not affecting triglyceride and HDL levels.

[0136] FIG. 17A shows a graph showing cholesterol levels when HG was administered; and FIG. 17B shows a graph showing LDL levels when HG was administered.

TABLE-US-00004 TABLE 4 HFD + HFD + HFD + HFD + HG 20 HG 40 HG 80 HG 80 CON HFD Orlistat mg/kg mg/kg mg/kg mg/kg CHOL 93.00 ± 129.75 ± 115.63 ± 126.78 ± 112.56 ± 110.14 ± 96.20 ± (mg/dl) 3.80 5.93 11.87 6.63 13.64 13.90 18.64 LDL 7.63 ± 9.00 ± 8.60 ± 7.78 ± 7.38 ± 7.13 ± 5.70 ± (mg/dl) 0.92 1.00 0.84 0.44 0.74 1.13 0.67 HDL 69.82 ± 84.73 ± 83.70 ± 80.78 ± 71.89 ± 71.20 ± 66.40 ± (mg/dl) 8.89 4.65 5.96 2.91 6.33 8.16 13.75 TG 46.09 ± 57.27 ± 76.40 ± 74.63 ± 58.89 ± 63.78 ± 80.10 ± (mg/dl) 7.08 9.55 9.47 10.68 11.92 9.94 14.16

[0137] 4-6 Analysis of Expression of Energy Metabolism-Related Proteins by HG

[0138] AMPK is activated when energy in hepatocytes decreases to maintain energy homeostasis in the liver, thereby inhibiting synthesis of fat and cholesterol, and conversely, promoting fatty acid oxidation. Therefore, to confirm whether administration of HG increased AMPK expression, an experiment was performed.

[0139] Specifically, adipose and liver tissues were mixed with a protein extraction solution, homogenized using a tissue homogenizer, and then centrifuged at 4° C., 15,000 rpm for 30 minutes to obtain a supernatant, and then a standard curve was created using the Bradford method to quantify protein. 6 X sample buffer was added to 30 μg of the protein, followed by heating in a water bath for 5 minutes. Electrophoresis was performed using a 10% SDS-PAGE gel, and immunoblotting was performed on a PVDF membrane for 1 hour and 20 minutes. After blocking for 1 hour with TBST buffer solution containing 5% (w/v) skim milk, anti-p-AMPK antibody was diluted to 1:1000 and reacted at 4° C. for 18 hours. After washing three times with the TBST buffer solution for 10 minutes, the membrane was reacted with a peroxidase-conjugated secondary antibody for 2 hours at room temperature. After washing three times for 10 minutes with the TBST buffer solution, a hyper film was color-developed and developed using an enhanced chemiluminescence kit (Amersham Life Sciences, Amersham, U.K.) to examine the change of AMPK phosphorylation in each control group and experimental group. As a result, in the HG-treated group, the amount of phosphorylated AMPK protein increased (FIG. 18).

[0140] These results indicate that HG has the AMPK phosphorylation-inducing effect which is critical in the anti-obesity effect.

[0141] FIG. 18A shows results of examining expression of p-AMPK protein in the adipose tissue when HG was administered; and FIG. 18B shows results of examining expression of p-AMPK protein in the liver when HG was administered.

PREPARATION EXAMPLE 1

Preparation of Tablet

[0142] A tablet was prepared by mixing components of Table 5 below with hydrangenol and tableting the mixture according to a common method of preparing a tablet.

TABLE-US-00005 TABLE 5 Name of raw material Unit weight (mg) Hydrangenol 10.0006 Silicon dioxide 15.3000 Magnesium stearate 10.8000 Crystalline cellulose 799.4945 Hydroxypropyl methylcellulose 29.0700 Calcium carboxymethyl cellulose 27.0000 Glycerin fatty acid ester 0.6930 Titanium dioxide 1.4697 Monascus red 4.4082 Caramel pigment powder 1.7640

PREPARATION EXAMPLE 2

Preparation of Capsule

[0143] A capsule was prepared by mixing components of Table 6 below with hydrangenol and packing a gelatin capsule with the mixture according to a common method of preparing a capsule.

TABLE-US-00006 TABLE 6 Name of raw material Unit weight (mg) Hydrangenol 2 Vitamin E 2.25 Vitamin C 2.25 Palm oil 0.5 Hydrogenated vegetable oil 2 Yellow wax 1 Lecithin 2.25 Soft capsule filling solution 387.75

PREPARATION EXAMPLE 3

Preparation of Jelly

[0144] A jelly was prepared by mixing components of Table 7 below with hydrangenol and packing a three-sided pack with the mixture according to a common method of preparing a jelly suitable for preference.

TABLE-US-00007 TABLE 7 Name of raw material Unit weight (mg) Hydrangenol 0.0030 Food gel 0.3600 Carrageenann 0.0600 Calcium lactate 0.1000 Sodium citrate 0.0600 Complex Scutellaria 0.0200 baicalensis extract Enzyme-treated stevia 0.0440 Fructooligosaccharide 5.0000 solution Red grape concentrate 2.4000 Purified water 13.9560

PREPARATION EXAMPLE 4

Preparation of Nourishing Cream

[0145] A nourishing cream was prepared using hydrangenol according to a composition of Table 8 below according to a common method.

TABLE-US-00008 TABLE 8 Raw material Content (%) Hydrangenol 0.01 Sitosterol 4.0 Polyglyceryl-2 oleate 3.0 3.0 Ceteareth-4 2.0 Cholesterol 3.0 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Sunflower oil 22.0 Carboxyvinyl polymer 0.5 Triethanolamine 0.5 Preservative Trace amount Flavoring Trace amount Purified water Residual quantity

[0146] The above composition ratio is generally formulated as a Preparation Example by mixing suitable ingredients, but the mixing ratio and raw materials may be arbitrarily changed, as needed.

[0147] Since the samples of the present disclosure are stable under the experimental conditions of all Preparation Examples, there is no problem in stability of the formulations.