KR101562325B1 - Composition for Anti-obesity Containing Dibenzoylmethane - Google Patents

Abstract

The present invention relates to a composition for the treatment or prevention of obesity or cholesterolemia comprising dibenzoylmethane as an active ingredient, and a health food for improving obesity or cholesterolemia.
According to the present invention, dibenzoylmethane is excellent in the effect of reducing body weight, decreasing body fat and reducing total cholesterol, and is useful for the treatment or prevention of obesity or cholesterol.

Description

TECHNICAL FIELD The present invention relates to a composition for treating or preventing obesity containing dibenzoylmethane,

The present invention relates to a pharmaceutical composition for treating or preventing obesity or cholesterolemia containing dibenzoylmethane as an active ingredient and a health food for improving obesity or cholesterolemia.

Obesity refers to the state of excess fat tissue accumulation in the body due to the increase in the size or number of adipocytes, which means that there is more fat than the amount of fat needed for the body.

The causes of obesity are food intake, lack of exercise, and genetic susceptibility. Obesity has negative consequences for health and quality of life, such as type 2 diabetes, hypertension, hypercholesterolemia, and cardiovascular disease, and the social costs are gradually increasing. In the United States, obesity causes more than 300,000 deaths annually, making it the second most preventable cause of death (McGinnis JM and Foege WH, JAMA 270: 2207-2212, 1993) (Colditz GA, Am. J. Clin. Nutr. 55: 503S-507S, 1992).

As such, the metabolic diseases caused by obesity are diverse and serious, and prevention and treatment are urgently needed to prevent them.

 Currently, obesity treatment drugs are largely classified into satiety suppressants, lipid absorption inhibitors, and psychotropic appetite suppressants depending on the action mechanism.

Saturation stimulants inhibit appetite by inhibiting the reuptake of the neurohormones serotonin and noradrenalin, which regulate appetite in the brain, and increase energy expenditure by increasing basal metabolic rate. Reductil, which is a representative product, was withdrawn from the market after the decision to discontinue the final sale at KFDA in October 2010 due to the adverse effects of cardiovascular factors such as myocardial infarction and stroke in the product, Sibutramine.

The fat absorption inhibitor suppresses the function of lipase, a digestive enzyme that absorbs fat into the body, and discharges the fat that is ingested to the outside of the body. As such a lipid absorption inhibitor, Orlistat component is Xenical. Xenical, which is known to have the least side effects, also shows adverse effects such as fat stasis, intestinal gas production, and abdominal bloating. Recently, patients who complain of kidney trouble have also appeared.

Psychotropic appetite suppressants treat obesity by promoting the production of the neurohormones norepinephrine and dopamine, which regulate appetite in the brain, and by reducing the appetite itself, and its components are phendimetrazine, (phentermine).

Leptin (leptin) is another candidate for obesity treatment. It releases blood from the adipocytes and passes through the blood-brain barrier, acts on receptors in the central nervous system, inhibiting food intake, reducing body weight, and promoting energy consumption. In obese patients, the hypothalamus is constantly exposed to leptin, resulting in resistance to leptin and hyperleptinemia. Although there is a high concentration of leptin in the blood, it is difficult to develop an obesity-controlling drug using leptin because it can not transmit an appetite suppression signal and a metabolic signal.

AMPK (AMP kinase) is an enzyme that acts as a sensor to maintain the energy homeostasis in the cell, and is activated when the ATP / ATP ratio is increased by depletion of ATP, thereby inhibiting the ATP consumption process and promoting the ATP production process. AMPK is known to play an important role in the regulation of liver fat metabolism by mediating the synthesis and degradation of fatty acids. Activation of AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), enzymes necessary for ATP-consuming fatty acid synthesis and cholesterol synthesis, acid synthase, pyruvate kinase, and ACC (Foretz M. et al., J Biol Chem 273: 14767-14771, 1998; Woods A. et al., Mol Cell Biol, 20: 6704-6711, 2000). In addition, AMPK activation has been reported to play a role in increasing fatty acid oxidation in muscle (Minokoshi Y. et al., Nature 415: 339-343, 2002).

On the other hand, dibenzoylmethane is a compound having a structure represented by the following general formula (1) as a component that can be extracted from licorice (Glycyrrhiza glabra), and a sunscreen (Nogueira MA et al., Farmaco, 58 2, 51 (14): 3977-9), antioxidants (Hegedus C. et al., Pharmacol Res, 72: 25-34, 2013), anticancer agents (Pan MH et al., J Agric Food Chem. (2): 41-9, 2003), but it has been shown to be effective in the treatment or prevention of obesity or cholesterolemia Is not known.

Accordingly, the present inventors have made extensive efforts to develop an obesity inhibitor, and as a result, it has been confirmed that dibenzoylmethane has an excellent effect of inhibiting obesity or cholesterolemia by increasing the expression or activity of AMPK (AMP kinase) .

It is an object of the present invention to provide a medicinal composition for treating or preventing obesity or cholesterol, which contains dibenzoylmethane as an active ingredient, and a health functional food for improving obesity or cholesterol.

To achieve the above object, there is provided a pharmaceutical composition for the treatment or prevention of obesity comprising dibenzoylmethane as an active ingredient.

The present invention also provides a health functional food for improving obesity containing dibenzoylmethane as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment or prevention of cholesterolemia comprising dibenzoylmethane as an active ingredient.

The present invention also provides a health functional food for improving cholesterolemia containing dibenzoylmethane as an active ingredient.

According to the present invention, dibenzoylmethane is excellent in the effect of reducing body weight, decreasing body fat and reducing total cholesterol, and is useful for the treatment or prevention of obesity or cholesterol.

FIG. 1 shows the results of experiments in which the phosphorylation of AMP-activated kinase (AMPK) was increased in skeletal muscle cell lines by DBM.
Figure 2 shows the results of experiments confirming that the increase in glucose uptake by DBM works through the AMPK signaling pathway.
FIG. 3 shows the results of experiments confirming that DBM-induced AMPK phosphorylation is increased by the release of intracellular calcium ions.
Figure 4 shows the results of experiments confirming that increased phosphorylation of p38 MAPK by DBM is driven by the AMPK signaling pathway.
Figure 5 shows the results of experiments confirming that GLUT4 gene expression and translocation are increased by DBM.
FIG. 6 is a graph showing the results of experiments in which weight loss and inhibition of body fat accumulation by DBM were confirmed.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a pharmaceutical composition for treating or preventing obesity containing dibenzoylmethane as an active ingredient.

The dibenzoylmethane of the present invention may be characterized in that the expression or activity of AMPK (AMP kinase) is increased to regulate lipid metabolism.

In one embodiment of the present invention, dibenzoylmethane was treated with dibenzoylmethane to analyze the activity of AMPK. As a result, it was found that the concentration of AMPK phosphorylated in muscle precursor cells by dibenzoylmethane treatment was higher than that of dibenzoylmethane (Fig. 1), and it was confirmed that phosphorylation of ACC regulated by AMPK and AMPK by dibenzoylmethane increases in a concentration-dependent manner (Fig. 1); It was confirmed that the concentration of intracellular calcium was increased by CaMKK (Calcium / calmodulin-dependent protein kinase kinase) when dibenzoylmethane was treated (Fig. 3)

In one embodiment of the present invention, it was confirmed that the dibenzoylmethane treatment increased the sugar uptake capacity by AMPK (Fig. 2), and the p38 mitogen-associated protein kinase (MAPK) (Fig. 4), and that it promotes the expression of GLUT4 gene, which is a sub-step in the signaling pathway of p38 mitogen-associated protein kinase (MAPK), and its migration to the surface of the cell surface (Fig. 5).

The dibenzoylmethane of the present invention increases the activity of AMPK through phosphorylation of AMPK and reduces weight gain, and thus can be used as an active ingredient of a composition for treating or preventing obesity and a health functional food for obesity improvement.

In another aspect, the present invention relates to a health food for improving obesity containing the composition for treating or preventing obesity as an active ingredient.

In another embodiment of the present invention, mice that received only high-fat diet and those that received dibenzoylmethane in combination with high fat diet were compared with mice given dibenzoylmethane, indicating that weight reduction, body fat reduction, and total cholesterol concentration (Fig. 6).

In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing cholesterol hemoglobin comprising dibenzoylmethane as an active ingredient.

In another aspect, the present invention relates to a health food for improving cholesterol hemoglobin, which comprises, as an active ingredient, a composition for treating or preventing cholesterol.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of the compositions.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select parenterally when injecting parenterally or intraperitoneally, intramuscularly, subcutaneously, intravenously, intramuscularly or intrasternally , But is not limited thereto.

The composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods . Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluent, liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / day, preferably 0.001 to 200 / day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The health food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of the food. Examples of the food include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, various soups, And a combination thereof, all of which include health foods in a conventional sense.

The dihydrogingerrone according to the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the dihidrogen ghreorone in the health food may be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, Can be added. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the above-mentioned dihydroxyzincoron as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

In addition, the dihydrogingerrone of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the dibenzoylmethane of the present invention.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

As used herein, the term "improvement" is meant to include preventing, ameliorating, treating, or delaying the onset of such symptoms.

As used herein, the term "improved obesity" means any action that improves or alleviates the symptoms of obesity with the administration of the composition.

As used herein, the term "functional food" means food prepared and processed using raw materials or ingredients having functionality useful to the human body according to Law No. 6727 on health functional foods, and " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

<Experimental Method>

The method commonly used in this experiment is as follows.

Use reagent

The CaMKK inhibitor STO-609 and metformin were purchased from Sigma Chemical Company (St. Louis, MO, USA). 5-Aminoimidazole-4-carboxamide-1-ribofuranoside (AICAR) was purchased from Toronto Research Chemical Incorporation (Toronto, ON, Canada). DBM (1, 7-bis [4-hydroxy-3-methoxyphenyl] -1,6-heptadiene-3, 5-dione) and p38 MAPK inhibitor SB203580 were purchased from Biomol International LP (Butler Pike, . Anti-phosphorylated p38 MAPK antibody, anti-AMPKa antibody, anti-ACC antibody, anti-p38 MAPK antibody, and anti-actin antibody were purchased from Millipore (MA, USA) Respectively. Compound C, an AMPK inhibitor, was purchased from Merck (RY 70-100; Rahway, NJ, USA). Hybond ECL nitrocellulose membranes were purchased from Amersham (Arlington Heights, IL, USA).

Cell culture

The mouse myoblast C2C12 cell line, rat myoblast L6 cell line, and pre-adipocyte 3T3-L1 cell line were treated with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic (100 U / mL penicillin and 100 g / mL streptomycin) The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% CO ₂ at 37 ° C. For myotube differentiation to test glucose uptake ability, rat myoblast L6 cell line was divided into 12 well plates at a density of 2 × 10 ⁴ cells / mL and cultured for 24 hours to show confluence of 80% or more. Then, 2% (v / v) FBS and cultured for 2, 4, and 6 days. After 7 days of differentiation into myotube, the glucose uptake ability was tested.

Western blot analysis

C2C12, L6, or 3T3-L1 cell lines were cultured in 6-well plates to have confluence of 60-70%. Prior to processing the selected agents, serum starvation at 37C was applied for 24 hours. After the agent was inoculated, the culture medium was inhaled and the cell line was washed twice with cold phosphate-buffered saline (PBS), followed by the addition of a protease inhibitor (0.5 M aprotinin, 1 M phenylmethylsulfonyl fluoride, and 1 M leupeptin) (SDS), 1% Nonidet P-40, 150 mM NaCl, and 50 mM Tris-HCl, [pH 8.0]) containing 100 L of lysis buffer (0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). The supernatant was sonicated by heating at 95 ° C for 5 minutes, centrifuged for 5 minutes, separated by electrophoresis using 8-16% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. The membrane was treated with primary antibody and incubated overnight at 4 ° C. The cells were washed 6 times with Tris-buffered saline containing 0.1% Tween 20. The secondary antibody conjugated with horseradish peroxidase (HRP) was then incubated at room temperature for 1 hour. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antiserum was used to normalize the loading of the protein. The wetsern blotting results were visualized using an ECL Gel Electrophoresis System (Amersham Biosciences; Buckinghamshire, UK). The membrane was scanned and densitometry analysis was performed using Image Labs.

RT-PCR

First-strand cDNA synthesis was carried out in a Thermoscript II one-step RT-PCR kit (Life Technologies; Paisley, UK) at 55 ° C for 20 min. The total RNA isolated from the C2C12 cell line was amplified by inactivating The reaction was carried out using a Gene Amp System 9700 thermocycler (Applied Biosystems; Warrington, UK) under the following conditions: 34 cycles 94C 30 sec, 55C 30 sec, and 72C 60 And incubation for the last 10 min at 72C. The number of PCR cycles was used to confirm that they were in the expotential phase of the amplification process. Ten μg of each sample was analyzed by electrophoresis. In electrophoresis, each band was stained with ethidium bromide and visualized using ultraviolet light. The intensity of the band was quantified using the gel documentation system (Gene Genius; Syngene, UK).

The primers used in the RT-PCR were as follows.

SEQ ID NO: 1: GLUT4-sense (5-TTG GAG AGA GAG CGT CCA AT-3)

SEQ ID NO: 2: GLUT4-antisense (5-CTC AAA GAA GGC CAC AAA GC-3)

SEQ ID NO: 3: FAS-sense (5-CAC ACA CAA TGG ACC CCC AG-3)

SEQ ID NO: 4: FAS-antisense (5-CAG AGG TGT TCG GCT TCA GG-3)

SEQ ID NO: 5: C / EBPa-sense (5-AGG TGC TGG AGT TGA CCA GT-3)

SEQ ID NO: 6: C / EBPa-antisense (5-CAG CCT AGA GAT CCA GCG AC-3)

SEQ ID NO: 7: C / EBPb-sense (5-CGG GGT TGT TGA TGT TTT TGG-3)

SEQ ID NO: 8: C / EBPb-antisense (5-TCG AAA CGG AAA AGG TTC TCA-3)

SEQ ID NO: 9: C / EBP-sense (5-CTT CGC CGA CCT CTT CAA C-3)

SEQ ID NO: 10: C / EBP-antisense (5-CGC ACA CCG CCA CTT G-3)

SEQ ID NO: 11: aP2-sense (5-AAG ACA GCT CCT CCT CGA AGG TT-3)

SEQ ID NO: 12: aP2-antisense (5-TGA CCA AAT CCC CAT TTA CGC-3)

SEQ ID NO: 13: SCD1-sense (5-CCT TCT TGC GAT ACA CTC TGG TG-3)

SEQ ID NO: 14: SCD1-antisense (5-TCA GCT ACT CTT GTG ACT CCC G-3)

SEQ ID NO: 15: PPAR-sense (5-GGT GAA ACT CTG GGA GAT TC-3)

SEQ ID NO: 16: PPAR-antisense (5-CAA CCA TTG GGT CAG CTC TT-3),

SEQ ID NO: 17: -actin-sense (5-CAG GAG GAG CAA TGA TCT TGA-3)

SEQ ID NO: 18: -actin-antisense (5-ACT ACC TCA TGA AGA TCC TCA-3).

Intracellular calcium concentration measurement

Concentrations of calcium ions were determined by treating the L6 cell line with fluo-3 AM, a calcium ion indicator, and using a confocal microscope, Zeiss LSM 510 Meta (Zeiss; Oberkochen, Germany). The cell lines were incubated at room temperature in a medium containing 5 mM of fluo-3 AM for 45 minutes, washed, and cultured in a medium without fluo-3 AM for 15 minutes. A culture plate was placed in a temperature controllable microscope at a magnification of 20X and then measured at 488 nm.

2-Deoxy-D [H ³ ] glucose (2-DG) ingestion ability

The glucose uptake capacity was measured by measuring the uptake capacity of 2-deoxy-D [H ³ ] glucose (2-DG) in differentiated L6 myotubes. Cell lines were washed with 37 PBS and incubated in serum-free DMEM for 3 h. After treatment of DBM, the cell lines were treated with Krebs Ringer Henseleit (KRH) buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 1.4 mM KCl, 1 mM CaCl ₂ , 1.2 mM MgSO ₄ , and 1 . ₂ mM KH ₂ PO ₄ ) at 37 for 15 min. The reaction plate was placed on ice and stopped and washed twice with cold PBS. After the cell line was pulverized with 0.5N NaOH, radioactivity was measured by scintillation counting with 400ul of pulverized material and 3.5ml of scintillation cocktail.

AMPKa2 silencing

Rat myoblast L6 cell line was plated on a 6-well plate and cultured for 24 hours to show 70% confluence. Transfection was performed using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA). Briefly, AMPK2 siRNA purchased from Dharmacon (Thermo Scientific; L-040809-00-0005) and Bioneer; Non-targeted control siRNA designed and synthesized by Daejon, Korea was used for transfection. 5 μl of each siRNA and 5 μl of Lipofectamine 2000 were mixed with 95 μl of reduced serum medium (Opti-MEM; Invitrogen), incubated for 30 minutes at room temperature, and placed in a well containing 800 μl of Opti-MEM siRNA concentration to be 100 nM. After 4 hours after transfection, the medium was replaced with fresh complete medium.

Myc-GLUT4 translocation assay

In order to quantitate the expression of GLUT4myc on the cell surface, a colorimetric absorbance assay combined with antibody was performed (N. Wijesekara, A. Tung, F. Thong, A. Klip. reduced endocytosis independently of AMPK. Am J Physiol Endocrinol Metab 290: E1276-1286). L6 cell line stably expressing GLUT4myc treated with DBM was treated with polyclonal anti-myc antibody (1: 1000) for 60 minutes and then cultured in PBS containing 4% paraformaldehyde for 10 minutes. -rabbit IgG antibody (1: 1000). After washing the cell line 6 times with PBS, 1 ml of OPD reagent (0.4 mg / ml) was treated for 30 minutes and the absorbance of the supernatant was measured at 492 nm.

High fat diet in mice

Dae Han Bio Link Co. (Chungbuk, Korea), 4 weeks old male C57BL / 6 mice were purchased. The rats were grown for 4 weeks using commercial feed. At the 8th week, they were randomly divided into three groups of 10 each, fed HFD, HFD and DBM (100 mg / kg), and fed diets for 12 weeks. Feed consumption and body weight were measured and recorded every week. After 12 weeks, Zoletil (Virvac Laboratories; Carros, France) and then used for further analysis.

Data Analysis

One-way ANOVA, Holm-Sidak comparisons and post-hoc Fisher test were used to measure the intensity of insulin release. The difference between the mean values was used as a statistically significant value when the p-value was less than 0.05.

Example 1. Increase of phosphorylated AMPK concentration in skeletal muscle cells by DBM

In order to analyze the effect of DBM on C2C12 mybolasts, activation of AMPK enzyme essential for glucose uptake was measured, and it was confirmed that phosphorylated AMPK was increased in proportion to the concentration and time of DBM (FIG. 1A, B). DBM is an analogue of curcumin and has a stronger effect on the phosphorylation of AMPK and the phosphorylation of ACC, which is a sub-step of its signal transduction step (Fig. 1C). As a result of comparing the glucose uptake capacity, DBM showed similar strength to metformin known as AMPK activator (FIG. 1D). These results confirm that DBM increases phosphorylation of AMPK in C2C12 cell line.

Example 2. Increase of 2-DG uptake by DBM

It is known that L6 myotubes are good targets for testing glucose uptake capacity (V. Sarabia, T. Ramlal, A. Klip. (1990) Glucose uptake in human and animal muscle cells in culture. Biochem Cell Biol 68: 536-542 .). Therefore, the 2-DG uptake by DMB using the differentiated L6 cell line was examined and it was confirmed that the uptake of 2-DG by DBM was increased (FIG. 2A). Pretreatment of the AMPK inhibitor coumpund C with 2 uM inhibited the uptake of 2-DG induced by DBM, suggesting that AMPK plays a key role in DBM-induced glucose uptake.

In order to confirm this, the sugar uptake ability by AMPK2 knockdown was confirmed. After transfection of AMPK2 short interfering RNA (siRNA), the inhibition of AMPK2 expression was confirmed by Western blotting using anti-AMPK antibody. DBM-induced glucose uptake was not observed in cell lines treated with AMPK2 siRNA (Fig. 2B). Inactivated mutants of AMPK2 K45R kinase also inhibited the effect of DBM (Fig. 2C). As shown in Figure 2D, (GFP) -tagged AMPK2 K45R mutants were transfected into approximately 30-40% of the cells. From the above results, it was confirmed that AMPK2 is important for DBM-induced glucose uptake ability.

Example 3. DBM-induced AMPK phosphorylation by intracellular calcium ion

In order to further analyze the upper level of the APMK signaling pathway, the concentration of intracellular calcium ion was measured using fluo-3 AM calcium-binding fluorescent staining material and it was confirmed that DBM increased fluorescence intensity in L6 cell line 3A). These results indicate that CaMKK plays an important role in the upper level of the AMPK signaling pathway. To confirm this, the addition of the CaMKK inhibitor STO-609 inhibited AMPK phosphorylation (FIG. 3B) Gt; 2-DG &lt; / RTI &gt; From these results, it was confirmed that the glucose uptake induced by DBM works through Ca - mediated CaMKK / AMPK signaling pathway.

Example 4. Increase of phosphorylation of p38 mitogen-associated protein kinase (MAPK) by DBM

To further analyze the APMK signaling pathway, the phosphorylation of p38 MAPK, which operates in the lower stages of AMPK, was measured. DBM treatment resulted in increased phosphorylation of p38 MAPK in the C2C12 cell line (Fig. 4A). As a result of treatment with compound C, an inhibitor of AMPK, it was confirmed that DBM-induced phosphorylation of p38 MAPK was inhibited (Fig. 4B). Furthermore, it was confirmed that the ingestion of 2-DG induced by DBM was inhibited when SB203580, a p38 MAPK inhibitor, was treated (Fig. 4C). These results confirm that the glucose uptake induced by DBM is regulated by activation of AMPK-dependent p38 MAKPK.

Example 5: Expression of GLUT4 by DBM and increase of GLUT4 translocation

We confirmed that DBM affects the activation of p38 MAPK and therefore also affects the expression of GLUT4 gene, which operates in its lower step. It was confirmed that the concentration of GLUT4 mRNA was increased in the DBC-treated C2C12 cell line (Fig. 5A), and the protein concentration was also increased (Fig. 5B). Using the colorimetric assay fused with the HRP-conjugated secondary antibody, the expression of GLUT4myc on the cell surface was found to increase, and it was confirmed that this result was not observed as a result of treatment with coumpund C (FIG. 5C). These results confirm that the glucose uptake induced by DBM works by promoting GLUT4 expression and translocation of GLUT4.

Example 5. Effect of DBM on weight loss and fat accumulation in obesity-induced obesity model

We confirmed the effect of DBM on weight gain induced by HFD (High-Fat Diet). As a result of steady ingestion of DBM, it was confirmed that the increase in body weight induced by HFD was suppressed (Fig. 6A). In order to confirm the storage of body fat, photographs of abdominal fat were taken and observed. As a result, it was confirmed that the amount of fat stored in the rats fed DBM with HFD was remarkably small (Fig. 6B, C). Liver storage in the liver was also inhibited by ingestion of DBM (Fig. 6D). It was also confirmed that the insulin concentration was also decreased in rats fed DBM together with HFD alone (Fig. 6E), and the blood glucose was also slightly lower (Fig. 6F). The concentration of Leptin was not significantly different (Fig. 6G). The phosphorylation of acetyl-CoA carboxylase (ACC), a key enzyme of fatty acid synthesis, was examined to confirm the effect of inhibiting lipid synthesis, and it was confirmed that ACC phosphorylation was increased by DBM (FIG. 6H). Since ACC phosphorylation exhibits a decrease in ACC activity, it was confirmed that DBM inhibited lipid synthesis.

To further analyze the mechanism of action of DBM, RT-PCR analysis of several genes involved in lipid synthesis was performed. The expression of the FAS gene was drastically reduced by treatment with DBM (Fig. 6I), which was not observed under AMPK2 knockdown conditions (Fig. 6J). From these results, it was confirmed that the inhibitory effect of DBM on lipid synthesis is inhibited by the expression of AMPK - mediated lipogenic gene.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Composition for Anti-obesity Containing Dibenzoylmethane <130> P14-B302 <150> KR 10-2013-0114230 <151> 2013-09-26 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GLUT4-sense <400> 1 ttggagagag agcgtccaat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GLUT4-antisense <400> 2 ctcaaagaag gccacaaagc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS-sense <400> 3 cacacacaat ggacccccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS-antisense <400> 4 cagaggtgtt cggcttcagg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C / EBPa-sense <400> 5 aggtgctgga gttgaccagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C / EBPA-antisense <400> 6 cagcctagag atccagcgac 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C / EBPb-sense <400> 7 cggggttgtt gatgtttttg g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C / EBPb-antisense <400> 8 tcgaaacgga aaaggttctc a 21 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> beta-actin-antisense <400> 9 cttcgccgac ctcttcaac 19 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> beta-actin-antisense <400> 10 cgcacaccgc cacttg 16 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> beta-actin-antisense <400> 11 aagacagctc ctcctcgaag gtt 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> aP2-antisense <400> 12 tgaccaaatc cccatttacg c 21 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SCD1-sense <400> 13 ccttcttgcg atacactctg gtg 23 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SCD1-antisense <400> 14 tcagctactc ttgtgactcc cg 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma-sense <400> 15 ggtgaaactc tgggagattc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma-antisense <400> 16 caaccattgg gtcagctctt 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin-sense <400> 17 caggaggagc aatgatcttg a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin-antisense <400> 18 actacctcat gaagatcctc a 21

Claims (5)

A pharmaceutical composition for the treatment or prevention of obesity comprising dibenzoylmethane as an active ingredient. The pharmaceutical composition for treating or preventing obesity according to claim 1, wherein the dibenzoylmethane modulates lipid metabolism by increasing the expression or activity of AMPK (AMP kinase). A health functional food for improving obesity containing dibenzoylmethane as an active ingredient. A pharmaceutical composition for the treatment or prevention of cholesterolemia comprising dibenzoylmethane as an active ingredient. A health functional food containing dibenzoylmethane as an active ingredient for improving cholesterol level.

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