KR102349724B1 - Composition for use of inhibition of obesity comprising Kahweol - Google Patents

Abstract

The present invention relates to a composition for preventing, treating and improving obesity comprising Kahweol as an active ingredient, and since the composition of the present invention has an effect of preventing or alleviating obesity by inhibiting the accumulation of body fat, it can be used as a treatment for obesity, etc. It can be usefully used.

Description

Anti-obesity composition comprising Kahweol {Composition for use of inhibition of obesity comprising Kahweol}

The present invention relates to an anti-obesity composition, and more particularly, to a composition for preventing, treating and improving obesity comprising Kahweol as an active ingredient.

As in developed countries in the West, the number of obese people is increasing in Korea due to the recent improvement in living standards and a westernized diet. Obesity is not a simple external problem, but is emerging as a serious health problem because it leads to adult diseases such as high blood pressure, hyperlipidemia, arteriosclerosis, heart disease, and type 2 diabetes.

Obesity refers to a state in which body fat is excessively increased, not simply a state of being overweight, but a state in which excess fat is accumulated in the body. Therefore, it is a disease in which calories consumed and remaining due to excessive calorie intake are excessively accumulated in adipose tissue in the form of triglycerides. It is known to be caused by action. Accordingly, studies on adipocyte differentiation and fat metabolism are being actively conducted to understand and treat obesity-related diseases (Kim JB. 2001. Korea J Lipidol. 9: 79-83; Grundy SM. 1998. Am J Clin Nutr. 67: 563S-572S; and Albu J, et. al. 1997. Nutr rea. 55: 150-156).

In general, methods for preventing or treating obesity include controlling food intake, inhibiting caloric absorption, promoting caloric expenditure, controlling energy metabolism, controlling signal transduction through the nervous system, and the like. Currently, anti-obesity drugs such as Xenical and Reductil are being used, but side effects such as heart disease and respiratory disease and continuity of efficacy are problematic. Therefore, there is a continuous need for a safe anti-obesity therapeutic agent that has an anti-obesity or alleviation effect and does not cause side effects, and although research on this is ongoing, the situation is still insufficient (refer to Korean Patent Registration No. 10-1326267).

As a result of the present inventors' diligent efforts to find a substance that has no side effects and has a fundamental preventive and therapeutic effect on obesity, Kahweol not only reduces the blood triglyceride concentration but also inhibits the differentiation of 3T3-L1 adipocytes. By showing the effect, it was confirmed that it can have anti-obesity activity, and the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity comprising Kahweol as an active ingredient.

Another object of the present invention is to provide a health functional food composition for improving obesity comprising Kahweol as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating obesity comprising Kahweol as an active ingredient.

In one embodiment of the present invention, the composition may have the effect of reducing visceral fat and reducing blood triglycerides.

In another embodiment of the present invention, the composition can inhibit the differentiation of preadipocytes.

In another embodiment of the present invention, the composition may inhibit the expression of PPARγ and C/EBPa.

On the other hand, the present invention provides a health functional food composition for improving obesity comprising Kahweol as an active ingredient.

In addition, the present invention provides a method for treating obesity, comprising administering a pharmaceutical composition comprising Kahweol to an individual in need of treatment for obesity.

In addition, the present invention provides the use of a pharmaceutical composition comprising Kahweol for the treatment of obesity.

Since Kahweol of the present invention exhibits anti-obesity activity, it can be used as a health functional food or pharmaceutical composition used for the purpose of preventing and treating obesity, and is expected to be utilized as a therapeutic agent for obesity.

1 is a view confirming the structure of Kahweol.
2 is a diagram confirming the cytotoxic effect of Kahweol in 3T3-L1 cells using MTT analysis.
3 is a view confirming the lipid accumulation effect of Kahweol.
4 is a diagram confirming the effect of Kahweol on mRNA and protein expression of fat cells-related genes.

The present inventors, based on the fact that when Kahweol is treated, can inhibit the differentiation of 3T3-L1 preadipocytes, the effect of the compound on reducing fat accumulation and suppressing the expression of adipocyte marker genes, etc. Confirmed, and based on this, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising Kahweol as an active ingredient.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of familial hypercholesterolemia by administration of the composition of the present invention.

As used herein, the term "treatment" refers to any action in which symptoms caused by familial hypercholesterolemia are improved or beneficially changed by administration of the composition of the present invention.

In the present invention, "Kahweol" is a compound having a structure represented by the following formula (1), and can be isolated from a natural product or synthesized artificially, and it is apparent to those skilled in the art that it has the same effect even when synthesized artificially.

[Formula 1]

In one embodiment of the present invention, in order to confirm the anti-obesity effect of Khaweol, as a result of confirming the intracellular lipid accumulation and TG (triglyceride) formation inhibitory activity of Khaweol, 40 μM Khaweol in 3T3-L1 cells It was found that upon treatment, it exhibited superior lipid accumulation and TG formation inhibitory activity than the positive control (see Example 3).

Therefore, Khaweol according to the present invention inhibits lipid accumulation and has TG formation inhibitory activity, so it can be used for various purposes and uses requiring an anti-obesity effect.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. may be additionally included in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, in general 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In addition, the present invention provides a health functional food composition for improving obesity comprising Kahweol as an active ingredient.

The health functional food composition according to the present invention may be used simultaneously with or separately from a medicament for the treatment of obesity, for the improvement of obesity.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms.

The health functional food composition according to the present invention may be added to health supplements such as food and beverages for the purpose of improving obesity.

There is no particular limitation on the type of food, and examples of food to which the active ingredient can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, and gums. , dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and all health functional foods in the ordinary sense.

In the health functional food composition according to the present invention, the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use thereof. In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be less than or equal to the above range.

The composition for a health drink of the present invention is not particularly limited in other ingredients except for containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. can 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 such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

In addition, the present invention provides a method for treating obesity comprising administering the pharmaceutical composition to a subject. In the present invention, "individual" refers to a subject in need of treatment for a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, dogs, cats, horses and cattle. .

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Reagent and Antibody Preparation

Kahweol was purchased from LKT Laboratories (St. Paul, MN, USA), and DMEM fetal bovine serum (FBS) and neonatal calf serum (NBCS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Insulin, indomethacin, dexamethasone, IBMX, and Oil Red O solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MTT was purchased from Amresco (Solon, OH, USA). Triglyceride quantitative color/fluorescence measurement kit was purchased from BioVision (Milpitas, CA, USA), primers were purchased from Macrogen (Seoul, Korea), and cDNA synthesis kit was purchased from TaKaRa Bio (Otsu, Shiga, Japan). Antibodies to C/EBPa and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively, and antibodies to PPARγ and adiponectin were obtained from Abcam plc (Cambridge, purchased in the UK).

1-2. Differentiation of 3T3-L1 cells

Mouse 3T3-L1 preadipocytes were purchased from the Korea Cell Line Bank (Seoul, Korea). They were maintained in DMEM supplemented with 10% NBCS, antibiotics (1% penicillin/streptomycin) (5% humidity, 37° C., CO ₂ incubator). After 2 days, cells were exposed to differentiation medium (MDI) consisting of DMEM containing 10% FBS, IBMX (0.5 mM), insulin (10 μg/ml), dexamethasone (1 μM) and indomethacin (100 μM). made it After 2 days, the culture medium was replaced with DMEM supplemented with 10% FBS and insulin every other day (day 2 to day 8).

1-3. MTT assay

3T3-L1 cells were seeded in 96-well plates at a concentration of ^{1×10 4 cells per well.} After incubation for 24 hours, cells were treated with Khaweol (4 or 40 μM). After 24 or 48 hours of incubation, respectively, the medium was removed and MTT solution (0.5 mg/ml) was added. After incubation for 3 hours, isopropanol was added and absorbance was measured at 495 nm using a spectrophotometer.

1-4. Oil Red O staining and triglyceride colorimetric analysis

3T3-L1 cells were treated or untreated with Kahweol (4 or 40 μM), and after 8 days, washed with 20% isopropanol and fixed with 5% formalin for 1 hour. The intracellular lipid droplets were stained with Oil Red O solution for 15 minutes and then dissolved in 100% isopropanol. The absorbance of each sample was measured at 495 nm.

Triglyceride (TG) quantification was confirmed by measuring the total amount of TG using a triglyceride quantitative colorimetric/fluorescence measurement kit (BioVision) according to the manufacturer's protocol. 3T3-L1 cells were homogenized in TG assay buffer containing 5% Nonidet P40 and then heated to 80° C. for the last 4 min. Then, it was centrifuged for 2 minutes to remove insoluble material, and diluted with _{dH 2 O.} The samples were mixed with the TG reaction mixture and incubated for 1 hour at room temperature. The colorimetric intensity was measured at 570 nm.

1-5. RT-PCR analysis

Total cellular RNA was extracted using RNAiso Plus (Takara). Reverse transcription and cDNA synthesis were performed using PrimeScript 1st strand cDNA synthesis kit (Takara) according to the manufacturer's protocol. As the PCR reagent, EmeraldAmp GT PCR Master Mix (Takara) was used. The PCR product was electrophoresed on a 1% agarose gel and stained with ethidium bromide. The sequences of the designed primers are shown in Table 1.

1-6. western blot analyze

3T3-L1 adipocytes were lysed using RIPA lysis buffer (Biosesang, Korea). Equal amounts of protein were separated on a 12% sodium dodecylsulfite polyacrylamide gel. The separated protein was transferred to a nitrocellulose membrane, and after blocking with 5% skim milk (10 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20) in TBST for 1 hour, PPARγ, C/EBPa, Incubate overnight at 4 °C with primary antibody against adiponectin. After washing with TBST, the membrane was incubated with an HRP-conjugated secondary antibody at room temperature for 1 hour, and the protein was detected using an enhanced chemiluminescence (ECL) reagent (GE Healthcare, Buckinghamshire, UK).

1-7. statistical analysis

Statistical analysis of all experiments was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL). Statistical differences were analyzed using one-way ANOVA. A P value of <0.05 was considered significant.

Example 2: Confirmation of 3T3-L1 cytotoxicity

In order to measure the cytotoxic effect of Kahweol on 3T3-L1 progenitor adipocytes, MTT analysis was performed according to the method of Examples 1-3.

As a result, as shown in FIG. 2 , no cytotoxic effect was observed in all Kahweol cells (4 or 40 μM) treated for 24 hours or 48 hours. Therefore, Kahweol was used at a concentration of 4 or 40 μM for subsequent experiments.

Example 3: Confirmation of the effect of lipid accumulation in 3T3-L1 cells

Oil Red O staining and TG detection were performed by the method of Examples 1-4 to confirm the effect of Kahweol differentiation and fat accumulation in 3T3-L1 adipocytes.

As a result, as shown in FIG. 3 , the control (non-Kahweol-treated) cells significantly increased the differentiation of pre-adipocytes into mature adipocytes and the formation of fat droplets, but when treated with 40 μM Kahweol, 3T3-L1 cells were differentiated. was confirmed to be inhibited. In addition, cells treated with 40 μM Kahweol exhibited a small spindle-shaped morphology similar to that of precursor adipocytes (see FIG. 3A ), and intracellular TG levels decreased by 43.3% after treatment with 40 μM Kahweol.

These results suggest that Kahweol inhibits intracellular lipid accumulation and TG formation.

Example 4: Confirmation of adipocyte marker gene expression

RT-PCR was performed by the method of Examples 1-5 to measure the mRNA expression levels of various adipocyte transcription factors including PPARγ and C/EBPa, and down-regulation of the protein corresponding to the mRNA strand was performed. As in Example 1-6, it was confirmed using Western blot.

As a result, as shown in FIG. 4 , it was confirmed that 40 μM Kahweol significantly reduced the mRNA levels of PPARγ and C/EBPa, and it was also confirmed that the mRNA level of adiponectin was reduced at the same concentration. In addition, it was confirmed that the expression of protein PPARγ, C/EBPa and adiponectin was inhibited by 40 μM kahweol, as in RT-PCR results.

These results suggest that Kahweol reduces lipid accumulation and expression of adipocyte marker genes through downregulation of PPARγ and C/EBPa.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (10)

delete delete delete delete delete delete delete A composition for inhibiting adipocyte differentiation in vitro comprising Kahweol as an active ingredient, the composition comprising:
The composition for inhibiting adipocyte differentiation in vitro, characterized in that the composition inhibits the expression of PPARγ and C/EBPa.
9. The method of claim 8,
The composition for inhibiting adipocyte differentiation in vitro, characterized in that it has the effect of reducing visceral fat and reducing intracellular triglycerides.
delete

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