KR101715592B1 - Composition for anti-obesity comprising ailanthoidol as effective component - Google Patents

Abstract

The present invention relates to an anti-obesity composition comprising ailanthoidol as an active ingredient. More specifically, it has an effect of inhibiting the accumulation of fat in adipose tissue treated with the composition of the present invention, and inhibiting the differentiation into adipocytes by inhibiting the expression of PPARγ and C / EBPα, which are transcription factors, in adipose precursor cells. Therefore, the composition of the present invention is effective for preventing, improving or treating obesity, and is useful not only for food but also for pharmaceutical use.

Description

[0001] The present invention relates to an anti-obesity composition comprising an anti-obesity agent,

The present invention relates to an anti-obesity composition comprising ailanthoidol as an active ingredient.

Obesity is spreading at a rapid pace, whether at home or abroad. According to a report by the World Health Organization (WHO), 2.6 million people die each year from obesity and overweight in the world. The World Health Organization currently reports an overweight adult population of over 25 million body mass index (BMI) and about 300 million people with an overweight obesity rate above 30 BMI. In addition, 44% of diabetic patients, 23% of ischemic heart disease patients, and 7 ~ 41% of various cancer patients were found to be caused by overweight and obesity. In Korea, 3 out of 10 adults were obese. According to statistics of the National Health Insurance Corporation, 32.8% (324,694 out of 987,854) who received health checkups in 2008 were diagnosed with obesity, and the percentage of obese persons in the medical checkups was 25.5% in 2006 and 24.1% For the first time in 2008, exceeding 30%.

Obesity in modern society is not just an external problem, but a serious health threat that causes various diseases. Therefore, studies for the prevention, improvement or treatment of obesity are being actively carried out. For example, Korean Patent Registration No. 0869856 discloses an obesity-suppressing composition containing a pepper extract and a health supplement containing the same, and Korean Patent No. 1018405 discloses an obesity- And a therapeutic composition are disclosed, but no mention is made of an anti-obesity composition comprising the ailanthoidol of the present invention as an active ingredient.

The present invention provides a composition for anti-obesity comprising ailanthoidol as an active ingredient, wherein the active ingredient of the present invention, ailanthoidol, Confirming that the accumulation of fat in the cells is inhibited and there is no cytotoxicity, the present invention has been completed.

In order to achieve the above object, the present invention provides a health functional food composition for preventing or ameliorating obesity, which comprises an ailanthoidol represented by Chemical Formula 1 as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity comprising an ailanthoidol represented by the general formula (1) as an active ingredient.

The present invention relates to an anti-obesity composition comprising ailanthoidol as an active ingredient. More specifically, since ailanthoidol has an effect of inhibiting accumulation of fat in adipose tissue, it can be very usefully used for preventing, ameliorating or treating obesity.

Fig. 1 shows the results of confirming the survival rate of 3T3-L1 adipocytes by treatment with ailanthoidol.
Figure 2 shows the effect of ailanthoidol on fat accumulation in 3T3-L1 adipocytes by concentration. (A) is an image showing that fat accumulation in 3T3-L1 adipocytes is reduced by treatment with 0 to 10 μM ailanthoidol using Oil-Red-O staining, and (B) (A 490 nm) of lipid accumulation according to the treatment concentration of 10 μM isalantoil, and (C) the cell viability after treatment with isalantoil at 0 to 10 μM (** p &Lt; 0.01, *** p < 0.0001).
Fig. 3 shows the result of confirming the lipid-lowering effect in 3T3-L1 adipocytes after treatment with islettoidol (* p < 0.05).
Fig. 4 shows the results of confirming the degree of secretion of adipokine in 3T3-L1 adipocytes by treatment with isletoyistol. (A) is leptin, (B) is IL-6, (C) is adiponectin and (D) is secretion amount of TNF-α.
Fig. 5 shows the results of confirming the effect of suppressing the expression of transcription factors in 3T3-L1 adipocytes by treatment with isletoyistol.

The present invention relates to a health functional food composition for preventing or ameliorating obesity, which comprises an ailanthoidol represented by the general formula (1) as an active ingredient.

The ailanthoidol represented by Formula 1 is characterized by inhibiting accumulation of triglyceride in adipose tissue.

The health functional food composition for preventing or ameliorating obesity according to the present invention may contain at least one selected from the group consisting of nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, Stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages.

The health functional food composition of the present invention may be added to food for the purpose of preventing or ameliorating obesity. The health functional food is not particularly limited, and may be a health functional food, a nutritional supplement, a nutritional supplement, It can be any type of food such as pharmafood, health food, nutraceutical, designer food, food additive, etc. Preferably, it can be meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza , Noodles, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages, and vitamin complexes. At this time, the method of adding is appropriately used according to a conventional method, and the amount to be added can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The present invention also relates to a pharmaceutical composition for preventing or treating obesity, which comprises an ailanthoidol represented by the general formula (1) as an active ingredient.

[Chemical Formula 1]

The pharmaceutical composition for prevention or treatment of obesity according to the present invention may contain pharmaceutically acceptable salts such as physiologically acceptable salts, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, The present invention also relates to a method for producing a microcrystalline cellulose composition comprising the steps of dissolving a microcrystalline cellulose, a cross, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Tartrate, magnesium stearate, and mineral oil, but is not limited thereto.

The pharmaceutical composition of the present invention may contain at least one auxiliary agent selected from a pharmaceutically acceptable antioxidant, a buffer, a bacteriostatic agent, a diluent, a surfactant, a binder, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, As shown in FIG. The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally. A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The pharmaceutical composition of the present invention may be manufactured in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules.

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 merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

[Experimental Method]

1. 3 T3 - L1  Cell culture and adipocyte differentiation

The mouse-derived 3T3-L1 fibroblasts used in this experiment were purchased from Korean Cell Bank. 3T3-L1 preadipocytes were cultured in DMEM containing 10% BCS and 100 U / ml of penicillin and 100 mg / ml of streptomycin at 37 ° C in 5% CO ₂ Lt; / RTI &gt;

To induce 3T3-L-1 cells to adipocytes, two days after all adipocytes became confluent, induction of differentiation with 10% FBS, 10 μg / ml insulin, 0.5 mM IBMX and dexamethasone The medium (DM) was used for 2 days, and the day when the medium was replaced with the differentiation inducing medium was designated as day 0. The medium containing 5 μg / ml of insulin was replaced with 10% FBS / DMEM from day 2 to day 4 after 4 days of differentiation and 10% FBS / DMEM And more than 90% of mature fat cells with lipids were used.

2. Cytotoxicity

In order to measure the ahilran toy stone in 3T3-L1 cell cytotoxicity CellTiter 96 ^® AQ _ueous One Solution Cell Proliferation Assay using the (Promega, Madison, WI) was measured on the number of cell viability by a colorimetric quantitation. 3T3-L1 cells were inoculated on a 96-well flat-bottom plate at 1 × 10 ⁴ cells / well and treated with Isolanti stone at each concentration.

After 24 hours of incubation, the number of viable cells was measured according to the manufacturer's instructions.

3. Oil Red -O staining

Oil Red-O staining was performed using cells that were differentiated on day 8. After the differentiation medium was removed, 3T3-L1 adipocytes were washed twice with phosphate buffered saline (PBS) and fixed with 10% formalin. After fixation, the cells were washed three times with distilled water and stained with Oil Red-O, which specifically reacted with liposomes. Then, the dye was removed, washed three times with distilled water, and the cells were photographed 200 times magnified using a digital camera connected to a phase contrast microscope (Leica DMI 4000B, GmbH Wetzlar, Germany). Lipids were eluted with isopropanol and absorbance was measured at 490 nm.

4. Fat dissolution ( Lipolysis )

The release of glycerol in the culture medium represents a change in degree of lipolysis (Green et al., 2004). The adult 3T3-L1 adipocytes (day 8) were treated with Isolanthiotol for 48 hours, and the culture was collected and centrifuged. After obtaining the supernatant, a free glycerol reagent was used to dissolve glycerol Was measured by a spectrophotometer at 540 nm.

5. Adipokine and  Cytokine ( Cytokine ) Measure

TNF-α, IL-6, adiponectin and leptin were measured by ELISA assay using cell culture supernatants. The supernatant was collected in the culture medium of the cells differentiated into adipocytes and the cells not treated with Isolan toilides after 8 days, and the assay was conducted according to the manufacturer's instructions.

6. Western Blotting ( Western Blotting ) analysis

3T3-L1 cells or untreated cells treated with Isolan toilestone were washed twice with cold phosphate buffered saline (PBS) and then treated with cold PRO-PREP ^TM Protein extraction solution (iNtRON Biotechnology, Korea). The extracted proteins were separated using 10% SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. Protein-transferred membranes were blocked with 3% skim milk containing Tris-buffer saline / Tween 20 solution and washed three times with TBS buffer.

Antibody was reacted with PPARγ antibody (Santa Cruz Biotechnology), C / EBPα (Santa Cruz Biotechology) antibody, phospho-AMPK (Cell Signaling Technology) antibody and β-actin (Sigma-Aldrich) antibody. To detect the immunoreactive band, Horseradish perosidase-conjugated secondary antibody was treated and detected with WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology).

7. Statistical Analysis

All data were expressed as mean ± standard deviation. Statistical analysis was performed using Graphpad Prism 4.0 software (GraphPad software Inc., San Diego, Calif.), And was analyzed by Bonferroni multiple comparison analysis with one-way ANOVA (AVOVA).

Example  One. Eyalan Toy Stone Of cytotoxicity

MTS analysis was performed on 3T3-L1 cells to confirm the cytotoxicity of the islet stones. As shown in Fig. 1, the isletoyl stearate treated for 24 hours had no significant effect on cell proliferation up to 20 [mu] M. Since the cells did not show up to 20 μM cell toxicity in 3T3-1 cells, the subsequent experiments set the concentration of the isletotyroids to 20 μM or less.

Example  2. Eyalan Toy Stone Inhibitory effect on fat accumulation

Oil-Red-O staining was performed to confirm the effect of inhibiting the accumulation of fat in the 3T3-L1 adipocytes. As a result, oil-red-O staining showed staining of triglycerides present in free fatty acids and phospholipids, confirming the formation of lipids. Insulin, dexamethasone and IBMX (3-isobutyl-1-methylxanthine) were used to differentiate 3T3-L1 cells into mature adipocytes. As shown in Fig. 2 (A), it was confirmed that the treatment with isalantoil reduced Oil-Red-O staining, and from these results, it was found that the isletoyl styrene reduced accumulation of fat. When DM cells were treated with 10 μM of islet rock, the size of lipid sphere was minimized. The OD value of Oil-Red-O staining decreased from 78.0% at 1 μM to 75.4% at 5 μM and to 74.2% at 10 μM when Isolan Toyo stone was treated at each concentration (FIG. 2 (B)). The proliferation of differentiated cells on day 8 was measured in order to confirm the cytotoxicity of Isolan toilast in differentiated 3T3-L1 cells. As shown in Fig. 3 (C), isletoyl stones did not show the effect on cell survival in differentiated 3T3-L1 cells. Thus, the islet toilide inhibited the differentiation of all adipocytes without cytotoxicity in adipocytes.

Example  3. Eyalan Toy Stone Lipid effect

By measuring the amount of glycerol present in the cell culture fluid following the treatment with the islet toilot, the lipid degradation effect of the isletoyl stearate was confirmed.

The triglyceride is activated by the hormone sensitive lipase (HSL) in the fat cells by the nutritional status or stimulation of various hormones and is decomposed and released by fatty acid and glycerol, so the amount of glycerol secreted indicates the degree of decomposition of triglyceride.

The results of this Example 3 show that when 5 μM and 10 μM of isletoyl stearate were treated, the fat breakdown increased to 22% and 25%, respectively, compared to the fat cells before treatment with isletoyl stones (FIG. 3) . From the above results, it can be seen that the islet of the present invention not only inhibits fat accumulation but also has an effect of decomposing fat.

Example  4. Eyalan Toi Stone Adipocaine  Inhibitory effect

Adipokines include TNF-α, leptin, IL-6, and aditonectin. Of these, adiponectin has an intrinsic property of increasing fat accumulation when secretion decreases, Concentration, and triglyceride levels in liver and muscle, thereby increasing insulin sensitivity. Adipokines such as TNF-α, leptin, and IL-6 are known to increase secretion during the differentiation of 3T3-L1 adipose precursor cells. In addition, leptin is secreted from adipocytes and functions to regulate food intake and energy consumption by transmitting signals to the central nervous system. The level of leptin mRNA in the adipocytes of obese patients and the amount of leptin in the serum Has been reported to have a very close relationship. It is also known that leptin reflects the size of adipocytes.

In Example 4, leptin, IL-6, adiponectin and TNF-alpha were detected in Isolatoid treated 3T3-L1 adipocytes in order to examine whether the release of adipocaine was controlled by isletoyl stones Was confirmed.

Leptin levels were decreased from 1 μM to 12%, from 5 μM to 57%, and from 10 μM to 62%, respectively, when the differentiated 3T3-L1 cells were treated with Isolan toilastol. 47%, 93% at 5 μM and 100% at 10 μM, respectively (FIGS. 4 (A) and 4 (B)). Adiponectin and TNF-α, on the other hand, showed a decrease in secretion compared with leptin and IL-6, but decreased about 40-50% when 5-10 μM isalantoil treatment 4 (C) and 4 (D)).

From the results of Example 4, it was judged that isletoyl stearate inhibited the accumulation of fat by reducing the release of adipocaine, indicating an anti-obesity effect.

Example  5. Transcription factors for adipocytes Eyalan Toy Stone Effect of

Expression of PPARγ and C / EBPα, the major transcription factors that regulate adipogenesis, was measured to determine if Islan toilides inhibit adipogenesis. As shown in FIG. 5, the expression levels of PPARγ and C / EBPα were decreased by 19% and 63%, respectively, at 10 μM in a concentration-dependent manner in the treatment with isletoyodol.

It was also confirmed that the degree of expression of pAMPK decreased when treated with Isolan toeotol. The AMP-activated protein kinase (AMPK) protein is a protein that is activated by sensing intracellular AMP. It inhibits the signal transduction pathway (lipogenesis, protein synthesis, gluconeogenesis) that consumes ATP and activates the signal transduction pathway oxidation) to protect cells from external stress. The first target protein of AMPK is acetyl-CoA carboxylase (ACC), and ACC is the main enzyme in the synthesis of malonyl-CoA from acetyl-CoA during lipogenic pathway. When the concentration of malonyl-CoA is decreased due to the inhibition of ACC, the activity of glycerol-acyl transferase involved in triglyceride synthesis is inhibited and lipogenesis is inhibited. When AMPK is activated, it is present in a phosphorylated form and phospho-AMPK is increased. As a result, phosphorylated AMPK was detected in 3T3-L1 adipocytes treated with Isolan to reduce the amount of phospho-AMPK in a concentration-dependent manner. From these results, it was concluded that isletoyl stearate in the 3T3-L1 adipocytes exhibits anti-lipogenic activity by the AMPK signaling pathway.

Claims (4)

A health functional food composition for preventing or ameliorating obesity comprising an ailanthoidol represented by Chemical Formula 1 as an active ingredient.
[Chemical Formula 1]

The health functional food composition according to claim 1, wherein the ailanthoidol represented by the formula (1) inhibits the accumulation of triglyceride in adipose tissue. A pharmaceutical composition for preventing or treating obesity, comprising an ailanthoidol represented by the general formula (1) as an active ingredient.
[Chemical Formula 1]

 4. The pharmaceutical composition for preventing or treating obesity according to claim 3, wherein the ailanthoidol of formula (1) inhibits the accumulation of triglyceride in adipose tissue.

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