KR20210062524A - Composition for preventing or treating obesity comprising Grateloupia elliptica - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating or preventing obesity, comprising an extract of Grateloupia elliptica or a fraction thereof as an active ingredient. It was confirmed that an ethyl acetate fraction of an ethanol or methanol extract of Grateloupia elliptica inhibits adipocyte differentiation and lipid accumulation, thereby exhibiting a weight loss effect. It was also confirmed that such ethyl acetate fraction thereof inhibits the expression of PPAR-γ, SREBP-1, and FABP-4, which are known to play an important role in fatty acid production, storage and transport in adipocytes, reduces the weight of epidermal fat, subcutaneous fat, peripheral fat and mesenteric fat, and lowers triglyceride (TG), total cholesterol (TC), leptin and insulin contents in high-fat diet-induced mouse serum, and thus can be used in pharmaceutical compositions and food compositions for the treatment or prevention of obesity.

Description

Composition for preventing or treating obesity, including true gambling {Composition for preventing or treating obesity comprising Grateloupia elliptica}

The present invention relates to a pharmaceutical composition for the treatment or prevention of obesity, comprising the extract of champignon gambling as an active ingredient.

Over the past 40 years, rates of overweight and obesity have been steadily increasing in some countries. The Global Health Observatory (GHO) reports that adult obesity worldwide has nearly tripled since 1975, and rates of overweight and obesity have increased in developed and developing countries. Obesity is a medical condition in which excess body fat is accumulated in fat cells, and the main cause of obesity is an imbalance between food intake and energy expenditure. Therefore, increasing energy consumption and suppressing adipogenesis are effective methods for treating obesity.

In addition, when obesity is prolonged, it is known to cause various metabolic diseases. Therefore, anti-obesity drugs have been developed to treat obesity and complications. Among synthetic anti-obesity drugs, Orlistat (Xenical) is the most commonly used drug for the treatment of obesity. However, this synthetic anti-obesity drug has side effects of gastrointestinal disorders and pain. There have been attempts to develop natural anti-obesity drugs that are not toxic to the human body due to the harmful side effects of these synthetic obesity drugs.

Marine organisms have the ability to search for various new physiologically active substances due to their unique metabolic processes and unique environments that are not found in terrestrial organisms. In addition, many studies on terrestrial organisms have already been conducted, but marine organisms have not yet been sufficiently studied, so it is also a field with high expectations for the development of natural materials with new usefulness.

Republic of Korea Patent Publication No. 10-2005-0078603

It is an object of the present invention to provide a pharmaceutical composition for the treatment or prevention of obesity, comprising an extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing obesity, comprising an extract or a fraction thereof as an active ingredient.

The present inventors have confirmed that the ethyl acetate fraction of the ethanol extract or methanol extract of Cham Gambling inhibits adipocyte differentiation and lipid accumulation, and exhibits a weight-reducing effect, and plays an important role in fatty acid production, storage and migration in adipocytes. It inhibited the expression of known PPAR-γ, SREBP-1, and FABP-4, reduced the weight of epidermal fat, subcutaneous fat, peripheral fat, and intestinal fat, and triglycerides (TG) in high-fat diet-induced mouse serum. By confirming that the content of cholesterol (TC), leptin and insulin was lowered, it was confirmed that it can be used in pharmaceutical compositions and food compositions for treating or preventing obesity, and the present invention was completed.

As an aspect for achieving the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of obesity, comprising an extract or a fraction thereof as an active ingredient.

The term "Grateloupia elliptica " of the present invention is a seaweed of the red algae genuarit family, and its height is 15-40cm, it is leaf-shaped, and is divided into several branches. It is red-purple, yellow, and green, but turns purple when it dries.

In the present invention, the term "extract" refers to an extract obtained by an extraction treatment, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract. It includes extracts of all formulations that can be formed by using.

In the present invention, drying of the extract may be performed by a known method within the range in which useful components are not destroyed from the harvested plant, for example, may be performed by natural drying in the shade. Further, the crushing or pulverization may be pulverized or pulverized to a degree that the useful components of the plant can be sufficiently extracted during the subsequent extraction process. The drying and crushing or pulverizing process may be performed in reverse order or repeatedly performed as necessary.

In the extraction of the present invention, the method of extracting is not particularly limited, and may be extracted according to a method commonly used in the art.

In the present invention, the extract of gambling (Grateloupia elliptica ) can be obtained by extraction with _{water, a lower alcohol of C 1} to C _{4 or a mixed solvent thereof.} In addition, the lower alcohol may be ethanol, methanol or butanol. The alcohol may be 20 to 100% alcohol, more specifically 20 to 100% ethanol, or methanol.

As used herein, the term "fraction" means a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing various various constituents.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. As a non-limiting example of the fractionation method, there may be mentioned a method of obtaining a fraction from the extract by treating an extract of Grateloupia elliptica with a predetermined solvent.

In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, alcohol having 1 to 4 carbon atoms, hexane (Hexane), chloroform (Chloroform), ethyl acetate (Ethyl acetate), butanol (buthanol), or a mixed solvent thereof.

In an exemplary embodiment of the present invention, an ethanol or methanol extract of champignon was obtained by extraction with ethanol or methanol in Grateloupia elliptica, which was hexane (hexane, Hex), chloroform, ethyl acetate, EtOAc), butanol (BuOH), water (water, H ₂ O) was sequentially fractionated to prepare a fraction of hexane, chloroform, ethyl acetate, butanol, and the remaining water layer.

In the present invention, the fraction is a hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction.

In one embodiment of the present invention, it was confirmed that the ethyl acetate fraction of the ethanol extract or methanol extract of Cham Gambling inhibits adipocyte differentiation and lipid accumulation, and exhibits a weight reduction effect, and is important for fatty acid production, storage and migration in adipocytes. It inhibited the expression of PPAR-γ, SREBP-1, and FABP-4, which are known to play a role, reduced the weight of epidermal fat, subcutaneous fat, peripheral fat, and intestinal fat, and triglycerides ( It was confirmed that it can be used in a pharmaceutical composition for treating or preventing obesity by lowering the content of TG), total cholesterol (TC), leptin and insulin.

The term "obesity" used in the present invention means a state in which adipose tissue is excessive in the body. At the time of diagnosis, obesity is defined if the body mass index (Body mass index: body mass index: weight (kg) divided by the square of height (m) is 25 to 30 or more.) Obesity refers to nutrients compared to energy expenditure over a long period of time. Excessive intake is caused by energy imbalance, and fatty acids and glucose introduced from plasma into fat cells are esterified and accumulated mainly in the form of triglycerides.

The term "prevention" as used in the present invention refers to all actions of inhibiting or delaying obesity by administering a composition comprising the extract or fraction according to the present invention.

The term "treatment" as used in the present invention refers to all actions in which the symptoms of obesity are improved or beneficially changed by administration of a composition comprising the extract or fraction according to the present invention.

The pharmaceutical composition comprising the extract or fraction of the champignon of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the preparation of a pharmaceutical composition. At this time, the extract or fraction included in the composition is not particularly limited thereto, but may include 0.001% by weight to 99% by weight, preferably 0.01% by weight to 50% by weight, based on the total weight of the composition.

The pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried agents, and suppositories. , May be oral or parenteral in various dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and disease. It can be determined according to the type, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of treating obesity, and anything can be applied. For example, non-human animals such as monkeys, dogs, cats, rabbits, mormons, rats, mice, cows, sheep, pigs, goats, etc., humans, birds and fish, etc. can be used, and the pharmaceutical composition may be parenterally or subcutaneously , Intraperitoneal, intrapulmonary and intranasal administration, and for local treatment, if necessary, may be administered by a suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the severity of the disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or cerebrovascular injection, but is not limited thereto.

A suitable total daily use amount can be determined by the physician within the range of correct medical judgment, and is generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/ The amount of kg can be divided and administered once to several times a day.

In another aspect, the present invention provides a food composition for improving or preventing obesity, comprising an extract or a fraction thereof as an active ingredient.

In the present invention, the description of gambling, extracts, fractions, obesity, and prevention are as described above.

The term "improvement" as used in the present invention refers to all actions in which obesity is improved or beneficially changed by administering the composition.

The food composition of the present invention may include the form of a pill, powder, granule, pill, tablet, capsule, or liquid, and there are different restrictions on the kind of food to which the extract or fractions thereof of the present invention can be added. There are no, for example, various beverages, gum, tea, vitamin complexes, and health supplements.

In addition to the extract or fractions thereof, other ingredients may be added to the food composition, and the kind is not particularly limited. For example, it may contain as an additional component, such as various herbal extracts, food additives or natural carbohydrates that are acceptable in food, as in ordinary foods, but is not limited thereto.

In the present invention, the term "food supplementary additive" refers to a component that can be added auxiliary to food, and is added to the manufacture of health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of the food additives of the present invention are not limited by the above examples.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides 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, etc.), stevia extract (rebaudioside A, glysir) Hibiscus) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used advantageously.

The food composition may include a health functional food, and in the present invention, the food composition may be a health functional food. The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and it may be excellent in portability.

The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food, the gambling extract of the present invention or a fraction thereof may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be used even below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and all health functional foods in the usual sense are included.

The present invention relates to a pharmaceutical composition for the treatment or prevention of obesity, comprising an extract or a fraction thereof as an active ingredient, wherein the ethyl acetate fraction of the ethanol extract or methanol extract of the extract inhibits adipocyte differentiation and lipid accumulation, It was confirmed to have a weight loss effect, and suppressed the expression of PPAR-γ, SREBP-1, and FABP-4, which are known to play an important role in fatty acid production, storage and migration in adipocytes, and epidermal fat, subcutaneous fat, and peripheral fat , Reduced the weight of intestinal fat, and confirmed that the content of triglycerides (TG), total cholesterol (TC), leptin and insulin in the serum of a high-fat diet-induced mouse was lowered to be used in pharmaceutical compositions and food compositions for the treatment or prevention of obesity. I can.

Fig. 1. Shows the cytotoxic effect of Ethanol Extract (GEE) of Cham Gambling on 3T3-L1 cells. The cytotoxicity of GEE was measured using MTT assay for 24 hours. Data are expressed as mean ± SD (n = 3) in each group. ^* p <0.05, ^** p <0.01 and ^*** p <0.001 indicate that there is a significant difference compared to the control group.
Figure 2. Shows the effect of GEE on intracellular lipid accumulation in 3T3-L1 cells. Lipid accumulation was measured by Oil Red O staining. 3T3-L1 cells were treated with a differentiation-inducing cocktail for 2 days and GEE was added to 3T3-L1 cells for 8 days. After 8 days, intracellular lipids were stained with Oil Red O and photographed using a microscope (A). Quantification of lipid content was measured by absorbance of lipid droplets stained at 500 nm (B). Data are expressed as mean±SD (n=3) in each group. ^* p <0.05, ^** p <0.01 and ^*** p <0.001 indicate that there is a significant difference compared to the control group.
Figure 3. It shows the process of preparing a fraction for each solvent of the methanol extract of chagamgam.
Figure 4. Oil red O staining results of the ethanol extract, methanol extract, hexane fraction of methanol extract, chloroform fraction, ethyl acetate fraction, and butanol fraction.
Fig. 5. It shows the result of quantification of Fig. 4.
Fig. 6. The effect of Ethanol Extract (GEE) of Cham Gambling on the expression of adipogenic proteins in 3T3-L1 cells is shown. Western blot analysis of SREBP-1, PPAR-γ, and FABP4 was performed, and the adipogenic protein band (A) was quantified and shown as a graph (B). Data are expressed as mean±SD (n=3) in each group. ^* p <0.05, ^** p <0.01 and ^*** p <0.001 indicate that there is a significant difference compared to the control group.
Fig. 7 shows the results of measuring the effect of GEE on body weight and fat weight gain in HFD-induced mice. The comparison of weight gain (A), weight change at week 7 (B), and fat weight (C) after treatment with GEE in HFD-induced obese mice for 7 weeks is shown. Data are expressed as mean±SD (n=3) in each group. ^* p <0.05, ^** p <0.01 and ^*** p <0.001 indicate significant differences compared to the control group, ^# p <0.05, ^## p <0.01 and ^### p <0.001 compared to the control group. It indicates that there is a significant difference.
Fig. 8. The results of histological analysis of white adipose tissue and liver tissue in HFD-induced mice are shown. At 8 weeks, all mice were euthanized, white adipose tissue and liver tissue were collected, fixed, and H&E stained to observe histological images. Lipid accumulation was indicated by measuring the area of lipid droplets in liver tissue (A), and the cell number and size of white adipocytes were measured through image J software. Data are expressed as mean±SD (n=3) in each group. ^* p <0.05, ^** p <0.01 and ^*** p <0.001 indicate significant differences compared to the control group, ^# p <0.05, ^## p <0.01 and ^### p <0.001 compared to the control group. It indicates that there is a significant difference.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example 1: Substance

Chamdobak ( G. elliptica ) was collected from Jeju Island, Korea. The collected champignons were washed with tap water to remove salt and epiphytic plants, and then stored in a freezer at -20 ℃ and freeze-dried. Subsequently, the dried champignon was homogenized for further experimentation.

3T3-L1 cell line was purchased from Korea Cell Line Bank (KCLB, Seoul), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), bovine serum (BS), phosphate buffered saline (pH 7.4; PBS) and penicillin- Streptomycin (PS) was purchased from Gibco (Grand Island, NY, USA). Anti-obesity monoclonal antibodies such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and fatty acid binding protein 4 (FABP4) were purchased from cell signaling technology (Bedford, MA, USA). sterol regulatory element-binding protein 1 (SREBP-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from santa cruz biotechnology (Santa Cruz, CA, USA). 3T3-L1 cell differentiation reagent including isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin was purchased from Sigma Chemical (St. Louis, Mo.). Serum insulin was measured by a commercial kit of Crystal Chem (Elk Grove Village, Illinois, USA), and serum triglyceride and total cholesterol content were measured by a colorimetric assay kit of Abcam (USA, Massachusetts, Cambridge). Serum leptin was measured by a commercial kit from Invitrogen (USA, New York, Grand Island). All chemicals and reagents used were for analysis and were obtained from commercial sources.

Example 2: Preparation of Ethanol Extract (GEE) of Cham Gambling

100 kg of dried champignons were mixed with a 60% ethanol solution and extracted under optimal conditions (70° C., 20 hours) in a shaking incubator. After extraction, 60% of the ethanol extract was filtered, and the filtrate was rotary evaporated at 37°C with a vacuum evaporator. The concentrated extract was sterilized at 90°C using a sterilizer. The sterilized extract was stored in a -80°C freezer and lyophilized. Garcinia Cambogi a Extract (GCE) was used as a positive control.

Example 3: Preparation of fractions of methanol extract of champignons

As in Example 2, a methanol extract of champignon was prepared, 6.0 g of the extract was suspended in distilled water, and fractionated sequentially according to polarity order, n -hexane ( n -hexane, n -Hex.), chloroform (Chloroform, CHCl ₃ ), ethyl acetate (EtOAc), n -butanol ( n -butanol, n -BuOH) and water (water, H ₂ O) to obtain a solvent fractionation layer in that order (Fig. 3). Each of the resulting fractions was concentrated under reduced pressure to prepare a powdered gambling fraction.

Example 4: Cell culture and differentiation

3T3-L1 cells were cultured in DMEM medium supplemented with 10% BS and 1% penicillin (100 units/ml ^-1 ) and streptomycin (100 μg/ml) under optimal culture conditions (37° C., 5% CO _{2 ).} . Subculture of 3T3-L1 cells was performed every 2 days. After 2 days, when passaged 3T3-L1 cells reached 60% confluence, they were passaged again. Differentiation of 3T3-L1 cells was performed 2 days after confluence of 3T3-L1 adipocytes. To induce differentiation, 3T3-L1 cell culture medium was added to 10% FBS, 1% PS and MDI solution [dexamethasone (0.25 μM), IBMX (0.5 mM) and insulin (5) for 2 days to induce adipocyte differentiation. μg/ml)]. After 2 days, the 3T3-L1 cell medium was further replaced for 2 days with DMEM medium containing 10% FBS, 1% PS and insulin (5 μg/ml). After MDI and insulin induction, the cells were then replaced with the culture medium every 2 days. After 8 days, the accumulated lipid content was measured by Oil Red O staining.

Example 5: Oil Red O staining

Oil red O staining was performed to quantify intracellular lipid accumulation in 3T3-L1 cells, and lipid staining was performed according to a method known by Kang (Anti-obesity effects of seaweeds of Jeju Island on the differentiation of 3T3- L1 preadipocytes and obese mice fed a high-fat diet, Food and chemical toxicology 90 (2016) 36-44). Intracellular lipids were stained with Oil Red O, which specifically stains lipids in differentiated 3T3-L1 cells. Oil Red O visualizes droplets of intracellular lipids in 3T3-L1 cells. To evaluate the anti-obesity activity, 3T3-L1 cells were cultured in 12 well plates and cell differentiation was induced. Subsequently, 25, 50, 100, 200 μg/ml of champignon ethanol extract (GEE) was treated 4 times during adipocyte differentiation except for the control group. After differentiation, the cells were washed with PBS and fixed with 4% formaldehyde for 1 hour to fix the cells on a well plate. After 1 hour, the cells were washed with 60% 2-propanol and dried at room temperature. After drying, the cells were stained with a 0.6% oil red O solution for 1 hour on a shaker. After staining with Oil Red O, the cells were washed several times with distilled water. After washing, the cells were dried and photographed using Lionheart™ (FX automated Microscope BioTek Instruments, Inc.) (Winuskey, Vermont, USA). For quantification of the lipid content, Oil Red O stained on 3T3-L1 cells on a shaker was eluted with 100% 2-propanol for 1 hour. After 1 hour, the relative oil red O content was measured by a microplate reader (Synergy HT Multi-Dection microplate reader, Bio-Tek, USA) at a wavelength of 500 nm.

Example 6: Animals

50 C57/BL 6J male mice were obtained from the Central Laboratory Animal Company (Seoul, Korea), and 5-6 weeks old, weighing 18 to 22 g, were used with approval from the Experimental Animal Center of Jeju National University. Experimental mice were housed under optimal conditions in a room controlled with food (normal diet, high fat diet), water, temperature (20-22° C.) and humidity (55%) on a 12:12 hour light/dark cycle. All mice were adapted to the environment for 2 weeks and then separated into 5 groups (n = 5 in each cage). Animals were fed a normal diet (Chow Diet, CD) group, high fat diet (HFD) group, GEE (125 mg/kg) + HFD group, GEE (250 mg/kg) + HFD group and GCE (125 mg/kg) + It was classified into 5 groups of the HFD group. Each morning test samples were infused by oral administration for 7 weeks.

Example 7: Cell viability assay

Cytotoxicity of Ethanol Extract (GEE) of Cham Gambling was analyzed by Kang (Kang) as described above by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). (Food and chemical toxicology 90 (2016) 36-44). 3T3-L1 cells were placed on 12 well plates at a density of 1 ^{×10 5} cells/well in DMEM medium containing 10% BS and 1% penicillin (100 units/ml ⁻¹ ) and streptomycin (100 μg/ml). Inoculated. Thereafter, the cells were treated as a test sample. The cells were then incubated for 24 hours. After 24 hours, MTT solution (100 Ml; 2 mg/ml) was added to each well and incubated for 3-4 hours. After incubation, the well plate was centrifuged at 800 xg for 5 minutes, and then the supernatant was removed. Formazan crystals were dissolved in dimethyl sulfoxide (DMSO) for 1 hour, and absorbance was measured at 540 nm using a micro plate reader.

Example 8: Western blot analysis

Differentiated 3T3-L1 cells were washed with PBS and collected. The collected cells were dissolved in lysis buffer [Tris (20mM), EDTA (5mM), NaP2O7 (10mM), NaF (100mM), Na3VO4 (2mM), NP-40 (1%), aprotinin (10 mg/ml), and Peptin (10 mg/ml) and PMSF (1 mM)] for 1 hour. Subsequently, the lysate was centrifuged at 12,000 xg for 20 minutes at 4° C. and the protein content was measured with a ^{BCA™ protein assay kit.} After protein quantification, the lysate was mixed with a 4X buffer containing dithiothreitol (DTT) and separated into a 12% SDS gel for 90 minutes at 120° C. before sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis, and separated. The resulting protein was transferred onto a nitrocellulose membrane. After transfer, the membrane was blocked with 5% skim milk in 1X TBST [Tris-HCl (25mM), KCl (2.65 mM), NaCl (137 mM), Tween 20 (0.05%)] for 2 hours and at low temperature with primary antibody. Mixed. The primary antibody was used at a 1:1,000 dilution. Subsequently, the membrane was washed 3 times with 1X TBST every 10 minutes and a secondary antibody was attached at a 1:3,000 dilution for 2 hours. After incubation, the target adipogenic protein band was detected on the X-ray film activated by the ECL Western Blotting Detection Kit.

Example 9: Serum analysis

Blood samples were collected by cardiac puncture into syringes rinsed with EDTA solution. Mouse serum was obtained by centrifuging the blood at 12,000 xg for 20 minutes at 4°C. Serum concentrations of triglyceride (TG), total cholesterol (TC), leptin and insulin levels were measured by a commercial colorimetric assay kit.

Example 10: Statistical Analysis

All results were analyzed in duplicate and provided as mean±SE. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) version 14 software. Statistical significance was found in Duncan's multiple range test and p-value ( ^* p <0.05, ^** p <0.01 and ^*** p <0.001 compared to control, ^# p <0.05, ^## p <0.01 and ^{## compared to the control group. #} p with <0.001) was determined by Student's t- test or a member (one-way) ANOVA (ANOVA).

Experimental Example 1. Measurement of Cytotoxicity of Ethanol Extract (GEE) of Cham Gambling in 3T3-L1 Adipocytes

Cytotoxicity of Ethanol extract (GEE) of Chamdobak in 3T3-L1 cells was measured by MTT assay. The cell viability of 3T3-L1 cells was analyzed after treatment with ethanol extract (GEE) of Cham Gambling for 24 hours. Figure 1 shows the cell viability of the ethanol extract (GEE) of Cham gambling compared to the control. As a result, there was no cytotoxic effect on 3T3-L1 cells at all GEE concentrations (50, 100, 200 μg/ml). Therefore, 50, 100, 200 μg/ml of GEE was selected to measure the inhibitory effect of GEE on lipid accumulation in differentiated 3T3-L1 cells.

Experimental Example 2. Effect of Cham Gambling on Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells

2-1. Effect of Ethanol Extract (GEE) of Cham Gambling on Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells

The anti-obesity effect of Ethanol extract (GEE) from Chagamgam was investigated in 3T3-L1 adipocytes. The inhibitory effect of Ethanol extract (GEE) on adipogenic differentiation and lipid accumulation was measured by oil red O staining dye. FIG. 2 shows microscopic and scan images of 3T3-L1 cells treated with ethanol extracts (GEE) of different concentrations (A), and results of quantification of lipid content (B). As a result, when 3T3-L1 cells were treated with ethanol extract (GEE), cell lipid accumulation was reduced, specifically 28, 34, 40, 61 at concentrations of 25, 50, 100, and 200 μg/ml, respectively. The lipid accumulation was inhibited in a concentration-dependent manner of% (FIG. 2). From these results, it was found that treatment with GEE reduced lipid accumulation in 3T3-L1 cells by inhibiting adipocyte differentiation and lipid accumulation.

2-2. Effects of Extracts and Fractions by Solvents of Cham Gambling on Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells

The effect of inhibiting the differentiation of adipogenesis and lipid accumulation by oil red O staining by using the methanol extract and the hexane fraction, chloroform fraction, ethyl acetate fraction, and butanol fraction of the methanol extract and methanol extract together with the ethanol extract identified in Experimental Example 2-1. Comparison was confirmed. Figure 4 is a result of oil red O staining of the ethanol extract, methanol extract, hexane fraction of methanol extract, chloroform fraction, ethyl acetate fraction, and butanol fraction of champignons, and Figure 5 shows the result of quantification of Figure 4.

As a result, it was found that the ethyl acetate fraction of the methanol extract of Cham Gambling had the best effect of inhibiting adipogenesis differentiation and lipid accumulation (FIGS. 4 and 5 ).

Experimental Example 3. Effect of Ethanol Extract (GEE) of Cham Gambling on the Expression of Fat-Specific Protein in 3T3-L1 Cells

To determine whether Ethanol extract (GEE) of Cham Gambling inhibits adipogenesis in 3T3-L1 cells, the expression of adipose-specific proteins involved in adipogenesis and lipid migration from the extracellular matrix to the cells was analyzed using Western blot analysis And analyzed. 6 is a result of Western blot showing the fat-specific protein levels of PPAR-γ, SREBP-1, and FABP-4 expression in 3T3-L1 cells. The PPAR-γ, SREBP-1, and FABP-4 are known to play an important role in fatty acid production, storage, and migration in adipocytes.

Thus, the purpose of the study was to investigate the effect of Ethanol Extract (GEE) of Chagamgam on the expression of fat-specific proteins. As a result, GEE (25, 50, 100, 200 μg/ml) significantly reduced the expression of fat-specific proteins such as PPAR-γ, SREBP-1, and FABP-4 compared to the control. From these results, it was found that GEE inhibits adipogenesis, lipid accumulation and lipid migration by down-regulation of adipose-specific protein expression in 3T3-L1 cells. Therefore, it was found that the ethanol extract (GEE) of Cham Gambling plays an important role in the formation of fat by regulating fat-specific proteins.

Experimental Example 4. Influence of Ethanol Extract (GEE) of Charming Gambling on Weight and White Adipose Tissue Weight Increase

All mice were orally administered with saline or ethanol extract (GEE) once a day for 7 weeks, and body weights were recorded every week (Fig. 7). As a positive control, Garcinia Cambogia Extract (GCE), which is known to have a weight loss effect, was used. The weight gain of the HFD group was significantly higher than that of the ND diet group, indicating that induction of a high fat diet caused obesity in the normal C57/BL mouse model. However, supplementation of GEE significantly attenuated HFD-induced weight gain compared to the HFD diet group.

Specifically, when the mice were orally administered GEE at a concentration of 125 or 250 mg/kg, the weight gain was significantly reduced. As shown in Figure 7A, significant weight loss was observed in the GEE and GCE treatment groups. Compared with HFD (38.74 ± 1.92 g), the body weight of the HFD + GEE (125 μg/ml) group decreased to 35.55 ± 2.25 g after 7 weeks, and the body weight of the HFD + GEE (250 μg/ml) group was 35.21 ± 1.93 g Decreased to. The weight loss effect was similar to that of Garcinia Cambogia Extract (GCE) used as a positive control.

7B shows the change in body weight over 7 weeks. As for the weight, CD showed a weight gain of 6.24 ± 1.08 g, and HFD showed a weight gain of 16.10 ± 2.30 g. However, a decrease in weight gain was observed in the GEE treatment group. The GEE (125mg/kg) + HFD group showed a weight gain of 13.23 ± 0.92g, and the GEE (250mg/kg) + HFD group showed a weight gain of 12.81 ± 0.69g, indicating a decrease in weight gain. . The weight gain reduction effect was similar to that of Garcinia Cambogia Extract (GCE) used as a positive control.

In addition, the weight gain of four types of white adipose tissue including epidermal (Epi), subcutaneous (Sub), mesenteric (Mes), and peripheral (Peri) adipose tissue was investigated. As a result, a significant increase in fat weight was observed in the HFD group compared to the control group. 7C shows the relative adipose tissue weight of each mouse group.

Compared with the CD group [Epi (0.43 ± 0.05 g), Sub (0.36 ± 0.07 g), Mes (0.31 ± 0.01 g), Peri (0.21 ± 0.01 g)], the HFD diet group [Epi (2.51 ± 0.19 g)] , Sub (2.12 ± 0.08 g), Mes (1.67 ± 0.11 g), Peri (0.44 ± 0.10 g)], high fat weight was measured. However, in the HFD + GEE (125 mg/kg) diet group, fat weight gain decreased with Epi (2.28 ± 0.52 g), Sub (2.00 ± 0.19 g), Mes (1.42 ± 0.07 g), and Peri (0.28 ± 0.01 g). Became. In addition, in the HFD + GEE (250 mg/kg) diet group, fat weight was reduced by Epi (2.11 ± 0.18 g), Sub (1.90 ± 0.00 g), Mes (1.31 ± 0.20 g), and Peri (0.22 ± 0.00 g). Became. In particular, oral administration of high concentration GEE (250 mg/kg) significantly reduced mouse Epi and Mes fat weight gain. These results showed that GEE effectively reduced HFD-induced body weight and fat gain in HFD-induced obese mice.

Experimental Example 5. Effect of Ethanol Extract (GEE) of Cham Gambling on Mouse Serum Biochemical Index in HFD Mice

In order to further confirm the anti-obesity effect of the ethanol extract (GEE) of Cham Gambling, the mouse serum biochemistry was investigated in HFD-induced mice. Specifically, serum triglyceride (TG), total cholesterol (TC), leptin and insulin levels were measured. Table 1 shows the results of the mouse serum biochemical studies of different dietary groups.

Compared to the CD group [TG (65.51 ± 2.03 nmol), TC (38.75 ± 0.00 μg/ml), leptin (212.50 ± 2.50 pg/ml), insulin (1.48 ± 0.02 ng/ml)], [TG (110.78 ± 1.62 nmol), TC (50.17 ± 0.05 μg/ml), leptin (4308.13 ± 59.37 pg/ml), insulin (4.50 ± 0.18 ng/ml)] significantly increased TG, TC, leptin and insulin levels. .

However, oral administration of GEE significantly reduced TG, TC, leptin and insulin levels. Specifically, by administration of GEE (250 mg/kg), TG (58.22 ± 0.14 mmol/μl), TC (29.33 ± 0.07 μg/μl), leptin (1896.88 ± 1.87 pg/ml), insulin (3.97 ± 0.00 ng/μl) ml), and by administration of GEE (125 mg/kg), TG (77.41 ± 0.14 mmol/μl), TC (89.53 ± 0.17 μg/μl), leptin (3176.88 ± 44.38 pg/ml) and insulin (4.10 ± 0.05 ng/ml). This effect is to reduce serum triglyceride (TG), total cholesterol (TC), leptin and insulin levels than Garcinia Cambogia Extract (GCE) used as a positive control. These results suggest that GEE may affect serum lipid and hormone levels in HFD-induced obese mice.

Parameters Groups CD HFD GEE
(125 mg/kg) +HFD GEE
(250 mg/kg) +HFD GCE
(125 mg/kg) +HFD Triglyceride (mmol/μl) 65.51 ± 2.03 ^#### 110.78 ± 1.62 77.41 ± 0.14 ^** 58.22 ± 0.14 ^*** 94.43 ± 0.14 ^** Total cholesterol
(μg/μl) 38.75 ± 0.00 ^## 50.17 ± 0.05 89.53 ± 0.17 ^** 29.33 ± 0.07 ^*** 53.66 ± 0.08 ^NS Leptin (pg/ml) 212.50 ± 2.50 ^#### 4308.13 ± 59.37 3176.88 ± 44.38 ^*** 1896.88 ± 1.87 ^*** 4561.88 ± 4.38 ^** Insulin (ng/ml) 1.48 ± 0.02 ^#### 4.50 ± 0.18 4.10 ± 0.05 ^NS 3.97 ± 0.00 ^NS 6.23 ± 0.18 ^**

Experimental Example 6. Histological analysis of mouse liver and white adipose tissue

Histological analysis of mouse liver and epidermal adipose tissue was performed by hematoxylin and eosin (H&E) staining after 7 weeks. 8 shows histological images of liver and white adipose tissue sections stained with H&E. According to histological analysis of liver tissue, the amount of lipid drops from the HFD group was higher than that of the CD group. However, these accumulated lipid droplets were reduced by treatment with GEE. That is, as a result of histological analysis in FIG. 8A, it was found that GEE administration reduced HFD-induced fat accumulation in liver tissue.

In addition, as a result of histological analysis of mouse white adipose tissue in FIG. 8B, the degree of lipid accumulation was proportional to the size of white adipose cells. Specifically, HFD increased lipid accumulation and white adipocyte size compared to the CD group, and GEE administration reduced HFD-induced fat accumulation in white adipose tissue. The average adipocyte size of the HFD group mice was larger than that of the CD group, and it was found that HFD induces an increase in the white adipocyte size. It was confirmed that the size of white fat was also reduced by the administration of GEE. In addition, GEE administration further reduced lipid droplet accumulation and reduced the size of white fat than Garcinia Cambogia Extract (GCE), which is known to have anti-obesity effects as a positive control.

In conclusion, it was confirmed that GEE can reduce lipid accumulation in mouse liver and white adipose tissue.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also present. It belongs to the scope of rights of

Claims (5)

A pharmaceutical composition for the treatment or prevention of obesity, comprising the extract or a fraction thereof as an active ingredient. The pharmaceutical composition according to claim 1, wherein the gambling extract is _{extracted with water, C 1} -C ₄ alcohol, or a mixed solvent thereof. The pharmaceutical composition according to claim 2, wherein the alcohol is ethanol or methanol. The pharmaceutical composition according to claim 1, wherein the fraction is a hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction. A food composition for improving or preventing obesity, comprising a gambling extract or a fraction thereof as an active ingredient.

Images