KR20210127655A - Composition for preventing or treating obesity comprising Grateloupia elliptica - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating obesity, including an extract of Grateloupia elliptica or a fraction thereof as an active ingredient. It is shown that an ethyl acetate fraction of an ethanol extract or methanol extract of Grateloupia elliptica inhibits fat cell differentiation and lipid accumulation, and shows an effect of reducing a body weight. It is also shown that the ethyl acetate fraction inhibits expression of PPAR-γ, SREBP-1 and FABP-4 known to play an important role in producing, storing and migrating fatty acids in fat cells, reduces the weight of epidermal fat, subcutaneous fat, peripheral fat and mesenteric fat, and reduces the content of triglycerides (TG), total cholesterols (TC), leptin and insulin in the high-fat diet-induced mouse sera, and thus can be used for a pharmaceutical composition and food composition for preventing or treating obesity.

Description

TECHNICAL FIELD [0001] 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 an extract of chamomile extract as an active ingredient.

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

In addition, when obesity is prolonged, it is known that various metabolic diseases are caused. Therefore, anti-obesity drugs have been developed to treat obesity and its 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 disturbance and pain. There has been an attempt to develop a natural anti-obesity agent that is not toxic to the human body due to the harmful side effects of these synthetic anti-obesity agents.

Marine organisms have the ability to search for a variety of new physiologically active substances due to their unique metabolic process and unique environment that terrestrial organisms do not have. In addition, terrestrial life has already been studied a lot, but marine life has not yet been sufficiently studied, so it is a field with high expectations for the development of natural substances 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, which includes an extract of chamomile 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, which includes an extract of chamomile 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 Chamgobak inhibits adipocyte differentiation and lipid accumulation, and exhibits a weight loss effect. It suppressed the known expression of PPAR-γ, SREBP-1, and FABP-4, reduced the weight of epidermal fat, subcutaneous fat, peripheral fat, and mesenteric fat, and triglyceride (TG), total By confirming that cholesterol (TC), leptin and insulin content can be lowered, it was confirmed that it can be used in pharmaceutical compositions and food compositions for the treatment or prevention of obesity, and the present invention was completed.

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

As used herein, the term “ Grateloupia elliptica ” is a seaweed of the red algae Ginuarit family, with a height of 15-40 cm, a leaf shape, and divided into several branches. The flesh is reddish-purple, yellow, and green, and turns purple when dried.

As used herein, the term "extract" refers to an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as the extract itself and the extract Contains extracts of all formulations that can be formed using

Drying of the extract in the present invention may be carried out by a known method in the range in which useful components from the collected plant are not destroyed, for example, it may be carried out by a method of natural drying in the shade. In addition, crushing or pulverization can be pulverized by crushing or pulverizing to an extent that useful components of plants can be sufficiently extracted in the subsequent extraction process. The drying, crushing, or pulverizing processes may be performed in reverse order or repeated if necessary.

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

In the present invention, the extract of Grateloupia elliptica _{may be obtained by extraction with water, C 1} to C ₄ lower alcohol 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" refers to a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing various various components.

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. Non-limiting examples of the fractionation method include a method of obtaining a fraction from the extract by treating an extract of Grateloupia elliptica with a predetermined solvent.

The kind of solvent used to obtain the fraction in the present invention 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, chloroform, ethyl acetate, butanol, or a mixed solvent thereof.

In an embodiment of the present invention, Grateloupia elliptica was extracted with ethanol or methanol to obtain an ethanol or methanol extract of Grateloupia elliptica, which was obtained by using hexane (hexane, Hex), chloroform, ethyl acetate, EtOAc), butanol (butanol, BuOH), and water (water, H ₂ O) were sequentially fractionated to prepare fractions 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 chamgambak inhibits adipocyte differentiation and lipid accumulation, and exhibits a weight loss effect, which is important for fatty acid production, storage and movement in adipocytes. It suppressed the expression of PPAR-γ, SREBP-1, and FABP-4, which are known to play a role, decreased the weight of epidermal fat, subcutaneous fat, peripheral fat, and mesenteric fat, and triglyceride ( TG), total cholesterol (TC), leptin and insulin content was confirmed to be lowered, it was confirmed that it can be used in a pharmaceutical composition for the treatment or prevention of obesity.

As used herein, the term "obesity" refers to a state in which adipose tissue is excessive in the body. At diagnosis, obesity is defined as body mass index (Body mass index: body mass index: weight (kg) divided by height (m) squared) is 25 to 30 or more. Obesity is defined as a decrease in nutrients compared to energy consumption over a long period of time. In case of excessive intake, it is caused by energy imbalance, and fatty acids and glucose flowing into adipocytes from plasma are esterified and are mainly accumulated in the form of triglycerides.

As used herein, the term “prevention” refers to any act of suppressing or delaying obesity by administering a composition comprising the extract or fraction of the true gambler according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of obesity are improved or beneficially changed by administration of the composition comprising the extract or fraction of the true gambler according to the present invention.

The pharmaceutical composition comprising the extract or fraction of the true gambler of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. In this case, the true gambling extract or fraction included in the composition is not particularly limited thereto, but may be included in an amount of 0.001 wt% to 99 wt%, preferably 0.01 wt% to 50 wt%, 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, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, freeze-drying agents and suppositories, , oral or parenteral formulations may be used. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal 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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term "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 dependent on the subject's type and severity, age, sex, and disease. It may be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and 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, and can be easily determined by those 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 any one is applicable. For example, any of non-human animals such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, human, bird and fish can be used, and the pharmaceutical composition is parenterally, subcutaneously , intraperitoneally, intrapulmonary and intranasally, and for local treatment, if necessary, by any 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 degree of disease, the drug form, the route and period 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 dural or intracerebrovascular injection, but is not limited thereto.

A suitable total daily amount may be determined by a treating physician within the scope of sound 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/kg. The amount of kg can be administered in divided doses from once to several times a day.

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

In the present invention, the description of the true gambling, extract, fraction, obesity, and prevention is the same as described above.

As used herein, the term “improvement” refers to any action in which obesity is improved or is advantageously changed by administering the composition.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, etc., and there is no particular limitation on the type of food to which the true gambling extract of the present invention or a fraction thereof can be added. There are, for example, various beverages, gum, tea, vitamin complexes, and health supplements.

Other ingredients may be added to the food composition in addition to the extract or a fraction thereof, and the type thereof is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food pharmaceutically acceptable food supplement additives or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as 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., but the above examples are not limited to the types of food supplement additives of the present invention.

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. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

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 food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects with respect to 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 during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, food is used as a raw material, so there are no side effects that may occur when taking the drug for a long time, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, during the production of food, the true gambling extract or a fraction thereof of the present invention 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 ingestion for health and hygiene purposes or for health control, the above amount may be used 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 includes all health functional foods in the ordinary sense.

The present invention relates to a pharmaceutical composition for the treatment or prevention of obesity, comprising the extract or a fraction thereof as an active ingredient. It was confirmed that it exhibits 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 transport in adipocytes, and epidermal fat, subcutaneous fat, and peripheral fat , reduced the weight of mesenteric fat, and confirmed that it lowered the triglyceride (TG), total cholesterol (TC), leptin and insulin contents in high-fat diet-induced mouse serum, which could be used in pharmaceutical compositions and food compositions for the treatment or prevention of obesity. can

Figure 1. Cytotoxic effect of ethanol extract (GEE) from Chammoth beetle on 3T3-L1 cells. The cytotoxicity of GEE was measured using the MTT assay for 24 hours. Data are presented 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.
Figure 2. 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 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 determined by absorbance of stained lipid droplets at 500 nm (B). Data are presented 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.
Fig. 3 shows the process of preparing the fractions for each solvent of the methanol extract of Chamdobak.
Fig. 4. Oil Red O staining results of ethanol extract, methanol extract, and hexane fraction, chloroform fraction, ethyl acetate fraction, and butanol fraction of the ethanol extract of Chamgamba.
Fig. 5 shows the results of quantification of Fig. 4;
Fig. 6. Effect of ethanol extract (GEE) from chamgamak on adipogenic protein expression in 3T3-L1 cells. Western blot analysis of SREBP-1, PPAR-γ, and FABP4 was performed, and adipogenic protein bands (A) were quantified and presented as a graph (B). Data are presented 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.
7 shows the results of measuring the effect of GEE on body weight and fat weight gain in HFD-induced mice. A comparison of body weight gain (A), body weight change at 7 weeks (B), and fat weight (C) after treatment with GEE in HFD-induced obese mice for 7 weeks is shown. Data are presented as mean±SD (n=3) in each group. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate significant differences compared to control, ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control indicates that there is a significant difference.
Figure 8. Shows the results of histological analysis of white adipose tissue and liver tissue in HFD-induced mice. At 8 weeks, all mice were euthanized and white adipose tissue and liver tissue were harvested, fixed, and then histologically imaged by H&E staining. 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 presented as mean±SD (n=3) in each group. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate significant differences compared to control, ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control 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 can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

Example 1: Materials

G. elliptica was collected from Jeju Island, Korea. The collected true gambles were washed with tap water to remove salt and epiphytic plants, and then stored in a -20 ℃ freezer and freeze-dried. Then, the dried chamomile was homogenized for further experiments.

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, USA). Serum insulin was measured by a commercially available kit from Crystal Chem (Elkgrove Village, Illinois, USA), and serum triglyceride and total cholesterol contents were measured by a colorimetric kit from Abcam (Cambridge, Massachusetts, USA). Serum leptin was measured by a commercially available kit from Invitrogen (New York, Grand Island, USA). All chemicals and reagents used were for analysis and were obtained from commercial sources.

Example 2: Preparation of Ethanol Extract (GEE)

100 kg of dried chamomile was mixed with 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 freeze-dried. Garcinia Cambogi a Extract (GCE) was used as a positive control.

Example 3: Preparation of Fractions of the Methanol Extract of Cham Gambaek

Carried out as in Example 2, and indeed were prepared gaming methanol extract, the extract was suspended in 6.0g of distilled water, and sequentially fractionated by the polarity order, n - hexane (. N -hexane, n -Hex) , chloroform (Chloroform, CHCl _3), ethyl acetate (ethyl acetate, EtOAc), n - butanol (n -butanol, n -BuOH) and water (water, H ₂ O) in order to obtain a solvent layer fraction (Figure 3). Each of the generated fractions was concentrated under reduced pressure to prepare a powdered true 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 the subcultured 3T3-L1 cells reached 60% confluence, they were subcultured again. Differentiation of 3T3-L1 cells was performed 2 days after confluence of 3T3-L1 preadipocytes. To induce differentiation, 3T3-L1 cell culture medium was mixed with 10% FBS, 1% PS and MDI solution [dexamethasone (0.25 μM), IBMX (0.5 mM) and insulin (5 μg/ml)]. After 2 days, the 3T3-L1 cell medium was further replaced with DMEM medium containing 10% FBS, 1% PS and insulin (5 μg/ml) for an additional 2 days. After MDI and insulin induction, cells were then replaced with culture medium every 2 days. After 8 days, the accumulated lipid content was determined 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 small droplets for intracellular lipids in 3T3-L1 cells. To evaluate anti-obesity activity, 3T3-L1 cells were cultured in 12 well plates and cell differentiation was induced. Then, 25, 50, 100, and 200 μg/ml of ethanol extract (GEE) from Chammoth root were 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, cells were stained with 0.6% Oil Red O solution for 1 hour on a shaker. After Oil Red O staining, cells were washed several times with distilled water. After washing, cells were dried and photographed using a Lionheart™ (FX automated Microscope BioTek Instruments, Inc.) (Winusky, VT, USA). For quantification of lipid content, Oil Red O stained on 3T3-L1 cells on a shaker was eluted with 100% 2-propanol for 1 h. 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

Fifty male C57/BL 6J mice were obtained from the Central Laboratory of Animals (Seoul, Korea), and 5-6 weeks of age, weighing 18-22 g, were used with the approval of the Experimental Animal Center of Jeju National University. Experimental mice were housed under optimal conditions in a controlled room 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 acclimatized to the environment for 2 weeks and then separated into 5 groups (n = 5 in each cage). Animals were treated in the normal diet (Chow Diet, CD) group, in the 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 HFD group. Test samples were infused by oral administration every morning for 7 weeks.

Example 7: Cell Viability Assay

Cytotoxicity of ethanol extract (GEE) from Chammoth beetle was assayed for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously described by Kang. (Food and chemical toxicology 90 (2016) 36-44). 3T3-L1 cells were cultured in DMEM medium containing 10% BS and 1% penicillin (100 units/ml ⁻¹ ) and streptomycin (100 μg/ml) at ^{a density of 1×10 5} cells/well in 12 well plates. inoculated. Thereafter, the cells were treated with 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×g for 5 min, 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 microplate reader.

Example 8: Western Blot Analysis

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

Example 9: Serum analysis

Blood samples were collected by cardiac puncture into syringes cleaned with EDTA solution. Mouse serum was obtained by centrifuging blood at 12,000 x g for 20 min at 4°C. Serum concentrations of triglyceride (TG), total cholesterol (TC), leptin and insulin levels were determined by commercial colorimetric assay kits.

Example 10: Statistical Analysis

All results were analyzed in duplicate and presented as mean ± SE. Statistical analyzes were performed using the Statistical Package for Social Sciences (SPSS) version 14 software. Statistical significance was determined by Duncan's multiple-range test and p-values ( ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 versus controls, ^# p < 0.05, ^## p < 0.01 and ^{## versus controls). #} p < 0.001) by Student's t-test or one-way analysis of variance (ANOVA).

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

The cytotoxicity of ethanol extract (GEE) from Chammoth beetle in 3T3-L1 cells was measured by MTT assay. The cell viability of 3T3-L1 cells was analyzed after treatment with ethanol extract (GEE) from Chammoth for 24 hours. Fig. 1 shows the cell viability of ethanol extract (GEE) from Chammoth beetle compared to the control group. As a result, there was no cytotoxic effect on 3T3-L1 cells at all GEE concentrations (50, 100, 200 μg/ml). Therefore, the inhibitory effect of GEE on lipid accumulation of differentiated 3T3-L1 cells was measured by selecting 50, 100, and 200 μg/ml of GEE.

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

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

The anti-obesity effect of ethanol extract (GEE) was investigated in 3T3-L1 adipocytes. The inhibitory effect of ethanol extract (GEE) of Chammoth root 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 different concentrations of ethanol extract (GEE) from Chammoth (A), and quantification of lipid content (B). As a result, when 3T3-L1 cells were treated with ethanol extract (GEE), cellular lipid accumulation was reduced, specifically, 28, 34, 40, 61 at concentrations of 25, 50, 100, and 200 μg/ml, respectively. % inhibited lipid accumulation in a concentration-dependent manner ( FIG. 2 ). Through these results, it was found that treatment of GEE inhibited adipocyte differentiation and lipid accumulation, thereby reducing lipid accumulation in 3T3-L1 cells.

2-2. Effect of extracts and fractions for each solvent in 3T3-L1 cells on adipocyte differentiation and lipid accumulation

The effect of inhibiting adipogenesis differentiation and lipid accumulation by using a methanol extract, a hexane fraction, a chloroform fraction, an ethyl acetate fraction, and a butanol fraction of the methanol extract, along with the ethanol extract of Chammoth root identified in Experimental Example 2-1 by Oil Red O staining comparison was confirmed. 4 shows the results of Oil Red O staining of the ethanol extract, methanol extract, and methanol extract, hexane fraction, chloroform fraction, ethyl acetate fraction, and butanol fraction, and FIG. 5 shows the quantification results of FIG.

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

Experimental Example 3. Effect of ethanol extract (GEE) from Chamgamakji on the expression of fat-specific proteins in 3T3-L1 cells

To determine whether ethanol extract (GEE) of chamomile extract (GEE) inhibits adipogenesis in 3T3-L1 cells, expression of fat-specific proteins involved in adipogenesis and lipid transport from the extracellular matrix into cells was analyzed using western blot analysis. and analyzed. 6 is a 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 movement in adipocytes.

Therefore, the effect of ethanol extract (GEE) of Chamgobak on fat-specific protein expression was investigated. 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 group. These results suggest that GEE inhibits adipogenesis, lipid accumulation, and lipid transport by down-regulation of adipose-specific protein expression in 3T3-L1 cells. Therefore, it could be seen that the ethanol extract (GEE) plays an important role in fat formation by regulating fat-specific proteins.

Experimental Example 4. Effect of Ethanol Extract (GEE) from Charcoal Root on increase in body weight and white adipose tissue weight

All mice were orally administered with saline or ethanol extract (GEE) from ginseng champagne (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, suggesting that induction of a high-fat diet induces 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 GEE was orally administered to mice at a concentration of 125 and 250 mg/kg, weight gain was significantly reduced. As shown in Figure 7A, significant weight loss was observed in the GEE and GCE treated 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 body weight changes over 7 weeks. As for body 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-treated 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, confirming that the weight gain decreased. . It showed similar weight gain reduction effect as Garcinia Cambogia Extract (GCE) used as a positive control.

In addition, the weight increase 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 group of mice.

Compared to 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)]. However, in the HFD + GEE (125 mg/kg) diet group, 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) decreased fat weight gain. became In addition, in the HFD + GEE (250 mg/kg) diet group, 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) reduced fat weight. became In particular, oral administration of a high concentration of GEE (250 mg/kg) significantly reduced mouse Epi and Mes fat weight gain. Through these results, it was found that GEE effectively reduced HFD-induced body weight and fat gain in HFD-induced obese mice.

Experimental Example 5. Effect of Ethanol Extract (GEE) from Charcoal Root on Mouse Serum Biochemical Index in HFD Mice

To further confirm the anti-obesity effect of ethanol extract (GEE) from chamomile extract, 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 mouse serum biochemical studies of different diet 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)], in the HFD diet group [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 / ml), also TG (77.41 ± 0.14 mmol/μl), TC (89.53 ± 0.17 μg/μl), leptin (3176.88 ± 44.38 pg/ml) and insulin by GEE (125 mg/kg) administration. (4.10 ± 0.05 ng/ml) level. This effect was shown to reduce serum triglyceride (TG), total cholesterol (TC), leptin and insulin levels compared to 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 from 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 shown 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 adipocytes. Specifically, HFD increased lipid accumulation and white adipocyte size compared to the CD group, and GEE administration decreased 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, suggesting that HFD induces an increase in white adipocyte size. It was confirmed that the size of white fat was also reduced by the administration of GEE. In addition, the administration of GEE reduced the accumulation of lipid droplets more than Garcinia Cambogia Extract (GCE), which is known to have an anti-obesity effect as a positive control, and reduced the size of white fat.

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

Although the preferred embodiment of the present invention has 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 as defined in the following claims are also provided. is within the scope of the

Claims (3)

A pharmaceutical composition for the treatment or prevention of obesity, comprising, as an active ingredient, an ethyl acetate fraction of an ethanol extract from Chamgamba. According to claim 1, wherein the concentration of the ethyl acetate (Ethyl acetate) fraction of the ethanol extract of Chamgobak is 125 ~ 250 ㎍ / ml of the pharmaceutical composition. A food composition for improving or preventing obesity, comprising, as an active ingredient, an ethyl acetate fraction of an ethanol extract from Chamgambaek.

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