KR101501380B1 - A composition comprising Euptelea Pleiosperma extracts having anti-obesity activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating obesity comprising Euptelea pleiosperma (EP) extract as an active ingredient, and a composition for preventing or improving obesity comprising the EP extract as an active ingredient will be. Since the composition comprising the EP extract of the present invention has the ability to inhibit the differentiation of adipose precursor cells into adipocytes and inhibit pancreatic lipase enzyme activity, it can be used as an anti-obesity composition, A pharmaceutical composition for preventing or treating diseases caused by unnecessary accumulation of fat, or a composition for preventing or ameliorating obesity.

Description

A composition comprising Euptelea Pleiosperma extracts having an anti-obesity activity comprising a < RTI ID = 0.0 >

The present invention relates to an anti-obesity composition comprising Yupe telereplus osperma , and more particularly, the present invention relates to an anti-obesity composition comprising an extract of Euptelea pleiosperma (EP) as an active ingredient, A composition and a composition for preventing or improving obesity comprising the EP extract as an active ingredient.

Obesity or obesity refers to the excess weight of a person's body weight, which is pathologically referred to as the World Health Organization's (WHO) standard body mass index (BMI) of 30 or more. If it is 25 or more, it is diagnosed as obesity. In most cases, weight means more than normal, but even if you do not have a lot of weight, the proportion of fat in your body is high. Obesity may not be a problem in itself, but it has serious social problems and secondary complications caused by excessive fat. For example, obesity can significantly increase the incidence of hyperlipidemia, hypertension, arteriosclerosis, diabetes, fatty liver, joint disorders, and the like.

As such, obesity causes various adult diseases. Increased weight can lead to diabetes and lower blood sugar, which can lead to hypertension. If the body weight is 20% higher than normal weight, the likelihood of developing diabetes increases to 10 times that of normal people. In addition, cardiovascular and pulmonary function may be impaired and life expectancy may be shortened. Complex factors such as hypertension, diabetes, hypercholesterolemia, and atherosclerosis may put pressure on the heart function and lead to heart failure and sudden death. In addition to women, the incidence of endometrial cancer is high, and cholelithiasis is more likely to occur. Men are more likely to develop colon cancer and prostate cancer. In addition, as the weight increases, people can not help but be conscious of the gaze that they are looking at, and if the obsession about it becomes worse, the symptoms of avoiding people and living alone are generated. The inferiority feeling that comes from the body also affects the self by losing confidence in his life.

In order to prevent obesity and maintain a healthy weight, balanced diet and regular exercise should be maintained throughout the lifetime. In particular, fat cells can be reduced in size once they are made, but it is impossible to remove them naturally and it is left permanently in the human body, so it is important to prevent obesity from beginning. Therefore, weight loss can be reduced primarily by reducing the amount of calories consumed and increasing activity and exercise.

However, as described above, since the fat cells once formed can not be removed, there are cases where a drug such as a diet meal intake and a fat absorption reduction is taken. However, such diet drugs can cause side effects such as cardiovascular disease and stroke, such as hypertension, and frequently increase in weight again when stopped. In the case of severe obesity, surgical treatment can be performed. Such surgery is performed by surgery to remove fat or gastroplasty or gastric banding to limit the amount of food that can be digested by the body.

Under these circumstances, the inventors of the present invention have made intensive studies to find natural products having an anti-obesity effect and few side effects. As a result, it has been found that Euptelea pleiosperma extract, one of Chinese native plants, inhibits the differentiation into adipocytes, The present invention has been accomplished by confirming that it inhibits the activity of pancreatic lipase enzyme as well as reducing the fat production rate.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of obesity which comprises an extract of Euptelea pleiosperma .

Another object of the present invention is to provide a composition for the prevention or amelioration of obesity, which comprises the above-mentioned extract of Yupe telapia pleurus as an active ingredient.

The present inventors focused on Euptelea pleiosperma (EP) while performing research on various natural products in order to find a natural product having an anti-obesity effect and a low side effect.

As a result of testing the anti-obesity effect of the EP extract, the EP extract showed inhibitory activity against the lipase secreted from the pancreas without showing cytotoxicity against lipid precursor cells, And inhibited the differentiation of adipose precursor cells into adipocytes and inhibited the production of triglycerides in adipocytes.

As described above, the EP extract exhibits an inhibitory activity against lipase, inhibits the differentiation of adipocytes and inhibits the production of triglycerides in adipocytes, and thus can be used for preventing, treating or improving obesity Could know.

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating obesity, which comprises an EP extract as an active ingredient.

The term "obesity " of the present invention refers to an excessive state of body fat, which means that the body mass index is clinically 25 in the case of Korea and 30 or more according to the World Health Organization (WHO). The body mass index (BMI) is calculated as the body weight (kg / m ² ) relative to the height of the kidney. Generally, it means that the body weight is higher than the normal value. However, when the body fat percentage of the body composition is high, even if the body weight does not go out, it is diagnosed as obesity. Obesity can shorten life and put you at risk for diseases like hypertension, diabetes, heart disease and some cancers. Obesity is likely to cause health problems, and as the degree of obesity increases, the risk also increases. Obesity is caused by a wide variety of reasons and many causes are often combined. The most frequent causes are genetic influences, physiological effects, food intake and eating disorders, lifestyle, weight, pregnancy or drugs. The first signs of obesity are above average weight and symptoms are as follows: sleep problems, sleep apnea, shortness of breath, varicose veins, skin problems, osteoarthritis due to gallstones or weight loss, Obesity is also associated with diseases such as hypertension, hyperglycemia (diabetes), hypercholesterolemia, and hypertriglyceridemia. Once you are overweight, it is not easy to get out of it, so obesity is a lifelong experience. However, diseases or problems caused by obesity can improve if weight is reduced.

Hereinafter, the term "anti-obesity " is used in the present invention to mean prevention, treatment, or improvement of obesity. Accordingly, the term "anti-obesity composition" used in the present invention means a pharmaceutical composition for preventing or treating obesity.

In the present invention, the term "prevention" means any action which inhibits or delays the onset of obesity by the administration of the pharmaceutical composition according to the present invention, and "treatment" Means any action that alters or alleviates symptoms of an onset individual.

The term " Euptelea pleiosperma (EP ) "of the present invention is a deciduous plant of Euptelea pleiosperma (EP ) belonging to the Eupteleaceae family, It is distributed from Assam province to China, and is distributed in Japan, and includes Euptelea pleiosperma and Euptelea polyandra . EP is distributed in East Asia, mainly in China and the Himalayas. Typical height is 2 to 9 m, flowering time is May, and seeds are bloomed from August to September. Leaves are edible, and the bark of flowers and branches is known to be medicinal, but no specific effects have been known yet, and there is no known specific anti-obesity effect. Therefore, the present inventors confirmed that the EP extract had an anti-obesity effect, and confirmed that it could be applied to foods or medicines.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating the liquid by evaporating the leaching solution, and is not limited thereto. The extract is obtained by drying the extract, diluted solution, Dried products, controlled products thereof or purified products thereof.

In the present invention, the extract may be prepared by extracting with an extraction solvent or extracting with an extraction solvent, followed by fractionation with a fraction solvent. The extraction solvent may be, but is not limited to, water, an organic solvent or a mixed solvent thereof. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloromethane Non-polar solvents or mixed solvents thereof may be used. Further, water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be preferably used. In one embodiment of the present invention, an extract using 95% methanol as the solvent was prepared.

The EP extract can be prepared by using common extraction, isolation and purification methods known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

The term " Yupe telereplasmus perma (EP) extract " of the present invention means an extract obtained by extracting from various parts of EP, i.e., samples containing some or all of leaves, stems and flowers of EP.

According to a specific embodiment of the present invention (Example 1), the EP extract of the present invention is prepared by disintegrating EP into a powder of a predetermined weight and extracting it with 5 to 10 times of a solvent. As the solvent, an organic solvent may be used, and the organic solvent may be a C1 to C4 lower alcohol or a mixture thereof, preferably methanol. Further, the extraction can be preferably carried out at a temperature of 65 to 85 캜, and more preferably at 75 캜. The extraction was repeated several times, and the extracted sample was filtered, concentrated by a vacuum concentrator, and lyophilized.

According to another embodiment of the present invention (Example 2), it was confirmed that the EP extract of the present invention significantly inhibited the activity of the lipase in a concentration-dependent manner through treatment of the concentration of the EP extract (Fig. 1). When the activity of lipase is inhibited, the inflow of the fat ingested through the food can be inhibited, and consequently, the effect of preventing or treating obesity can be exhibited.

According to another embodiment of the present invention (Example 3), it has been confirmed whether the anti-obesity composition of the present invention can be safely used in vivo. Specifically, the cytotoxicity analysis of the EP extract was carried out and the cell survival rate was expressed as a percentage of the carrier treated with the carrier. As a result, it was confirmed that the EP extract was a safe substance which did not induce cytotoxicity to a concentration of 100 μg / ml 2).

According to another embodiment of the present invention (Example 5), the effect of the anti-obesity composition of the present invention on lipogenesis-related proteins was analyzed. Specifically, the amount of expression of C / EBPα, C / EBPβ and PPARγ, which are key factors involved in lipogenesis in fat cells treated with various concentrations of EP extract, was analyzed by Western blot analysis, C / EBP?, C / EBP? And PPAR? Were detected in a large amount in the case of the control (Con) in which the EP extract was not treated, And the content of each protein was decreased as the treatment concentration was increased (FIG. 5). This suggests that the EP extract of the present invention inhibits the expression of key factors involved in fat production and thus exhibits anti-obesity activity.

According to another embodiment of the present invention (Example 6), the effect of the anti-obesity composition of the present invention on already produced adipocytes was analyzed. Specifically, the amount of triglyceride (TG) in fat cells and the secreted amount of glycerol secreted in the medium were measured by treating EP extracts with adipocytes differentiated from adipose precursor cells. As the concentration of EP extract increased It was confirmed that the content of triglyceride (TG) in adipocytes was decreased and the amount of secreted glycerol in the medium was increased (Figs. 6A to 6C). This suggests that the EP extract of the present invention has anti-obesity activity by decomposing TG in the already formed adipocyte.

The anti-obesity composition of the present invention has effects of weight loss, decrease of body fat, decrease of blood triglyceride content and decrease of blood cholesterol content. According to a specific example of the present invention (Example 4), when the differentiation of 3T3-L1 adipose precursor cells was induced using an adipogenic differentiation medium containing IBMX (3-isobutyl-1-methylxanthine), dexamethasone and insulin , It was confirmed that the content of triglyceride was decreased according to the concentration of the extract by inhibiting adipocyte production by adding the EP extract of the present invention (FIGS. 3 and 4).

In addition, the anti-obesity composition of the present invention can inhibit fat production, thereby showing effects of preventing or treating not only obesity but also fatty liver or lipid-related cardiovascular diseases directly or indirectly related to obesity.

The term " fatty liver "of the present invention is a disease in which fat, especially triglyceride, accumulates in hepatocytes and accounts for more than 5% of liver weight. However, the most common cause of fatty liver disease is alcohol. Alcohol reduces various metabolic functions of the liver, especially fatty acid oxidative degradation, and accumulates fat in the liver. Fatty liver often occurs in obese people, and it can also be caused by endocrine diseases such as diabetes, hyperthyroidism, overuse of steroid hormone, or severe nutritional deficiency. There are no special symptoms, so it is not possible to diagnose it by symptoms alone. Fatigue, anorexia and lethargy, which are common symptoms of liver disease, can occur and feel discomfort or dullness in the lower part of the right rib.

The term "lipid-related cardiovascular disease" of the present invention is a disease caused by accumulation of fatty acid or cholesterol due to excessive intake or metabolic disorder. Hypertriglyceridemia, in which the concentration of triglycerides in the blood is abnormally high, is defined by the American Heart Association as a risk factor for cardiovascular disease. Plasma triglyceride increases rapidly after meals and returns to the pre-meal level after about 8 hours. Triglycerides increase in diabetes, obesity, and drinkers, and increase in people who consume a lot of sugar. Other hypothyroidism, nephrotic syndrome, kidney failure, birth control pills, and large amounts of diuretics and beta blockers can also increase triglycerides. As the blood triglyceride increases, LDL (low density lipoprotein) - cholesterol increases and the LDL promotes atherosclerosis. Examples of the lipid-related cardiovascular diseases include hyperlipidemia, heart disease, hypertension, arteriosclerosis, and the like.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants. In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The content of the EP extract contained in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but is preferably 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the final composition can do.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administering" of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, rectal.

The term "individual " refers to all animals, including mice, mice, livestock, etc., including humans who have developed or are capable of developing obesity. Preferably, it may be a mammal, including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Preferably, the pharmaceutical composition of the present invention may be administered at a dose of 0.0001 to 100 mg / kg of body weight per day, preferably 0.001 to 100 mg / kg of body weight per day based on the EP extract contained therein. The administration may be such that the recommended dose is administered once a day or divided into several times.

Since the extract of Yupei Teliaplus osperma extract of the present invention is used as a food composition, a cosmetic composition, or a pharmaceutical composition, it can be safely contained and usefully used because its side effects are less than general synthetic compounds.

In another aspect of the present invention, there is provided a composition for preventing or improving obesity comprising an EP extract as an active ingredient.

Since the EP extract of the present invention not only exhibits an anti-obesity effect but also has proven safety derived from natural products, it can be used as a composition for food addition and can be used for the production of a health functional food for preventing or improving obesity.

When used as a food additive, the EP extract may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment), and may further include a food-acceptable food-aid additive. The content of the EP extract of the present invention derived from a natural product is not particularly limited because it has no problem in terms of stability, but is preferably 0.01 to 90% by weight, more preferably 1 to 20% by weight, 60% by weight. When the food is a beverage, it may be contained in a proportion of 1 to 30 g, preferably 3 to 20 g based on 100 ml. In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like. In addition, it is also possible to use antiseptics (such as potassium sorbate, sodium benzoate, salicylic acid, and sodium dehydroacetate), disinfectants (such as bleaching powder and highly bleached white powder, sodium hypochlorite), antioxidants (butylhydroxyanilide (BHA), butylhydroxytoluene BHT), coloring agent (tar color), coloring agent (sodium nitrite, sodium nitrate), bleaching agent (sodium sulfite), seasoning (MSG, sodium glutamate, etc.), sweetener (dicin, cyclamate, saccharin, ), Food additives such as perfumes (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, emulsifiers, thickeners, encapsulating agents, gum bases, foam inhibitors, additives may be added. The additives are selected according to the type of food and used in an appropriate amount.

The food additive composition of the present invention can be used as an additive in the production of a conventional food, and can be used in the production of food having an effect of preventing or improving obesity. The food to which the food additive composition is added is a functional food, Food, and the like.

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

The term "health functional food" of the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in a food raw material, Refers to a food which is designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease and the like can be sufficiently exhibited to the living body by the above components. Recovery, and so on.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the extract of Yupe teliaplus osperma according to the present invention as an active ingredient can be prepared by admixing other suitable auxiliary ingredients that can be contained in the food and known additives according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice creams, soups, beverages, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

Examples of foods that can be used in the present invention include special nutritive foods (such as crude oil, infant formula, etc.), processed meat products, fish meat products, tofu, jelly, noodles (such as ramen, (Such as fermented milk, cheese), other processed foods, kimchi, pickled foods (various kinds of kimchi, pickles, etc.), seasonings (such as soy sauce, soybean paste, , Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

Since the composition containing the extract of Euptelea pleiosperma (EP) of the present invention has an ability to inhibit the differentiation of lipid precursor cells into adipocytes and inhibit the activity of pancreatic lipase enzyme, it can be useful as an anti-obesity composition, , Or lipid-related cardiovascular diseases, or a composition for preventing or ameliorating obesity or for adding a food for improving obesity.

1 is a graph showing the inhibitory activity of pancreatic lipase enzyme activity of the EP extract of the present invention.
2 is a graph showing cytotoxicity of the EP extract of the present invention.
FIG. 3 is a graph showing the effect of the extract of EP on the inhibition of the concentration-dependent adipogenesis of the EP extract of the present invention by the addition of 3T3- L1 cells were observed under a microscope by oil red o staining.
FIG. 4 is a graph showing the content of triglyceride triglyceride calculated by measuring the absorbance of the stained oil-red dyed area in FIG.
FIG. 5 is a Western blot analysis image showing changes in expression amounts of C / EBP?, C / EBP? And PPAR? According to the treatment concentration of the EP extract of the present invention.
FIG. 6A is a graph showing the amount of glycerol secreted in the medium according to the treatment concentration of the EP extract of the present invention. FIG.
FIG. 6B is a photograph showing the change of adipocytes according to the treatment concentration of the EP extract of the present invention. FIG.
FIG. 6C is a graph showing changes in content of triglyceride (TG) in adipocytes according to the treatment concentration of the EP extract of the present invention. FIG.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of the extract of Yupe telera flourospora (EP)

10 g of EP was crushed, and 95% methanol of 5 to 10 times the volume of the sample was added thereto, followed by extraction at 75 캜, followed by filtration to obtain a liquid component, which was repeated three times to obtain an extract. The extract was applied to a vacuum concentrator (N-1000S-W, EYELA, Japan) and concentrated. The concentrate was applied to a lyophilizer (FDU2100, EYELA, Japan) to obtain an EP extract in the form of a dry powder.

Example 2: Inhibition effect of EP extract on pancreatic lipase enzyme activity

To investigate the inhibitory effect of EP extract on the lipase activity, the activity of lipase was analyzed by treatment with each concentration of each extract. Lipase is an enzyme that hydrolyzes triglyceride (TG), which is secreted from the pancreas and triglyceride (TG) into glycerol and fatty acid. The inhibitory activity of pancreatic lipase, a lipid digesting enzyme, This is a very useful test method to predict. In this example, the inhibitory activity of the lipase enzyme activity of the sample was measured as follows before analyzing the anti-obesity activity of each sample in a cell assay system.

First, an enzyme solution and a substrate composed of 0.25 M Tris buffer (pH 7.7), 250 mM CaCl _2, and 5 mM 4-nitrophenyldodecanoate (PNPD) were added to a 1.5 ml tube and mixed thoroughly. The mixture was preheated at 37 ° C for 5 minutes and diluted with 0.25 M Tris buffer (pH 7.7) and various concentrations of EP extract were added to the reaction mixture at 37 ° C for 10 minutes, followed by the addition of 20% sodium dodecyl sulfate (SDS). The reaction solution was centrifuged at 15,000 rpm for 20 minutes at 4 ° C. The separated supernatant was dispensed into a 96-well tissue culture plate and absorbance was measured at 412 nm using a multi-plate reader. The value obtained by subtracting the absorbance of the reaction sample at 0 minutes from the absorbance of the sample reacted for 10 minutes was expressed as a percentage of the sample untreated control group. The measured value was expressed as an average value of three repeated experiments (Fig. 1).

As a result, as shown in FIG. 1, it was confirmed that the lipase activity was decreased compared to the sample-untreated control group (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 1 / / It was confirmed that the extract significantly inhibited the lipase activity in a concentration-dependent manner. These results indicate that the sample acts as a lipase inhibitor to lower the absorption of fat in the body and thus possess antiobesity activity.

Example 3: Cytotoxicity analysis of EP extract

To determine whether the EP extract of the present invention can be used safely in vivo, the cytotoxicity analysis of the EP extract was conducted. 3T3-L1, a mouse preadipocyte cell line, was purchased from the American Type Culture Collection (ATCC, USA) and cultured with 10% calf serum (BCS) and penicillin / streptomycin And cultured in DMEM (Dulbecco's modified eagle medium) medium. 10 ⁵ cells per well were plated on 24-well tissue culture plates for 24 h and treated with various concentrations of EP extract solution (0, 1, 5, 10, 25, 50, 75, 100 ug / ml) . After 72 hours, the medium containing the EP extract was removed and replaced with fresh medium containing water-soluble tetraolium salt (WST) reagent. The plate was incubated at 37 ° C for 30 minutes, and the plate was covered with a multiplate reader (Paradigm, Beckman, Absorbance at 480 nm was measured (Fig. 2). At this time, the cell survival rate was expressed as a percentage of the vehicle treated with vehicle (dimethylsulfoxide, DMSO).

As a result, as shown in FIG. 2, it was confirmed that the EP extract was a safe substance which did not induce cytotoxicity at all up to a concentration of 100 μg / ml.

Example 4: Inhibitory effect of EP extract on lipogenesis

In order to confirm the effect of inhibiting the lipogenesis of the EP extract, induction of differentiation into adipocytes and lipogenesis and its inhibitory effect were confirmed.

First, 3T3-L1 preadipocytes were incubated with 0.5 μM 3-isobutyl-methylxanthine (IBMX), 1 μM dexamethasone (DEX) and 10 μg / mL insulin ) To induce differentiation into adipocytes. 10 ⁵ cells per well were plated on 12 well tissue culture plates and replaced with medium containing 10% fetal bovine syndrome (FBS) 2 days later. After 2 days, the EP extract was treated at different concentrations in a medium containing IBMX, DEX, and insulin, and the insulin and the extract were treated 4 times at 2-day intervals, and the inhibitory effect on fat production was measured 2 days later.

The cells were washed with 1X phosphate buffered saline (PBS) and fixed with 10% formalin. The cells were washed with 1 ml of phosphate buffered saline (PBS) Red ocher solution and incubated for 30 minutes. After the dyeing was completed, the dye deposited on the cells was extracted with 100% isopropanol and the absorbance of the dye extract was measured at 520 nm (FIGS. 3 and 4).

As a result, as shown in FIGS. 3 and 4, it was confirmed that the EP extract reduced triglyceride production in a concentration-dependent manner.

From the above results, it was found that the EP extract of the present invention had an effect of inhibiting lipogenesis.

Example 5: Analysis of lipogenesis-related proteins

In Example 4, the cells that had been induced to differentiate into adipocytes and treated with the sample were disrupted, proteins were separated from the cells, and then the respective antibodies against C / EBP?, C / EBP? And PPAR? The Western blot analysis was used to analyze the expression amount of each protein according to the treatment of the extract of the present invention (FIG. 5). At this time, actin was used as an internal control group.

As shown in FIG. 5, in the case of the control (Con) not treated with the EP extract of the present invention, the C / EBPα, C / EBPβ and PPARγ were all detected in a large amount. However, as the treatment concentration of the EP extract increased, And the protein content was decreased.

Thus, the EP extract of the present invention showed anti-obesity activity by inhibiting the expression of key factors involved in fat production.

Example 6: Fat resolution analysis

10 ⁵ 3T3-L1 preadipocytes were each divided into 12-well plates, cultured for 2 days, replaced with medium containing 10% FBS, incubated again for 2 days, and then treated with 0.5 μM IBMX, 1 μM DEX And 10 쨉 g / ml insulin, for 2 days, exchanged with adipogenic differentiation medium, and incubated for 2 days. At this time, when changing to the fat-forming differentiation medium, 0, 5, 10, 25 or 50 μg / ml of the EP extract was separately added. After completion of the final culture, a culture supernatant was obtained and the content of glycerol secreted into the culture supernatant was quantitated (Fig. 6A). The differentiated fat cells were stained with Oil Red O staining (Fig. 6B) (TG) was quantitated (Fig. 6C).

As shown in FIG. 6A, it was confirmed that the secretion amount of glycerol secreted to the medium was increased in proportion to the treatment concentration of the EP extract, and as shown in FIG. 6B, in proportion to the treatment concentration of the EP extract, As shown in FIG. 6C, it was confirmed that the content of triglyceride (TG) in adipocytes was decreased in proportion to the concentration of EP extract.

The results of FIGS. 6A to 6C show that glycerol, which is a degradation product, is released to the outside of the cell through the treatment of the EP extract of the present invention by decomposition of triglyceride (TG) in adipocytes.

Therefore, the EP extract of the present invention not only inhibits the differentiation of normal cells into adipocytes, but also decomposes triglyceride (TG) in already differentiated adipocytes, thus showing more effective anti-obesity activity.

Claims (6)

A pharmaceutical composition for preventing or treating obesity comprising an extract of Euptelea pleiosperma (EP) as an active ingredient.
The method according to claim 1,
Wherein the content of the EP extract is 0.0001 to 50% by weight, based on the total weight of the final composition.
The method according to claim 1,
Wherein the extract is obtained by extracting with a solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, hexane, dichloromethane or a mixed solvent thereof.
The method according to claim 1,
Wherein the extract is obtained by extraction at a temperature of 65 to 85 캜 using 95% methanol as a solvent.
The method according to claim 1,
Wherein the extract inhibits the lipid precursor cells from differentiating into adipocytes.
A composition for preventing or improving obesity comprising an extract of Euptelea pleiosperma as an active ingredient.

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