WO2017030419A1 - Composition including euphorbia supina raf. extract or fraction as active ingredient for preventing or treating obesity - Google Patents

Abstract

The present invention relates to an antiobesity pharmaceutical composition including, as an active ingredient, an Euphorbia supina Raf. extract or fraction, or an antiobesity food composition and a composition for weight loss including as an active ingredient, an Euphorbia supina Raf. extract or fraction. The Euphorbia supina Raf. extract or fraction according to the present invention inhibits adipocyte differentiation and lipid accumulation and also has excellent effects for inhibiting obesity such as reducing the weight of adipose tissue and body weight in an obesity-induced group, thereby being usefully applicable in preventing, alleviating, or treating obesity or various obesity-associated diseases caused by obesity.

Description

Composition for the prevention or treatment of obesity, including Arabidopsis extract or fraction as an active ingredient

The present invention is Euphorbia supina Raf . The present invention relates to an anti-obesity pharmaceutical composition comprising a extract or a fraction as an active ingredient or an anti-obesity food composition and a weight loss composition comprising an extract or a fraction as an active ingredient.

Obesity refers to a condition with excess body fat, which means that the body fat is more than 25% (man), 30% (woman) of body weight. In modern society, the dietary environment has been improved and the nutritional intake has been improved, and the average life span has been extended and the quality of life has been improved.However, due to the excessive consumption of food, the reduction of physical exercise caused by the change of transportation, and the westernization of eating habits, etc. As a result, obese patients are increasing rapidly every year. The World Health Organization (WHO) explains that obesity is a cause of obesity worldwide, resulting in increased frequency of foods with low nutrients but high calorie intake, and decreased physical activity due to changes in transportation and lifestyle. That is, the root cause of obesity is the energy inequality that energy accumulation is because the calorie intake is greater than the calorie consumption. Obesity is also a risk factor for adult diseases and is involved in the development of diseases such as diabetes, arteriosclerosis, high blood pressure, stroke, heart abnormalities, and various inflammatory tumors. Obesity, in particular, is accompanied by serious weight gain. Excessive weight gain also affects joints and bones, resulting in skeletal abnormalities. For this reason, obesity is not only an external problem but also a serious problem for physical health.

The prevention of obesity is controlled through balanced food and regular eating habits, it is desirable to increase the physical exercise to increase the basic metabolism to ensure that the body has a normal metabolism. As obesity is known to be a problem in modern society, the word diet has attracted much attention. There are many ways to lose weight by taking advantage of the fact that people who are obese are interested in weight loss, among which are fasting and fasting, enzymes, liposuction, diarrhea, diuretics, treatments, and the like. These methods provide easy and comfortable weight loss for modern people, but side effects are common. In general, modern people are only interested in diet effects that appear easily and quickly, but are not interested in fundamental treatments, which overlooks the mechanisms of metabolism and nutritional physiological approaches that have clear and scientific causes and fundamental therapeutic purposes. If you focus only on weight loss, you will have another problem and a yo-yo phenomenon. Obesity can be seen as an independent disease, but the symptoms that cause other diseases than the interest is increasing, easy to use, easy to lose weight, fewer side effects such as yo-yo phenomenon It is time to develop the therapeutics that appear.

The anti-obesity agents currently used for weight loss are largely classified into appetite suppressants, fat absorption inhibitors, and energy consumption promoters. Among them, drug therapy using an appetite suppressant such as amphetamine inducer is temporary and the appetite suppressing ability is not sustained, and there are side effects such as headache, insomnia, blood pressure rise, tension, anxiety, hallucinations, dizziness, and decreased vision. Cause. Representative drugs include sibutramine. It is known to act on the hypothalamus of the brain causing appetite suppression. It suppresses resorption of serotonin and norepinephrine, the neurohormones that act on appetite control, reduces appetite, prevents overeating, and reduces weight by inhibiting high energy intake. Most of these appetite suppressants prohibit cardiovascular patients from using these drugs because they can strain the heart due to side effects of increased blood pressure. In addition, the fat absorption inhibitor is Orlistat (Orlistat). Orlistat is a lipase inhibitor secreted by the pancreas and digestive system, which inhibits the absorption of fat by inhibiting lipase secreted by the pancreas and digestive system, and suppresses the accumulation of fat in the body, thereby exerting a weight loss effect. However, olistat may cause fat imbalance by inhibiting absorption of fat-soluble vitamins, causing stomach cramps, gastric bloating and oily stool, causing discomfort in the digestive system.

Therefore, in recent years, there is a need for the development of a composition that allows ingestion of herbal medicines derived from natural products, etc., in the form of a general food, rather than a therapeutic agent used by artificial methods. In particular, attention is focused on the treatment of obesity derived from natural products without side effects.

On the other hand, Euphorbia supina Raf . ) Is an annual herb belonging to the Euphorbiaceae, and its main stem spreads along the ground, its length is 10-25cm, some leaves are hairy, some have reddish brown spots and a white latex. The leaves are long ovals, 5-10mm long and 4-6mm wide, with fine teeth on the edges, spread horizontally and arranged in two rows. The upper side of the leaf is dark green and glossy, the back side is green white, and the petiole is very short. Aegean bedbugs are widely distributed in temperate and tropical regions such as Korea, China, Japan, Southeast Asia, and North and South America. Baby bugs have excellent antioxidant, anti-cancer, detoxification and sedative effects, so they can be widely used for various cancers, asthma, diabetes, heart disease, kidney disease, malignant headache, and mental anxiety. It is known to be effective in treating blood stools and hematuria. As mentioned above, although various pharmacological effects of A. aeruginosa are known, it is not yet known whether the extracts and fractions of A. aureus have anti-obesity activity, and there are no studies on this.

Therefore, the present inventors completed the present invention by confirming that the extract or fraction of the Arabidopsis bug extract has excellent anti-obesity effect in order to develop a new drug.

It is an object of the present invention to provide an anti-obesity pharmaceutical composition comprising the extract or fraction of Arabidopsis as an active ingredient.

Still another object of the present invention is to provide an anti-obesity food composition comprising the extract or fraction of A. vulgaris as an active ingredient, and a composition for weight loss comprising the extract or fraction as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for anti-obesity comprising the extract or fractions of Arabidopsis as an active ingredient.

In another aspect, the present invention provides a food composition for anti-obesity comprising the extract or fractions of Arabidopsis as an active ingredient.

In another aspect, the present invention provides a composition for weight loss, comprising the extract or fractions of Arabidopsis as an active ingredient.

In addition to inhibiting the differentiation and lipid accumulation of adipocytes in the Arabidopsis vulgaris extract or fraction according to the present invention, the obesity-induced obesity-induced obesity or obesity, such as weight loss and fat tissue is excellent It can be usefully used to prevent, ameliorate or treat various obesity related diseases.

1 is a diagram briefly showing the manufacturing process of the Arabidopsis extract or fraction.

Figure 2 is a diagram showing the cell viability of fat cells when treated with Arabidopsis extract (Vehicle: normal control, EtOH: Arabidopsis extract treated group, EGCG: positive control).

Figure 3 is a diagram showing the cell viability of fat cells when treated with Arabidopsis fraction (Vehicle: normal control group, CH ₂ Cl ₂ : Arabidopsis dichloromethane fraction treatment group, EtOAc: Arabidopsis chloroacetate fraction treatment group, H ₂ O: Arabidopsis water fraction treated group, EGCG: positive control).

Figure 4 is a diagram showing the fat accumulation rate in fat cells during treatment with Arabidopsis extract (Vehicle: normal control, MDI: negative control, EtOH: Arabidopsis extract treatment group, DW: Arabidopsis extract water treatment group, EGCG: positive control).

Figure 5 is a diagram showing the fat accumulation rate in fat cells during treatment with Arabidopsis fraction (Vehicle: normal control, MDI: negative control, CH ₂ Cl ₂ : Arabidopsis dichloromethane fraction treatment group, EtOAc: Ethyl acetate fraction treated group, H ₂ O: Arabidopsis water fraction treated group, EGCG: positive control).

6 is a diagram showing the results of measuring the leptin level of the mouse blood when treated with Arabidopsis extract (Normal: normal control, HFD: obesity-induced group, EtOH: Arabidopsis extract treated group, EtOAc: Arabidopsis; Ethyl acetate fraction treated group, H ₂ O: Arabidopsis water fraction treated group, GC: positive control).

Figure 7 is a diagram showing the result of measuring the adiponectin level of mouse blood when treated with Arabidopsis fraction fraction by ELISA.

8 is a diagram showing the results of PPAR-γ levels measured in real-time PCR in mouse liver tissue when treated with Arabidopsis extract or fraction.

Figure 9 is a diagram showing the results of measuring the C / EBP α level in real mouse liver tissue when treated with Arabidopsis extract or fraction.

10 is a diagram showing the results of measuring the PPAR-γ level in the mouse adipose tissue treated with Arabidopsis extract or fraction by Real-time PCR.

Figure 11 is a diagram showing the results of measuring the C / EBP α level in the mouse adipose tissue when treated with Arabidopsis extract or fraction by real-time PCR.

Hereinafter, the present invention will be described in detail.

The present invention is Euphorbia supina Raf . ) Provides an anti-obesity pharmaceutical composition comprising an extract or fraction as an active ingredient.

Euphorbia of the present invention supina Raf . ) Is an annual herb belonging to the Euphorbiaceae, and is widely distributed in temperate and tropical regions such as Korea, China, Japan, Southeast Asia, and North and South America. Aegean bugs are known to have good anti-oxidant and anti-cancer effects, detoxification, sedative and hemostatic effects.

In the present invention, the term "extract" refers to a preparation obtained by squeezing a crude drug into an appropriate leach solution and evaporating the leach solution, and extracts obtained by the extraction treatment, dilutions or concentrates of the extracts, and dried products obtained by drying the extracts, and their crude preparations. Or a purified product.

The baby bug bed can be used without limitation, such as cultivated or commercially available and can be used by washing or drying as it is, the drying method can be used both dry, shade, hot air drying and natural drying.

The Arabidopsis extract or fraction may be extracted or fractionated from various sites of Arabidopsis, preferably from the flowers, leaves, stems, roots or outposts of Arabidopsis, more preferably baby It is extracted or fractionated from the leaves, stems or outposts of the bedbugs, and most preferably is extracted or fractionated from the outposts of the Arabidopsis.

The Arabidopsis extract or fraction can be obtained from nature using methods of extraction, separation and fractionation known in the art. The Arabidopsis extract or fraction may be obtained according to various extraction solvents and extraction methods. Water or an organic solvent may be used as a suitable solvent for obtaining the extract or fraction from the Arabidopsis, and any pharmaceutically acceptable organic solvent may be used. For example, water, methanol (ethanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), butanol (butanol), such as alcohol having 1 to 4 carbon atoms, etc. may be used alone or in combination Can be. Preferably methanol or ethanol (alcohol) can be used and more preferably ethanol can be used.

Extraction temperature for obtaining the Arabidopsis extract is preferably 50 ~ 100 ℃. In one embodiment of the present invention, the Arabidopsis bed bug extract was obtained at a temperature of 70 ° C., but is not limited thereto. In addition, the extraction method includes, but is not limited to, a variety of methods such as hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, compression method, preferably hot water extraction or reflux cooling Extraction methods.

The Arabidopsis bed fraction can be obtained as each solvent fraction through the process of fractionating the organic Arabidopsis extract from non-polar to polar. Suitable solvents for fractionating Arabidopsis fraction may be water, ethanol, methanol, hexane, chloroform, dichloromethane, ethyl acetate, butanol or a mixed solvent thereof, preferably water, dichloromethane, ethyl acetate And most preferably water, ethyl acetate or a mixed solvent thereof.

The Arabidopsis extract or fractions may be obtained by cooling, heating and filtration at room temperature in a conventional manner known in the art to obtain a liquid, or may further evaporate, spray-dried, or lyophilize the solvent. In addition, the Arabidopsis herb extract or fraction may be prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying. In addition, the extract or fractions may be further purified by various chromatography such as silica gel column chromatography, thin layer chromatography, high preformance liquid chromatography, and the like. Can also be obtained.

Therefore, the Arabidopsis extract or fraction used in the present invention is a concept including all the extracts, fractions and purified products obtained in each step of extraction, fraction or purification, their dilutions, concentrates or dried products.

In the present invention, the term "obesity" refers to a state having excessive body fat, and means a state in which body fat is 25% (male) or 30% (female) or more of body weight.

As used herein, the term "anti-obesity" means treating, preventing or ameliorating a condition that is obese.

According to one embodiment of the present invention, Arabidopsis extract or fraction inhibits the proliferation of adipocytes into adipocytes, decreases leptin expression in blood and increases adiponectin expression. In addition, it reduces the expression level of PPAR-γ protein, C / EBP α protein, C / EBP β protein, and C / EBP δ protein, which are involved in the early and middle fat cell differentiation, and inhibits weight gain in obesity-induced mouse models. Has an excellent effect. Therefore, Arabidopsis extract or fraction may be usefully used as a pharmaceutical and food composition for the prevention or treatment of obesity-related diseases or complications caused by obesity.

Extracts or fractions of the Arabidopsis of the present invention is not limited thereto, but metabolic syndrome, hypertriglyceridemia, high density lipidemia, low density lipidemia, angina pectoris, myocardial infarction, hypogonadism, sleep apnea, premenstrual syndrome, stress urinary Urinary incontinence including incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, weight-related cancers, orthostatic hypotension, pulmonary hypertension, menstrual disorders, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, sleepiness, depression, anxiety , Psychosis, tardive dyskinesia, drug addiction, substance abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive compulsive behavior, panic attack, social phobia, bulimia, atherosclerosis, gallbladder disease such as cholelithiasis, anorexia, polycystic ovary disease and Reproductive disorders, infections, varicose veins, epidermal growth and skin diseases such as eczema, insulin resistance, chronic arterial obstruction, orthopedics May be used to prevent or treat injury, thromboembolic events, cardiovascular disease, urinary disorders, lipid syndromes, hyperglycemia, and stress at least one obesity-related disorder selected from the group consisting of.

In addition, the extract or fraction of the Arabidopsis of the present invention can be used for the prevention, improvement or treatment of obesity, abdominal obesity or visceral fat accumulation in each part of the body, preferably for the prevention, improvement or treatment of visceral fat accumulation Can be used. The abdominal obesity or visceral fat accumulation includes, but is not limited to, those caused by lack of exercise, alcohol (alcohol) intake, stress or the like sitting on a chair for a long time.

The Arabidopsis extract or fraction based on the total weight of the pharmaceutical composition of the present invention is preferably included in 0.01 to 95% by weight, more preferably 1 to 80% by weight.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the art. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are preferably, but not limited to, those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. The carrier, excipient, and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xyltone, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like. When the pharmaceutical composition is formulated or formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. The components may be added independently or in combination to the active ingredient, ie, Arabidopsis extract or fraction.

As used herein, the term "administration" means providing a subject with a pharmaceutical composition of the present invention in any suitable manner.

The present invention relates to a pharmaceutical composition comprising an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinic, i.e., the symptoms of a disease or disorder being treated. It may be administered in a therapeutically effective amount that is an amount that induces remission. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the pharmaceutical compositions of the invention will vary depending upon the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It can be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and drugs used simultaneously. The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intradural, transdermal, intestinal, subcutaneous, sublingual or topical administration may be, but is not limited to.

The pharmaceutical composition of the present invention may be administered at 1 to 10,000 mg / kg / day, preferably 1 to 2000 mg / kg / day, may be administered once a day, or may be administered several times.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, orally, rectally or intravenously, intramuscularly, subcutaneously.

In addition, the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers for the prevention or treatment of obesity, obesity-related diseases or complications. .

In another aspect, the present invention provides a food composition for anti-obesity comprising the extract or fractions of Arabidopsis as an active ingredient.

Extracts or fractions of Arabidopsis of the present invention can be used as a dietary supplement, food additives or dietary supplements.

When the Arabidopsis extract or fraction of the present invention is used as a food additive, the Arabidopsis extract or fraction may be added as it is, or may be appropriately used according to conventional methods such as being used in combination with other foods or food ingredients. .

In addition, the mixed amount of the extract or fractions of the active ingredient, which is the active ingredient may be appropriately changed depending on the purpose of use (prevention, health or therapeutic treatment), the extract or fraction of the extract is based on the total weight of the food composition It is preferably included in 0.01 to 95% by weight, more preferably 1 to 80% by weight. As a specific example, in the preparation of food or beverage, the Arabidopsis extract or fraction of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material.

There is no particular limitation on the type of the food, but examples of the food to which the extract or fraction of the present invention may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, Dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes and the like, and includes all of the health food in the usual sense.

When the food composition of the present invention is made into a beverage, it may contain various ingredients such as various flavoring agents or natural carbohydrates as in the general beverage. The natural carbohydrate includes monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is included in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

The food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonate used in the beverage may include, but may include, but is not limited to, a natural fruit juice, fruit juice for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The additive ratio is not particularly limited, but is preferably included within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In the case of long-term intake for health and hygiene or health control, the food composition of the present invention can be taken for a long time because there is no problem in terms of safety.

In another aspect, the present invention provides a composition for weight loss, comprising the extract or fractions of Arabidopsis as an active ingredient.

The weight loss composition according to the present invention may be a pharmaceutical composition or may be a food composition. When formulated into pharmaceutical compositions, various auxiliaries used in medicine, such as carriers or other additives such as stabilizers, emollients, emulsifiers and the like, can be added as necessary, so long as they do not adversely affect the active ingredient. The formulation may be any formulation suitable for pharmaceutical preparations of herbal ingredients, including tablets, pills, capsules, granules, powders, extracts, premises, acupuncture and the like.

As used herein, the term "prevention" means any action that inhibits obesity or delays progression by administration of a composition of the present invention.

As used herein, the term "treatment" refers to any action in which obesity is improved or beneficially altered by administration of a composition of the present invention.

Terms not defined otherwise in this specification are intended to have a meaning commonly used in the art to which the present invention pertains.

Hereinafter, the present invention will be described in detail by way of examples and preparation examples. However, the following Examples and Formulation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Formulation Examples.

Example  One. Baby Bedbugs ( Euphorbia supina Raf . ) Preparation of Extract

Euphorbia dried in the air supina Raf . ) 2.5 kg of plants were repeatedly extracted three times at 70 ° C. with 70% ethanol using a reflux extractor to obtain 250 g of Arabidopsis extract (10.06%), which was then used in Examples.

Example 2 Preparation of Celandine Bedbug Fractions

After 250 g of the Arabidopsis extract obtained in Example 1 was suspended in distilled water, the process of separating the dichloromethane (CH ₂ Cl ₂ ) layer was repeated three times. The dichloromethane layers thus obtained were combined and concentrated under reduced pressure to obtain 128 g of a dichloromethane fraction in powder form (yield 51.2%). Ethyl acetate (CH ₃ COOC ₂ H ₅ ) was added to the remaining water layer, and fractionated in the same manner as above. After concentration under reduced pressure, lyophilization was carried out to obtain 48 g of ethyl acetate fraction (powder 19.2%) and water. 74 g (29.6% yield) of fractions were obtained and used in the examples below.

The preparation process of Arabidopsis vulgaris extract and fractions is briefly shown in FIG.

Example 3. Cytotoxicity and Viability Assay

3-1. Preparation and Culture of Adipose Progenitor Cells (3T3-L1)

3T3-L1 cells are adipose progenitor cell lines widely used for the study of metabolic processes of fat cells, and the more active the differentiation of 3T3-L1 cells, the more fat accumulation in fat cells occurs. 3T3-L1 cells were distributed from ATCC (American Type Culture Collection, USA) and used. Cell lines obtained from ATCC were incubated in a DMEM medium containing 10% bovine calf serum (BCS) and 1% penicillin in a 5% CO ₂ and 37 ° C. incubator. The cells were used in the experiment when the cells were stabilized through two or more passages.

3-2. Cytotoxicity and Viability Analysis

MTT (3- (4,5-dimethylthiazol-2-yl) to confirm the cytotoxicity of the Arabidopsis extract or fraction obtained in Example 1 or 2 and the appropriate concentration of the Arabidopsis extract and fraction without toxicity -2,5-diphenyl tetrazolium bromide) assay was performed. 3T3-L1 cells were inoculated in 48 well plates at a concentration of 2 × 10 ⁵ cells / well, cultured once every two days and incubated until 100% confluent. In the cultured cells, Arabidopsis extract was treated at concentrations of 4, 20, and 100 ㎍ / ml, Arabidopsis dichloromethane fraction, Arabidopsis ethyl acetate fraction, and Arabidopsis extract water fractions 0.8, 4, respectively. The cells were treated at 20 μg / ml and incubated at 37 ° C., 5% CO ₂ for 8 days. After incubation, the absorbance was measured using MTT buffer. As a normal control group, a cell treated with only median (Vehicle) and a positive control group with epigallocatechin gallate treated group (EGCG) were used, and the experiment was repeated three times. It was performed by. Cell viability was calculated by Equation 1 below, and the results are shown in FIGS. 2 and 3.

[Equation 1]

% Cell viability = (absorbance of sample treated / absorbance of control) × 100

As shown in Figures 2 and 3, the extract of Aegi bed bug extract, Aegi bed bug, which is an active ingredient of the present invention was confirmed that there is no toxicity at high concentration.

Example 4 Confirmation of the Inhibitory Effect of Arabidopsis Extract or Fraction of Adipocyte Differentiation

The fat accumulation rate was checked to determine whether the Arabidopsis extract or fraction has an effect of inhibiting the differentiation of 3T3-L1 cells into adipocytes. To confirm this, stabilized 3T3-L1 cells were dispensed into 48-well plates at a density of 2 × 10 ⁵ cells / well and incubated for 2 more days at the 100% confluent time point. 3T3-L1 cells were treated with 10% Fetal Bovine Serum (FBS), 1% containing MDI (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 1 μg / mL insulin) Differentiation was induced in adipocytes for 2 days by culturing in DMEM medium containing penicillin, and cultured for 2 days in 10% FBS containing 1 μg / mL insulin and 1% penicillin-containing DMEM medium after 48 hours of culture. During the adipocyte differentiation process, Arabidopsis ethanol extract and Arabidopsis ethanol extract were treated to culture medium at concentrations of 4, 20, and 100 μg / mL, Arabidopsis dichloromethane fraction, Arabidopsis diethylmethane acetate, Arabidopsis larvae Water fractions were treated in culture at concentrations of 0.8, 4, 20 μg / mL. Then, the degree of adipocyte differentiation was observed on day 8, when the differentiation was completed. The normal control group was treated with media only (Vehicle), and the negative control group was treated with IBMX, Insulin, Dexamethasone to induce lipid accumulation (MDI).

In order to observe the degree of differentiation of adipocytes, Oil Red O staining was performed to specifically react with lipid droplets. Specifically, the medium was removed from the cells induced by adipocyte differentiation as described above, and fixed by treating 10% formalin for 1 hour at room temperature. It was then washed with 60% isopropanol and each well was completely dried. Oil Red O dye (60% Oil Red O stock, 40% sterile distilled water) was treated for 1 hour, then washed twice with 60% isopropanol and once with sterile distilled water. For elution of the combined Oil Red O was treated with 100% isopropanol for 10 minutes, the eluate was measured at 520nm.

The fat accumulation rate in the adipocytes was calculated by the following Equation 2, and the results are shown in FIGS. 4 and 5.

[Equation 2]

% Fat accumulation = (absorbance of sample treated / absorbance of control) × 100

As shown in Figures 4 and 5, it was confirmed that fat accumulation in fat cells was inhibited in a concentration-dependent manner by the treatment of Aegipeta nymph extract, ethyl acetate fraction, dichloromethane fraction and water fraction as the active ingredient of the present invention. Therefore, it can be seen that the Arabidopsis extract or Arabidopsis fraction of the present invention inhibits the differentiation of adipocytes into adipocytes and thus has an excellent effect of preventing or treating obesity.

Example 5. Confirmation of the weight loss effect of Arabidopsis extract or fraction

5-1. Construction of Obesity-induced Mouse Model

To build the obesity induction group model for the experiment, five-week-old C57BL / 6J male mice (20-25 g) were purchased from Samtaco (Osan, Korea) and fed with solid feed and water for one week. ± 10%), constant temperature (22 ± 2 ° C) and 12 hours cycle, the experimental environment was adjusted for 1 week. After one week of adaptation, divided into 12 groups, normal control group (Normal), obesity-induced group treated with 45% high fat diet (HFD), Aegean fungus extract treated group prepared in Example 1 (2, 10, 50 mg / kg, ESEE + HFD), Aegean soil bed ethyl acetate fraction treatment group prepared in Example 2 (0.8, 4, 20 mg / kg, ESEA + HFD), Aegean soil bed water fraction treatment group (0.8, 4, 20 mg / kg, ESH ₂ O + HFD) and the positive control Garcinia cambogia extract treatment (200 mg / kg, GC + HFD) group.

Obesity-induced, Arabidopsis extract treated, Arabidopsis ethyl acetate fraction treated, Arabidopsis water fraction treated and positive controls were 0.5% carboxymethyl in the Arabidopsis extract, Arabidopsis fraction and Garcinia cambogia extract. After dissolving with cellulose (sterile distilled water + carboxymethyl cellulose), 45% high fat diet was ingested freely and the extract or fractions were orally administered once a day for 6 weeks. In normal control, the diet was freely consumed and the obese group was orally administered 0.9% saline solution. A mouse model was constructed in the same manner as above, and used in the following examples.

5-2. Check your weight change

In order to confirm the anti-obesity effect of the Arabidopsis vulgaris extract or fractions, in the mouse experimental group constructed by the method of Example 5-1, the body weight was measured at weekly intervals after the start of the experiment and the results, and the results are shown in Tables 1 to 3 below. Table 3 shows.

As shown in Tables 1 to 3, it was confirmed that the body weight of the obesity-induced group was significantly increased compared to the normal control group, in contrast, the garcinia cambogia extract treated group was reduced by 34.13% compared to the obesity-induced group.

Specifically, as shown in Table 1, the weight gain of the Arabidopsis extract extract group was 13.52% and 34.54% decreased, respectively, compared to the obesity-induced group, and as shown in Table 2, Arabidopsis vs. ethyl acetate fraction treatment group Compared with the obesity-induced group, weight gain decreased by 23.02% and 52.27%, respectively. In addition, as shown in Table 3, it was confirmed that the body weight treatment group Aegi than the obesity-induced weight loss 20.21% and 52.28%.

Therefore, extracts or fractions of the extract, which is an active ingredient of the present invention, reduce the body weight to a significant level in a concentration-dependent manner, especially in the group treated with 20 mg / kg of ethyl acetate fraction showed significantly greater weight loss than the positive control group. In addition, the 20 mg / kg group of Arabidopsis water fraction showed a greater weight loss effect than the positive control group. From these results, it was confirmed that the extract or fraction of the Arabidopsis of the present invention is excellent in anti-obesity effect by causing weight loss.

Example  6- Baby Bedbugs  Extract or Fraction  Confirmation of organ weight and fat tissue weight reduction effect

In order to confirm the anti-obesity effect of the extract or fraction of the Arabidopsis of the present invention, the weight of liver tissue, kidney tissue, white epididymal adipose tissue and spleen tissue of the experimental group constructed in the above 5-1 was measured. Specifically, after the experiment period in 5-1, the mouse model was sacrificed by ether anesthesia, the abdomen was cut, each tissue was removed, blood and foreign substances were removed using physiological saline, and the weight was measured. Are shown in Tables 4 to 6 below.

As shown in Tables 4 to 6, the white epididymal adipose tissue was significantly increased in the obesity-induced group compared to the normal control group, while the garcinia cambogia extract treatment group was significantly reduced in weight. . In addition, it was confirmed that the weight of the white epididymal adipose tissue was significantly dependent on the concentration in the Arabidopsis vulgaris extract or fraction treatment group.

Therefore, the Arabidopsis oleracea extract or fraction of the present invention has an effect of reducing the weight of adipose tissue can be usefully used as an anti-obesity pharmaceutical composition, an anti-obesity food composition or a composition for weight loss.

Example 7 Determination of Triglyceride Concentrations in Serum

In order to confirm the anti-obesity effect of the extract of Arabidopsis of the present invention, the concentration of triglycerides in the blood of the mouse model was measured after the end of the experiment of the above example.

Specifically, the blood was fasted and the heart was collected after anesthesia after the experiment was terminated, and the collected blood was left at room temperature for 30 minutes and then centrifuged at 3000 rpm at 4 ° C. for 10 minutes to obtain serum (triglyceride). ), Total cholesterol, high density lipoprotein-cholesterol and low density lipoprotein-cholesterol concentrations were measured using an Asanpharm (Korea) kit. Table 9 shows.

As shown in Table 7 to Table 9, the concentration of triglyceride, total cholesterol and LDL-cholesterol in the serum of the obesity-induced group was higher than that of the normal control group, and the concentration of HDL-cholesterol was lowered. On the other hand, compared to the obesity-induced group, Arabidopsis extract or fraction treatment group, the active ingredient of the present invention, the concentration of triglyceride, total cholesterol and LDL-cholesterol in the serum was lower, and the concentration of HDL-cholesterol was higher. Confirmed.

Accordingly, the extract or fraction of the Arabidopsis of the present invention significantly reduced the concentration of triglycerides, total cholesterol, and LDL-cholesterol in experimental animals, and increased the concentration of HDL-cholesterol in a concentration-dependent manner, Obesity effect is excellent.

Example  8 - Baby Bedbugs  Extract or Fraction  In serum Leptin  And Deponectin  Check the effect on concentration

In order to confirm the anti-obesity effect of Arabidopsis extract or fraction, leptin and adiponectin concentrations were measured in the blood of the animal model after the experiment was terminated. Leptin and adiponectin are both proteins secreted by fat cells.

Serum was obtained by centrifuging blood collected by heart blood collection from mice as in Example 7, and the concentrations of leptin and adiponectin were measured using an ELISA kit, and the results are shown in FIGS. 6 and 7.

As shown in FIG. 6, the concentration of leptin in the serum was significantly increased in the obesity induction group compared to the normal control group, and the positive control group was significantly decreased in comparison with the obesity induction group. In addition, it was confirmed that the concentration of leptin in the serum concentration group-dependent compared to the obesity-induced group in the Arabidopsis extract or fraction administration group as an active ingredient of the present invention.

As shown in FIG. 7, the adiponectin concentration in serum was significantly decreased in the obesity-induced group compared to the normal control group, and the garcinia cambogia extract treated group, which was a positive control group, was significantly increased in comparison with the obesity-induced group. In addition, it was confirmed that the concentration of adiponectin in the serum of the Arabidopsis extract or fraction administration group, which is an active ingredient of the present invention, compared to the obesity-inducing group.

Thus, Arabidopsis extract or fraction may have an anti-obesity effect due to the effect of reducing leptin levels in blood and increasing adiponectin levels.

Example  9- Baby Bedbugs  Extract or Fraction  Obesity expression gene inhibitory effect confirmed

In order to confirm whether the Arabidopsis extract or fraction of the present invention inhibits obesity-related genes, RNA is extracted from liver tissue and white epididymal adipose tissue, and then expression of PPAR-γ and C / EBP α genes using real-time PCR. The level was confirmed.

Specifically, first, the lysis buffer was homogenized in liver tissue and white epididymal adipose tissue of the mouse, and then the supernatant was obtained using a centrifuge. CHCl ₃ solution was added to the supernatant, and the supernatant was obtained again using a centrifuge. After isopropanol was added to the supernatant thus obtained, the supernatant was removed using a centrifugal separator and washed with 75% ethanol to obtain precipitate RNA. This precipitate was used after melting with water without Rnase. RNA was synthesized cDNA using a cDNA synthesis kit was used to perform real-time PCR.

Real-time PCR is StepOnePlus ^TM Instrument instrument was used. First mix 10 μl of SYBR® Green Reagents, 0.4 μl of forward primer (10 pmol / l), 0.4 μl of reverse primer (10 pmol / μl), 7.2 μl of water without Rnase, 2 μl of template (30 ng / μl) After dispensing the plate for -time PCR was performed using StepOne Softewear 2.3v. PCR conditions were 10 cycles at 95 ℃, 10 seconds at 95 ℃ and 1 minute at 60 ℃ 40 cycles, liver tissue results are shown in Figures 8 and 9, adipose tissue results are shown in Figures 10 and 11.

As shown in Figures 8 to 11, the Arabidopsis bed extract or fraction according to the present invention was confirmed that the concentration of the PPAR-γ, C / EBP α inhibits the gene expression in liver and white epididymal adipose tissue.

Therefore, it can be seen that the extract and fractions of the Arabidopsis of the present invention to suppress the expression of obesity factors, it will be excellent in the prevention or treatment of obesity, obesity-related diseases or complications.

Formulation Example 1 Preparation of Pharmaceutical Formulations

Powder  Produce

Powder was prepared by mixing 20 mg of Arabidopsis extract or fraction, 100 mg of lactose and 10 mg of talc and filling into an airtight bag.

Tablet manufacturing

Tablets were prepared by mixing 10 mg of Arabidopsis vulgaris extract or fraction, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, and then compressing them according to a conventional method for preparing tablets.

Capsule  Produce

According to a conventional capsule preparation method, capsules were prepared by mixing 10 mg of Arabidopsis extract or fraction, 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate, and filling into gelatin capsules.

Injection manufacturing

According to a conventional method of preparing an injection, 10 mg of Arabidopsis extract or fraction per ampule (2 mL), mannitol 180 mg, 2,974 mg of sterile distilled water for injection, and 26 mg of Na ₂ HPO _{4 ·} 2H ₂ O were prepared.

Liquid  Produce

According to the conventional method for preparing a liquid solution, 20 mg of Arabidopsis extract or fraction, 10 g of isomerized sugar, and 5 g of mannitol were added to the purified water to dissolve it, and then a suitable amount of lemon flavor was added, and the above ingredients were mixed. Then, purified water was further added to adjust the total volume to 100 mL, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 2 Preparation of Food Formulations

Health food manufacturing

Extract or fractions 100 mg, vitamin A proper amount, vitamin A acetate 70 g, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 g, vitamin C 10 mg, biotin 10 g, nicotinic acid Amide 1.7mg, folic acid 50g, calcium pantothenate 0.5mg, mineral mixture appropriate amount, ferrous sulfate 1.75mg, zinc oxide 0.82mg, magnesium carbonate 25.3mg, potassium phosphate monobasic 15mg, calcium diphosphate 55mg, potassium citrate 90mg, carbonate After mixing 100 mg of calcium and 24.8 mg of magnesium chloride, granules were prepared and health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is mixed composition of a component suitable for a health food in a preferred embodiment, the formulation ratio may be arbitrarily modified.

Health drink manufacturing

According to the usual method of preparing healthy beverages, extracts or fractions of Arabidopsis extract 100 mg, vitamin C 15 g, vitamin E (powder) 100 g, iron lactate 19.75 g, zinc oxide 3.5 g, nicotinic acid amide 3.5 g, vitamin A 0.2 g, vitamin B1 0.25 g, 0.3 g of vitamin B2 and a fixed amount of water were mixed, and then stirred and heated at 85 ° C. for about 1 hour. The resulting solution was filtered, sterilized in a 2 L container, sealed, sterilized, and refrigerated, thereby preparing a healthy beverage. . In this case, the composition ratio is a relatively suitable composition for the preferred drink in a preferred embodiment, but the composition ratio may be arbitrarily modified according to the regional and ethnic preferences such as the demand hierarchy, the demand country, the intended use.

Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the spirit of the invention.

Claims (12)

Euphorbia supina Raf .) An anti-obesity pharmaceutical composition comprising an extract or fraction as an active ingredient. The anti-obesity pharmaceutical composition according to claim 1, wherein the extract of Arabidopsis is an extract of one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixtures thereof. According to claim 2, wherein the C1-C4 alcohol is characterized in that at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol and butanol, anti-obesity pharmaceutical composition. The anti-obesity pharmaceutical composition according to claim 1, wherein the Arabidopsis fraction is a fraction of at least one solvent selected from the group consisting of water, dichloromethane, ethyl acetate, and mixtures thereof. According to claim 1, The Arabidopsis extract or fraction is characterized in that to inhibit the differentiation of fat cells, anti-obesity pharmaceutical composition. According to claim 1, The Arabidopsis extract or fraction is characterized in that to reduce the expression of leptin (leptin) in the blood or increase the expression of adiponectin (adiponectin), anti-obesity pharmaceutical composition. The method of claim 1, wherein the Arabidopsis extract or fraction is to reduce the expression level of one or more selected from the group consisting of PPAR-γ protein, C / EBP α protein, C / EBP β protein and C / EBP δ protein Characterized in that the anti-obesity pharmaceutical composition. According to claim 1, wherein the Arabidopsis extract or fraction is metabolic syndrome, hypertriglyceridemia, high density lipidemia, low density lipidemia, angina pectoris, myocardial infarction, sexual dysfunction, sleep apnea, premenstrual syndrome, stress urinary incontinence Including urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, weight-related cancers, orthostatic hypotension, pulmonary hypertension, menstrual disorders, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, sleepiness, depression, anxiety, psychosis Retardant dyskinesia, drug addiction, drug abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive-compulsive behavior, panic attack, social phobia, bulimia, atherosclerosis, gallbladder diseases such as gallstones, reproductive problems such as anorexia, polycystic ovarian disease Disorders, infections, varicose veins, skin diseases such as epidermal growth and eczema, insulin resistance, chronic arterial occlusion, orthopedic injury, thromboembolism Anti-obesity pharmaceutical composition, characterized in that preventing or treating one or more obesity-related diseases selected from the group consisting of syndrome, heart disease, urinary diseases, lipid syndrome, hyperglycemia and stress. According to claim 1, The Arabidopsis extract or fraction is characterized in that to prevent or treat the accumulation of visceral fat, anti-obesity pharmaceutical composition. Anti-obesity food composition comprising the extract or fractions of Arabidopsis as an active ingredient. The food composition of claim 10, wherein the food composition is a dietary supplement, a food additive, or a dietary supplement. A composition for weight loss, comprising the extract or fraction of Arabidopsis as an active ingredient.

Images