WO2011062458A2

WO2011062458A2 - Composition containing homoisoflavanone as active ingredient for preventing and treating obesity or metabolic syndrome

Info

Publication number: WO2011062458A2
Application number: PCT/KR2010/008283
Authority: WO
Inventors: 권혁상; 고종희; 유종수; 윤종민; 장현수; 유정수; 연승우; 강재훈
Original assignee: 일동제약주식회사
Priority date: 2009-11-23
Filing date: 2010-11-23
Publication date: 2011-05-26
Also published as: WO2011062458A3; KR101157028B1; KR20110056686A

Abstract

The present invention relates to a composition for preventing and treating obesity, obesity-related complications or metabolic syndrome, containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient. More specifically, the present invention relates to: a pharmaceutical composition for preventing and treating obesity, obesity-related complications or metabolic syndrome by inhibiting differentiation of adipocytes and maintaining a high level of SIRT1, thereby reducing bodyweight, abdominal fat and glucose tolerance; a method for preventing and treating same comprising the step of administering to a subject in need thereof an effective amount of homoisoflavanone or a pharmaceutically acceptable salt thereof; and uses of homoisoflavanone or a pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent. The composition of the present invention inhibits differentiation of adipocytes and maintains a high level of SIRT1, thereby reducing bodyweight, abdominal fat and glucose tolerance, and can thus be useful for the prevention and treatment of obesity, obesity-related complications or metabolic syndrome.

Description

[Specification】

[Name of invention]

Obesity or metabolic syndrome prevention and treatment composition containing homoisoflavanone as an active ingredient

Technical Field

The present invention relates to a composition for the prevention and treatment of obesity, obesity complications or metabolic syndrome containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient, more specifically, inhibits the differentiation of fat cells, A pharmaceutical composition for the prevention and treatment of obesity, obesity complications or metabolic syndrome, homoisoflavanone or a pharmaceutically acceptable salt thereof, which maintains SIRT1 at high levels to reduce weight, abdominal fat and glucose tolerance. A method for the prophylaxis and treatment comprising administering to a subject in an effective amount, and the use of homoisoflavanone or a pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent.

Background Art

The prolonged life expectancy due to the development of medicine and the improvement of living standards and the decrease in the amount of exercise due to the increase of the living population in urban centers, the hyperglycemia caused by obesity, hypertension, lipid metabolism and insulin resistance, which are adult metabolic diseases, Due to this, fatal diseases such as type 2 diabetes and its complications, myocardial infarction and stroke due to arteriosclerosis are on the rise.

Metabolic Syndrome refers to a syndrome that is accompanied by risk factors such as hypertriglyceridemia, hypertension, abnormal glucose metabolism, abnormal blood height, and obesity. The symptom itself is not fatal, but because it is predisposed to developing serious diseases such as diabetes and ischemic cardiovascular disease, it is the most threatening condition for modern people. At one time it was not known for its cause, but it was called by several names, including Syndrome X, but it was established by the World Health Organization and the US National Institute of Health's Cardiopulmonary-Blood Institute (NHLBI). HKAdult Treatment Program ΠΙ: ATP m) is officially referred to as metabolic syndrome or Insulin resistance syndrome.

According to the criteria of the National Cholesterol Education Program (NCEP) published in 2001: ① Abdominal obesity with waist circumference of 40 inches (102 cm) for men and 35 inches (88 cm) for women, ② Triglycerides 150 mg / dL or more ， ③ HDL cholesterol is 40 mg / dL for men, 50 mg / dL for women, ④ blood pressure 130/85 mmHg or more ， ⑤ fasting blood glucose (fasting If one of the five risk factors, such as glucose or more, 110 mg / dL or more, indicates three or more, metabolic syndrome is determined. In Asians, when waist circumference is more than 90 cm for men and 80 cm for women, abdominal obesity is somewhat adjusted. According to this rule, Koreans report about 25% of the population shows symptoms of metabolic syndrome. There is also.

<5>

<6> The cause of metabolic syndrome was insulin resistance, which was first pointed out. Insulin secreted to lower blood sugar does not regulate blood sugar normally, but rather induces blood vessel cell proliferation to thicken blood vessel walls or reabsorb sodium in the kidneys. Metabolism such as hypertension, hyperlipidemia, abdominal obesity, etc. by increasing the blood pressure or lipolysis to increase triglycerides in the blood and at the same time lowering high density cholesterol and promoting the storage of fat in the intestines. Syndrome is known to develop. The reason for such insulin resistance is not yet clearly identified, but the biggest factor affecting is known to be abdominal obesity, excessive stress and aging is also known as the cause. In addition, in the past, insulin resistance was considered as a single cause of metabolic syndrome. In recent years, insulin resistance has been estimated as three main causes by adding the combined effects of obesity, fat cell disorders, and metabolic syndrome factors. '

<7>

<8> Metabolic syndrome is rapidly increasing incidence and prevalence due to the aging of the population and environmental changes, resulting in very high mortality rates and complications, resulting in huge economic and social losses. In addition to low efficacy and high incidence of side effects when taking long-term, it is urgent to develop therapeutic drugs in terms of improving quality of life. ： ^"

<9>

<ιο> Currently, in the West, including the United States, Europe, and Japan, the number of obese people is estimated at more than 300 million, and in developing countries it is increasing by more than 1% per year. Even the World Health Organization (WHO) defines obesity as the second most common cause of death in the United States, after smoking, which has cost more than $ 110 billion annually. This is high and socially serious.

<ιι> Global patent applications related to the treatment of obesity have increased from 1999 to 2004. In particular, in the United States, the rate of increase was rapidly increasing at an early age, but gradually decreased since 2005, which is related to the fact that the actual development of obesity treatments is not visible. About 90% of obesity-related patents are related to appetite suppression, dietary control and metabolism of jibing ¹ , but recently, the share of patents related to the regulation of fat growth and energy metabolism is gradually increasing. This is because the limitations of medical conditions and the necessity of developing a new mechanism of obesity treatment to improve them are recognized. ^'

<i2> Sirtuins are known to induce life-long effects in yeast

(Sir2-related enzymes) belong to the histone deacetylase enzymes (histone deacetylase) of the class III stream requiring励⁺ as cofactor to mammalian homologs (~ 110kD) of the protein. Sirtuins found in animals so far are a total of seven species from SIRT1 to SIRT7, and they differ from each other in their position and function, of which SIRT1 is activated in the nucleus and metabolized,

^It modulates the activity of various substrates associated with inflammatory reactions and degenerative brain diseases.

When dietary restriction increases the expression and activity of SIRT1 in various tissues such as liver, fat, and pancreas, it is directly related to transcription factors such as PPARY and F0X01 and proteins related to energy metabolism of mitochondria such as UCPl, UCP2, and UCP3. Indirect activity control by deacetylation or deacetylation of coregulators such as p300, NcoR, and PGCla prevents glycolysis in the liver, reduces fat accumulation in adipose tissue, increases insulin sensitivity in the pancreas, and increases energy in muscle tissue Enhancing metabolism causes weight loss by heat dissipation.

<14>

<15> Leptin and adiponectin are new mechanisms for the treatment of obesity.

(diponectin), ciliary neurotrophic factor (CNTF), glucagon pentide, and other biological proteins classified into adipokine, or fragments thereof, and antagonists of hormones and novel obesity-related receptors. In the case of leptin, most obese patients are resistant and Axokine (hCNTF), which was expected to be a promising anti-obesity agent, stops developing in Phase III. I'm not doing it.

<16> In the current situation where the obese population is rapidly increasing, the demand for therapeutic agents is decreasing, and this is due to the dissatisfaction with the side effects and efficacy of the existing drugs. This is a desperate situation.

Therefore, the expression and activity of SIRT1 increased by dietary restriction such as news in vivo. Developing a drug that can replace the disease could lead to new treatments for obesity, obesity complications, or metabolic syndrome.

<18> Polygonatum odoratum is a perennial herb belonging to the family Liliaceae, and is widely used for the purpose of nourishment and tonic in oriental medicine and private life. Cough, diabetes caused by various weaknesses, malnutrition and pulmonary tuberculosis It is used as a medicinal herb due to the severity of the disease. However, there is no known effect of enhancing the expression of SIRT1 or the effects of obesity or metabolic syndrome.

[Detailed Description of the Invention]

[Technical work problem]

While the present inventors studied natural substances that increase the activity of SIRT1, the homoisoflavanone of the present invention increases the activity of SIRT1 and the homoisoflavanone of the present invention has an effect on obesity and metabolic syndrome. It was found that the present invention was completed.

Accordingly, an object of the present invention is to prevent and treat one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient thereof. It is to provide a pharmaceutical composition. ᅳ „

Another object of the present invention is one selected from the group consisting of obesity, obesity complications and metabolic syndrome comprising an extract extracted from the one or more solvents selected from the group consisting of acetone, ethyl acetate and methylene chloride as an active ingredient It is to provide a pharmaceutical composition for the prevention and treatment of the above diseases.

Still another object of the present invention is to provide a method for treating obesity, obesity, and complications comprising administering to a subject in need thereof an homoaflavanone or a pharmaceutically acceptable salt thereof represented by Formula 1 or Formula 2; It provides a method for preventing and treating at least one disease selected from the group consisting of metabolic syndrome.

<23> A further object is homo iso flavanone or pharmaceutically represented by obesity, obesity, complications, and the formula (1) or (2) for at least one ^preventive and producing a therapeutic agent for a disease selected from the group consisting of metabolic syndrome of the present invention To provide acceptable salt usage.

<24> A further object is such confines of acetone, the ethyl acetate and methylene chloride fluoride extract the need ^thereof, and extracted with one or more solvents selected from the group consisting of Loja of the present invention is obesity, comprising administering an effective amount to the object To prevent and treat one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome. Will be. ^. It is another object of the present invention to prepare a roundworm for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome in the group consisting of acetone, ethyl acetate and methylene chloride. To provide a use of the extract extracted with one or more solvents selected.

Technical Solution

In order to achieve the above object, the present invention consists of obesity, obesity complications and metabolic syndrome containing homoisoflavanone represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a pharmaceutical composition for the prevention and treatment of one or more diseases selected from the group.

In order to achieve the other object of the present invention, the present invention is made of an isomer consisting of acetone, ethyl acetate and methylene chloride _. It provides a pharmaceutical composition for the prophylaxis and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic group including extracts extracted with one or more solvents selected from the group as an active ingredient.

In order to achieve another object of the present invention, the present invention comprises the steps of administering an homoisoflavanone or a pharmaceutically acceptable salt thereof represented by the formula (1) or (2) in an effective amount to an individual in need thereof It provides a method for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome.

In order to achieve another object of the present invention, the present invention is homoiso is represented by the formula (1) or formula (2) for the preparation of a prophylactic and therapeutic agent for one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome It provides the use of flavanones or pharmaceutically acceptable salts thereof.

In order to achieve the another object of the present invention, the present invention is to administer the extract extracted from an isophore with at least one solvent selected from the group consisting of acetone, ethyl acetate and methylene chloride to an individual in need thereof It provides a method for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome comprising the steps.

In order to achieve the another object of the present invention, the present invention is one or more selected from the group consisting of obesity, obesity complications and metabolic syndrome and a roundabout for the production of a prophylactic and therapeutic agent of acetone, ethyl acetate and methylene chloride It provides a use of the extract extracted with one or more solvents selected from the group consisting of. <32>

<33>. Hereinafter, the present invention will be described in detail.

<34>

The composition of the present invention comprises homo. Isoflavanone or a pharmaceutically acceptable salt thereof (or a salt thereof) represented by Formula 1 or Formula 2 as an active ingredient, obesity, obesity It may be used for the purpose of preventing and treating one or more diseases selected from the group consisting of complications and metabolic syndrome.

<36> [Formula 1] ^'

<40> Polygonatum odoratum is a perennial herb belonging to the family Liliaceae, often grows in heights of 30-60 cm in the shades or alpine meadows of the country. Green flowers bloom around June and July, and the seeds ripen in August. It is widely used for the purpose of nourishment and tonic in oriental medicine and private medicine, and it is used for various weakness symptoms, malnutrition, cough caused by pulmonary tuberculosis and diabetes. ᅳ

The extract of the present invention can be extracted by a known solvent extraction method. For example, from 1 to 6 carbon atoms such as water, ethanol, methanol, propanol, isopropanol and butanol and alcohol, acetone, ether, chloroform, ethyl acetate, methylene chloride, nucleic acid , Selected from organic solvents such as cyclonucleic acid, petrolem ether, diethyl ether and benzene It can be extracted using one or a combination of these solvents. Preferably acetone, ethyl acetate, or may be extracted using at least one solvent or the solvents selected from the group consisting of methylene chloride, the combined common and other known solvents ^added to,

<42>

The extract of the present invention can be used as it is extracted by the solvent extraction method, or can be fractionated by a known method to remove impurities and increase the concentration of the active ingredient. The extract can be concentrated under reduced pressure for efficiency of the fraction, and solvents for fractionation include alcohols of 1 to 6 carbon atoms such as water, ethanol and methanol, ethyl acetate, dichloromethane, chloroform, n-nucleic acid and diethyl ether. , Any one selected from organic solvents such as acetone, benzene, or a mixed solvent thereof may be used, and preferably, any one selected from water, n-butanol, and ethyl acetate, or a mixed solvent thereof may be fractionated. Fractions can be used as is or refractionated, preferably ethyl acetate in case of refraction.

<44>

In an embodiment of the present invention, after extracting the isophore extract under reduced pressure and fractionating it with n-butanol and water to obtain a water fraction and adding ethyl acetate thereto, the isophore extract is obtained by obtaining an ethyl acetate layer. An ethyl acetate fraction of was prepared. As a result of measuring the increased expression efficiency of SIRT1 of the prepared fraction, it was confirmed that the expression of SIRT1 was high in the ethyl acetate fraction of the present invention (see Example 3).

<46>

The homoisoflavanone of the present invention can be isolated and purified from nature, commercially available and used, or prepared by chemically synthesized rice known in the art. Separation and purification from nature can be separated and purified from isophores by solvent extraction and chromatographic separation methods known in the art _. have. The extraction method is as described above, and the separation is performed by separation method using chromatography known in the art from the extract, for example, silica gel column chromatography, to obtain fractions according to polarity, and to separate the separated specific fractions again. It can be separated by reverse phase column chromatography and high performance liquid chromatography (HPLC).

<48>

In an embodiment of the present invention, the herbal extracts having the effect of increasing the SIRT1 expression were searched, and the ethyl acetate extract was found to have the effect of increasing the expression of SIRT1 (see Example 1). Using HPLC Homoisoflavanone of the invention was isolated (see Example 4).

<50>

The composition of the present invention is excellent in preventing and treating obesity, obesity complications or metabolic syndrome.

Obesity is a condition of excessive body fat. Metabolic syndrome

(Metabolic Syndrome) refers to a syndrome that is accompanied by risk factors such as hypertriglyceridemia, hypertension, abnormal glucose metabolism, abnormal coagulation, and obesity.

<53> ：

This efficacy of the present invention has been demonstrated through in vivo and in vitro experiments.

<55>

In one embodiment of the present invention, the extract of the present invention in the HEK 293 cell line

Whether to increase the expression of SIRT1 and the degree of activity of the expressed SIRT1;

P53 deacetylation was examined as a method of measuring the degree of deacetylation.

<57>^"As a result, Polygonatum extract of the present invention increases the expression of SIRT1, the SIRT1 expression was confirmed to exhibit a high activity (see Example 2).

In addition, the expression change of SIRT1 was observed in various tissues related to obesity after 5 hours after the administration of the rattan extract of the present invention to the mice.

As a result, it was confirmed that the expression of SIRT1 was increased in the adipose and muscle tissues of the mice administered the rattan extract of the present invention (see Example 2).

<60>

In one embodiment of the invention the homoisoflavanone of the present invention is a fat precursor cell

Treatment with 3T3L1 and pancreatic β-cell HIT-T15 inhibited the expression of PPARy and increased insulin secretion. As a result, it was confirmed that the expression of PPARY in the group to which the composition of the present invention was administered and insulin secretion was increased (see Example 5).

<62>

Meanwhile, the composition of the present invention has the effect of increasing the level of SIRT1 in vivo. SIRT1 is known to induce a prolonged life effect in yeast

Sirtuins (Sir2—related enzymes) are mammalian homologues (~ 110kD) of the class III hi stone deacetylases that require NAD ⁺ as a cofactor.

<64> In one embodiment of the present invention, the effect of increasing the level of SIRT1 by the composition of the present invention was confirmed through in vivo experiments. As a result of measuring the amount of SIRT1 protein in muscle, liver and adipocytes by regenerating the mice and the control group administered the composition of the present invention over time, it was confirmed that the amount of SIRT1 was increased in fat and muscle (see 'Example 6). ).

<66>

In addition, in one embodiment of the present invention, a high-fat obese feed was supplied to mice to prepare a obese model, and after administration of the composition of the present invention, the changes in body weight and abdominal fat were observed. As a result, the group fed the obese feed supplemented with the composition of the present invention had a weight loss, a decrease in the size of abdominal fat, and a decrease in fat even though there was no difference in feed intake compared to the group fed the obese feed only. It confirmed that it decreased. Insulin resistance, glucose tolerance, blood total cholesterol, triglyceride, free fatty acid levels, and metabolic syndrome were measured. As a result, the glucose tolerance was improved by more than 40¾> by the composition of the present invention, and the total cholesterol in blood was reduced by 12%, triglyceride and free fatty acid by 44% and 15%, respectively. ).

<68>

In addition, in one embodiment of the present invention by supplying a high fat 만 obese feed to mice to produce a obese model and after administration of the extract of the present invention, the change in body weight and glucose tolerance due to insulin resistance was measured.

As a result, the group administered the obese feed added with the composition of the present invention was confirmed that the weight was reduced despite the difference in feed intake compared to the group administered only the obese feed. In addition, it was confirmed that the improvement over ^"the glucose Chengdu by 30% in the compositions of the present invention (see Example 10). ·

<71> ^■ -

It was confirmed that the composition of the present invention is excellent in the prevention and treatment of obesity, obesity complications and metabolic syndrome.

<73>

The composition of the present invention not only exhibits the efficacy described above, but also contains the herbal extract as an active ingredient, so that the side effects on the human body are extremely less than that of the chemical synthetic medicine.

In one embodiment of the present invention was tested for toxicity to the composition of the present invention. As a result, significant changes in indicators related to hepatotoxicity or kidney toxicity can be observed. (See Example 9).

<76>

Therefore, the homoisoflavanone of the present invention inhibits the differentiation of adipocytes and maintains SIRT1 at a high level, thereby reducing abdominal fat in a high fat diet obesity-induced mouse model and significantly reducing weight to obesity, Prevent and treat obesity complications or metabolic syndrome. It can be seen that there is an effect.

<78>

Therefore, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, obesity complications or metabolic syndrome, which contains the extract of Hungle, or Homoisoflavanone represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient. To provide a composition.

In addition, the present invention is an obesity comprising the step of administering an isogulle extract, or homoisoflavanone represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof to an individual in need thereof, Provided are methods of preventing and treating one or more diseases selected from the group consisting of obesity complications and metabolic syndrome.

In addition, the present invention is homoisoflavanone represented by the formula (1) or (2) for the preparation of a prophylactic and therapeutic agent for one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome Provide for the use of acceptable salts.

<82>

In addition, the present invention is one selected from the group consisting of obesity, obesity complications and metabolic syndrome comprising an extract extracted from the at least one solvent selected from the group consisting of acetone, ethyl acetate and methylene chloride as an active ingredient Provided is a pharmaceutical composition for preventing and treating the above diseases.

In addition, the present invention is an obesity, obesity complication comprising the step of administering an extract extracted from the at least one solvent selected from the group consisting of acetone, ethyl acetate and methylene chloride to an individual in need thereof And it provides a method for preventing and treating at least one disease selected from the group consisting of metabolic syndrome.

In addition, the present invention is a method for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome is selected from the group consisting of acetone, ethyl acetate and methylene chloride Provided is the use of an extract extracted with one or more solvents.

<86> In the present invention, the term 'effective amount' refers to the effect of preventing or treating one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome in a subject to which the composition or formulation of the present invention is administered. Refers to the amount indicating

A 'subject' may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ or the like derived from an animal. The diffuse individual may be a patient in need of treatment. ^"" <88> The compositions of the present invention may have such a chain extract, wherein homo or iso-flavanone or pharmaceutically acceptable salts of the tank containing the carrier and the remaining amount of 0.001 to 99.999 wt ¾ thereof. Castle.,

<89>

The extract of the gorilla or homoisoflavanone according to the present invention may be used on its own or in the form of a pharmaceutically acceptable salt. As used herein, 'pharmaceutically acceptable' refers to a physiologically acceptable and normally does not cause allergic reactions or similar reactions when administered to humans. acid addition salts formed by free acid) are preferred. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4- Luenesulfonic acid, glutanoic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

<91>

The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of an extract of gulose or homoisoflavanone ^ alone or in addition to one or more pharmaceutically acceptable carriers. It can be included as. The pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long time. As used herein, the term “pharmaceutically effective amount” refers to an amount that exhibits more reaction than the negative control group, and preferably refers to an amount sufficient to treat or prevent obesity. Pharmaceutically effective amounts of isoflavanone include, but are not limited to, 0.001 to 1000 mg / day / kg body weight, but the pharmaceutically effective amount may include disease and symptoms thereof, age, weight, health status and sex of the patient. ， Such as route of administration and duration of treatment It may vary according to the factor.

<93>

The composition of the present invention may be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. Routes of administration may be administered orally or parenterally, without limitation thereto. Parenteral routes of administration include, for example, transdermal, nasal, abdominal, muscle, subcutaneous or intravenous routes. ：

<95>

In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention, together with a suitable carrier for oral administration, may be prepared according to methods known in the art in powder, granule, tablet, pill, dragee, capsule, It may be formulated in the form of a liquid, gel, syrup, suspension, wafer, and the like. Examples of suitable carriers include sugars and corn starch, wheat starch, rice starch and potato starch, including lactose, dextrose, sucrose, solbi, mannitl, xili, erysri, malty, etc. Fillers such as saloses, gelatin, polyvinylpyridone, and the like, including starch, salose, methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-salrose, and the like. In some cases, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, and an antiseptic.

<97>

In addition, when administered parenterally, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injections, transdermal and nasal inhalants together with suitable parenteral carriers. Such injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. In the case of injectables, examples of suitable carriers include, but are not limited to, water, ethanol, polyols (e.g. glycerin, propylene glycol and liquid polyethylene glycols), their mixtures and / or solvents comprising vegetable oils, or It may be a dispersion medium. More preferably, suitable carriers include Hanks' solution, Ringer's solution, triethane such as phosphate buffered saline (PBS) containing amines or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Isotonic solutions can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, the injection Part may further include isotonic agents, such as sugars or nat "chlorides. For transdermal administration, ointments, creams, lotions, gels, external preparations, pasta preparations, liniments, air Forms such as agents etc. As used herein, "transdermal administration" means the topical administration of the pharmaceutical composition to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

<99>

<ιοο> These formulations are formulated in Remington's, a commonly known formula in pharmaceutical chemistry.

Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, East on, Pennsylvania.

<ιοι>

In the case of inhaled dosages, the compounds used according to the invention are pressurized using a suitable layering agent, for example, dichlorofluorometrichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in aerosol spray form from a pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or blowers may be formulated to contain a compound and a powder mixture of a suitable powder base such as lactose or starch.

<103>

<104> with the other pharmaceutically acceptable ^carriers, may be (Remington 'as a reference that is described in the following literature s Pharmaceutical Sciences, 19th ed., 1995, Mack Publishing Company, Easton, PA).

<105>

Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with known compounds having the effect of preventing and treating obesity, obesity complications or metabolic syndrome.

<107>

In addition, the rattan extract or homoisoflavanone or a salt thereof according to the present invention may be provided in the form of a food composition for the purpose of improving obesity, obesity complications or metabolic syndrome, the food composition of the present invention Includes all forms of functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

<109> [Favorable effect]

Therefore, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, obesity complications or metabolic syndrome, etc. containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient. Homoisoflavanone or the composition of the present invention can be useful for preventing and treating obesity, obesity complications or metabolic syndrome by inhibiting adipocyte differentiation and maintaining SIRT1 at a high level to reduce weight, abdominal fat and glucose tolerance. -

[Brief Description of Drawings]

<ιιι> Figure 1 shows the results of treatment of herbal extracts in the ΗΕΚ 293 cell line to measure the SIRT1 protein expression change by Western blot (C: negative control group; 1209: decane ethyl acetate extract).

<ιΐ2> Figure 2 shows the crude extract of SIRT1 obtained by treating the herbal extracts in the ΗΕΚ 293 cell line

The degree of deacetylation of p53 by (crude extract) was measured by ELISA and compared with the negative control (ID1209: Dongleul ethyl acetate extract).

FIG. 3 shows the results of Western blot analysis of the increase in SIRT1 expression in adipose tissue of mice by the administration of herbal extracts (ID1209: decane ethyl acetate extract; Vehicle: negative control group).

Figure 4 is a Western blot result of measuring whether SIRT1 expression and PPARY expression increase in each tissue of the mouse by the administration of isophore ethyl acetate extract. Figure 4A is the liver tissue, Figure 4B is the adipose tissue, Figure 4C is a measure of the expression of SIRT1 in muscle tissue (Vehicle: negative control group; ID1209: isomeric ethyl acetate extract).

FIG. 5 shows the results of the process of determining the extraction solvent of the composition including the present invention from an isomer. FIG. 5A is a schematic diagram of a process for obtaining each solvent extract from an isomer (MeOH: methanol extract; BuOH: n-butane extract; EtOAc: ethyl acetate extract; CHC1 ₃ : chloroform extract; Et ₂ 0: ethyl ether extract Hex: n-nucleic acid extract;

¾0: water extract; The parenthesis shows the amount of each extract obtained from 10 g of canopy

). FIG. 5B is a result of measuring the SIRT1 protein expression change by Western blot after treating the solvent extracts with HEK 293 cell line for each concentration (C; negative control group treated with DMS0 only).

FIG. 6 is a schematic diagram of a process for separating and purifying homoisoflavanone of the present invention from an isomer (MeOH: methane; EtOH: ethanol; Si0 ₂ CC: silica gel column chromatography). Photography; Prep IX: preparative liquid chromatography; ID2244: homo isoflavanone represented by the formula (1); ID2252: homoisoflavanone represented by the formula (2).

7 shows the results of measuring whether the expression of SIRT1 is increased by the composition of the present invention.

Figure 7A is the result of the SIRT1 protein expression change by the treatment of each composition for each concentration in the HEK 293 cell line 'western blot. FIG. 7B shows SIRT1 protein levels in adipocyte tissue 5 hours after administration of each composition (Vehicle: Negative Control; ID1209: Isogul ethyl acetate extract; ID2244: Homoisoflavanone represented by Formula 1; ID2252: Formula 2 Homoisoflavanone displayed).

Fig. 8 shows the results of testing the effect of inhibiting adipocyte differentiation by the composition of the present invention in 3T3L1, an adipocyte cell line. FIG. 8A is a measure of the amount of peroxysomal proliferative receptor (PPARY) mRNA (GAPDH: Glyceraldehyde-3 Phosphate Dahai Hlogogenase (Glyceraldehyde-3) used as internal control. -phosphate dehydrogenase); ID2244: homoisoflavanone represented by formula (1); ID2252: homoisoflavanone represented by formula (2). 8B is a result of measuring the amount of PPARY protein (ID2244: homoisoflavanone represented by formula 1; ID2252: homoisoflavanone represented by formula 2). FIG. 8C is a photograph showing the degree of differentiation into adipocytes using an oil red o dye and a graph showing the numerical value thereof (ID2244: homoisoflavanone represented by Chemical Formula 1; ID2252: represented by Chemical Formula 2). Homoisoflavanone).

9 is a graph showing the degree of increased glucose-induced insulin secretion by the homoisoflavanone of the present invention in pancreatic cell (HIT-T15) strain (ID2244: homoisoflavanone represented by Formula 1; ID2252: homoisoflavanone represented by the formula (2).

FIG. 10 is a Western blot result of measuring whether SIRT1 expression and PPARY expression increase in each tissue of the mouse by the composition of the present invention. FIG. 10A shows the expression of SIRT1 in adipocyte tissue, FIG. 10C in liver tissue, and FIG. 10D shows muscle tissue, and FIG. 10B shows the expression of PPARY in adipocyte tissue (Vehicle: negative control group; ID2244) Homoisoflavanone represented by 1, ID2252: homoisoflavanone represented by the formula (2).

11 is a result of measuring the SIRT1 protein and mRNA levels in each tissue of the mouse by the composition of the present invention over time. 11A and 11B show adipocyte tissue and muscle tissue. The SIRT1 protein level and the ratio of the internal standard, a -act in, respectively, Figure 11C and Figure 11D shows the ratio of the SIRT1 mRNA level of the adipose tissue and muscle tissue and the internal standard GAPDH, respectively (GAPDH: Glyceraldehyde-3-phosphate dihydrogenase: ID2244 (homoisoflavanone represented by Formula 1, 50 mg / kg body weight); ♦: ID2252 (Homoisopolarbanon represented by Formula 2, 50 mg / kg body weight)). 12 is a result of testing the anti-obesity effect of the composition of the present invention in obesity-induced animal model. Figure 12A is a measure of the weight of the mice over time, Figure 12B is a measure of feed intake. * Indicates p <0.05 and ** indicates p <0.01, which is significant when compared with high fat diet (： normal diet; 画: high fat diet; A: high fat diet + resveratrol (4000 mg / kg feed); High fat diet + ID2244 (homoisoflavanone represented by Formula 1, 500 mg / kg); ♦ high diet diet + ID2252 (homoisoflavanone represented by Formula 2, 500 mg / kg) ·

Figure 13 is a result of testing the anti-obesity effect of the composition of the present invention in obesity-induced animal model. Figure 13A is a macroscopic observation of the mouse, Figure 13B is a graph measuring the weight of the fat cell tissue. The values are mean mean standard deviation of 9-10 rats, and * is p <0.05 and ** is p <0.01, which is significant when compared with the high fat diet (ND: normal diet; HF: high diet). Resveratrol: high fat diet + resveratrol (4000 mg / kg feed); ID2244: high fat diet + formula 1 Homoisoflavanone (500 mg / kg feed); ID2252: high fat diet + formula 2 Homoisoflavanone (500 mg / kg feed)).

FIG. 14 is a micrograph of changes in adipocyte tissue and liver tissue according to administration of the composition of the present invention in an animal model of obesity (WAT: adipocyte tissue, Liver: liver tissue; ND: normal diet; HF). : High fat diet; HF + RES: High fat diet + resveratrol (4000 mg / kg feed); HF + ID2244: High fat diet + Formula 1 Homoisoflavanone (500 mg / kg feed); HF + ID2252: Homoisoflavanone (500 mg / kg feed) represented by high fat diet + formula 2).

15 is a cold test result associated with the anti-obesity effect of the composition of the present invention in an obesity-induced animal model. The values are mean mean standard deviation of 6 rats, * is p <0.05, ** is ρθ.01, and *** is ρ 0.0 () 1, which is significant when compared with the high fat diet. High-fat diet; 參: high-fat diet + ID2244 (homoisoflavanone represented by formula 1, 200 mg / kg feed); ♦: high-fat diet + ID2252 (homoisoflavanone represented by formula 2, 200 mg / kg feed)). ^'

16 is a result of measuring the blood glucose concentration by oral glucose administration in the animal model of obesity (X: normal diet; 画: high-fat diet; A: high-fat diet + resveratrol (4000 mg feed: Homoisoflavanone, 500 mg / kg feed, represented by formula + ID2244 (Formula 1); ♦: High fat diet + ID2252 (Homoisoflavanone, 500 mg / kg feed).

17 shows the effects of metabolic syndrome on the composition of the present invention in an obesity-induced animal model. Test result. Figure 17A is the total cholesterol, Figure 17B is a triglyceride, Figure 17C is the result of measuring the concentration of free fatty acids in the blood, respectively. The values are mean mean standard deviation of 9-10 rats, and * is p <0.05, ** is p <0., Which is significant when compared with the high fat diet (ID2244: high fat diet + formula 1). Homo isoflavanone represented by: ID2252: Homoisoflavanone represented by the high-fat diet formula (2).

FIG. 18 is a graph measuring mRNA levels of various indicator substances in each tissue in an obesity-induced animal model. Figure 18A is measured in adipocytes, Figure 18B in muscle tissue, Figure 18C in liver tissue (HF: high fat diet; ID2244: high fat diet + Formula 1 Homoisoflavanone represented by Formula 1; ID2252: Homoisoflavanone represented by high fat diet + Formula 2; PGC-la: PPARY coactivator; F0X01: Forkhead Box 01 ， Forkhead transcription factor; PPAGg: PPAGy, peroxysomal proliferative agent Answer receptor; UCP-2, UCP-3: uncoupling protein 2, ^' 3).

FIG. 19 is a chromatogram measured by HPLC to quantify the content of homoisoflavanone in an isocol ethyl acetate fraction. 19A is an isocol ethyl acetate fraction, FIG. 19B is a chromatogram of ID2244, and FIG. 19C is ID2252.

20 is a result of testing the anti-obesity effect of the present foot composition in obesity-induced animal model. Figure 20A is a measure of the weight of the mouse hourly, Figure 20B is a measure of feed intake over time. Values are mean 土 standard deviation of 6 rats, * is p <0.05, ** is ρ <0 · 01, *** is ρ <0.0 () 1, which is significant when compared with the high fat diet. (画: normal diet; ♦: high-fat diet; high-fat diet + sibutramine (100 _{mg g} / kg feed); ·: high-fat diet + isgulale ethyl acetate extract (5000 mg / kg feed)).

Figure 21 is a low temperature test associated with the antibaman effect of the composition of the present invention in obesity-induced animal model

(Cold test) results. Values are mean 土 standard deviation of 6 rats, and *** means p <0.0, which is significant when compared with high fat diet (♦: High fat diet; ·: High fat diet + Ethofole ethyl acetate extract (5000mg / kg feed)).

22 is a result of measuring the blood glucose concentration by oral glucose administration in obesity-induced animal model. Values are the mean 土 standard deviation of 6 rats (♦: high fat diet; note: high fat diet + isolegulate ethyl acetate extract (5000 mg / kg feed)).

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples. <135>

<136> <Example 1>

<i37> SIRT1 expression layer natural extract selection

<138>

<i39> <1-1> SIRT1 Expression Enhancement Natural Extract 1st Screening

<i40> SIRTKSirtuin 1) In a medium added 0.5% (v / v) of serum-free Dulbecco's modified Eagle's medium (DMEM) at a concentration of drug dissolved in dimethyl sulfoxide (DMS0) to select expression enhancers. After incubating the HEK 293 cells for 16-20 hours, the obtained protein was electrophoresed on a ¾ SDS-PAGE gel and transferred to the PVDF membrane for immunoblotting with SIRT1 specific antibody (Cell Signaling Technology, USA). The stem was established. As a control group, drugs with no drug-containing DMS0 treatment group were selected to increase the expression of SIRT1 than the control group.

<i4i> As a result, eight kinds of herbal extracts were selected from the library of more than 320 kinds of herbal extracts of the inventors' research team, including Baekganjam, Astragalus, Jade Juk, Baekji, Aloe, Geumumhwa, Floured Wheat, Seaweed (see Table 1). ). ^"

<142>

<i44> Confirmation of efficacy of SIRT1 enhanced natural extract (Secondary selection)

<145>

The final material was selected through experiments to determine whether the SIRT1 activity, in vivo) SIRT1 expression is enhanced for the natural extract selected in Example 1.

<147>

<148> <2-1> SIRT1 Level Increasing Efficacy Test by Natural Extracts (in vitro)

In the same manner as in Example 1, the primary extract of HEK 293 cell line was first administered to determine whether the expression of SIRT1 protein was increased.

As a result, as shown in Figure 1 ID1209 was confirmed that the excellent expression of SIRT1 protein.

<151>^'

<i52> SIRT1 protein is known to promote deacetylation of p53. The degree of deacetylation of p53 by crude extract of SIRT1 obtained by treatment of HEK 293 cell line first selected natural extract was measured by ELISA using Fluor de Lys Kit (BI0M0L # AK-555). <153>

As a result, as shown in FIG. 2, ID1209, ID1210, ID1211 and ID1212

It was confirmed that the activity of SIRT1 was high.

<155>

<156> <2-2> In vivo SIRT1 Level Increasing Effect Test by Natural Extracts

All animal procedures were performed according to the guidelines of the Ildong Pharmaceutical Animal Ethics Committee. Male rats, 8 weeks old C57BL / 6, were purchased from Oriental Bio. The room temperature was maintained at 23 ± 1 ^° C, the humidity at 50 ± 10%, and the contrast was 12 hours (day light 08: 00-20: 00). . To aid in absorption, fasted mice were administered with 500 mg / kg of natural extract, respectively. Five hours after administration, rats were sacrificed and liver, fat, and muscle tissues were removed. The extracted tissue was crushed with Na ₃ V0 ₄ and protease inhibitor.

. Protein was extracted with (50 mM Tris-HCl, pH7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X—100) and the protein was quantified using BCA quantification. To confirm the expression of SIRT1, muscles separated 100 / g of protein and tissues other than muscles separated 60 proteins from 8% SDS-PAGE gel, transferred to PVDF membrane and blocked with 5% skim milk for 10 minutes. Western blot was performed with anti _SIR2 (Mi 11 ipore) antibody which can identify the SIRT1 protein of the mouse.

<158>

As a result, as shown in FIG. 3, ID1209 was found to increase SIRT1 protein expression most in vivo. As shown in FIG. 4, ID1209 increased SIRT1 protein expression in all liver, adipose and muscle tissues associated with obesity and metabolic syndrome.

<160>

<i6i> ID1209 was finally screened based on the results of Example 2 (see Table 1).

ID1209 is an extract of the ethyl acetate of jade jade which is a herbal medicine, and jade is the root of Po / ygc aium odoraiura. <162>

<163>

<164> <Example 3>

<165> SIRT1 Activity Test of Fractions of Extracts

Various solvent extraction tests were performed to select compositions having effective efficacy from ID1209 selected as herbal extracts with high potency of enhancing SIRT1 expression.

In order to confirm the presence of an active ingredient in the solvent fraction which enhances the expression and activity of SIRT1 from isogule, the active fraction according to the extraction solvent was identified.

PoJygonatum odoratum is extracted and separated in the order shown in FIG. 5A.

It was used in the next step to measure SIRT1 activity.

After crushing 10 _g of isocraps, 30 ml of 100% methanol was immersed, and the extraction solution extracted three times for 24 hours from the top was filtered through a filtering device and methane was removed using a depressurizing device to obtain a methanol extract. It was. 20 ml of n-butanol and water were added to the methanol extract obtained by the above process, and the mixture was mixed. The upper layer was concentrated to obtain n-butanol extract (BuOH), and the same amount of ethyl acetate was added to the aqueous layer. After mixing, the upper layer was concentrated to give an ethyl acetate extract (EtOAc). Equivalent amount of chloroform was added to the aqueous layer, followed by concentrating, and the lower layer was concentrated to obtain chloroform extract (CHC1 ₃ ). The same amount of ethyl ether was added thereto, followed by mixing and concentrating the upper layer. (Et ₂ 0) was obtained. Finally, after homogeneous mixing of n-nucleic acid in the aqueous layer The upper and lower layers were concentrated to obtain n-nucleic acid extract (Hex) and water extract (0).

Each of the extracts obtained in the above process was washed twice with ethanol to remove residual solvent, concentrated under reduced pressure and dissolved in DMS0 at a certain concentration to test the activity of SIRT1 by the method of Example 1.

As a result, only ethyl acetate extract was found in the solvent extracts obtained from isophore.

It was found that SIRT1 expression was increased up to 2.5 ug / ml. In contrast, the expression of SIRT1 was not increased even at concentrations of 10 ug / ml or more in other solvent extracts (see FIG. 5B). Therefore, the components that increase the activity of SIRT1 in the bridle extract was found to be limited to the ethyl acetate fraction.

<174>- ^'

<175> <Example 4>

<176> Separation of Active Ingredients from Extracts

A separate purification was performed to identify the active ingredient from the roundworm selected as a herbal extract with high potency of enhancing SIRT1 expression. ― ^'

20 kg of dried and crushed isophore w7j o / 3aiuffl odor a turn) was immersed in 40 L of methanol, and extracted three times at room temperature for 12 hours, followed by filtration and concentration under reduced pressure to obtain a methanol extract. . The methanol extract was suspended in distilled water, and then ethyl acetate and solvent fractions were used to remove the water fraction. The ethyl acetate fraction was again suspended in 90% methanol and divided into "nucleic acid fractions" to obtain 90% methanol fractions with increased SIRT1 activity compared to the other fractions.

Silica gel column chromatography was performed to elute the 90% methanol fraction with methylene chloride and a methanol mixed solvent. Start eluting with 100% methylene chloride solvent, methylene chloride: methanol = 100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 1: 1, 100% methanol in order of solvent polarity. Similar fractions were combined into one, resulting in a total of seven subfractions (PSM-1 to PSM-7 fractions). Silica gel column chromatography was performed on the PSM-2 fraction, which is an SIRT1 activity enhancing fraction, in the same manner as above to obtain a total of eight subfractions (PSM-2-1 to PSM-2-8 fractions). The PSM-2-2 fractions were subjected to silica gel column chromatography using; 2-nucleic acid, ethyl acetate and methanol in a common solvent (10: 1: 1 → 5: 1: 1 → 1: 1: 1). Fractions were obtained (PSM-2-2-1 to PSM-2-2-9 fractions).

<i8i> For the PSM-2-2-4 fraction, ID2244 was isolated by preparative HPLC. Preparatory

To separate by HPLC, a YMC ODS Pack ODS-A preparative column was used and 45% aceto Nitrile was eluted at a rate of 3 ml / min under a constant solvent composition and confirmed by a 254 nm UV detector.

Preparative HPLC was performed on the PSM-2-2-5 fractions. ID2252 * was isolated. Preparatory

For separation by HPLC, a YMC ODS Pack 0DS-A preparative column was used and eluted at a rate of 3 ml / min under a constant solvent composition of 40% acetonitrile and confirmed by a 254 nm UV detector (see FIG. 6).

<183> [S 2]

§ 1ΐΕ (刹 S 咖: 難 ¾ character

<185>

<186>

<187>

<188> <i89> The isolated fractions were tested for SIRT1 activity in the same manner as in Example 1. ^'

As a result, a total of six SIRT1 active substances were identified (see Table 2).

Two substances (ID2244, ID2252), which strongly enhances SIRT1 expression in vitro on iro and in vivo wVo, were obtained as an active ingredient of decanter extract (see FIG. 7). The two identified substances were subjected to structural identification in the following manner.

<i92> ID2244 showed a molecular ion peak of m / z 314 in EIMS, a molecular weight of 314, and a hydoxyt ropy Hum fragment of m / z 107 _. The presence of the phenyl group was confirmed. In the -NMR spectrum, two methyl signals (δ δ .93, δ 1.95) in the high magnetic field are represented by δ 4.18 (1H, dd, J = 4.4, which represents -CH ₂ -CH-CH ₂ -residues).

11.2 Hz), 4.02 (1H, dd, J = 7.2, 11.2 Hz), 2.71 (1H, m), 3.04 (1H, dd, = 4.4, 13.6 Hz), and 2.56 (1H, dd, J = 10.4, 13.6 Hz) signal and aromatic AA 'ΒΒ' system signal (δ 6.73 and 7.05, J = 8.4Hz) were detected. In the C-NMR spectrum, a total of 18 signals were detected and assigned to each (see Table 3). ID2244 represented by the formula (1) is 4 ', 5, 7-trihydroxy-6, 8- dimethyl- arc of molecular weight 314 _. Cattle feeders flavanone (4 have been identified ', 5,7 eu trihydroxy-6,8-dimethyl-homoisof lavanone ) ^to'

<i93> ID2252 was expected to have a skeleton similar to that of ID2244, and the molecular ion peak was observed at m / z 330 in the EIMS positive mode. It was confirmed that there is.

In the H-NMR spectrum, one methyl group signal (δ 1.96, 3Η, s) and one methoxy group signal (53.72, 3H, s) were identified in the high magnetic field. ^A total of 18 signals were detected in the ¹³ C- 證 spectrum and assigned to each (see Table 3). ID2252 represented by the formula (2) is 4 ', 5,7-trihydroxy-6-methyl-8-methoxy-homoisoflavanone having a molecular weight of 330 (4', 5, 7—tr ihydroxy-6-methyl-8 -methoxy- homoisof lavanone).

<194> [Table 3]

<195> 證 assignment of ID2244 and ID2252

<196> <Example 5>

<197> (/ fl vitro) Efficacy Test of Homoisoflavanone (ID2244. ID2252) in vivo

<198>

In vitro in vitro efficacy tests were performed on cell lines related to obesity and diabetes in order to determine whether two homoisoflavanones isolated from dorsum were effective against diseases targeted to SIRT1.

<200> 3T3L1 and ΗΙΤ-ΊΊ5 cells were supplied by the Korea Cell Line Bank and 10% fetal bovine serum

It was grown at 37 ^° C with 5% carbon dioxide in DMEM medium containing (fetal bovine serum). These cultures contained 2 ¾ g / ml gentamycin.

<201> <202> Measurement of Adipocyte Differentiation and PPARy Expression by <5-l> Homoisoflavanone

The fat precursor cell line 3T3L1 cells were dispensed by 5xi0 ⁵ / well in 6-well plates and incubated for 3 days to maintain post-confluent status. 2 ~ 2 in DMEM (including 10¾ FBS) culture containing 1 mM dexamethasone, 1.7 mM insulin and 1-methyl-3-isobutylxanthine (MIX) Differentiation was induced by incubation for 3 days. ID2244 and ID2252 were first treated 1 hour before the differentiation inducing factor. While maintaining eruption for three days. To confirm the expression level of PPARy (Peroxisome Prol iferator-Activated Receptor γ) over time, RNA and protein were extracted by date. RNA was extracted with one step RNA reagent (Biosesang), and each RNA was synthesized cDNA using M-MLV polymerase (Promega). Primers used for PCR were synthesized by Intergrated DNA Technologies (see Table 4).

<204>

<205> [4] ^' _.

<206> Primer for confirming the amount of PPARY expression

<207>

PPARy has a predenaturation of 2 minutes at 94 ^° C and denaturation

1 minute at 94 ^° C, annealing at 30 ^° C at 65 ^° C, elongat ion at 40 ^° C at 72 ^° C, post-elongation at 7 ^° C at 72 ^° C, 25 Cycles were performed. After completing the PCR, the PCR product was confirmed on a 1.5% agarose gel.

The protein was TEN buffer (50 mM Tris-HCl, pH7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) with Na ₃ V0 ₄ and a protease inhibitor. After extraction from cells and protein quantification, 100 / zg protein was subjected to Western blot with anti-PPARy antibody.

<2io> 3 days after inducing differentiation and ^further, replaced with only 1.7mM insulin DMEMCL0% FBS) culture solution was carried out for 2 days differentiation. After that, it was replaced with DMEM containing only 1OT FBS and maintained for 2 days, followed by oil red 0 staining. Cells were treated with 25% glutaraldehyde for oil red staining. After being left for a minute, it was left to shake for 30 minutes with 1 ml of an oil redo stain solution dissolved in 60% isopropanol. After rinsing with distilled water until clear water appeared, the microscope was observed, the plate was slightly dried, and eluted using DMSO lm £. It was transferred to 96-well piate and quantified by measuring absorbance at 505 nm.

<211>

The anti-obesity effect of ID2244 and ID2252 using 3T3L1 cell line of adipose tissue origin showed that both substances reduced PPARY mRNA and protein levels, and triglyceride production decreased in both groups. It was confirmed. As a result, both substances effectively inhibited mast cell and differentiation by inhibiting the expression of PPARY (see FIG. 8).

<213>

<214> Insulin secretion promoting test by <5 "2> homoisoflavanone

<2i5> Pancreatic Cells to Determine if ID2244 and ID2252 Promote Insulin Secretion

. The test was conducted using (pancreas cell) HIT-T15. HIT-T15 cells were divided into 24-well plates at 2 × 10 ⁵ / well and incubated for 24 hours. Krebs- Ringers bicarbonate (KRB) buffer (115mM NaCl, 4.7mM KC1, 2.56mM CaCl ₂ , 1.2mM

After washing twice with KH ₂ P0 ₄ , 1.2mM MgS0 ₄ , 20mM NaHC0 ₃ , 16mM HEPES, 0.3% bovine serum albumin (BSA), pH7.4), it was left in the incubator for 30 minutes. Each sample was replaced with KRB buffer (containing 5 mM glucose) and then incubated in a 37 ^° C. incubator for 2 hours. Mouse insulin ELISA kit (Shibayagi) was used to determine the amount of insulin secreted.

<216>

As a result, it was confirmed that when the ID2244 was treated with the pancreatic cell line HIT-T15, insulin secretion was increased by adding glucose. As a result, it was confirmed that the composition of the present invention has excellent metabolic syndrome treatment efficacy (see FIG. 9).

<218>^'

<219> Example 6

<220> In vitro SIRT1 Level Increase Potency Test by Homoisopolar Banon

<221>

In order to determine whether homoisofulabanones of the present invention increase SIRT1 levels in vivo, W57, the amount of SIRT1 expression in each organ of C57BL / 6 mice was analyzed. Phase confirmed. ^'

The experiment was conducted in the same manner as in Example 2-2, and ID2244 and ID2252 were respectively applied.

Liver, fat, and muscle were harvested at 3, 5 and 8 hours after the administration to 50 mg / kg. Extracted tissue. Some were rapidly immersed in liquid nitrogen for RNA extraction and stored at -70 ^° C until use, and the rest of the extracted tissue was placed in crushing buffer for protein extraction and then stored at -70 ^° C until use. ,

<224> V

<225> <6-1> In vivo SIRT1 protein level measurement by homoisoflavanone

The extracted tissue was subjected to Western blot after extracting the protein as in Example 2-2.

<227>

As a result, it was confirmed that both ID2244 and ID2252 increased SIRT1 protein expression in fat and muscle (see FIG. 10).

<229> There was a difference in the expression increase time according to each organ, and the expression level of SIRT1 increased with time after the administration of the substance and then immediately decreased to return to the normal state, which is a side effect of SIRT1 overexpression at all times. It is thought to be an in vivo mechanism of minimization.

In addition, in the adipose tissue, it was confirmed that the expression of PPARY was decreased with the increase of the expression of SIRT1 by the two substances, and in the liver, there was no difference or the decrease in the expression of SIRT1. It is similar to.

<231>

<232> <6-2> Measurement of SIRT1 mRNA Level Increase in vivo by Homoisoflavanone

In order to determine whether homoisoflavanones of the composition of the present invention increase SIRT1 expression in vivo, RNA was extracted from tissues and the amount thereof was measured.

RNA was extracted from the extracted tissue using a one step RNA reagent (Biosesang).

CDNA was made using extracted RNA 2 / g and the polymerase used was prepared according to the method provided using M-MLV polymerase (Promega). Quantitative polymerase chain reaction (quantitative PCR) was performed using the prepared cDNA. The primers shown in Table 5 were used to measure the expression level of each gene.

<235>-<236> [Table 5] ^' <237> mRNA measurement

<238>

As a result, there was no difference in the expression of SIRTl mRNA by all organs and time (see FIG. 11). This confirms that these substances affect the stability of the protein rather than inducing the production of SIRT1 protein.

<240>

<241><Example7>. . ^'

242 Pharmacokinetics Test of Homoisoflavanone

<243>

<244> In order to confirm the availability of the composition of the present invention

Pharmacokinetic tests were performed on ID2244 and ID2252, and the absorption and removal rates of the active ingredients in the blood were calculated for each of the six C57BL / 6 mice by oral and intravenous administrations, respectively.

<245>,

Each active ingredient (ID2244 and ID2252) contains Cremophor ELP, ethanol, and distilled water.

It melt | dissolved in the mixed solvent in the ratio of 1: 1: 10, and used. As the experimental animals, male ICR mice of 7 weeks old were used, and mice fasted for 12 hours or more were orally administered by gastric nutrient (gavage) at a dose of 50 mg / kg or intravenously injected at a dose of 25 mg / kg. Blood samples collected at 15, 30, 60, 120, 240, and 480 minutes 0 = 4 to 6) after administration of the active ingredient were separated from the supernatant (plasma) after centrifugation under conditions of lOOrpm, lOrain, and 4 ^° C. . The separated plasma samples were compared with the standard sample for calibration curve used to determine the active ingredient concentration in pre- and post- plasma plasma by protein precipitation method. Samples were analyzed using a triple quadropole LC / MS (Agilent 6460). Plasma concentrations obtained after calculating the concentrations of each sample are displayed as time-concentration graphs _. The parameters were calculated using the non-compartment model of the Winnolin 5.0.1 program.

<247> <248> [Table 6]

<250> As a result of measuring the pharmacokinetic power of two homoisoflavanones, Cm _a x was about 40% higher in ID2252 and terminal T _1/2 was more than twice as long in ID2244 as oral administration. The difference could be related to the pharmacokinetic differences of homoisoflavanone. The bioavailability of the two homoisoflavanones was similar to 45¾ and 4¾, respectively, because ID2252 had high absorption rate of drug and ID2244 had high stability in the body. It was confirmed to be saint (see Table 6).

<251>

<253> Efficacy test for obesity and metabolic syndrome of homoisoflavanone

<254>

<255> The active ingredient of ID1209 through pharmacokinetic test and in vivo SIRT1 expression test

ID2244 and ID2252 were determined to exhibit somewhat different properties in vivo, and the two active ingredients (ID2244 and ID2252) of the composition of the present invention were tested for efficacy against obesity and metabolic syndrome.

<256>

<257> <8-1> Model of obesity-induced mouse by high fat diet

The test showed that 8-week-old C57BL / 6 mice had ID2244, per kg feed containing 40% fat.

Body weights were measured for 7 weeks with feed supplemented with 500 mg of ID2252, and after 7 weeks various changes in obesity and related indicators were compared with those of the normal and obese diet groups. Resveratrol, which enhances SIRT1 activity and showed anti-obesity effect, was used as a positive control group that received a diet containing 4,000 mg of resveratrol per kg obese feed.

<259> In the high-fat diet containing more than 40% fat, resveratrol was mixed with 0.4% of silver and 0.05% of ID2244 and ID2252, respectively, and stored for a long time before use. The composition of the feed is shown in Table 7. -

<261>-<262> [Table 7]

<263> Feed Ingredients ^''

<264>

<265>

Preparation of the animal was performed in the same manner as in Example 2-2. Mice were freely fed high fat diet for 7 weeks. The mouse groups were the normal feed group (ND) without Uji, the untreated high-fat diet group (HF), the group containing ID2244 and ID2252 500 mg / kg high-fat diet (ID2244, ID2252), high-fat diet Ten rats were divided into groups containing 4 g of resveratrol per kg (Resveratrol).

<267>

<268> <8 "2> Weight, Feed Intake and Abdominal Fat Change

Body weight was measured once a week and feed intake was measured twice a week. After the end of the experiment, all groups of mice recovered and dissected plasma to determine the weight of liver and abdominal fat. Some liver and adipose tissues were observed by staining.

<270>

As a result, the obese feed group added with ID2244 and ID2252, respectively, lost 24% and 11% after 7 weeks, respectively, compared with the obese feed group, and the positive control group received 17%. There was no difference in feed intake (Fig. 12A, B true article). After 7 weeks of autopsy, the drug group not only reduced the size of abdominal fat cells compared to the fat diet group (see FIG. 13A), but also reduced the amount of fat by 62¾ and 42%, respectively, compared to the fat diet group. In the resveratrol-administered group, the amount of fat was reduced by 46% compared to the fat diet group, confirming a direct correlation between body weight loss and abdominal fat amount (see FIG. 13B).

Histological Analysis of Liver Tissue As a result, it was confirmed that fat, which was widely and densely distributed in the liver tissue of the obese diet group, was locally and mildly relieved in the ID2244 and ID2252 administration groups (see FIG. 14).

<273>

<274> <8-3> Cold test

To determine the cause of weight loss, a cold test was performed. The live rats were placed in a cold place at 4 ^° C. and measured and recorded in rectal temperature every 1-2 hours for 8 hours to compare with the initial body temperature.

ID2244, ID2252 administration group significantly overcome the decrease in body temperature seen in the obese diet group, it can be seen that the effect of inhibiting weight gain by ID2244, ID2252 administration is due to increased heat dissipation through active metabolism in the body (Fig. 15). Reference).

<277>

<278> <8-4> Glucose Tolerance Measurement by Insulin Resistance

<279> To measure glucose tolerance due to insulin resistance, blood was obtained from the tail of a mouse fasted for 16 hours or more (0 min), and glucose was injected into the tail of the mouse at 2 g / kg. Blood was obtained at 30, 45, 60, 90 and 120 minutes and the allocation was measured and recorded.

<280>

As a result, the values of the area under curves (AUC) of the obese diet group and the resverat group were 7421 ± 2726, 14912 ± 2815, 10502 ± 2037, 8859 ± 1969, respectively. Compared with the obese diet group, ID2244 and ID2252 administration groups showed 40 and 49% reduction in glucose tolerance (p <0.01), whereas resverat administration group was not significant (see FIG. 16).

<282>

<283> <8 "5> Blood Test

After the experiment, blood was collected from each group of mice to separate plasma. Triglyceride assay kit (Cayman) is used for triglycerides. Free fatty acids are obtained using NEFA (Wako). Measured.一

As a result, the total cholesterol level in the blood was decreased by 12% in the ID2252 administration group as an indicator of metabolic syndrome, but the triglyceride and free fatty acids were 44% in the ID2244 administration group. %, Reduced by 15% and 45%, 23% in the ID2252 administration group was confirmed (see Fig. 17).

<286> '

<287> <8-6> Measurement of mRNA Expression Change

In order to investigate the anti-obesity effects of ID2244 and ID2252 and the causes of alleviation of various metabolic syndrome symptoms, mRNAs of various marker genes related to fat differentiation, energy metabolism, mitochondria biosynthesis, etc. Expression change was confirmed. Primers used are shown in Table 8 and the method was performed in the same manner as in Example 6-2.

<289>

<290> [Table 8]

<29i> Primer for mRNA Measurement

<292>

<293>^'■ ,

<294> Measurement _. As a result, mRNA expressions of PGC-l a and UCP-2 and UCP-3 in all tissues increased more than twofold by the treatment of each drug, so that the expression of SIRT1 increased with the biosynthesis of energy and mitochondria. It was confirmed that it is related to the promotion of the degradation of adipocytes by the expression of related genes (see Fig. 18).

<295>

<296> <Example 9>

<297> Toxicity Test <298>

<299> For two substances _: histological analysis of liver tissue and biochemical analysis of blood were performed to determine toxicity.

<300> AST, creatine, BUN _. Bilirubin and bilirubin were analyzed using a cholesteric automatic dry chemistry analyzer (A25 analyzer, Biosystems).

As a result, as shown in FIG. 14, abnormal morphology was not observed in the microscopic observation of liver tissue. As a result of blood analysis, as shown in Table 7, both substances showed hepatotoxicity, such as ALT and AST, and creatinine, There were no significant changes in indicators related to renal toxicity such as BUN et al. (See Table 9).

<302>

<303>

<305>

In summary, the results of the efficacy test for obesity and metabolic syndrome for the active ingredients of ID1209 of the above example, ID2244 and ID2252 is determined to be nontoxic when administered orally at a dose of 50 mg / kg, 8 times or more It was found that the high dose of resveratrol showed the same or more anti-obesity effect as compared to the administration group and improved various markers related to obesity and metabolic syndrome. In addition, the two substances have different characteristics and slightly different effects on various indicators, and therefore, the combination-related administration may be able to more effectively improve the symptoms related to obesity and metabolic syndrome.

<307>

<308> <Example 10>

<309> Efficacy Test on B ᅵ Only of Emulsion Ethyl Acetate Fraction ―

<310> <3 i i> The above embodiment. Only the ethyl acetate fraction of 3 increased the activity of SIRT1, and as shown in Example 8, the major active ingredient ID2244 and

• _; ID2252 has been shown to inhibit obesity and metabolic syndrome in vitro and in vivo experiments. As shown in FIG. 4, isobutyl acetylacetate extract was shown to inhibit the expression of SIRT1 in tissues associated with obesity and metabolic syndrome in mice. The present invention is increased. Efficacy test for obesity and metabolic syndrome was performed on the composition of isogulet ethyl acetate fraction.

<312>

<313> <10-1> Analysis of Homoisoflavanone Contents in the Fraction of Ethyl Acetate

HPLC analysis was performed to quantify the contents of the active ingredients ID2244 and ID2252 contained in the isophore ethyl acetate fraction. Ten grams of crushed isomers were extracted three times with 100 ml of methanol, filtered, and concentrated. The solvents were partitioned with ethyl acetate and water. Ethyl Arthurate, which is an organic layer, was concentrated, and the resultant was prepared at a concentration of lmg / ml. The instrument used in the analysis was a Thermo Accel HPLC system (PDA detector, Pump, Autosampler). YMC's Pack Pro-C18 column (150x4.6mm, 5um) was used and the active ingredient was detected at 295nm. The eluting solvent was eluted with distilled water and acetonitrile in a gradient elution (0-5min 20% acetonitrile, 15min tilt elution 100% acetonitrile, 15-17min 100% acetonitrile. 20m in 20% acetonitrile Trill). It was confirmed that the active ingredients, respectively 0.1, 1, 10, lOOug / ml was measured at different concentrations of the absolute geuryeoteumyeo ryangseon gum in a concentration per area ratio, each of the R ² value of 0.999 to secure the quantitation.

In the above analysis conditions, ID2244 was detected at 12.41 min and ID2252 at 12.17 min, and the two samples were separated in the extract sample and used for quantification (see FIG. 19).

As a result, it was confirmed that the active ingredient ID2244 and ID2252 contained 74 and 94mg, respectively, in 5,000mg of isobutyl acetate acetate for use in obesity efficacy test.

<317>

<318> <10-2> Model of obesity-induced mouse by high fat diet

The test was conducted on 8 week-old C57BL / 6 mice with dietary supplements containing 5000 mg of isogulet acetylacetate extract per kg feed containing 40% fat, measuring weight changes for 8 weeks, and various obesity and associated symptoms after 8 weeks. Indicators were compared with those of the normal and obese diet groups. Anti-obesity sibutramine (sibutramine (HC1 · ¾0)) was used as a positive control group fed the diet supplemented with lOOmg per kg obese feed. In the high-fat diet containing more than 40% fat, sibutramine was mixed at 0.0% and isophore ethyl acetate extract at 0.5%, respectively, and stored for a long time before use. The composition of the feed is shown in Table 7. .

<321>

All animal procedures were performed as in Example 8-1. Mouse groups were treated with ¾ semi-fed diet (ND), untreated drug-free high fat (HF), and ragweed ether acetate extract (kg). Party. Six groups were tested for each group, divided into a group containing 5000 mg (ID1209) and a feed group added with 100 mg of sibutramine (Sibutramine).

<323>

<324> <10-3> Determination of Changes in Weight and Feed Intake by Ethyl Extracts

Body weight was measured once a week and feed intake was measured twice a week.

<326>

As a result, the weight loss was 6% after 8 weeks compared to the group administered only obese diet. The sibutramine-treated group, a positive control group, decreased by 5%. There was no difference in feed intake between the groups (see Figure 20).

<328>

<329> <10-4> Cold test

To determine the cause of weight loss, a low temperature test (cold test) was performed, and the experiment was carried out in the same manner as in Example 8-3.

<331>

As the group was significantly overcome the decrease in body temperature in the obese group, etc., the effect of suppressing weight gain by the administration of the extract was influenced by the increased heat dissipation through active body metabolism. Fig. 21

<333>

<334> <10-5> Determination of Changes in Sugar Tolerance by Extract of Ethyl Gule Extract

Changes in glucose tolerance due to insulin resistance were carried out in the same manner as in Example 8-4.

<336>

As a result, after 8 weeks, the area under curves (AUC) of the Baman's group and the Dong'le extract were reduced by 30% compared to the obese group's group. It was found that glucose tolerance was improved. (ρ <0 · 01) (see FIG. 22).

Example 11

Establishment of Optimal Extraction Conditions of Toggle Extracts To develop a method of increasing the content of active ingredients such as homoisoflavanone ID2244 and ID2252 of the present invention in extracts of the present invention, it is easy to be fair in mass production and lower the production cost. Optimal extraction conditions were tested.

The method for obtaining isotle ethyl acetate extract is the same as that in Example 3. After finely crushing 10 g of the rake, 30 ml of 100% methanol was immersed, and the extraction solution was repeatedly extracted three times for 24 hours at room temperature with a strainer and methane was removed by a depressurizer to obtain a methanol extract. To the methanol extract obtained by the above process, 20 ml of ethyl acetate and water were added to the methanol extract, respectively, and the mixture was mixed, and the upper layer was concentrated to obtain an ethyl acetate extract (EtOAc).

The extraction method using the single solvent used in the present Example is as follows. 100 g of methanol, ethanol, acetone, ethyl acetate and methylene chloride were immersed in 30 ml of finely crushed 10 grams each, and the extraction solution extracted three times for 24 hours was filtered through a filtering device, and each solvent was removed using a decompression device. ^' Were removed to obtain each solvent extract. Each extract obtained in the above process was washed twice with ethanol to remove residual solvent, concentrated under reduced pressure and dissolved in DMS0 at a certain concentration to test whether the SIRT1 activity by the method of Example 1. -As a result, when methanol or ethanol is used as a single solvent, the yield of the extract is higher than that of the conventional extraction method, but the content of the active ingredient in the extract is too low, 0.1-0.4%, whereas acetone, ethyl acetate and methylene chloride are used as the single solvent. The process was used to confirm that the content of the active ingredient is increased to about 4-5%. Although the pharmaceutical compositions and formulation examples of the present invention will be described, the present invention is not intended to be limited thereto but merely to be described in detail.

Preparation Example 1 Preparation of Powder

Tung Acetyl Acetate Extract 20 mg _.

Lactose 100 rag <352> talc 10 mg

The above ingredients are mixed and layered in an airtight cloth to prepare a powder.

<354>

Preparation Example 2 Preparation of Tablet

<356> 10 mg of isocol ethyl acetate extract,

<357> corn starch 100 mg

<358> Lactose 100 mg

<359> Magnesium Stearate 2 mg

The tablets are prepared by mixing the above components and then tableting according to a conventional method for preparing tablets.

<361>

<362> <Preparation Example 3> Preparation of the capsule agent 〜

10 mg of homoisoflavanone represented by the formula (1)

<364> Crystalline Cellulose 3 mg

<365> Lactose 14.8 mg

<366> 0.2 mg magnesium stearate

<367> The capsules are prepared by mixing the above ingredients according to a conventional capsule preparation method and filling the gelatine capsules.

<368>

Preparation Example 4 Preparation of Injection

Homoisoflavanone 1 rag represented by Formula 2

<37i> Mantle 180 mg

Sterile distilled water for injection 2793 mg

<373> Na ₂ HP0 ₄ 12H ₂ 0 26 mg

According to the conventional method of preparing an injectable drug, the above-mentioned ingredient is prepared per ampoule (2 ml).

<375>

Preparation Example 5 Preparation of Liquid

<377> Homoisoflavanone 2 rag represented by the formula (1)

<378> 10 g of isomerized sugar

<379> 5 g of Manni

<380> amount of purified water After dissolving each component in purified water according to a conventional method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to purified water to a brown bottle. Fill and sterilize to prepare a liquid.

Industrial Applicability

As described above, the present invention provides a pharmaceutical composition for preventing and treating obesity, obesity complications or metabolic syndrome containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of the present invention inhibits adipocyte differentiation and maintains SIRT1 at a high level to reduce body weight, abdominal fat and glucose tolerance, and thus can be usefully used for preventing and treating obesity, obesity complications or metabolic syndrome. This is high. ^.

Claims

[Scope of Claim]

[Claim 1]

A pharmaceutical composition for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome containing homoisoflavanone or a pharmaceutically acceptable salt thereof as an active ingredient represented by the following formula (1) or (2) .

[Claim 2]

Pharmaceutics for the prevention and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome comprising an extract extracted from a backbone with at least one solvent selected from the group consisting of acetone, ethyl acetate and methylene chloride Composition. -

[Claim 3]

At least one disease selected from the group consisting of obesity, obesity complications and metabolic syndrome, comprising administering an effective amount of homoisoflavanone or a pharmaceutically acceptable salt thereof represented by Formula 1 or Formula 2 to an individual in need thereof Prevention and treatment methods.

【Claim 4】.

Homosoflavanone represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof for the preparation of an agent for the prophylaxis and treatment of one or more diseases selected from the group consisting of obesity, obesity complications and metabolic syndrome.

[Claim 5]

A step of administering an effective amount of the extract extracted from the agglutinate with one or more solvents selected from the group consisting of acetone, ethyl acetate and methylene chloride to an individual in need thereof. A method for preventing and treating at least one disease selected from the group consisting of obesity, obesity complications and metabolic syndrome.

[Claim 6]

One or more diseases selected from the group consisting of obesity, obesity ^'s complications and metabolic syndrome and acetone, ethyl acetate for the manufacture of prophylactic and therapeutic agents. And extracts extracted with one or more solvents selected from the group consisting of methylene chloride.