KR20070008228A - Composition for the prevention and treatment of obesity and type 2 diabetes comprising a tussilago farfara extract or tussilagone - Google Patents

Abstract

A composition comprising a Tussilago farfara extract or a tussilagone is provided to be able to effectively inhibit acyl CoA:diacyl glycerol acyl transferase, thereby being effectively used for treating obesity and type 2 diabetes. The composition having acyl CoA:diacyl glycerol acyl transferase inhibition activity comprises a Tussilago farfara extract or a tussilagone represented by the formula(1) or a pharmaceutically acceptable salt thereof, wherein the extracts is extracted by water, organic solvent such as ethanol and chloroform, or mixture thereof. The composition has an activity of preventing or treating obesity or type 2 diabetes.

Description

Composition for the prevention and treatment of obesity and type 2 diabetes comprising a Tussilago farfara extract or Tussilagone}

    1 is a result of measuring the concentration of acyl coay: diacylglycerol acyl transferase enzyme activity inhibitory effect of the compound represented by the formula (1).

The present invention relates to a composition having an acyl coA: diacylglycerol acyltransferase (DGAT) inhibitory activity, including a Tussilago farfara extract or a tussilagone or a pharmaceutically acceptable salt thereof. More specifically, the composition relates to a composition having a prophylactic and therapeutic effect on obesity and type 2 diabetes.

Acyl CoA: diacylglycerol acyltransferase (abbreviated as DGAT) is an enzyme that catalyzes the final process of the glycerol 3-phosphate pathway. ( sn -1,2-diacylglycerol) and Fetyacyl CoA (Fatty acyl CoA) is used as a substrate to synthesize triglycerides. Biosynthesis of triglycerides is via the glycerol 3-phosphate pathway (such as liver and adipose tissue) and the monoacylglycerol pathway (intestinal epithelial cells of the small intestine).

Recently, selective inhibition of DGAT, an enzyme that catalyzes the process of triglyceride biosynthesis in liver, adipose tissue, skeletal muscle, etc., has emerged as effective in the prevention and treatment of obesity and type 2 diabetes (Chen HC, et al ., Trends Cardiovasc . Med, 10, p.188-192, 2000 ;....... Farese Jr. et al, Curr Opin Lipidol, 11, p.229-234, 2000, A. Subauste et al, Currunt Drug Target- Immun , Endocrine & Metabol Disorders 3, p. 263-270, 2003, Y. Yu et. Al. Anals of Medicine 36. p.252-261, Hubert C. et al., Arterioscler. Thromb . Vasc . Biol . 25 , p.1-5, 2005), a team led by Dr. Robert V. Farese Jr. of the Gladstone Heart Disease Research Center in the United States, studied diets using mice that knocked out the DGAT gene. by providing evidence that obesity has been effectively suppressed and that sensitivity to insulin and leptin has been increased to improve glucose metabolism. Selective DGAT inhibitors have been shown to be useful in the treatment of obesity and type 2 diabetes (Smith SJ. Et al., Nature genetics, 25, 87-90, 2000).

To date, synthetic compounds of DGAT enzyme inhibitors are known as pyrrolecarboxylic acid derivatives (JP05213985A, Mitsubishi Kasei Corp, Japan) and phosphonic acid ester derivatives (JP2004067635A, Otsuka Pharmaceut Factory INC, Japan). The researches to be developed were first reported by the team (Korea Research Institute of Biotechnology and Geological Metabolism Laboratory) for the first time to find new substances with anti-obesity activity from ginseng (Korean Patent Application No. 2001-75636, Lee et al. Planta Med. 70, p. 179-200, 2004), reported quinolones alkaloids, tanshinones, and flavonoids (Prenylflavonoids) isolated from herbal resources such as sesame oil, salvia, and ginseng. Korean Patent Application No. 2002-64744, Ko et al. Arch.Phar.Res. 25, p. 446-448, 2002, Korean Patent Application No. 2003-00524). In foreign countries, as an inhibitor reported by the Omura group of Kitasato Institute of Japan, roselipins [ Gliocladium roseum KF-1040, IC ₅₀ ; 15-22 μM, US6432682 (2002), US6608185 (2003), cochlioquinone A and A1, L. Antibiot. 56, 967 (2003), 57, 59 (2004)], amidepsines [ Humicola sp. FO-2942, IC ₅₀ ; 10-50 μM] and xanthohumols [ Humulus lupulus , IC ₅₀ ; 50-194 μM,], other eicosapentaenoic acid, 2-bromooctanoate, etc. have been reported (Rustan et al .; J. Lipid. Res. , 29, p. 1417-1426, 1988).

In addition to the conventional DGAT enzyme inhibitors described above, as part of a study to obtain a substance having excellent DGAT inhibitory activity from natural products, the present inventors search for an active substance that inhibits acyl coay: diacylglycerol acyltransferase. In the process, it was confirmed that the extract of Kantoin and sesquiterpenoid compound Tucilagone isolated therefrom have excellent inhibitory activity against DGAT, and the Kanto Extract and Tucilagone for the prevention and treatment of obesity and type 2 diabetes The present invention has been completed by revealing what can be used.

It is an object of the present invention to provide a composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity comprising a Kanto extract.

It is still another object of the present invention to provide a composition having an acyl coei: diacylglycerol acyltransferase inhibitory activity comprising a tussilagone or a pharmaceutically acceptable salt thereof.

      In one aspect, the present invention relates to a composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity comprising a kantoin extract.

As used herein, the term "extract" refers to an active ingredient isolated from natural products, wherein the substance is isolated from Kantoin and has acyl coay: diacylglycerol acyltransferase inhibitory activity. The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes an extract, a dry powder thereof, or any form formulated using the same.

As used herein, the term “Tussilago farfara” is meant to include all organs of natural, hybrid or variegated mandarins, eg, roots, stems, leaves, flowers, but preferably buds of mandarins. Kwandonghwa is a perennial herb belonging to the chrysanthemum family. In oriental medicine, buds are called Kwandonghwa, which has the effect of strengthening the lungs and coughing and sputum, and the lungs are weak and coughing, sputum, and sometimes blood is mixed. It is known to be used when there is seawater and asthma due to symptoms or colds. Its ingredients are known to contain sterols, fullavonoids and tannin compounds (Kim Chang-min et al., Medicinal Metabolism Dictionary, Vol. 1, pp 439-444, 1997, Jungdam, Seoul). Japanese Patent JP2002255803 discloses a composition containing kantoon extract, used for cosmetics, food, etc., which discloses Kantoin, in particular its activity as a phototoxic inhibitor of leaf extract. Japanese Patent Application Nos. JP2004196731, JP2003342122, JP2003277226, JP2003261417 and the like describe cosmetic compositions containing Kanto extract, external skin preparations and the like. However, prior to the present invention, the inhibitory activity of the kantoin extract against acyl coay: diacylglycerol acyltransferase was not examined.

As used herein, the term “acylcoa: diacylglycerol acyltransferase (DGAT) inhibition” refers to a phenomenon in which the enzyme-catalyzed reaction for synthesizing triglycerides by DGAT is blocked or the synthesis efficiency is reduced. In the case of inhibiting the enzyme activity, various reactions may be induced in cells. Specific examples of reactions induced by inhibiting DGAT enzyme activity are the prevention and treatment of type 2 diabetes through the inhibition of obesity and the suppression of insulin privatity.

Inhibiting the biosynthesis of triglycerides by inhibiting DGAT, a final enzyme involved in triglyceride synthesis, inhibits the accumulation of fat in adipose tissue, decreases the size of fat cells and promotes the release of ediffonectin from fat cells. It has been studied as a target for the treatment of obesity, as it has been found that the increase in exercise volume and weight gain induced by high fat diet are suppressed. In addition, DGAT inhibition is known to improve insulin resistance by preventing fat accumulation in non-adipoise tissues such as muscle, liver and pancreas. When the cells are stimulated by insulin, serine inhibitory phosphorylation of insulin receptor substance-1 (IRS-1) decreases, resulting in phosphatidylinositol-3 kinase (PI-3K), protein kinase B (PKB), and PKC _λ ( Through signal transduction through protein kinase C _λ ), GLUT-4 (glucose transporter-4) is exocytosis into the membrane and glucose is introduced into the cell. Inhibition of DGAT activity in cells results in an increase in PI-3K, PKB and PKC _λ activity resulting in an increase in the number of GLUT-4 exocytosed into the membrane, resulting in an increase in the number of glucose finally introduced into the cell. . In other words, inhibition of DGAT activity increases insulin sensitivity. Chen et al., Arterioscler Thromb Vasc Biol. 25 (3): 482-486, 2005; Chen et al., J Clin Invest. 111 (11): 1715-22, 2003; Chen et al., J Clin Invest 109 (8): 1049-1055, 2002; Chen et al., Diabetes. 51 (11): 3189-3195, 2002; Subauste and burant., Curr Drug Targets Immune Endocr Metabol Disord. 3 (4): 263- 270, 2003). As the direct relationship between DGAT inhibition and insulin resistance is revealed, it can be seen that DGAT inhibition acts as a therapeutic target for type 2 diabetes, in which glucose uptake is impaired while insulin secretion is normal or insulin resistant.

In a specific embodiment of the present invention, the ethanol extract of Kantoin inhibited the activity of DGAT by 59.3% at a concentration of 100ug / ml, and inhibited the chloroform fraction of the ethanol extract by 76.7%. The above results demonstrate that the Kanto extract of the present invention has excellent DGAT inhibitory activity, and further has activity for treating obesity and type 2 diabetes.

Thus, as a preferred embodiment, the present invention provides a composition having prophylactic and therapeutic activity against obesity and type 2 diabetes.

As used herein, the term “obesity” refers to a condition or disease with excessive body fat due to energy imbalance. By administering the composition of the present invention, weight can be reduced and cholesterol in the blood , Triglycerides, low density lipoprotein (LDL) -cholesterol, etc. are reduced.

As used herein, the term “type 2 diabetes” refers to a disease or condition in which hyperglycemia is caused by failure to maintain proper blood glucose levels due to metabolic disorders in the production and use of glucose. . Diabetes is divided into type 1 diabetes, which is caused by insulin deficiency that controls glucose utilization, and type 2 diabetes, which occurs because normal or cells do not respond properly to insulin. For the purposes of the present invention, diabetes relates to type 2 diabetes with insulin resistance, and by administering the composition of the present invention, it is possible to prevent and treat symptoms or complications caused by diabetes. Diabetes symptoms that can be prevented and treated with Kantoin extract include polydipsia, polyphagia, hyperinsulinemia, hyperglycemia, and complications include hypertension, kidney disease, vision disorders, periodontal disease, nerves Disorders, vascular disorders, diabetic necrosis, extensor infarction, and the like.

As used herein, the term "prevention" means any action that inhibits or delays the development of obesity and type 2 diabetes by administration of the composition. As used herein, the term "treatment" means any action that improves or advantageously alters the symptoms of obesity and type 2 diabetes by administration of the composition.

In the present invention, the Kantoin extract may be extracted by using water, an organic solvent, or a mixed solvent thereof. Preferably it is extracted using an organic solvent. The extracted solution can be used directly or can be concentrated and / or dried.

When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof may be used and extracted by room temperature or warming under conditions where the active ingredient of the herbal medicine is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may vary, so select an appropriate organic solvent. The extraction method is not particularly limited, and examples thereof include cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like.

Filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon or the like, or may be used, such as ultrafiltration, cryofiltration, centrifugal separation, but is not limited thereto.

Concentration of the extract may be used, such as concentrated under reduced pressure, reverse osmosis concentration. The drying step after concentration includes freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. If desired, a process of grinding the final dried extract may be added.

In addition, the extract can perform an additional fractionation process. Preferably, the extract is suspended in distilled water to obtain a nonpolar solvent soluble layer by extraction and separation with a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. It can be used by concentrating and / or drying it.

In one specific embodiment, the Kwandonghwa extract of the present invention is chopped dried Kwanhwa, three to 20 times the weight (kg), preferably 5 to 15 times the water, C ₁ to C ₄ lower alcohol or Mixed solvents thereof, preferably ethanol solvents; At 20 to 70 ° C., preferably at 20 to 30 ° C. extraction temperature; 1 hour to 10 days, preferably 3 days to 8 days; By chilling, ultrasonic extraction or reflux cooling extraction method; The extract obtained by extracting 1 to 5 times, preferably 1 to 3 times, was obtained by filtration, concentration under reduced pressure, and drying. In addition, the extract was suspended in distilled water, and then extracted 1 to 5 times with chloroform and concentrated under reduced pressure to obtain a chloroform fraction with high DGAT inhibitory activity.

In still another aspect, the present invention relates to a composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity comprising a tusylagone represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

In order to identify the active ingredient exhibiting DGAT inhibitory activity in the extract, the present inventors fractionate the soluble extract of Kantoin non-polar solvent by performing chromatography, followed by gel filtration chromatography and high performance liquid chromatography. Analysis of light spectrum, electron shock mass spectrometry, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum and the like. As a result, it was found that the active ingredient having DGAT activity was tusilagon, a sesquiterpenoid compound of Formula 1, and published in S. Hwang et al. Eur. J. Pharm. 141, 269, 1987, F. Bohlmann, et al. Phytochemistry, 20, 2024, 1981). Tucilagone is known to be an antagonist of platelet activating factor (Hwang SB et al. Eur. J. Pharmacol. 141 (2): 269-281, 1987), but prior to the present invention the DGAT inhibitory activity of tucilagone It is not known about.

In the specific embodiment of the present invention, the purely isolated tucilagone of Formula 1 had higher DGAT inhibitory activity as the concentration was increased, and when treated at a concentration of 50 µg / ml, the DGAT activity was suppressed to 96.2%, IC ₅₀ Was found to be 4.2 μg / ml. The results demonstrate that tucilagone has excellent DGAT inhibitory activity and has therapeutic activity for obesity and type 2 diabetes.

Thus, as a preferred embodiment, the present invention provides a composition comprising tucilagone or a pharmaceutically acceptable salt thereof having prophylactic or therapeutic activity against obesity and type 2 diabetes.

In the present invention, the term “pharmaceutically acceptable salts” means salts derived from pharmacologically or physiologically acceptable inorganic acids, organic acids and bases. Examples of suitable acid include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, Formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium and the like.

Tucilagones of Formula 1 may be isolated from natural substances, preferably from plants. Various organs, roots, stems, leaves, flowers, as well as plant tissue cultures of natural, hybrid, and mutant plants can be extracted and isolated. In addition, chemical synthesis is possible by methods known in the art.

The method of obtaining tucilagone from a plant is by various well-known extraction methods. For example, the plant is ground and then extracted with a lower alcohol solvent such as methanol, ethanol, and the like, and then suspended in distilled water, for example, in a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethylacetate. It can be obtained by extracting and separating the nonpolar solvent soluble layer with a tide or a mixed solvent thereof. From the obtained nonpolar organic solvent fraction, the tusilagon of formula (1) can be obtained. The extract can be further purified by performing one or more chromatographic chromatography, in which the type of column and the developing solvent can be controlled in various ways (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed.pp. 6-7, 1998).

In one specific embodiment, the inventors have extracted the buds of mandarins with ethanol and their distilled water suspensions are 1 to 10 times, preferably 1 to 5 times the volume of chloroform 1 to 10 times, preferably 2 times to The chloroform fraction obtained by extracting 5 times was subjected to adsorption chromatography, gel filtration chromatography, high-performance liquid chromatography, and the like, by silica gel column chromatography, and pure tucilagone was separated.

As described above, since the Kantoin extract or Tucilagone of Formula 1 has excellent DGAT inhibitory activity, it can be seen that it has excellent prophylactic and therapeutic activity against obesity and type 2 diabetes. Since the above-mentioned Kantoin extract and Tucilagone of Formula 1 contain ingredients obtained from natural extracts as active ingredients, they are safe and do not cause toxicity, side effects and resistance, and thus have long-term intake. Indeed, mouse acute toxicity experiments showed that when administered intravenously, 28.9 mg / kg LD ₅₀ did not cause toxicity (Li YP Gen. Pharmacol. 19 (2): 261-3, 1988). In addition, the composition can be used not only in humans, but also in cattle, horses, sheep, pigs, goats, camels, antelopes, dogs and the like, which can cause obesity and diabetes.

The pharmaceutical composition for preventing and treating obesity and type 2 diabetes disease of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition. In addition, the composition does not increase the efficacy, but may include additional ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the composition is inorganic, organic, such as vitamins B ₁ , B ₂ , B ₆ , C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, mixtures thereof Additives may further be included. In addition, the composition may include a substance having a therapeutic activity against obesity and diabetes alone or used previously.

The composition may comprise a pharmaceutically acceptable carrier and be formulated for human or veterinary use in oral or parenteral use.

When formulating the composition of the present invention, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate ( Calcium carbonate, sucrose or lactose, and gelatin are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, styrate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and the like. As the non-aqueous solvent and suspending solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used.

Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

In another aspect, the present invention relates to a method for preventing and treating obesity and type 2 diabetes by administering to a patient a composition comprising a organoderma extract or tucilagone of formula (1).

As used herein, the term "patient" refers to humans, horses, sheep, and pigs having obesity and diseases caused by type 2 diabetes and its direct and indirect causes, and whose symptoms may be improved by administering the composition of the present invention. Means animals such as goats, camels, antelopes and dogs. By administering to the patient a composition comprising the Kantoin extract of the present invention or the tucilagone of the formula (1), it is possible to effectively prevent and treat the aforementioned type 2 diabetes. The composition of the present invention can be administered in parallel with existing diabetes therapeutics.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as the target tissue can be reached. May be administered. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to a patient's sexually transmitted disease, age, severity, and drug activity. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The method of administering the composition containing the extract or compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. In general, when the effective dose is oral administration, it is usually 1 to 1 time per adult 500 mg / kg is preferred, and in the case of intravenous administration, 1 to 100 mg / kg is preferred. Dosage levels for a particular patient may vary depending on gender, age, health condition, diet, time of administration, method of administration, drug mixture and severity of disease.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the following examples.

Reference Example . Analyzer

The UV spectrophotometric (UV) instrument used in the present invention used Shimadzu UV-260 spectrophotometer (Shimadzu Co., Ltd.), and the hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) instrument was a barley. An Unity 300 NMR (Varian UNITY 300 NMR, Varian) was used, and the electron shock mass spectrometer (EI-MS) was investigated using Hewlettpackard 5989A (Hewlettpackard 5989A, Hewlettpackard).

Example  One. Kanto Crude extract  Produce

3 kg of the Kwandonghwa purchased from Daejeon medicinal herbs were chopped, immersed in 30 l of ethanol, left to stand at room temperature for 7 days, filtered, and the extract was concentrated under reduced pressure and dried to give 625 g of the total extract.

Example  2. Kanto  Preparation of Chloroform Soluble Extract

625 g of the total extract was suspended in 2 L of distilled water, followed by extraction three times by adding 10 L of chloroform, thereby obtaining a crorolform fraction having a high diacylglycerol acyltransferase inhibitory activity. The chloroform fractions obtained above were concentrated under reduced pressure to give 154 g of a chloroform soluble part.

Example  3. Kanto  Isolation of Chemical Formula 1 from Chloroform Soluble Extracts

154 g of the chloroform soluble part of Example 2 was adsorbed onto 700 g of silica gel (230-400 mesh, Art. 9385, Merck) and silica gel column chromatography (10 × 80 cm) was performed. At this time, using a normal hexane: ethyl acetate mixed solvent (15: 1) as the developing solvent to increase the amount of ethyl acetate to increase the polarity to the normal hexane: ethyl acetate mixed solution (1: 1), it was carried out at 500ml per hour The diacylglycerol acyltransferase inhibitory substance was eluted and divided into seven fractions. The enzyme inhibitory activity of these fractions was measured, and the active fraction was purified by secondary silica gel chromatography and gel filtration chromatography using fraction 5 with high inhibitory activity. Finally, 1 g of pure tucilagon was purified by preparative high performance liquid chromatography. Got it. In this case, YMC-Pack SIL (250 × 20 mm I.D., S-5um) was used as a column, and hexane: acetone (10: 1) was used as a solvent.

Example  4. Chemical formula 1 to  Physicochemical Properties of Compounds Displayed

       The physicochemical properties of Formula 1 isolated from Kantoin are as follows.

Compound of Formula 1

(1) Property of material: white powder

(2) Molecular formula and molecular weight of substance: C ₂₃ H ₃₄ O ₅ , 390

(3) UV spectrophotometric spectrum [λ _max (nm) MeOH]: 223

(4) ESI-mass spectrometry: m / z = 413 [M + Na] ⁺

(5) Hydrogen nuclear magnetic resonance spectrum [300 MHz, CDCl ₃ , δ (ppm)]: 0.78 (d, J = 6.6), 0.98 (d, J = 6.6 Hz), 1.07 (t, J = 7.2 Hz), 1.22 (d, J = 6.6 Hz), 1.46 (m), 2.10 (s), 2.14, 2.15 (d, J = 1.2 Hz), 2.18, 2.31 (m), 2.37 (dd, J = 4.8, 16.5 Hz), 2.50 (dd, J = 3.3, 10.6), 2.59 (m), 4.79 (s), 5.14 (s), 5.58 (br.t, J = 3 Hz), 5.63 (d, 1.1 Hz)

(6) Carbon nuclear magnetic resonance spectrum [300 MHz, CDCl ₃ , δ (ppm)]: 42.6 (C-1), 214.8 (C-2), 57.2 (C-3), 49.1 (C-4), 43.9 ( C-5), 31.2 (C-6), 73.3 (C-7), 146.1 (C-8), 42.3 (C-9), 110.1 (C-10), 27.6 (C-11), 21.6 (C -12), 15.4 (C-13), 69.6 (C-14), 15.2 (C-15), 21.4 ( C H ₃ CO), 170.9 (CH ₃ C O), 166.0 (C-1 '), 114.6 (C-2 '), 162.0 (C-3'), 33.8 (C-4 '), 11.9 (C-5'), 18.9 (C-6 ')

Example  5. Acyl Koei : Diacylglycerol Acyl transferase  Active black

5-1. Acyl Koei : Diacylglycerol Acyl transferase  Active measurement

In order to measure the activity of acyl coay: diacylglycerol acyltransferase, the inventors have carried out microsomal protein isolated from rats according to the method of Coleman et al. (Coleman et al, Methods Enzymol ., Pp98-103, 1992). by using, as a substrate 1, 2-diacylglycerol (Sigma Co., D0138) and ^[14 C] palmitoyl-after the enzyme reaction using Koei (Amersham Inc., CFA583) produced ^[14 C] triacylglycerol The amount of radioactivity of was measured. Specifically, reaction solution (175 mM Tris-HCl, pH 8.0), 20 μl of bovine serum albumin (10 mg / ml), 100 mM MgCl ₂ , 30 μM of [ ¹⁴ C] palmitoyl coei (0.02 μCi, 10.0 μl of the sample solution dissolved in dimethyl sulfoxide (DMSO) was added to Amersham) and 200 μM 1,2-dioleoyl glycerol), and 100 to 200 μg of the isolated microsomal protein was added and reacted at 25 ° C. for 10 minutes. Thereafter, 1.5 ml of reaction terminator (2-propanol / heptane / water = 80/20/2, v / v / v) was added to stop the reaction. In order to separate [ ¹⁴ C] triacylglycerol produced by the reaction, 1 ml of heptane and 0.5 ml of distilled water were added thereto, followed by shaking. Then, 1 ml of supernatant was added thereto and 2 ml of alkaline ethanol solution (ethanol / 0.5 N sodium hydroxide) was added. / Water = 50/10/40, v / v / v) and shaken. 0.65 ml of the supernatant of the shake was taken and the amount of radioactivity was measured by a liquid scintillation counter (LSC). The inhibitory activity of diacylglycerol acyltransferase was calculated by the following equation.

T: cpm value of the test sphere in which the sample was added to the enzyme reaction solution,

C: cpm value of the control without the sample in the enzyme reaction solution and

B: cpm value of the control in which the sample was added without adding the enzyme source.

5-2. Of the extract of Kantoin and the compound represented by DGAT  Inhibitory activity measurement

According to the method for measuring diacylglycerol acyltransferase activity of 5-1, diacylglycerol was added by adding ethanol extract of Example 1, chloroform fraction and water fraction of Example 2 at concentrations of 100 and 50 ug / ml, respectively. Acyltransferase enzyme inhibitory activity was measured and the results are shown in Table 1.

According to the method for measuring diacylglycerol acyltransferase activity of 5-1, diacylglycerol was added by adding ethanol extract of Example 1, chloroform fraction and water fraction of Example 2 at concentrations of 100 and 50 ug / ml, respectively. Acyltransferase enzyme inhibitory activity was measured and the results are shown in Table 1.

Solvent fractionation of the ethanol extract showed that the diacylglycerol acyltransferase inhibitory active material was transferred to the chloroform fraction.

Column chromatography was performed to separate and purify the active substance from this fraction, and the compound of formula (I), a purified active substance, isolated by diacylglycerol acyltransferase inhibitor, was added for each concentration of tusilagon, and DGAT inhibitory activity and IC ₅₀ was measured and the results are shown in Table 2.

As described above, the Tungguan extract of the present invention and the sesquiterpenoid compound of the present invention, the silylagon, effectively inhibit acylcoay: diacylglycerol acyltransferase to prevent and treat obesity and type 2 diabetes. Can be used effectively.

Claims (6)

A composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity, comprising an extract of Kwanshu (Tussilago farfara). The composition of claim 1 having prophylactic or therapeutic activity against obesity or type 2 diabetes. The composition according to claim 1, which is extracted using water, an organic solvent or a mixed solvent thereof. The composition of claim 3, which is extracted using ethanol and chloroform. A composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity, including cedrol or a pharmaceutically acceptable salt thereof. Formula 1

A composition according to claim 5 having prophylactic or therapeutic activity against obesity or type 2 diabetes.

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