KR100668172B1 - Composition for the prevention and the treatment of obesity and type 2 diabetes comprising the extract of Alnus hirsuta or betulinic acid - Google Patents

Abstract

The present invention is directed to a composition having an acyl coA: diacylglycerol acyltransferase (DGAT) inhibitory activity, including an Alnus hirsuta S. extract or betulinic acid. In particular, the composition has a prophylactic and therapeutic effect, in particular for obesity and type 2 diabetes.

Teal, Betulinic Acid, Acyl Coai: Diacylglycerol Acyl Transferase, Obesity, Type 2 Diabetes

Description

Composition for the prevention and the treatment of obesity and type 2 diabetes comprising the extract of Alnus hirsuta or betulinic acid}

1 is a graph showing the results of measuring the activity inhibitory effect of the diacylglycerol acyltransferase enzyme of the compound of Formula 1 by concentration.

The present invention is directed to a composition having an acyl coA: diacylglycerol acyltransferase (DGAT) inhibitory activity, including an Alnus hirsuta S. extract or betulinic acid. The present invention relates to compositions 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 Fatty acyl CoA are used as substrates to synthesize triglyceride 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 catalyzing triglyceride biosynthesis in liver, adipose tissue and skeletal muscle, has emerged to be effective in the prevention and treatment of obesity and type 2 diabetes (Chen HC, et al., Trends Cardiovasc Med., 10, 188-192, 2000; Farese Jr. et al., Curr.Opin. Lipidol., 11, 229-234, 2000; A. Subauste et. Al., Currunt Drug Target-Immun, Endocrine & Metabol Disorders 3, p. 263-270, 2003; Y. Yu et. Al. Ann of Medicine 36, 252-261; Hubert C. et al., Arterioscler. Thromb. Vasc. Biol, 25, 1-5, 2005 ), Robert V. Farese Jr., Gladstone Institute for Heart Disease. The team used mice that knocked out the DGAT gene to effectively suppress diet-induced obesity induced by high-fat diets and increased susceptibility to insulin and leptin. Evidence of improved glucose metabolism has shown that selective DGAT inhibitors are useful in the treatment of obesity and type 2 diabetes (Smith SJ. Et al. Nature genetics, 25, 87-90, 2000). In addition, due to the polymorphism of the DGAT promoter, the population with low DGAT expression was reported to have a low risk of cardiovascular diseases such as lean, high HDL-cholesterol, and low diastolic blood pressure. Therefore, the search for selective inhibitors of DGAT enzymes is expected to contribute to the creation of a leading substance that can effectively prevent or cure obesity and type 2 diabetes diseases.

To date, synthetic products 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 research to be developed was first reported by the team (Korea Research Institute of Bioscience and Biotechnology, 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, 179-200, 2004), reported quinolones alkaloids, tanshinones, and flavonoids (Prenylflavonoids) substances isolated from the herbal resources, sorghum, salvia, and ginseng (Korean patent) Application 2002-64744, Ko et al. Arch.Phar.Res. 25, p. 446-448, 2002, Korean Patent Application No. 2003-524). In foreign countries, as an inhibitor reported by the Omura group of the Kitasato Institute of Japan, roselipins (Gliocladium roseum KF-1040, IC50; 15-22 μM, US6432682 (2002), US6608185 (2003), Coclioquinone A and Aone [Lee et al., J Antibiot (Tokyo). 56, 967-969, 2003; Yoganathan et al., J Antibiot (Tokyo). 57, 59-63, 2004], amidepsines [Humicola sp. FO-2942, IC 50; 10-50 μM] and xanthohumols [Humulus lupulus, IC50; 50-194 μM], other eicosapentaenoic acid, 2-bromooctanoate, etc. have been reported (Rustan et al .; J. Lipid. Res. 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. It was confirmed that the extract of Tealwood and betulinic acid, which is a triterpenoid compound isolated therefrom, has excellent inhibitory activity against DGAT, and the Tealwood extract and betulinic acid are obesity and type 2 diabetes mellitus. The present invention has been completed by revealing that it can be used for the prevention and treatment of the.

One object of the present invention to provide a composition having an acyl coay: diacylglycerol acyl transferase inhibitory activity comprising a teal extract.

Still another object of the present invention is to provide a composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity comprising betulinic acid.

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

As used herein, the term “extract” refers to an active ingredient separated from natural products, and here the substance is separated from a teal and has acyl coay: diacylglycerol acyltransferase inhibitory activity. It can be obtained by an extraction process using a 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 “ Alnus hirsuta S. ” means all organs of natural, hybrid or varietal teal, including all roots, branches, stems, leaves, and flowers, but preferably Teal is a branch and bark of the teal, which is a tree belonging to the birch (Betulaceae), and is used for harvesting light branches, leaves, and bark in the spring and autumn, and drying them in the sun. It is used for the prevention of diarrhea, trauma, etc., and it is known to contain triterpene, sterol, and tannin compounds (Kim Chang-min et al., Medicinal Metabolism Dictionary, Vol. 8, pp4837-4838, 1997, Book Publishing Jungdam, Seoul). Japanese Patent Application Laid-Open No. 10-109920 describes a composition containing a teal extract having a testosterone-5-alpha-reductase activity inhibitory effect. Koei room: no bar looked inhibitory activity of teal tree extract for diacylglycerol acyltransferase.

As used herein, the term "acyl CoA: diacylglycerol acyltransferase (DGAT) inhibition" refers to a phenomenon in which DGAT blocks the enzymatic catalytic reaction for synthesizing triglycerides or decreases the synthesis efficiency. 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 weight gain inhibition and increased insulin sensitivity as described above.

Obesity is a disease caused by energy imbalance. When energy influx increases above energy release, excess calories are converted to triglycerides and stored in adipose tissue. Overexpressing DGAT that DGAT, the final enzyme involved in triglyceride synthesis, is directly related to obesity, increases the size of adipocytes or promotes energy expenditure in Dgat ^-/- mice. The size and fat size were decreased, the triglyceride level of tissue was decreased, and the body weight was decreased. In fact, transplantation of white adipose tissue (WAT) derived from Dgat ^{− / −} mice into wild type receptor mice imparts resistance to obesity, suggesting that DGAT inhibition acts as a target for treating obesity (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).

Diabetes mellitus is a disorder in which hyperglycemia is caused by failure of the body to maintain proper blood sugar levels. DGAT affects blood sugar levels. When the cells are stimulated by insulin, serine inhibitory phosphorylation of insulin receptor substance-1 (IRS-1) is reduced, resulting in phosphatidyl inositol-3 kinase (PI-3K), protein kinase B (PKB), and PKCλ ( Through signal transduction through protein kinase Cλ), GLUT-4 (glucose transporter-4) is exocytosis into the membrane and glucose is introduced into the cell. When the activity of DGAT is inhibited in cells, the activity of PI-3K, PKB and PKCλ is increased, and the number of GLUT-4 exocytosed into the membrane is increased, and finally the number of glucose introduced into the cell is increased. In other words, inhibition of DGAT activity increases insulin sensitivity. Indeed, transplantation of white adipose tissue (WAT) derived from Dgat ^{− / −} mice into wild type receptor mice has been shown to reduce insulin and glucose resistance (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 methanol extract of teal inhibited the activity of DGAT by 50.4% and the chloroform fraction of methanol extract by 67.3%. The above results demonstrate that the teal extract of the present invention has excellent DGAT inhibitory activity, and further has activity for treating obesity and type 2 diabetes.

Therefore, 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, neutral May reduce fat, high density lipoprotein (HDL) -cholesterol in the blood.

As used herein, the term “type 2 diabetes” refers to a condition or disease in which hyperglycemia is induced 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 Tealwood extract include polydipsia, polyphagia, hyperinsulinemia, hyperglycemia, and complications include hypertension, kidney disease, vision disorders, periodontal disease, Neurological 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.

Teal extract in the present invention can be used to extract 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.

The solvent may be water, an organic solvent, or a mixed solvent thereof. 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 suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon or the like, or ultrafiltration, cryofiltration, centrifugal separation, etc., but is not limited thereto. In addition, the method may further include a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity, etc.).

Drying the filtrate includes, but is not limited to, lyophilization, 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, an additional fractionation process may be performed on the extract. 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 teal extract of the present invention, by cutting the dried teal, about 3 to 20 times the weight (kg), preferably about 5 to 10 times the water, C ₁ to C ₄ Lower alcohols or mixed solvents thereof, preferably methanol, at 20 to 70 ° C., preferably at 20 to 30 ° C. for about 1 hour to 10 days, preferably about 3 to 8 days The extract obtained by reflux cooling extraction, preferably cold needle extraction, was filtered and concentrated under reduced pressure to obtain a teal extract soluble in water, lower alcohols and mixed solvents thereof. In addition, the extract was suspended in distilled water, and then extracted with chloroform 1 to 10 times 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 betulinic acid represented by the following general formula (1).

In order to identify the active ingredient exhibiting DGAT inhibitory activity in the teal extract, the inventors fractionate the teal non-polar solvent soluble extract by performing chromatography using reversed phase chromatography, and then obtain a compound obtained by performing reversed phase chromatography. Analysis of impact mass spectrometry, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum and the like were performed. As a result, it was found that the active ingredient having DGAT activity was betulinic acid, which is a triterpene-based compound of the lupine type of Chemical Formula 1, and published in Chen Peng. Computer-Assisted structure Elucidation: Magnetic resonance in chemistry. 36, 267-278, 1998]. Betulinic acid is known to have apoptosis-inducing activity in human melanoma (Pisha. E. et al, Nature Med. 1, 1046-1051, 1995), but DGAT inhibition of betulinic acid prior to the present invention. No activity is known.

In a specific embodiment of the present invention, the purely isolated betulinic acid of Formula 1 had higher DGAT inhibitory activity as the concentration was increased, and when treated at a concentration of 250 µg / ml, the DGAT activity was inhibited to 90.13%. IC50 was 4.5 μg / ml. The results demonstrate that betulinic acid has excellent DGAT inhibitory activity, and furthermore has obesity and type 2 diabetes therapeutic activity.

Accordingly, as a preferred embodiment, the present invention provides a composition comprising betulinic acid having prophylactic or therapeutic activity against obesity and type 2 diabetes.

Betulinic acid 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.

The method for obtaining betulinic acid from a plant is by various 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. The betulinic acid of formula 1 can be obtained from the obtained nonpolar organic solvent fraction. 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 present inventors extract the branches and bark of the teal with methanol and distilled water suspension thereof from 1 to 100 times, preferably 1 to 5 times the volume of chloroform 1 to 10 times, preferably 2 Chloroform fractions obtained by extraction 5 times were subjected to adsorption chromatography such as silica gel column chromatography, reversed phase chromatography such as Rp-18 (ODS) column chromatography, gel filtration chromatography, etc. An acid was obtained.

As described above, the Tealwood extract or betulinic acid of Formula 1 has excellent DGAT inhibitory activity, and thus it can be seen that it has excellent prophylactic and therapeutic activity against obesity and type 2 diabetes. Since the Tealwood extract and betulinic acid of Chemical 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 experiments have shown no toxicity. 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 type 2 diabetes.

The pharmaceutical composition 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 diabetes alone or used previously.

The composition comprises a pharmaceutically acceptable carrier and can be formulated for human or veterinary use.

Pharmaceutical compositions for oral administration may be presented as discrete units such as capsules or tablets; Powder or granules; Solvents, syrups or suspensions (in aqueous or non-aqueous liquids; edible blowing agents or whips; or emulsions).

That is, the composition comprising the extract or compound of the present invention can be administered in the form of oral and parenteral administration during actual clinical administration, when formulated, fillers, extenders, binders, wetting agents, disintegrating agents and It is prepared using diluents or excipients such as 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 are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. 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. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin 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.

As 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 teal extract or betulinic acid of formula (1).

As used herein, the term "patient" refers to humans, horses, sheep and pigs with diseases caused by obesity and type 2 diabetes and their direct and indirect causes, and whose symptoms may be improved by administration of the composition of the present invention. Means animals such as goats, camels, antelopes and dogs. By administering to the patient a composition comprising the teal extract of the present invention or betulinic acid of Formula 1, the above-mentioned type 2 diabetes can be effectively prevented and treated. 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. In consideration of all the above factors, 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. Specifically, the composition of the present invention is preferably oral administration or intravenous administration. In general, when the effective dose is oral administration, 1 to 500 mg / kg is preferred once per adult, 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 UV-260 spectrophotometer, Shimadzu Co., Ltd.), and the hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) instrument was Barry. 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 1: Preparation of the teal crude extract

Branches and 10kg of the Teal trees collected from Yasan, Chungnam, Korea were chopped and immersed in 100 liters of methanol, and left to stand at room temperature for 7 days to obtain an extract, and then extracted once more by adding 50 liters of methanol. After the respective extracts were filtered, the filtrates were mixed, concentrated under reduced pressure and dried to give 625 g of the total extract.

Example 2: Preparation of Teal Chloroform Soluble Extract

625 g of the total extract was suspended in 2 L of distilled water, and then extracted three times by adding 10 L of chloroform, thereby obtaining a chloroform fraction having a high acyl coco: diacylglycerol acyltransferase inhibitory activity. The obtained chloroform fractions were concentrated under reduced pressure to obtain 154 g of a chloroform soluble part.

Example 3: Isolation of Chemical Formula 1 from Teal Chloroform Soluble Extract

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, by using chloroform as an initial solvent, the amount of methanol was increased to increase the polarity until the chloroform: methanol mixing ratio was 1: 1, and the reaction was carried out at 500 ml per hour to inhibit acyl coa: diacylglycerol acyl transferase. The active substance was eluted and divided into six fractions. The enzyme inhibitory activity of these fractions was measured, and fractions 2 and 3 with high inhibitory activity were collected. 46 g of the collected fractions were subjected to secondary silica gel chromatography to obtain 23 g of highly active fractions, and the active fractions were purified by reverse phase chromatography using an ODS gel. At this time, the elution solvent was eluted in the order of 70%, 80%, 100% using distilled water / methanol (v / v), and the fraction with high enzyme inhibitory activity was obtained in 100% methanol eluate. Sephadex LH-20 (Sephadex LH-20) was then performed with chloroform: methanol (1: 1) to obtain 1 g of betulinic acid.

Example 4: Physicochemical Properties of Formula 1

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

Formula 1

(1) Property of material: white powder

(2) Molecular formula and molecular weight of substance: C ₃₀ H ₄₈ O ₃ , 456

(3) UV spectrophotometric spectrum [λ _max (nm) MeOH]: 210

(4) Electron impact mass spectrometry (70 eV): m / z (rel. Int) = 479 [M + Na] ⁺

(5) Hydrogen nuclear magnetic resonance spectrum [500 MHz, pyridine-d ₅ , δ (ppm)]: 0.998 (m), 1.675 (broad, d, J = 12.6 Hz), 1.880 (m), 3.482 (t, J = 7.8 Hz), 0.838 (m), 1.571 (m), 1.391 (m), 1.473 (m), 1.335 (m), 1.410 (m), 1.452 (m), 1.228 (m), 1.220 (m), 1.959 (m), 2.766 (m), 1.270 (m), 1.894 (m), 1.551 (m), 2.644 (m), 1.787 (t, J = 11.4 Hz), 3.574 (m), 1.535 (m), 2.270 (m), 1.595 (m), 2.282 (m), 1.250 (s), 1.034 (s), 0.849 (s), 1.085 (s), 1.095 (s), 4.972 (s), 4.795 (s), 1.817 (s)

(6) Carbon nuclear magnetic resonance spectrum [500 MHz, pyridine-d ₆ , δ (ppm)]: 15.244 (q, C-27), 16.774 (q, C-26), 16.774 (q, C-25), 16.693 (q, C-24), 19.129 (t, C-6), 19.817 (q, C-30), 21.549 (t, C-11), 26.461 (t, C-12), 28.654 (t, C- 2), 29.011 (q, C-23), 30.338 (t, 15), 30.629 (t, C-21), 33.243 (t, C-16), 35.169 (t, C-7), 37.864 (s, C-10), 37.953 (t, C-22), 38.949 (d, C-13), 39.629 (t, C-1), 39.871 (s, C-4), 41.458 (s, C-8), 43.190 (s, C-14), 48.126 (d, C-19), 50.109 (d, C-18), 51.299 (d, C-9), 56.260 (d, C-5), 56.988 (s, C -17), 78.468 (d, C-3), 110.298 (t, C-29), 151.702 (s, C-20), 179.316 (s, C-28)

Example 5: Acyl Coei: Diacylglycerol Acyl Transferase Activity Assay

5-1: Acyl Coay: Determination of Activity of Diacylglycerol Acyl Transferase

The present inventors used microsomal protein isolated from rats according to Coleman et al., Methods Enzymol. 98-103, 1992 to measure the activity of acyl coay: diacylglycerol acyltransferase. using, as the substrate 1, 2-diacylglycerol (Sigma Co., D0138) and ^[14 C] palmitoyl-Koei of the ^[14 C] triacylglycerol produced after enzymatic reaction using (Amersham Inc., CFA583) The amount of radioactivity was measured. Specifically, the reaction solution (175 mM Tris-HCl (pH 8.0), 20 μl bovine serum albumin (10 mg / ml), 8 mM MgCl ₂ , 30 μM [ ¹⁴ C] palmitoyl coei (0.02 μCi, Amersham) and 200 μM 10.0 μl of the sample solution dissolved in methanol or dimethyl sulfoxide (DMSO) was added to 1,2-dioleoyl glycerol), 100-200 μg of the isolated microsome protein was added thereto, followed by reaction at 25 ° C. for 10 minutes. Ml of reaction termination solution (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 to the mixture, followed by shaking. Then, 1 ml of supernatant was added thereto and 2 ml of alkaline ethanol solution (ethanol / 0.5N 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 acyl coay: 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: Determination of Teal Extract and Acyl Coai: Diacylglycerol Acyltransferase Inhibitory Activity of Chemical Formula 1

According to the DGAT activity measuring method of 5-1, the Teal Methanol extract prepared by the method of Example 1 and the chloroform fraction prepared by the method of Example 2 were each added at a concentration of 125 µg / ml to acyl coay: dia Silglycerol acyltransferase enzyme inhibitory activity was measured, and the results are shown in Table 1.

As a result of the solvent fractionation of the methanol extract, it can be seen that the enzyme-inhibiting active substance was transferred to the chloroform fraction.

Column chromatography was performed to separate and purify the active substance from this fraction, and the DGAT inhibitory activity and IC ₅₀ were measured by adding the compound of formula 1, which is a purified active substance separated and purified by DGAT inhibitor, and the results are shown in Table 2 is shown.

Example 6: Acute Toxicity Test

Mice (20 ± 5 g, central laboratory animals) and rats (235 ± 10 g, central laboratory animals) were used for the acute toxicity test of teal extract and compounds. ICR-based mice and Sprague-Dawley rats were divided into four groups of 10 rats each, followed by oral administration of Tealwood extract and compound of the present invention at doses of 100, 250 and 500 mg / kg, respectively. As a result, none of the four groups died, and no symptoms were apparent from the control group.

As discussed above, the teal extract of the present invention and its active ingredient betulinic acid can be effectively used to prevent and treat type 2 diabetes by effectively inhibiting acyl coay: diacylglycerol acyltransferase. .

Claims (6)

Obesity and type 2 diabetes prevention and treatment composition comprising a teal extract. delete The method for preventing and treating obesity and type 2 diabetes, according to claim 1, wherein the teal extract is a teal methanol extract obtained by extracting teal with methanol. The method for preventing and treating obesity and type 2 diabetes, according to claim 1, wherein the teal extract is a teal chloroform fraction obtained by further extracting the teal methanol extract with chloroform. delete delete

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