KR20080037854A - A composition for preventing or treating obesity or type 2 diabetes - Google Patents

Abstract

A composition comprises the extract of Piper nigrum or amide compound isolated therefrom is provided to inhibit activity of acyl CoA: diacylglycerol acyltransferase(DGAT) and improve energy metabolism and glucose metabolism, so that the composition is useful for preventing or treating obesity or type 2 diabetes. A composition for preventing or treating obesity or type 2 diabetes comprises the extract of Piper nigrum prepared by using water, organic solvent including hexane, ether, dichloromethane, chloroform and ethylacetate and a mixed solvent, or amide compound isolated therefrom represented by the formula(1) or formula(2) which is obtained by extracting Piper nigrum with lower alcohol, extracting the extract of Piper nigrum with non-polar organic solvent to obtain a fraction, and subjecting the fraction to chromatography.

Description

A composition for preventing or treating obesity or type 2 diabetes}

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

The present invention is an acyl CoA: diacylglycerol acyltransferase (DGAT) comprising a Piper nigrum extract, or an amide compound derived therefrom or a pharmaceutically acceptable salt thereof. It relates to a composition having an inhibitory activity and a composition for the prevention or treatment of obesity or type 2 diabetes comprising the composition.

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 catalyzing triglyceride biosynthesis in liver, adipose tissue and skeletal muscle, 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), and Dr. Robert V. Farese Jr. of the Gladstone Heart Disease Research Center in the United States have studied diets using high-fat diet-induced weight gain in mice that knocked out the DGAT gene. Selective DGAT inhibitors have been shown to be effective in the treatment of obesity and type 2 diabetes by providing evidence that induced obesity has been effectively suppressed and insulin sensitivity and leptin have increased sensitivity to improved glucose metabolism. Usefulness (Smith SJ. Et al., Nature genetics , 25, 87-90, 2000).

To date, synthetic products of DGAT enzyme inhibitors are known as pyrrolecarboxylic acid derivatives (JP JP05213985A, Mitsubishi Kasei Corp, Japan) and phosphonic acid ester derivatives (JP JP2004067635A, Otsuka Pharmaceut Factory INC, Japan). In the case of DGAT enzyme inhibitors, the research team first started looking for 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 quinolone alkaloids, tanshinones, and flavonoid-flavoroids isolated from herbal resources such as sesame oil, sweet ginseng, and ginseng (Korean patent application) 2002-64744; Ko et al. Arch.Phar.Res. 25, p.446-448, 2002; Korean Patent Application No. 2003-00524). Overseas, as an inhibitor reported by the Omura group of the Kitasato research institute in 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 and 2-bromooctanoate 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. It was confirmed that the extract and the amide compounds separated therefrom have excellent inhibitory activity against DGAT, and the extracts and the amide compounds isolated therefrom prevent metabolic diseases such as obesity, type 2 diabetes mellitus and insulin resistance. And the present invention has been completed by revealing that it can be used for treatment.

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

Still another object of the present invention is an amide compound selected from the group consisting of piperrolein B and dehydropipernonaline of formulas 1 and 2 isolated from the extract, or a pharmaceutically acceptable salt thereof. Acyl coay comprising: a diacylglycerol to provide a composition having an acyl transferase inhibitory activity.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of obesity or type 2 diabetes comprising the composition.

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

As used herein, the term “piper nigrum” refers to a fruit including natural, hybrid or varieties of pepper trees, which are evergreen vines belonging to the Black Pepper family.

As used herein, the term "extract" refers to a substance having an acyl coa: diacylglycerol acyltransferase inhibitory activity isolated from natural products. In the present invention, the extract is used as a meaning including not only the extract, but also its dry powder or any form formulated using the same.

In the present invention, the grass and the extract may be extracted using water, an organic solvent or a mixed solvent thereof, and preferably may be extracted using an organic solvent. The extracted liquid may be used in liquid form or may 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, ultrafiltration, cryofiltration, centrifugal separation, etc., but is not limited thereto. It may also further comprise a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity).

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, the extract can perform an additional fractionation process. Preferably, the extract is suspended in distilled water to extract and separate a nonpolar solvent soluble layer with a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. It can be concentrated and / or dried.

In one specific embodiment, the reef extract of the present invention is a dry reef as a powder, 3 to 20 times the weight (kg), preferably 5 to 15 times the water, C ₁ to C ₄ lower Alcohols or mixed solvents thereof, preferably ethanol solvents; 20 to 70 ° C., preferably 20 to 30 ° C. extraction temperature; A time of 1 hour to 10 days, preferably 3 days to 8 days; One to five times, preferably one to three times; The extract obtained by cold needle, ultrasonic extraction, reflux cooling extraction, preferably cold needle extraction method was obtained by filtration and concentration under reduced pressure. 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 having high DGAT inhibitory activity.

As used herein, the term “acylcoa: diacylglycerol acyltransferase (DGAT) inhibition” refers to a phenomenon in which DGAT blocks an enzyme catalytic reaction for synthesizing triglycerides or decreases synthetic efficiency. Say. When the enzyme activity is inhibited, various reactions may be induced in cells. One example of a response induced by inhibiting DGAT enzyme activity is the prophylactic and therapeutic effect of type 2 diabetes through suppressing obesity and improving insulin resistance.

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. Increasing the amount of exercise, weight gain induced by high-fat diets has been found to be suppressed, and has been studied as targets for the treatment of obesity and type 2 diabetes. In addition, DGAT inhibition is known to improve insulin resistance by preventing the accumulation of fat in non-adipoise tissue, such as muscle, liver, 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 exocytosed into the membrane and glucose is introduced into the cell. When DGAT activity is inhibited in cells, the activity of PI-3K, PKB and PKC _λ increases, increasing the number of GLUT-4 exocytosed into the membrane and finally increasing the number of glucose 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 the vinegar inhibited the activity of DGAT by 59.3% at a concentration of 100 ug / ml, and inhibited the chloroform fraction of the ethanol extract by 76.7%. The above results indicate that the reef extract of the present invention has excellent DGAT inhibitory activity, and further has the prophylactic and therapeutic activity of metabolic diseases such as obesity and type 2 diabetes, insulin resistance.

In one preferred embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity or type 2 diabetes, comprising a chorus extract.

As used herein, the term “obesity” refers to a condition or disease with excess body fat due to energy imbalance. By administering the composition of the present invention, body weight can be reduced and blood Cholesterol, triglycerides and low density lipoprotein (LDL) -cholesterol in the blood 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. it means. 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 purpose of the present invention, diabetes relates to type 2 diabetes having insulin resistance, and by administering the composition of the present invention, diabetes resistance is improved to prevent metabolic diseases such as diabetes, hyperlipidemia, hypertension, arteriosclerosis, and cardiovascular diseases. Can be prevented and treated.

As used herein, the term "prevention" means any action that inhibits or delays the onset of the disease by administration of a composition. As used herein, the term "treatment" means any action that improves or advantageously alters the symptoms of the disease by administration of the composition.

The reef extract of the present invention contains a component obtained from an edible natural extract as an active ingredient and is safe, and does not cause toxicity, side effects and resistance, thus allowing long-term administration.

The pharmaceutical composition of the present invention 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 or diabetes.

As another aspect, the present invention relates to a composition having an acyl coay: cholesterol acyltransferase inhibitory activity, including an amide compound of formula 1 or 2 or a pharmaceutically acceptable salt thereof.

In order to identify the active ingredient showing the DGAT inhibitory activity in the extract of the reef, the inventors fractionated the repellent non-polar solvent soluble extract by performing chromatography, followed by gel filtration chromatography and high performance liquid chromatography. As a result of the analysis of ultraviolet spectrophotometric spectra, electron impact mass spectrometry, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, and the like, the active ingredient having DGAT activity was an amide compound of Formulas 1 and 2 It was found to be piperrolein B and dehydropipernonaline.

Amide-based compounds of formula 1 or 2 of the present invention can be isolated from pepper plants, preferably reefs, and extract various organs, roots, stems, leaves, flowers, as well as plant tissue cultures of natural, hybrid, varieties of reefs. Can be separated. In addition, chemical synthesis is possible by methods known in the art.

The method for obtaining the amide compound of the general formula (1) or (2) from a plant may be 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 to give a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethyl. It can be obtained by extracting and separating the nonpolar solvent soluble layer with an acetate or a mixed solvent thereof. From the obtained nonpolar organic solvent fraction, the amide compounds of the formulas (1) and (2) can be obtained. The extract can be further purified by performing one or more chromatographic chromatography, where 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 fruit of the vinegar is extracted with ethanol and its distilled water suspension is extracted 1 to 10 times, preferably 2 to 5 times with 1 to 100 times, preferably 1 to 5 times the volume of chloroform. The obtained chloroform fraction was subjected to adsorption chromatography such as silica gel column chromatography, gel filtration chromatography, high-performance liquid chromatography, etc. sequentially to obtain piperrolein B of formula (I) and dihydropiperin of formula (2). Dehydropipernonaline can be separated.

As used herein, the term “pharmaceutically acceptable salts” means salts derived from pharmacologically or physiologically acceptable inorganic acids, organic acids and bases. Examples of suitable acids 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 include alkali metals such as sodium and the like; Alkaline earth metals such as magnesium, ammonium and the like.

In a specific embodiment of the present invention, the purely isolated amide compounds of Formula 1 or 2 had higher DGAT inhibitory activity as the concentration increased, and when treated at a concentration of 30 ㎍ / mL, the DGAT activity was increased to 80 to 90%. Inhibition and IC ₅₀ was found to be 6.9 and 7.2 μg / ml, respectively. The above results indicate that the amide compound of Formula 1 or 2 of the present invention has excellent DGAT inhibitory activity and has therapeutic activity for obesity and type 2 diabetes.

Accordingly, as one preferred embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity or type 2 diabetes, comprising an amide compound of formula 1 or 2 or a pharmaceutically acceptable salt thereof.

The amide compound of Formula 1 or 2 of the present invention or a pharmaceutically acceptable salt thereof has excellent DGAT inhibitory activity, and thus has excellent prophylactic or therapeutic activity against obesity or type 2 diabetes.

As a result of toxicity experiments performed by oral administration of the amide compounds of Formulas 1 and 2 of the present invention to rats, the 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is determined to be a safe substance of at least 1 g / kg or more.

The pharmaceutical composition may also be used not only in humans but also in cattle, horses, sheep, pigs, goats, camels, antelopes, dogs and the like, which may cause obesity or 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 pharmaceutical composition of the present invention may further include a component that does not increase the efficacy but is commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the composition is inorganic or organic, such as vitamins B ₁ , B ₂ , B ₆ , C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, and 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 pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and may be formulated for human or veterinary use for oral or parenteral use. When formulating the composition of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, and such solid form preparations contain at least one excipient such as starch, calcium carbonate in a composition comprising the extract or compound of the present invention. (Calcium carbonate), sucrose (Sucrose) or lactose (Lactose) and gelatin are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, styrate, and talc may be 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.

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

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating obesity or type 2 diabetes, comprising a narcotic extract, or an amide compound of Formula 1 or 2 or a pharmaceutically acceptable salt thereof. The present invention relates to a method for preventing or treating obesity or type 2 diabetes by administration.

As used herein, the term “individual” has a disease caused by obesity or type 2 diabetes, or a direct or indirect cause thereof, and a condition in which symptoms may be improved by administering the pharmaceutical composition of the present invention. Means mammals such as horses, sheep, pigs, goats, camels, antelopes, and dogs, including humans.

As used herein, the term “administration” means introducing a pharmaceutical composition of the invention to a subject in any suitable manner. The route of administration may be oral or parenteral via any general route so long as it can reach the desired tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows migration to target cells.

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 the effective dose level is the sex, age, severity, drug of the patient. Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. 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 merely to illustrate the invention, but the content of the present invention is 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), and the hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) instrument was a 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. Extracts Fraction  Preparation and separation and purification of enzyme inhibitory active substances

The reef used in the present invention was purchased from Daejeon Herbal Medicine Market, washed thoroughly with water, dried in the shade, and then powdered with a blade grinder.

Three times of ethanol (relative to the dry weight of the reef) was added to 5 kg of the powdered reef and left for 7 days at room temperature for extraction. The filtrate was concentrated under reduced pressure to obtain a crude extract. In order to separate and purify the active substance from the crude extract, normal hexane, chloroform, ethyl acetate and water were separated into their respective fractions to measure DGAT inhibitory activity by the method of Example 3 below.

A portion of each fraction was dried to prepare a sample at 1 mg / ml, and the inhibitory activity was measured. As a result, normal hexane 25%, chloroform 76.6%, ethyl acetate 55%, and water 15% showed the best DGAT in the chloroform layer. Inhibitory activity was shown.

The chloroform layer (157.7 g) obtained above was concentrated under reduced pressure, and column chromatography of silica gel was carried out using a step gradient solvent system composed of n-hexane: ethyl acetate = (50/1 to 0/100). The active fractions were separated. The DGAT inhibitory activity of the separated active fractions was measured, and the 10th fraction having the highest inhibitory activity was used as the eluting solvent. 50%, 60%, 70%, 80%, 90%, 100% methanol was used as a reversed phase column. The active fraction was separated by chromatography (reversed-phase column chromatography, ODS gel). The low activity liquid chromatography (Lchroprep RP-) was measured while measuring the DGAT inhibitory activity of the separated active fraction and flowing the fifth fraction having the highest inhibitory activity to 8 ml / min using 80% methanol. 18 Lobar column, 40-63 mm, 310x25mm, UV 210nm). The second fraction having high DGAT inhibitory activity was flowed at 4 ml / min using 80% methanol, and high performance liquid chromatography (HPLC, YMC Jsphere ODS H-80, 250 × 20 mm) was used. The pure compound 1 (42.4 mg, 45 minutes) was obtained. After the first column chromatography, the 11th active fraction was subjected to reverse phase column chromatography, low pressure liquid chromatography, and high performance liquid chromatography with fraction 10 to obtain Compound 2 (123 mg, 43 minutes). The physicochemical properties of compounds 1 and 2 are as follows.

Compound 1

(1) Appearance of substance: Colorless oil

(2) Molecular formula and molecular weight of substance: C ₂₁ H ₂₉ NO ₃ , 343

(3) Electron impact mass spectrometry (70 eV): m / z (rel. Int) = 366 [M + Na] ⁺

(4) Hydrogen Nuclear Magnetic Resonance Spectrum [300 MHz, Chloroform-d ₃ , δ (ppm)]: 6.88 (1H, br s, H-2 '), 6.74 (1H, m, H-5'), 6.73 (1H) , br s, H-6 '), 6.27 (1H, d, J = 15.3 Hz, H-9), 6.03 (1H, dt, J = 15.9, 6.9 Hz, H-8), 5.92 (2H, s, H-7 '), 5.75 (1H, d, J = 15.3 Hz, H-2), 3.54 (2H, t, J = 5.4 Hz, H-1 "), 3.38 (2H, t, J = 5.5 Hz, H-5 "), 2.30 (2H, t, J = 7.5 Hz, H-2), 2.16 (2H, q, J = 6.6 Hz, H-7), 1.61 (4H, m, H-2" and 4 "), 1.54 (4H, m, H-4 and 3"), 1.45 (2H, m, H-6), 1.35 (4H, m, H-3 and 5)

(5) Carbon nuclear magnetic resonance spectrum [300 MHz, Chloroform-d ₃ , δ (ppm)]: 24.57 (t, C-3 "), 25.37 (t, C-4"), 25.56 (t, C-4) , 26.55 (t, C-2), 28.95 (t, C-5), 29.24 (t, C-6), 29.34 (t, C-3), 32.80 (t, C-7), 33.39 (t, C-2), 42.54 (t, C-1 "), 46.67 (t, C-5"), 100.85 (t, C-7 '), 105.34 (d, C-2'), 108.15 (d, C -5 '), 120.14 (d, C-6'), 129.27 (d, C-8), 129.30 (d, C-9), 132.42 (s, C-1 '), 146.48 (s, C-3 '), 147.87 (s, C-4'), 171.37 (s, C-1)

Compound 2

(1) Appearance of material: colorless plate

(2) Molecular formula and molecular weight of substance: C ₂₁ H ₂₅ NO ₃ , 339

(3) Electron impact mass spectrometry (70 eV): m / z (rel. Int) = 362 [M + Na] ⁺

(4) Hydrogen Nuclear Magnetic Resonance Spectrum [300MHz, Chloroform-d ₃ , δ (ppm)]: 7.23 (1H, dd, J = 15, 10.5 Hz, H-3), 6.88 (1H, s, H-2 ' ), 6.74 (1H, br s, H-6 '), 6.73 (1H, br s, H-5'), 6.31 (1H, d, J = 15.9 Hz, H-2), 6.27 (1H, d, J = 14.7 Hz, H-9), 6.21 (1H, dd, J = 15.0, 10.8 Hz, H-4), 6.07 (1H, dt, J = 15.6, 6.0 Hz, H-5), 6.02 (1H, m, H-8), 5.93 (2H, s, H-7 '), 3.61 (2H, br s, H-5 "), 3.49 (2H, br s, H-1"), 2.31 (4H, m , H-6 and H-7), 1.52-1.68 (6H, m, H-2 ", H-3" and H-4 ")

(5) Carbon nuclear magnetic resonance spectrum [300 MHz, Chloroform-d ₃ , δ (ppm)]: 24.7 (t, C-3 "), 25.6 (t, C-4"), 26.8 (t, C-2 " ), 32.2 (t, C-6), 32.8 (t, C-7), 43.2 (t, C-1 "), 46.7 (t, C-5"), 100.9 (t, C-7 '), 105.4 (d, C-2 '), 108.2 (d, C-5'), 119.1 (d, C-2), 120.4 (d, C-6 '), 127.8 (d, C-4), 129.4 ( d, C-8), 130.1 (d, C-9), 132.1 (s, C-1 '), 141.1 (d, C-3), 142.5 (d, C-5), 146.7 (s, C- 3 '), 147.9 (s, C-4'), 165.6 (s, C-1)

Example  2. Determination of structure of active substance

Compound 1, which was completely isolated and purified, was a colorless oil. [M + Na] ⁺ was m / z 366 as a result of molecular weight measurement, and the molecular formula was estimated to be C ₂₁ H ₂₉ NO ₃ in high resolution FAB-MS. As a result of measuring the ultraviolet absorbance of the compound, the maximum absorption was found at 260 nm, and the same absorption as that of Compound 1. ^{In the 1} H-NMR spectrum, two olefin protons are observed at δ 6.03 (1H, dt, J = 15.9, 6.9 Hz, H-8) and 6.27 (1H, d, J = 15.3 Hz, H-9) Methylenedioxy protons (δ 5.92, s) were observed. From the observation of methylene protons binding -N- at δ 3.54, δ 3.38, the presence of a piperidine ring could be estimated. In addition, protons overlaid with six methylene protons and piperidine rings were observed at δ 2.16-2.30 (4H) and δ 1.35-1.61 (14H), respectively. ^{From the 13} C-NMR spectrum, one carbonyl carbon, one methylenedioxy carbon, two olefin carbons, six methylene carbons and piperidine, carbon of benzene ring were observed. The above instrumental analysis spectra and previously reported data (Park IK, Lee SG, Shin SC, Park JD, Ahn YJ. Larvividal activity of isobuthylamides identified in Piper nigrum fruits against three mosquito species.J. Agric.Food Chem. 2002; 50: 1866-1870), Compound 1 was found to be piperrolein B having one olefinic group near the benzene group instead of the carbonyl group.

Compound 2 was a pale colorless plate. [M + Na] ⁺ was m / z 362. The molecular formula was estimated to be C ₂₁ H ₂₅ NO ₃ in ESI-MS. Was born is displayed absorption at 211 and 268 nm results of measuring the UV absorbance of the compound, ratio recording infrared spectrophotometer (Bio-Rad Digilab Division, FTS -80) As a result of measuring the IR absorption by using a carbonyl group (1660㎝ ^- The presence of ¹ ) was confirmed. During the NMR test to determine the structure of the compound, the signals of the six olefinic protons in the ¹ H-NMR spectrum were δ 6.31 (1H, d, J = 15.9 Hz, H-2), 7.23 (1H). , dd, J = 10.5, 15.0 Hz, H-3), 6.21 (1H, dd, J = 10.8, 15.0 Hz, H-4), 6.07 (1H, dt, J = 15.3, 6.0 Hz, H-5) , 6.02 (1H, m, H-8) and 6.27 (1H, d, J = 14.7 Hz, H-9), where the position of this olefinic proton was ¹ H- ¹ H COSY and 2D of HMBC. It was able to confirm exactly from -NMR spectrum. Instrumental analysis spectra and previously reported data (Shoji N, Umeyama A, Saito N, Takemoto T, Kajiwara A, Ohizumi Y. Dehydropipernonaline, an amide possessing coronary vasodilating activity, isolated from Piper longum LJ Pharmaceu. Sci. 1986; 75: 1188-1189), Compound 2 was found to be dehydropipernonaline having a piperidine group.

Example  3. Acyl Koei : Diacylglycerol Acyl transferase  Active black

3-1. Acyl Koei : Diacylglycerol Acyl transferase  Active measurement

We use Colman et al., Methods to determine the activity of acyl coay: diacylglycerol acyltransferase. Enzymol ., Pp98-103, 1992), using microsomal proteins isolated from rats as an enzyme source, 1,2-diacylglycerol (Sigma, D0138) and [ ¹⁴ C] palmitoyl-coei as substrates (Amersham, CFA583) was used to measure the amount of radioactivity of [ ¹⁴ C] triacylglycerol produced after the enzyme reaction. 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.

3-2. Coral  Of the extract with the compound of formula 1 or 2 DGAT  Inhibitory activity measurement

According to the method for measuring diacylglycerol acyltransferase activity of 3-1, diacylglycerol acyltransfer was added by adding ethanol extract, chloroform fraction, and water fraction of the reef prepared in Example 1 at 100 and 50 ug / ml, respectively. The enzyme activity inhibitory activity was measured and the results are shown in Table 1.

Diacylglycerol Acyltransferase Inhibitory Activity of Algae Ethanol Extract, its Chloroform and Water Fraction Concentration (μg / ml) DGAT inhibition rate (%) Ethanol extract 100 59.3 50 20.0 Chloroform fraction 100 76.7 50 54.4 Water fraction 100 7.1 50 -

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

Column chromatography was carried out to separate and purify the active substance from this fraction, and the compound of formula 1 or 2, the active substance isolated and purified by diacylglycerol acyltransferase inhibitor, was added at different concentrations to inhibit DGAT enzyme activity and IC. ₅₀ was measured and the results are shown in Table 2.

Diacylglycerol Acyltransferase Enzyme Inhibitory Activity of the Compounds of Formulas 1 and 2        Concentration compound DGAT inhibition rate (%) IC ₅₀ (ug / ml) 100 (ug / ml) 30 10 3 One Compound 1 98.4 85.0 65.1 27.0 11.9 6.9 Compound 2 94.7 92.2 66.3 11.5 2.9 7.2

Example  4. On the rat  Acute toxicity test for oral administration

In order to determine the acute toxicity of the extracts and compounds of the present invention, the acute toxicity test was carried out as follows.

Acute toxicity test was conducted using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were administered once orally with an extract and compound of the present invention at a dose of 1 g / kg / ml each. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

Therefore, the extracts and compounds of the present invention do not show a change in toxicity even in rats up to 1 g / kg, oral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of more than 1 g / kg compound.

As described above, the edible extract of the present invention, or an amide compound of Formula 1 or 2, which is an active ingredient thereof, effectively inhibits acyl coay: diacylglycerol acyltransferase to prevent obesity or type 2 diabetes or It can be effectively used for treatment.

Claims (9)

A composition having acyl coay: diacylglycerol acyltransferase inhibitory activity comprising a herb extract. The composition according to claim 1, which is extracted using water, an organic solvent or a mixed solvent thereof. A composition comprising a fraction obtained by extracting the extract of the vinegar with an organic solvent selected from the group consisting of hexane, ether, dichloromethane, chloroform, ethyl acetate and a mixed solvent thereof. A pharmaceutical composition for the prevention or treatment of obesity or type 2 diabetes, comprising the composition of any one of claims 1 to 3. A composition having an acyl coay: diacylglycerol acyltransferase inhibitory activity comprising a compound of formula 1 or 2 or a pharmaceutically acceptable salt thereof. Formula 1

Formula 2

The composition of claim 5, wherein the compound is isolated from the pepper plant. 7. The composition of claim 6, wherein said plant is a grass. A pharmaceutical composition for preventing or treating obesity or type 2 diabetes, comprising the composition of claim 5. Obtaining a lake extract using a lower alcohol solvent; Obtaining a fraction from the extract using a nonpolar organic solvent; And separating the compound of Chemical Formula 1 or 2 having acyl coay: diacylglycerol acyltransferase inhibitory activity from the vinegar comprising purifying the fraction by chromatography. Formula 1

Formula 2

Images