KR100607585B1 - New enoic acid amide compounds having inhibitory activity against acyl-CoA:cholesterol acyltransferase, process of preparation thereof and composition comprising the same - Google Patents

Abstract

The present invention relates to a novel enoic acid amide compound having an acyl coei: cholesterol transferase inhibitory activity, a method for preparing the same, and a composition containing the same, wherein the enoic acid amide compound is Mylacen 2 or Mylacen 2 It is characterized by that it is Larsen 3 (Mylacen 3).

Myracene 2 or myracene 3, an enoic acid amide compound of the present invention, effectively inhibits the activity of acyl-CoA: cholesterol transferase, and thus, hyperlipidemia associated with acyl-CoA: Cholesterol transferase, coronary heart disease, and arteries. It can be usefully used for the prevention and treatment of cardiovascular diseases such as sclerosis and myocardial infarction.

Description

New enoic acid amide compounds having inhibitory activity against acyl-CoA: cholesterol acyltransferase, process of preparation details and composition configuring the same}

The present invention relates to a novel anoic acid amide compound having an acyl-CoA: cholesterol acyltransferase (ACAT), a preparation method thereof, and a composition containing the same. Is characterized in that Mylacen 2 (Mylacen 2) or Mylacen 3 (Mylacen 3).

Cardiac circulatory disease is currently the number 1 mortality disease in the world, and in developed countries such as Gumi, more than one-third of all deaths occur in Korea.

Atherosclerosis, a representative disease of the cardiac circulatory system, is one of the major diseases that occur with aging, as the artery thickens and hardens, loses elasticity, and becomes weak. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral arteriosclerosis, headache, dizziness, and mental disorders are indicated and cause encephalopathy, and in the case of coronary arteriosclerosis, pain and arrhythmia in the heart cause angina and myocardial infarction. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading cause of death in modern society, especially among men in their 50s and 60s.

Cardiac circulatory diseases are caused by high plasma cholesterol levels, and research has been actively conducted to develop drugs that can lower plasma total cholesterol levels for the purpose of preventing cardiovascular diseases. As a result, a number of substances that inhibit the biosynthesis of cholesterol in the human body have been developed and commercialized. However, in order to improve the side effects reported by these drugs, research has recently been focused on finding compounds that can only selectively regulate blood cholesterol, and one of them is related to ACAT inhibitors.

ACAT is an enzyme that generally esterifies cholesterol, and its mechanism of action occurs largely in three parts of the body (intestinal, liver, and vascular wall cells).

First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol absorbed from the outside or biosynthesized in the body is accumulated in a carrier called VLDL (very low density lipoprotein) in the liver, and then supplied to each organ through the blood vessels, whereby ACAT converts cholesterol into cholesteryl ester form. This enables the accumulation of cholesterol in the carrier. Third, in the cells that make up the arterial vessel wall, ACAT converts cholesterol into its ester form to promote the accumulation of cholesterol in the cell, which is a direct cause of atherosclerosis.

Therefore, the drug that inhibits the activity of ACAT may first reduce the amount of cholesterol that enters the body by inhibiting the absorption of intestinal cholesterol, and secondly, by inhibiting the release of cholesterol into the blood vessels in the liver to reduce blood cholesterol levels. Third, it is expected to prevent arteriosclerosis by preventing cholesterol from accumulating in blood vessel wall cells.

ACAT activity inhibitors reported to date are activity inhibitors for rat hepatic microsome ACAT or rat hepatic macrophage (J774) ACAT.

Human ACAT includes human ACAT-1 and human ACAT-2. Human ACAT-1 (50 kDa) acts mainly on the liver, adrenal glands, macrophages and kidneys of adults. Human ACAT-2 (46 kDa) is a small intestine. (Rudel, LL et al ., Curr. Opin. Lipidol 12 , 121-127, 2001). Substances that inhibit human ACAT activity are targets for the prevention and treatment of hypercholesterolemia, cholesterol stones or atherosclerosis through mechanisms that inhibit the absorption of cholesterol from food and the accumulation of cholesteryl esters in the blood vessel walls. (Buhman, KK et al. , Nature Medicine 6 , 1341-1347, 2000).

On the other hand, Mylabris phalerate Pallas is a carcass of the Meloidae assistant, and has been used in China as an analgesic, diuretic and anti-inflammatory drug since ancient times. Since it was first discovered in Jeju Island about 10 years ago in Korea, it has been used in the private sector for the treatment of chronic adult diseases such as cancer treatment supplements, nervous system diseases and diabetes.

Therefore, the present inventors, while studying the traditional Korean medicine and folk medicine in order to develop a new hyperlipidemia, arteriosclerosis treatment, confirmed that the action of inhibiting ACAT activity in the enoic acid amide compound isolated and purified from Banmyo extract The present invention has been completed.

The present invention aims to provide a novel enoic acid amide compound having an acyl coei: cholesterol transferase inhibitory activity.

In addition, the present invention is to provide a method for producing the enoic acid amide compound.

In addition, the present invention is to provide a composition for the prevention and treatment of cardiovascular diseases comprising the enoic acid amide compound as an active ingredient.

The present invention is a novel anoic acid amide compound represented by the following formula (1) having an acyl coei: cholesterol transferase inhibitory activity (myrasen 2) and a novel anoic acid amide compound represented by the following formula (2) ).

The enoic acid amide compounds of the present invention (myrasen 2, myracene 3) can be used in the form of pharmaceutically acceptable salts, and include all salts, hydrates and solvates prepared by conventional methods.

The present invention also provides a method for preparing the enoic acid amide compound.

The enoic acid amide compound of the present invention (myrasen 2, myracene 3) was used by extraction, separation and purification from half seedlings, and can be obtained by any conventional method.

Extraction, separation and purification of the enoic acid amide compound (myrasen 2, myracene 3) from the seedling according to the present invention is as follows.

The dried half seedling powder was extracted with methanol, concentrated, extracted with water and n-hexane, and separated into hexane and water layers. The water layer is extracted again with chloroform and separated into a chloroform layer and a water layer. The water layer is extracted again with ethyl acetate and separated into an ethyl acetate layer and a water layer.

As a result of measuring the ACAT activity of the hexane layer, the chloroform layer, the ethyl acetate layer and the water layer obtained in the above process, it was confirmed that the ACAT activity was strongly inhibited in the ethyl acetate layer. (oily) get the substance.

The ethyl acetate concentrate is separated by silica gel column chromatography using a mixed solvent of chloroform and methanol. At this time, it is preferable to use a solvent of chloroform: methanol = 100-0: 0-100 (v / v), and the active substance is most effectively separated when a solvent having 100% of chloroform is used.

The active fraction is separated by silica gel column chromatography using a mixed solvent of n-hexane and ethyl acetate. At this time, it is preferable to use a solvent having n-hexane: ethyl acetate = 100-50: 0-50 (v / v), and a solvent having n-hexane: ethyl acetate = 2: 1 (v / v) is used. In this case, the active substance is most effectively separated.

The active fraction is separated again using C-18 reverse phase column chromatography (C-18; Merck, column size: Φ2.0 x 15.0 cm) while varying the ratio of water and methanol solution. At this time, the active material is most effectively separated when using a solvent of water: methanol = 1: 4 (v / v).

The active fractions were separated using high reversed phase chromatography (HPLC; hydrosphere C-18: s-5 μm, column size: Φ 250 × 20.0 mm I.D.) with varying ratios of water and methanol solution. The active substance is most effectively separated using a solvent with water: methanol = 1: 9 (v / v).

By the above method, 8 mg of myracene 2 and 6 mg of myracene 3, which are pure active substances, can be obtained from 500 g of dried seedlings.

The present invention also provides a composition for the prevention and treatment of cardiovascular diseases, comprising the enoic acid amide compound as an active ingredient.

Banmyo separated from the extract, a purified Mai Larsen 2 according to the present invention jwigan ACAT, human ACAT-1 and human and IC ₅₀ value of 162 μM, 85 μM, 61 μM each of the ACAT-2, MY Larsen 3 jwigan ACAT, IC ₅₀ values for human ACAT-1 and human ACAT-2 are 150 μM, 72 μM and 54 μM, respectively, effectively inhibiting ACAT activity.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction caused by the synthesis and accumulation of cholesteryl esters.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the enoic acid amide compound (myrasen 2 or myracene 3).

The composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. Are prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, water It is prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. The daily dosage is about 0.1-100 mg / kg of myracene 2 (or myracene 3), preferably 0.5-10 mg / kg, and more preferably, administered once or several times a day.

As a result of toxicity experiments by orally administering the enoic acid amide compound of the present invention (myrasen 2, myrasen 3) to mice, 50% lethal dose of the enoic acid amide compound (myrasen 2, myrasen 3) ( LD ₅₀ ) appears to be at least 1 g / kg and is considered a safe substance.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of cardiovascular diseases.

The composition of the present invention can be added to health foods for the purpose of improving cardiovascular disease. When the enoic acid amide compound of the present invention (myrasen 2 or myracene 3) is used as a food additive, the myracene 2 or myracene 3 may be added as it is or may be used together with other food or food ingredients, and a conventional method It can be suitably used according to. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the enoic acid amide compound (myrasen 2 or myracene 3) of the present invention is added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight based on the raw materials. . However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, and drinks. Alcoholic beverages and vitamin complexes, and includes all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example  : Extraction, Separation and Purification of Enoic Acid Amide Compounds (Myrasen 2, Myracene 3)

1. Extraction from Semimyo

500 g of the dried half seedlings were washed and dried, and then ground using a grinder to obtain a powder.

The half seedling powder was placed in 1.5 L of methanol and left at room temperature for 24 hours. Then, the methanol solution was stirred and filtered through a filter paper to separate the liquid phase. The above extraction process was repeated twice to collect only the liquid phase and concentrated under reduced pressure.

The methanol extract obtained above was suspended in 500 ml of water, and again 500 ml of n-hexane was added thereto. The separatory funnel was separated into a hexane layer (9.4 g) and a water layer.

The water layer was extracted again by adding chloroform, and separated into a chloroform layer (17.2 g) and a water layer using a separatory funnel.

The separated water layer was extracted again by adding ethyl acetate and separated into an ethyl acetate layer (22.0 g) and a water layer using a separatory funnel.

As a result of measuring the ACAT activity of the hexane layer, the chloroform layer, the ethyl acetate layer and the water layer obtained in the above process, the ACAT activity was strongly inhibited in the ethyl acetate layer.

Thus, the ethyl acetate layer was concentrated and dried under reduced pressure to give a dark brown oily substance.

2. Isolation and Purification of Ethyl Acetate Extract

The ethyl acetate extract obtained in step 1 was separated using silica gel column chromatography (silica gel; Merck, Art 9385, column size: φ 7.5 x 21.5 cm) by varying the ratio of chloroform and methanol. When the solvent with 100% chloroform was used, the active substance was most effectively separated, and 8.8 g of the active fraction was obtained.

The active fraction was separated using silica gel column chromatography (silica gel; Merck, Art 9385, column size: Φ 3.5 x 19.0 cm) with varying ratios of n-hexane and ethyl acetate solution. In the case of using a solvent of n-hexane: ethyl acetate = 2: 1 (v / v), the active substance was most effectively separated, and 1.2 g of the active fraction was obtained.

The active fraction was separated again using reverse phase C-18 column chromatography (C-18; Merck, column size: Φ2.0 x 15.0 cm) with varying ratios of water and methanol solution. The active substance was most effectively separated using a solvent of water: methanol = 1: 4 (v / v).

The active fraction was separated again using high reversed phase chromatography (HPLC; hydrosphere C-18: s-5 μm, column size: Φ 250 × 20.0 mm I.D.) with varying ratios of water and methanol solution. In the case of using a solvent of water: methanol = 1: 9 (v / v), the active substance was most effectively separated. At this time, 8 mg of myracene 2 and 6 mg of myracene 3 were obtained.

3. Structural Analysis of Enoic Acid Amide Compounds (Miracen 2, Myracene 3)

The material obtained through the above example was determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima, and the molecular weight and molecular formula were determined by using a polarizer (Jasco DIP-). 181 digital polarimeter). Nuclear Magnetic Resonance (NMR) analysis (Bruker AMX 300) also revealed ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC ( ¹ H-Detected heteronuclear Multiple-Quantum Coherence) and Heteronuclear Multiple- Bond Coherence (DEP), Distortionless Enhancement by Polarization (DEPT) spectra were obtained, and molecular structure was determined.

The measurement results are as follows. The compound of formula 1 was identified as myracene 2, and the compound of formula 2 was identified as a novel compound of myracene 3.

[Mirasen 2]

1) Physical property: amorphous powder

2) Luminous intensity: [α] _D ²⁵ 0 (c = 0.6, CHCl ₃ )

3) Molecular weight: 279

4) Molecular formula: C ₁₈ H ₃₃ NO

5) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 5.24 (4H, m) 2.67 (2H, t, J = 6.5 μs), 2.09 (2H, t, J = 7.5 μs) 1.96 (4H, m), 1.50 (2H, m), 1.28-1.19 (14H, m), 0.81 (3H, t, J = 6.5 μs)

6) ¹³ C-NMR (CDCl ₃ , 125 MHz) δ 179.3 130.9, 130.8, 129.1, 129.0, 36.5, 32.7, 30.7, 30.5, 30.4, 30.3, 30.2, 28.1, 28.1, 26.9, 26.5, 23.5, 14.4

7) FAB-MS: m / z [M + H] ⁺ 279 (80), 220 (20), 150 (25), 109 (35), 95 (60), 81 (80), 67 (90), 59 (100)

[Mirasen 3]

1) Physical property: amorphous powder

2) Luminous intensity: [α] _D ²⁵ 0 (c = 0.6, CHCl ₃ )

3) Molecular Weight: 218

4) Molecular formula: C ₁₈ H ₃₅ NO

5) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 5.24 (2H, m), 2.09 (2H, t, J = 7.5 Hz), 1.93 (4H, m), 1.50 (2H, m), 1.23-1.19 ( 20H, m), 0.81 (3H, t, J = 6.6 Hz)

6) ¹³ C-NMR (CDCl ₃ , 125 MHz) δ 179.3, 130.9, 130.8, 36.5, 33.1, 30.84, 30.81, 30.62, 30.64, 30.61, 30.45, 30.35, 30.24, 28.1, 28.1, 26.9, 23.7, 14.5

7) FAB-MS: m / z [M + H] ⁺ 281 (50), 264 (25), 126 (30), 98 (25), 83 (20), 72 (90), 59 (100)

Experimental Example 1  : Effect on ACAT Activity of Enoic Acid Amide Compounds (Miracen 2, Myracene 3)

In order to determine the effect on the ACAT activity of myracene 2 and myracene 3 extracted, separated and purified from the seedlings according to the present invention, the following experiment was performed.

1. Preparation of ACAT Enzyme Source

1) Rat liver ACAT

Liver of white rat (male Sprague-Dawley 150-200g) was extracted as ACAT enzyme source. 1 g of liver was homogenized in 5 ml of homogenization solution (0.1 M KH ₂ PO ₄ , pH 7.4, 0.1 mM EDTA and 10 mM β-mercaptoethanol). The homogenate was centrifuged at 3,000 × g for 10 minutes at 4 ° C., and the obtained supernatant was centrifuged at 15,000 × g for 15 minutes at 4 ° C. to obtain a supernatant. The supernatant was placed in an ultracentrifuge tube (Beckman) and centrifuged at 100,000 xg for 1 hour at 4 ° C. to obtain microsomal pellets, which were then suspended in 3 ml of homogenization solution at 100,000 ° C. at 4 ° C. for 1 hour. Centrifuged. The obtained pellet was suspended in 1 ml of homogenization solution. The protein concentration in the obtained suspension was measured by the method of Lowry et al., And the protein concentration was adjusted to 4-8 mg / ml. The suspension obtained was stored in a deep freezer (Forma Scientific Inc.).

2) Human ACAT-1 and Human ACAT-2

Human ACAT-1 and ACAT-2 proteins were obtained using baculovirus expression systems to investigate the effects on human ACAT-1 and ACAT-2 activity.

The cDNAs of hACAT-1 and hACAT-2 obtained through human cDNA library screening were inserted into baculovirus delivery vectors and introduced into insect cells, sf9 cells, to prepare viruses. Subsequently, the recombinant viruses of hACAT-1 and hACAT-2 were isolated by plaque purification, and the titer of the viral stock was increased through three amplification processes. Recombinant virus was infected to Hi5 insect cells having good protein expression efficiency so as to have a multiplicity of infection (Mutiplicity of Infection) of 1, followed by shaking culture for one day at 27 ° C.

The cultured hACAT-1 and hACAT-2 were collected by centrifugation at 500xg for 15 minutes to separate microsomal fractions from overexpressed Hi5 cells, and then quenched by rapid freeze thawing in hypotonic buffer. After breaking the ultracentrifugation for 100,000 hours at 100,000xg.

The obtained microsome fractions were suspended in storage buffer so that the protein concentration was 8 mg / ml and stored in a cold freezer until use.

2. ACAT activity measurement

6.67 μl of a cholesterol solution dissolved in acetone at a concentration of 1 mg / ml was mixed with 6 μl of 10% triton WR-1339 (Sigma Co.) in acetone, and acetone was removed by evaporation using nitrogen gas. It was. Distilled water was added to the obtained mixture to adjust the concentration of cholesterol to 30 mg / ml.

10 μl of aqueous solution of cholesterol, 10 μl of 1M KH ₂ PO ₄ (pH 7.4), 5 μl of 0.6 mM bovine serum albumin (BSA), 10 μl of microsome solution obtained in 1, 10 μl A sample (Mirasen 2, Myracene 3) and 45 μl of distilled water were added (total 90 μl). The mixture was pre-reacted for 30 minutes in a 37 ° C. water bath.

10 μl of (1- ¹⁴ C) oleyl-CoA solution (0.05 μCi, final concentration: 10 μM) was added to the pre-reacted mixture and the resulting mixture was reacted for 30 minutes in a 37 ° C. water bath. To this was added 500 μl isopropanol: heptane mixture (4: 1 (v / v)), 300 μl heptane and 200 μl 0.1 M KH ₂ PO ₄ (pH 7.4) and the mixture was vigorously vortexed. After mixing, the mixture was left at room temperature for 2 minutes.

The resulting 200 µl supernatant was placed in a scintillation bottle, and 4 ml of scintillation liquid (Lumac) was added. The radiation dose of this mixture was measured with a 1450 Microbeta liquid scintillation counter, Wallacoy, Finland.

ACAT activity was calculated from cholesteryl oleate picolol (picomol / min / mg protein) synthesized per mg of protein for 1 minute by calculating the radiation dose per hour from the measured radiation dose.

The results are shown in Table 1 and Table 2.

Inhibitory Activity of Myracene 2 on ACAT Enzyme IC ₅₀ (μM) Rat liver ACAT 162 Person ACAT-1 85 Person ACAT-2 61

Inhibitory Activity of Myracene 3 on ACAT Enzyme IC ₅₀ (μM) Rat liver ACAT 150 Person ACAT-1 72 Person ACAT-2 54

As shown in Table 1 and Table 2, Mai Larsen 2 of the present invention jwigan ACAT, human ACAT-1 and human 162 μM IC for ACAT-2 ₅₀ values each, 85 μM, 61 μM, and the My Larsen 3 jwigan IC ₅₀ values for ACAT, human ACAT-1 and human ACAT-2 were 150 μM, 72 μM and 54 μM, respectively.

Accordingly, it can be seen that myracene 2 and myracene 3 effectively inhibit ACAT activity, and the cardiovascular system such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction caused by synthesis and accumulation of cholesteryl ester It can be usefully used for the prevention and treatment of diseases.

Experimental Example 2  : Acute Toxicity Test

In order to determine the acute toxicity of myracene 2 and myracene 3 of the present invention, an acute toxicity test was conducted in the following manner.

Six-week-old SPF rats were used as experimental animals and divided into two groups per group. Compounds obtained in the above examples (myrasen 2 and myracene 3) were each suspended in 0.5% methylcellulose solution and administered orally at a dose of 1 g / kg / 15 ml.

After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed. The necropsy was performed to visually observe the abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms were observed in all animals treated with the test substance, no animals died, and no toxicity change was observed in weight change, blood test, blood biochemical test, autopsy findings, etc.

As a result, all the tested compounds did not show toxicological changes up to 1000 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 1000 mg / kg or more.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1  : Preparation of Pharmaceutical Formulations

1. Preparation of Tablets

Tablets containing the enoic acid amide compound of the present invention (myrasen 2, myracene 3) or a pharmaceutically acceptable salt as an active ingredient were prepared by the following method.

The compounds of the above examples were sieved and mixed with lactose, starch and pregelatinized corn starch, then a suitable volume of purified water was added and granulated into a powder. The granules were dried and then mixed with magnesium stearate and compressed to obtain tablets.

The components of the tablet are as follows.

Myracene 2 (or Myracene 3) 5.0 mg

Lactose BP 150.0mg

Starch BP 30.0mg

Pregelatinized Corn Starch BP 15.0mg

1.0 mg magnesium stearate

2. Preparation of Capsule

Capsules containing the enoic acid amide compound of the present invention (myrasen 2, myracene 3) or a pharmaceutically acceptable salt as an active ingredient were prepared by the following method.

The compound of the above example was mixed with an amount of excipient and magnesium stearate. The resulting mixture was filled into gelatin capsules to obtain capsules.

The components of the capsule are as follows.

Myracene 2 (or Myracene 3) 5.0 mg

Starch 1500 100.0mg

Magnesium Stearate BP 1.0mg

3. Preparation of Injection

Injection solution containing the enoic acid amide compound of the present invention (myrasen 2, myracene 3) or a pharmaceutically acceptable salt as an active ingredient was prepared by the following method.

The compound of this example was dissolved in an appropriate volume of sodium chloride BP for injection, and the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, then the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, and the glass was dissolved and enclosed under an upper grid of air, and then sterilized by autoclaving at 120 ° C. for at least 15 minutes to obtain an injection solution.

The components of the injection solution are as follows.

Myrasen 2 (or myrasen 3) 100 μg / ml

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Formulation Example 2  : Manufacture of food

Foods containing myracene 2 (or myracene 3) were prepared as follows.

1. Preparation of Cooking Seasonings

Myracene 2 (or myracene 3) 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.

2. Preparation of Tomato Ketchup and Sauce

Health promotion tomato ketchup or sauce was prepared by adding 0.2-1.0 wt% of myracene 2 (or myracene 3) to tomato ketchup or sauce.

3. Manufacturing of Flour Foods

0.1-5.0% by weight of myracene 2 (or myracene 3) was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare foods for health promotion.

4. Preparation of soups and gravy

0.1-1.0 wt% of myracene 2 (or myracene 3) was added to the soup and gravy to prepare health promoting meat products, soups of noodles and gravy.

5. Preparation of Ground Beef

10% by weight of myracene 2 (or myracene 3) was added to the ground beef to prepare a ground beef for health promotion.

6. Manufacture of Dairy Products

0.1-1.0 wt% of myracene 2 (or myracene 3) was added to the milk, and various milk products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, yulmu was alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

Myracene 2 (or myracene 3) was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with spray and hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the dry powder of myracene 2 (or myracene 3) prepared above were combined and prepared in the following ratio.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of Myracene 2 (or Myracene 3) (1% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3  : Preparation of Beverages

1. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 After sterilizing at 20 ° C. for 20 seconds to 180 seconds, the mixture was mixed with cooling water at a ratio of 1: 4, and 0.5 to 0.82% of carbon dioxide was injected to prepare a carbonated beverage containing myracene 2 (or myracene 3).

2. Manufacture of health drinks

Instant sterilization by homogeneously mixing subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and myracene 2 (or myracene 3) After this, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

0.5 g of myracene 2 (or myracene 3) was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

0.1 g of myracene 2 (or myracene 3) was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

Myracene 2 (or myracene 3) of the present invention effectively inhibits the activity of acyl-CoA: cholesterol transferase, and thus, hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction associated with acyl-CoA: cholesterol transferase. It can be useful for the prevention and treatment of cardiovascular diseases such as.

Claims (11)

A pharmaceutical composition for the prevention and treatment of cardiovascular diseases comprising the enoic acid amide compound represented by the following Chemical Formula 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula 1>

<Formula 2>

The pharmaceutical composition of claim 1, wherein the cardiovascular disease is hyperlipidemia, coronary heart disease, arteriosclerosis, and myocardial infarction. The pharmaceutical composition for preventing and treating cardiovascular diseases according to claim 1, wherein the enoic acid amide compound is obtained by extracting, separating and purifying from a seedling. 1) extracting the dried half seedling powder with methanol, extracting sequentially using n-hexane, chloroform, ethyl acetate, and 2) separating the extract obtained in step 1) into the enoic acid amide compound of claim 1 or 2 using column chromatography, A method for extracting, separating and purifying an enoic acid amide compound according to claim 1. The method of claim 4, wherein step 2) (1) separating the ethyl acetate fraction obtained in step 1 by column chromatography using a mixed solvent of chloroform and methanol, (2) separating the active fraction by column chromatography using a mixed solvent of n-hexane and ethyl acetate, (3) separating the active fraction using reverse phase column chromatography while changing the proportion of the aqueous methanol solution again, and (4) separating the active fraction using high speed reverse phase chromatography while changing the proportion of the methanol solution again; A method for extracting, separating and purifying an enoic acid amide compound according to claim 1. delete delete delete A food composition useful for improving a cardiovascular disease containing an enoic acid amide compound represented by Formula 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula 1>

<Formula 2>

10. The food composition of claim 9, wherein the cardiovascular disease is hyperlipidemia, coronary heart disease, arteriosclerosis, and myocardial infarction. delete