KR100758263B1 - New n-acetyldopamine compounds, and the composition comprising periostracum cicadae extract or n-acetyldopamine compounds isolated from the same for prevention and treatment of cardiovascular disease - Google Patents

Abstract

The present invention relates to a novel acetyldopamine compound, and a composition for the prevention and treatment of cardiac circulatory diseases, which contains a sesame extract or an acetyldopamine compound isolated therefrom as an active ingredient. Since the acetyldopamine-based compound has excellent antioxidant activity against low-density lipoprotein, the composition comprising the same may be useful for the prevention and treatment of cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low-density lipoprotein. .

Description

Novel acetyldopamine-based compound, and a composition for the prevention and treatment of cardiovascular diseases, containing the sesame extract or acetyldopamine-based compound isolated therefrom as an active ingredient {NEW N-ACETYLD OPAMINE COMPOUNDS -ACETYLDOPAMINE COMPOUNDS ISOLATED FROM THE SAME FOR PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASE}

The present invention relates to a novel acetyldopamine-based compound, and a composition for the prevention and treatment of cardiac circulatory disorders, which contains a sesame extract or an acetyldopamine-based compound isolated therefrom as an active ingredient.

Recently, vascular disorders such as arteriosclerosis have increased significantly as adult diseases increase. Atherosclerosis is a typical vascular disorder. Atherosclerosis is a disease in which the arteries are hardened by genetic factors related to lipid metabolism and environmental factors such as eating habits, smoking, and lack of exercise, and are likely to occur in the cerebral artery or coronary artery. It develops into circulatory diseases such as cerebrovascular diseases. In the case of cerebral arteriosclerosis, headache, dizziness, and mental disorders are indicated, which is a cause of encephalopathy, and in the case of coronary arteriosclerosis, it is known that it causes pain and arrhythmia in the heart, causing angina and myocardial infarction. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and atherosclerosis is the leading cause of death in modern society, especially among men in their 50s and 60s.

The hypothesis of the early onset of atherosclerosis is the response-to-injury hypothesis, which causes vascular endothelial cells due to genetic variations, peroxides, hypertension, diabetes, increased plasma homocysteine levels, and microbial infections. Is a dysfunction that does not maintain normal homeostasis. When endothelial cells are in a dysfunctional state, cell adhesion substances are greatly expressed and cell permeability is increased, and adhesion to immune cells, platelets, lipids, etc. and permeability to tissues are increased, and inflammatory mediators by these immune cells and Atherosclerotic lesions develop and develop due to inflammatory responses such as growth factor secretion [Ross, R., N. Engl. J. Med. , 340: 115-126, 1999. At this time, low-density lipoprotein (LDL) in the blood is transformed into modified-LDL (MLDL) through oxidation, glycosylation, integration, glycoprotein binding, etc., which stimulate and damage vascular endothelial cells and smooth muscle. Cause [Steinberg, D., J. Biol. Chem. 272: 20963-20966, 1997; Griendling, KK et al. , Circulation , 96: 3264-3265, 1997; Bavab, M. et al. , Arterioscler. Thromb. Vasc. Biol. , 16: 831-42, 1996. As a result, when the expression of vascular cell adhesion molecule-1 (VCAM-1) of endothelial cells and the release of inflammatory mediators of inflammatory cells are promoted, LDL enters and accumulates under the endothelial cells, LDL and oxidized MLDL in turn promote the inflammatory response of the lesion by repeating the process of inducing the influx and activation of immune cells, such as macrophages, T lymphocytes [Rajavashisth, TB et al. , Nature , 344: 254-257, 1990; Quinn, MT et al. , Proc. Natl. Acad. Sci. USA , 84: 2995-2998, 1987. Next, the lesions are necrotic by the action of the introduced macrophages or lymphocytes released from hydrolysases, inflammatory mediators, growth factors, etc., and monocytes are introduced into the necrotic lesions, smooth muscle migration and differentiation, fibrous tissue Iterative process such as the formation of a. Through this process, the lesion tissue develops into a fibrous lesion of a complicated structure in which the fibrous cover is applied to the necrotic tissue with the MLDL nucleus [Fuster, V. et al. , Artherosclerosis and coronary artery disease , vol 1, 539-555, 585-594; vol. 2, 492-510, 1st Edition, Lippincott, 1996], the formation of thrombi from developed lesion tissue and the hardening of the arteries result in circulatory diseases such as blood flow disorders [Ross, R., N. Engl. J. Med. , 340: 115-126, 1999.

LDL oxidation is most important as an early cause of atherosclerosis. Oxidative stress produced in vivo and in vivo transforms LDL in the blood into oxidized-LDL, which enters the intima through an adhesion molecule. Monocytes are introduced into the oxidized-LDL to form foam cells, producing fat streak, an early lesion of atherosclerosis. The early lesions of atherosclerosis are characterized by the expression of VCAM-1, intracellular adhesion molecule-1 (IMC-1) and monocyte chemoattractant protein-1 (MCP-1), which are produced by arterial endothelial cells. It is induced by the transcription factor (NF-κB). NF-κB is a heterodimer composed of p50 and p65 and is a transcription factor involved in regulating the target gene of many cells. Activated NF-κB binds to specific promoter genes such as IL-1, VCAM-1, ICAM-1, and other factors involved in the progression of atherosclerosis, resulting in the expression of various inflammatory factors. It is activated by various factors such as reactive oxygen species and cytokines.

Antioxidants and free radical scavengers since been shown to inhibit this NF-κB activity in case of ingestion of antioxidants adequately inhibit LDL oxidation in vivo (in vivo), and inhibiting the expression of adhesion molecules, and NF-κB It is expected to inhibit the atherosclerosis by reducing the activity of, and studies on this are in progress (Korean Patent Publication No. 2003-0014155). Therefore, there is a growing interest in the combination therapy of administering a lipid-lowering agent together with LDL antioxidant in hyperlipidemia or atherosclerosis patients.

In addition, high blood cholesterol levels are susceptible to coronary cardiovascular disease. Therefore, in order to reduce blood cholesterol levels, it is necessary to reduce dietary intake of cholesterol and fat or to inhibit cholesterol absorption and biosynthesis by inhibiting enzymes related to lipid metabolism. Attempts have been made to reduce LDL levels by inhibiting (Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, D. et al. , N. Engl. J. Med) ., 320: 915-924, 1989).

Probucol, N, N'-diphenylenediamine (N, N'-diphenylenediamine), phenolic synthetic antioxidants BHA (butylatedhydroxyanisol) and BHT (butylated hydroxy toluene), which are currently being used for the treatment of hyperlipidemia, use LDL cholesterol. The antioxidant power of reducing, weakening the degree of oxidation and reducing lesion formation is excellent, but its use is limited because of its many side effects.

On the other hand, Periostracum cicadae means the fault of cicada and horse cicada, also known as 'chosun'. Terminations are curved oval-like cicada-shaped, about 3.5cm long, 2cm in diameter, and the outer surface is yellowish white to yellowish brown, translucent and glossy, almost no smell, and tastes dull. Herbal medicine has been used as an antipyretic and analgesic since ancient times, and it has a calming effect and pain relief effect, so it is used to tickle five grains of sesame seeds that have been removed from the wings and legs. It is also used for the treatment of fever due to cold, chronic relieving asthma, children's race, sore throat, tetanus, itching, ocular, swelling, urticaria. The components reported and isolated from medicinal insect streaks include acetyldopamine compounds [Naoki, N. et al ., Chem. Pharm. Bull. , 48: 1794-1752, 2000].

Therefore, the present inventors, while searching for a new hyperlipidemia and atherosclerosis treatment agent with fewer side effects in natural products, confirmed the excellent antioxidant activity against low-density lipoproteins in extracts of medicinal sesame and acetyldopamine-based compounds isolated therefrom. The invention has been completed.

The present invention seeks to provide novel acetyldopamine compounds.

In addition, the present invention is to provide a method for preparing the acetyldopamine-based compound.

In addition, the present invention is to provide a composition for the prevention and treatment of cardiac circulatory diseases, which comprises the sesame extract or acetyldopamine-based compounds isolated therefrom as an active ingredient.

The present invention provides novel acetyldopamine compounds.

The present invention also provides a method for preparing the acetyldopamine compound.

In another aspect, the present invention provides a composition for the prevention and treatment of cardiovascular disease, which comprises a sesame extract or an acetyldopamine-based compound isolated therefrom as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an acetyldopamine compound represented by the following formula (1).

The acetyldopamine compound of Formula 1 is (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene) -1 , 4-benzodioxane [(2R, 3R) -2- (3 ', 4'-Dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene) -1,4-benzodioxane] It can be used in the form of pharmaceutically acceptable salts and includes all salts, hydrates and solvates prepared by conventional methods.

In addition, the present invention provides a method for preparing an acetyldopamine-based compound represented by Formula 1,

(1) preparing a crude extract by adding a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to the lead;

(2) adding water to the crude extract obtained in the step (1) and suspending, sequentially dividing with n-hexane, chloroform and ethyl acetate to obtain an ethyl acetate soluble extract; And

(3) separating and purifying the ethyl acetate soluble extract obtained in step (2) by chromatography to obtain an acetyldopamine compound.

Specifically, the limbs are washed with water to remove foreign substances, and then dried in the shade. An appropriate amount of solvent is added to the dried lead to complete immersion. The extraction solvent is preferably a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, most preferably ethanol. In addition, the extraction time is preferably 3 days, and the extract obtained above is filtered and concentrated to obtain a crude extract.

Suspended by adding water to the crude extract obtained above, and fractionated sequentially with a nonpolar solvent, preferably n-hexane, chloroform and ethyl acetate, to obtain n-hexane soluble extract, chloroform soluble extract and ethyl acetate soluble extract. Antioxidant effect was measured for each soluble extract and it was confirmed that the antioxidant effect was the best in ethyl acetate soluble extract.

The ethyl acetate soluble extract obtained above was filtered, and then separated into nine fractions (fractions 1 to 9) by silica gel chromatography. At this time, chloroform, methanol, and a mixed solvent thereof are used as the mobile phase solvent. Antioxidant effect was measured for each fraction, and the most effective fractions 7 and 8 were collected to form a reversed phase C-18 (Licroprep RP-18) column as a fixed phase and a mixed solvent of methanol and water, preferably 1: 1 (v / v). Eight fractions are obtained by column chromatography using a mixed solvent mixed with a mobile phase. Antioxidant effect was measured for each fraction to collect the most effective fractions 5 and 6, and preparative high-performance liquid chromatography (prep-HPLC) was performed. At this time, the purified compounds 1 and 2 are obtained by performing column chromatography using a mixed solvent of methanol and water, preferably a mixed solvent mixed at 1: 1 (v / v), as a mobile phase.

Compound 1 is (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene) -1,4-benzodioxane And compound 2 is (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethyl) -1,4-benzo Dioxane, represented by the following formula (2).

는 단일결합 또는 이중결합이다.)(In Formula 2,

Is a single bond or a double bond)

The acetyldopamine-based compound of Formula 2 was used by extraction, separation, and purification from lead, can be prepared by conventional methods, and commercially available reagents can be used.

In another aspect, the present invention provides a composition for the prevention and treatment of cardiovascular disease, which comprises an acetyldopamine-based compound represented by Formula 2, or isolated from it.

Antioxidant activity of low-density lipoproteins of sesame extract and acetyldopamine-based compounds isolated from the present invention was measured by TBARS (Thiobarbituric Acid Reactive Substances) method, and the inhibitory effect on the oxidation of low-density lipoproteins was used as a positive control. It is superior to probucol, and therefore has an excellent antioxidant effect. In addition, as a result of the acute toxicity test conducted in mice, there were no dead individuals and showed no change by toxicity. Therefore, the sesame extract according to the present invention and the acetyldopamine-based compound isolated therefrom can be usefully used for the disease, treatment and prevention of cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low density lipoprotein.

The composition of the present invention comprises 0.0001 to 50 parts by weight of the compound, based on the total weight of the composition.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the sesame extract or acetyldopamine-based compound separated therefrom.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. have.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage is 10-2,000 mg / kg, preferably 50-500 mg / kg for the sesame extract. In addition, in the case of the acetyldopamine compound, it is about 0.1-100 mg / kg, Preferably it is 0.5-10 mg / kg, It is more preferable to divide and administer once or several times a day.

As a result of oral administration of the compound of the present invention to mice, 50% lethal dose (LD ₅₀ ) by oral administration toxicity test is judged to be a safe substance of at least 1,000 mg / kg or more.

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 may be a health food for the purpose of improving cardiovascular disease.

When the sesame extract or acetyldopamine-based compound separated therefrom according to the present invention is used as a food additive, the sesame extract or the acetyldopamine-based compound separated therefrom may be added as it is or may be used together with other food or food ingredients, and It can be suitably used according to the phosphorus method. 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 prior art extract of the present invention or the acetyldopamine compound separated therefrom is added in an amount of 1 to 20 parts by weight, preferably 5 to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. have.

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, drinks, Alcoholic beverages and vitamin complexes, and the like and include 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 natural carbohydrates described above 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 said 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 usually 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 1 Preparation of Sesame Extract of the Invention and Acetyl Dopamine Compound Isolated therefrom

1-1: Preparation of Piping Crude Extract

500 ml of water was added to 200 g of dried sesame (Jeju-do, South Korea), and boiled at 90 ° C. for 4 hours, followed by filtration to obtain an extract and a solid residue. The obtained extract was concentrated under reduced pressure to obtain 7 g of hot water extract.

In addition, 500 ml of 95% ethanol (EtOH) was added to 200 g of dried sesame (Jeju-do, Korea), and the mixture was left at room temperature for 3 days, filtered through a filter paper, and concentrated to obtain 2.9 g of an oily substance as a sesame ethanol extract.

As a result of measuring the antioxidant activity according to the method of Experimental Example 1 for the hot water extract and the ethanol extract, the ethanol extract was used in the following examples because the antioxidant activity of the ethanol extract was excellent.

1-2: Preparation of Pulmonary Nonpolar Solvent Soluble Extract

200 ml of water was added to the first ethanol extract obtained in Example 1-1, suspended, n-hexane ( n -hexane), chloroform (CHCl ₃ ) and ethyl acetate (EtOAc), and then n-hexane soluble. 0.56 g of an extract ( n- hexane-soluble), 0.43 g of chloroform soluble extract (CHCl ₃ -soluble) and 1.25 g of ethyl acetate soluble extract (EtOAc-soluble) were obtained, respectively.

As a result of measuring the antioxidant effect in the same manner as in Experimental Example 1 for each fraction, it was confirmed that the antioxidant activity is excellent in the ethyl acetate soluble extract and used in Example 1-3 below.

1-3: Separation of Compounds of the Invention from Fused Ethyl Acetate Fusible Extract

1.25 g of the ethyl acetate soluble extract obtained in Example 1-2 were separated in two steps using column chromatography.

First, silica gel column chromatography (silica gel: Merk, Art 9385, column size: φ7 × 40 cm) was 100% chloroform, chloroform: methanol = 95: 1, 90: 1, 60: 1, 30: 1, 10: 500 mL of each of 1, 5: 1, 1: 1 (v / v) and 100% of mobile phase solvent conditions of methanol was developed to obtain nine fractions (Fr. 1 to 9). As a result of measuring the antioxidant activity of each fraction by the same method as in Experimental Example 1, 7 to 8 fractions (chloroform: methanol = 5: 1 and 1: 1 solvent conditions, yield 287 mg) showing the best antioxidant activity were isolated. Then, a second column chromatography was performed.

Reverse column C-18 column chromatography (Licroprep RP-18: Merck, Art 13900, column size: φ 2.5 × 15 cm) was performed. At this time, 50 ml each of the solvents were developed under a methanol: water = 1: 1 (v / v) mobile phase solvent condition to obtain 8 fractions, and the antioxidant activity of each fraction was measured in the same manner as in Experimental Example 1. As a result, 5-6 fractions (Fr. 5-6, 138 mg) which were the most excellent in antioxidant activity were obtained.

Finally, two purified from fractions 5-6 obtained by preparative high-performance liquid chromatography (preparative HPLC, Hydrosphere C-18 column, 250 x 20 mm, methanol / water = 1: 1 solvent conditions). Compound was obtained, compound 1 yield was 6.3 mg, compound 2 yield was 5.4 mg.

1-4: Structural Analysis of Compounds of the Invention

The following analysis was performed for structural analysis of the two compounds obtained in Examples 1-3.

Molecular weight and molecular formula were determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima, Micromass, UK, and the fluorescence was determined using a polarizer (DIP-181 digital polarimeter, Jasco, Japan). Nuclear magnetic resonance (NMR) analysis (AMX 500, Bruker, Germany) also revealed ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC (1H-Detected heteronuclear Multiple-Quantum Coherence) and HMBC (Heteronuclear). Multiple-Bond Coherence (DEP) and Distortionless Enhancement by Polarization (DEPT) spectra were obtained and the molecular structure was determined.

The results of the above instrumental analysis are published in Naoki, N. et al ., Chem. Pharm. Bull , 48: 1749-1752, 2000], the compound of the present invention is represented by the formula (2), compound 1 is (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl ) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene) -1,4-benzodioxane [(2R, 3R) -2- (3 ', 4'-Dihydroxyphenyl) -3-acetylamino -6- (N-acetyl-2 "-aminoethylene) -1,4-benzodioxane] identified a novel compound that has not yet been reported. In addition, compound 2 is (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethyl) -1,4-benzo Dioxane [(2R, 3R) -2- (3 ', 4'-Dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethyl) -1,4-benzodioxane] was identified.

[Compound 1]

(2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene) -1,4-benzodioxane.

1) Physical property: white powder

2) Radiance: [α] _D ²⁵ + 33.8 ° ( c = 0.1, MeOH)

3) Molecular Weight: 384

4) Molecular Formula: C ₂₀ H ₂₀ N ₂ O ₆

5) ¹ H NMR (CD ₃ OD, 500 MHz) δ 1.86 (3H, s, H-3b), 2.02 (3H, s, H-5 "), 4.69 (1H, d, J = 7.1 Hz, H- 2), 5.68 (1H, d, J = 7.1 Hz, H-3), 6.09 (1H, d, J = 14.7 Hz, H-1 "), 6.73-6.81 (3H, m, H-8, H- 5, H-6), 6.84-6.89 (3H, m, H-5 ', H-2', H-6 '), 7.29 (1H, d, J = 14.7 Hz, H-2 ").

6) ¹³ C NMR (CD ₃ OD, 125 MHz) δ 22.5 (C-3b), 22.6 (C-5 "), 78.3 (C-3), 78.4 (C-2), 113.9 (C-1") , 173.2 (C-2b), 114.7 (C-2 '), 115.6 (C-5'), 116.3 (C-5), 118.3 (C-8), 120.3 (C-6 '), 120.6 (C- 6), 122.9 (C-2 "), 128.7 (C-1 '), 128.7 (C-1'), 131.9 (C-7), 142.6 (C-4a), 144.6 (C-8a), 146.5 ( C-3 '), 147.2 (C-4'), 170.6 (C-4 ").

7) EI-MS m / z : 384 [M] ⁺ , 51 (100), 63 (45), 76 (50), 104 (50), 123 (20), 132 (40), 148 (15), 384 (5).

[Compound 2]

(2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethyl) -1,4-benzodioxane.

1) Physical property: yellow powder

2) Radiance: [α] _D ²⁵ + 20.2 (c = 0.8, MeOH)

3) Molecular Weight: 386

4) Molecular Formula: C ₂₀ H ₂₂ N ₂ O ₆

5) ¹ H NMR (CD ₃ OD, 500 MHz) δ 1.86 (3H, s, H-3b), 1.89 (3H, s, H-5 "), 2.68 (2H, t, J = 7.2 Hz, H- 1 "), 3.21 (2H, t, J = 7.2 HZ, H-2"), 4.68 (1H, d, J = 7.1 Hz, H-2), 5.66 (1H, d, J = 7.1 Hz, H- 3), 6.71-6.76 (3H, m, H-8, H-5, H-6), 6.78-6.88 (3H, m, H-5 ', H-2', H-6 ').

6) ¹³ C NMR (CD ₃ OD, 125 MHz): 22.5 (C-3b), 22.6 (C-5 "), 35.8 (C-1"), 42.1 (C-2 "), 78.2 (C-3 ), 78.3 (C-2), 115.6 (C-2 '), 116.3 (C-5'), 117.9 (C-5), 118.1 (C-8), 120.6 (C-6 '), 123.2 (C -6), 128.8 (C-1 '), 134.1 (C-7), 142.2 (C-4a), 144.3 (C-8a), 146.4 (C-3'), 147.1 (C-4 '), 173.2 (C-4 "), 173.2 (C-2b).

7) EIMS (rel. Int.) M / z : 386 [M] ⁺ , 51 (20), 77 (22), 94 (5), 104 (15), 123 (75), 136 (100), 151 (85), 193 (60), 327 (15), 386 (65).

Experimental Example 1 Determination of Antioxidant Activity of the Compound of the Present Invention by TBARS (Thiobarbituric Acid Reactive Substances) Method

In order to measure the antioxidant activity of the acetyl dopamine-based compound of the present invention on low-density lipoprotein, the following experiment was performed.

Antioxidant activity was determined by TBA (thiobarbituric acid) method, which is a aldehyde (dialdehyde), an oxidation product of unsaturated fatty acids produced by Cu ²⁺ , which is known to induce oxidation of low density lipoprotein (Cu ²⁺ -mediated LDL-oxidation). Packer, L. Ed., Methods in Enzymology . Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego, 1994].

300 ml of plasma was collected from humans, and centrifuged at 100,000 × g for 24 hours using an ultracentrifuge to remove the high-density lipoprotein (VLDL) / chylomicron layer suspended in the upper layer. After adjusting to g / ml, 25 ml (1.5-2.5 mg protein / ml) of low-density lipoprotein suspended in the upper layer was again separated by centrifugation at 100,000 × g for 24 hours. 20 μl of the low-density lipoprotein isolated above (protein concentration, 50-100 μg / ml) was mixed with 210 μl of 10 mM phosphate-buffered saline (PBS), and each solvent extract prepared in Example 1 above. 10 μl of each fraction and a solution of the compounds were added. At this time, the compounds were dissolved in DMSO (dimethylsulfoxide) and used, and diluted to various concentrations before using in the experiment. As a negative control, only the solvent DMSO was added, and as a positive control, probucol was added. 10 μl of 0.25 mM CuSO ₄ was added to the solution for 4 hours at 37 ° C., and 1 ml of 20% trichloroacetic acid (TCA) solution was added to stop the reaction. Next, 1 ml of a 0.67% TBA solution dissolved in 0.05N NaOH solution was added, stirred for 10 seconds, heated at 95 ° C. for 5 minutes to cause a color reaction, and the solution was cooled with ice water. Thereafter, the supernatant was separated by centrifuging the solution at 3000 rpm for 5 minutes, and the absorbance at 540 nm was measured by an ultraviolet-vis spectrophotometer to measure the amount of malondialdehyde (MDA) produced by the color reaction. It was.

On the other hand, to obtain the standard curve of malondialdehyde, dilute the stock solution of malonaldehyde bis (dimethylacetal) to make 250 µl of PBS standard solution containing 0-10 nmol malondialdehyde. Color development was carried out in the same manner as described above, and the absorbance at 540 nm was measured to obtain a standard curve of malondialdehyde. The standard curve was used to quantify the amount of malondialdehyde produced from the compounds of the present invention.

The results are shown in Table 1.

Antioxidant Activity of Low Density Lipoproteins of Compounds of the Present Invention sample % Inhibition or IC ₅₀ (μM) Occurred Hot Water Extract (40 ㎍ / mL) 45% inhibition Leaded Ethanol Extract (40 μg / ml) 60% inhibition n-hexane (40 μg / ml) 8% inhibition Chloroform (40 μg / ml) 11% inhibition Ethyl acetate (40 ㎍ / ml) 85% inhibition Compound 1 2.1 μM Compound 2 1.5 μM Positive control (Probucol) 3.6 μM

As shown in Table 1, the hydrothermal extract obtained in Example 1-1 showed an antioxidant effect of 45% at 40 ㎍ / ㎖, while the ethanol extract showed an antioxidant effect of 60% at the same concentration, the antioxidant effect of the ethanol extract Confirmed better.

In addition, the ethyl acetate solvent extract of each solvent extract obtained in Example 1-2 showed an antioxidant effect of 85% against the low density lipoprotein at a concentration of 40 ㎍ / ㎖, the most excellent antioxidant activity compared to the soluble extract of n-hexane, chloroform It was confirmed that there is.

Furthermore, (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-aminoethylene which is the compound 1 obtained in Example 1-3 ) -1,4-benzodioxane and compound 2 (2R, 3R) -2- (3 ', 4'-dihydroxyphenyl) -3-acetylamino-6- (N-acetyl-2 "-amino ethyl) antioxidant activity of the 1,4-benzodioxane confirmed the antioxidant activity is superior to the IC ₅₀ is shown below the IC ₅₀ of a positive control with 2.1 μM and 1.5 μM, respectively low density lipoprotein.

Therefore, the sesame extract of the present invention and the acetyldopamine-based compound isolated therefrom can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low density lipoprotein.

Experimental Example 2: Acute Toxicity Test

In order to determine the acute toxicity of the sesame extract and acetyldopamine compounds of the present invention, the following experiment was performed.

SPF (specific pathogens free) ICR mice were divided into 4 groups (3 males and 3 females / experimental group) at 4 weeks of age, temperature 22 ± 3 ℃, humidity 55 ± 10%, illumination 12L / 12D It was bred in the animal room of the. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (for mice and rats, Cheil Jedang Co., Seoul, South Korea) and negative water were supplied after sterilization and freely ingested.

Compounds 1 and 2 prepared in Examples 1-3 were prepared at a concentration of 50 mg / ml in 0.5% tween 80, and then 0.04 ml (100 mg / kg) and 0.2 ml (500 mg) per 20 g of mouse body weight. / Kg) and 0.4 ml (1,000 mg / kg) orally. Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least 1 hour, 4 hours, 8 hours, 12 hours after the administration, and at least once every morning and afternoon from the day after the administration. In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. Changes in body weight were measured at intervals of 1 day from the day of administration to observe the weight loss phenomenon of animals caused by Compounds 1 and 2.

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

Therefore, the compounds of the present invention did not show a change in toxicity in all mice up to 1,000 mg / kg, it was determined that oral administration minimum dose (LD ₅₀ ) is a safe substance of 1,000 mg / kg or more.

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

Formulation Example 1 Preparation of a Pharmaceutical Formulation

1-1. Manufacture of powder

Sesame extract or acetyldopamine compound isolated therefrom according to the present invention

                                                                     2 g

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Sesame extract or acetyldopamine compound isolated therefrom according to the present invention

                                                                  100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of Capsules

Sesame extract or acetyldopamine compound isolated therefrom according to the present invention

                                                                  100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Preparation of Injection

Sesame extract or acetyldopamine compound isolated therefrom according to the present invention

                                                               10 μg / ml

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

In a suitable volume of injectable sodium chloride BP, the sesame extract or isolated acetyldopamine compound according to the present invention is dissolved, and the pH of the resulting solution is adjusted to pH 3.5 using dilute hydrochloric acid BP, and the injectable sodium chloride BP is Volume to adjust and thoroughly mix. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Formulation Example 2 Preparation of Food

Foods containing the compound of the present invention were prepared as follows.

2-1. Preparation of Cooking Seasonings

0.2 to 10 parts by weight of the sesame extract or acetyldopamine-based compound isolated from the extract according to the present invention was prepared for cooking for health promotion.

2-2. Preparation of Tomato Ketchup and Sauce

Health care tomato ketchup or sauce was prepared by adding 0.2-1.0 parts by weight of the sesame extract or acetyldopamine-based compound isolated therefrom to tomato ketchup or sauce.

2-3. Manufacture of Flour Food

0.1 to 5.0 parts by weight of the sesame extract or acetyldopamine-based compound isolated therefrom according to the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

2-4. Preparation of soups and gravy

0.1-1.0 parts by weight of the sesame extract or acetyldopamine-based compound isolated therefrom according to the present invention was added to soups and broths to prepare meat products for health promotion, soups and broths of noodles.

2-5. Preparation of Ground Beef

The ground beef for health promotion was prepared by adding 10 parts by weight of the sesame extract or acetyldopamine-based compound isolated therefrom to the ground beef.

2-6. Manufacture of dairy products

0.1 to 1.0 parts by weight of the sesame extract or acetyldopamine-based compound isolated therefrom according to the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

2-7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were 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.

The decanted extract according to the present invention or the acetyldopamine-based compound separated therefrom was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh by a grinder to obtain a dry powder.

The grains, seeds and the dry powder of the sesame extract according to the present invention or the acetyldopamine-based compound separated therefrom were prepared by blending the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

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

Dry powder (1 part by weight) of the sesame extract or acetyldopamine-based compound isolated therefrom according to the present invention,

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

Formulation Example 3 Preparation of Beverage

3-1. 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 therein to make a syrup, and the syrup is 85-98. Sterilizing at 20 ℃ for 180 seconds, mixed with cooling water at a ratio of 1: 4, and then injected with 0.5 to 0.82% of carbon dioxide gas to prepare a carbonated beverage containing a sesame extract or an acetyldopamine compound separated therefrom according to the present invention. It was.

3-2. Manufacture of health drinks

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and sesame extracts according to the present invention or acetyldopamine compounds isolated therefrom After sterilization by the homogeneous formulation, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.

3-3. Preparation of Vegetable Juice

0.5 g of acetyldopamine compounds isolated from the sesame extract according to the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-4. Preparation of Fruit Juice

Health promotion fruit juice was prepared by adding 0.1 g of the sesame extract or acetyldopamine-based compound isolated therefrom to 1,000 ml of apple or grape juice.

Since the extract of the present invention and the acetyldopamine-based compound isolated therefrom have excellent antioxidant activity against low-density lipoproteins, the composition comprising the same can be used for cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low-density lipoproteins. It can be usefully used for prevention and treatment.

Claims (11)

delete delete (1) preparing a crude extract by adding a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to the lead; (2) adding water to the crude extract obtained in the step (1) and suspending, sequentially dividing with n-hexane, chloroform and ethyl acetate to obtain an ethyl acetate soluble extract; And (3) A method for preparing an acetyldopamine compound represented by the following Chemical Formula 1 comprising the step of separating and purifying the ethyl acetate soluble extract obtained in step (2) by chromatography to obtain an acetyldopamine compound. <Formula 1>

4. The method of claim 3, wherein the alcohol in step (1) is selected from methanol, ethanol and butanol. A composition for the prevention and treatment of hyperlipidemia or arteriosclerosis disease, which comprises the extract of the vertebrae as an active ingredient. 6. The composition for preventing and treating hyperlipidemia or atherosclerosis disease according to claim 5, wherein the sesame extract is obtained by extracting sesame from water, methanol, ethanol, butanol or a mixed solvent thereof. 7. The composition for preventing and treating hyperlipidemia or atherosclerosis disease according to claim 6, wherein the sesame extract is an ethanol extract of sesame. 8. The composition for preventing and treating hyperlipidemia or atherosclerosis disease according to claim 7, wherein the sesame extract is an ethyl acetate soluble extract obtained by sequentially dividing the sesame ethanol extract with n-hexane, chloroform, and ethyl acetate. A composition for the prevention and treatment of hyperlipidemia or atherosclerosis disease comprising the acetyldopamine compound represented by the following formula (2) as an active ingredient. <Formula 2>

(In Formula 2,

Is a single bond or a double bond) A composition for the prevention and treatment of hyperlipidemia or arteriosclerosis disease comprising the acetyldopamine compound represented by the formula (1) of claim 3 as an active ingredient. Prevention and improvement of hyperlipidemia or arteriosclerosis disease containing a prior art extract extracted from water, methanol, ethanol, butanol or a mixed solvent thereof or the compound represented by the formula (1) or the compound represented by the formula (2) of claim 3 as an active ingredient Dietary supplements.