KR100724088B1 - Composition containing flavonoid compounds for the prevention and treatment of cardiovascular disease - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of cardiac circulatory diseases, including a flavonoid compound, and more particularly, the flavonoid compound isolated and purified from Cudrania japonica may have excellent antioxidant activity against low density lipid protein (LDL). In addition, it effectively inhibits the activity of acyl COA: cholesterol transferase (ACAT). Therefore, the flavonoid compound according to the present invention is for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction, which are known to be caused by oxidation of low density lipid protein or synthesis and accumulation of cholesteryl esters. It can be usefully used in the composition.

Description

Composition containing flavonoid compounds for the prevention and treatment of cardiovascular disease

The present invention relates to a composition for the prevention and treatment of cardiovascular diseases comprising a flavonoid compound.

Recently, vascular disorders such as arteriosclerosis have increased significantly along with the increase in adult disease. 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 and cause cerebral pneumoconiosis, and in the case of coronary arteriosclerosis, pain and arrhythmia in the heart are known to cause angina pectoris and myocardial infarction. 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.

The hypothesis of the early onset of atherosclerosis is a chronic inflammatory process for damage of the vessel wall, which is suggested as a response-to-injury hypothesis [Circ. Res. 2001, 89, 298-304. This is a dysfunction state in which vascular endothelial cells fail to maintain normal homeostasis due to genetic variations, peroxides, hypertension, diabetes, increased plasma homocysteine concentrations, and / or microbial infections.

More specifically, these causes cause low-density lipoprotein (LDL) in the blood to change to highly modified-LDL (HM-LDL) through oxidation, glycemic binding, integration, glycoprotein binding, and the like. They stimulate and cause damage to vascular endothelial cells and smooth muscle. 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 HM-LDL in turn to induce the influx and activation of immune cells, such as macrophages, T lymphocytes to promote the inflammatory response of the lesion. 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, and fibrous tissues. Iterative process such as the formation of a. Through this process, the lesion tissue develops into a fibrous lesion of a complex structure covered with fiber in the necrotic tissue that has HM-LDL as its nucleus, and circulatory disorders such as blood flow disorder are generated due to the formation of blood clots and hardening of the arteries. Will appear.

LDL oxidation is most important as an early cause of atherosclerosis, including atherosclerosis [Circulation, 1995, 91, 2488-2496; Arterioscler. Thromb. Vasc. Biol., 1997, 17, 3338-3346. 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. Atherosclerotic early lesions 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). In addition, NF-κB causes plaque formation and rupture in the vessel wall. NF-κB is activated by various factors such as reactive oxygen species and cytokines, and is a heterodimer composed of p50 and p65, which is a transcription factor involved in regulating the target gene of many cells. to be. 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. Adjust

Antioxidants and free radical scavengers, so is found to inhibit this NF-κB activity in case of ingestion of antioxidants as appropriate, inhibit LDL oxidation in vivo (in vivo), and inhibiting the expression of adhesion molecules and NF- It is expected to inhibit the atherosclerosis by reducing the activity of κB, and studies are underway (Korea Patent Publication No. 2003-0014155). In addition, studies on the factors and the removal of LDL peroxide in hyperlipidemia and atherosclerosis patients [Curr. Atheroscler. Res., 2000, 2, 363-372 are also actively progressing.

In addition, high blood cholesterol levels are likely to cause coronary cardiovascular disease. Therefore, in order to reduce blood cholesterol levels, it is necessary to suppress the absorption of cholesterol by dieting to reduce the intake of cholesterol and fat or by inhibiting enzymes related to lipid metabolism. . Thus, many studies have been conducted on acyl-CoA: cholesterol acyltransferase (ACAT), an enzyme that esterifies cholesterol.

The mechanism of action of ACAT occurs largely in three parts of the intestinal, liver and vascular wall cells of the body.

First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol that is absorbed from the outside or biosynthesized in the body is accumulated in a carrier called very low-density lipoprotein (VLDL) in the liver and then supplied to each organ of the body through blood vessels. The conversion allows for 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. In addition, since foam cells contain a large amount of cholesteryl ester derived from cholesterol by ACAT activity, the formation of foam cells derived from macrophages and smooth muscle cells is very important from an experimental and clinical aspect. Since the proliferation of foam cells in the vessel wall is directly related to increased ACAT activity, the development of ACAT inhibitors as a potent antiarterial agent is desirable.

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 from 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 microsomal ACAT or rat hepatic macrophage (J774) ACAT. Human ACAT includes hACAT-1 (50 kDa) and hACAT-2 (46 kDa), hACAT-1 mainly acts on the liver, adrenal glands, macrophages and kidneys of adults and hACAT-2 acts on the 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 Med. 6, 1341-1347, 2000.

Probucol, N, N'-Diphenylenediamine (N, N'-Diphenylenediamine), a phenolic synthetic antioxidant, BHA (Butylatedhydroxyanisol) and BHT (Butylated hydroxy toluene), which are currently used to treat hyperlipidemia, use LDL cholesterol. It is reduced, weakens the degree of oxidation, and reduces the lesion formation, the antioxidant power is excellent, but many side effects are limited use.

Therefore, attempts have been made to reduce plasma low-density lipoprotein (LDL) levels by inhibiting cholesterol absorption and inhibiting biosynthesis for the purpose of preventing such diseases [Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, N. Engl. J. Med., 1989, 320, 915-924. In addition, in patients with hyperlipidemia or atherosclerosis, there is a growing interest in the concurrent administration of lipid-lowering agents with LDL antioxidants.

Meanwhile, Cudrania tricuspidata is a deciduous arborescent or shrub belonging to the Mulberry family, and is mainly distributed in Korea, China, and Japan. Components reported and isolated from cucurbita include cudraxanthone, Hano, Y. et al. , Planta Med ., 56: 478, 1990; Zou, Y.-S. et al. Bioorg. Med. Chem. , 12: 1947, 2004), and flavonoids (Lee, IK et al . J. Nat. Prod ., 58: 1614, 1995). The leaves of Cudrania japonica are used for the treatment of eczema, mumps, pulmonary tuberculosis, chronic low back pain, bruises and acute arthritis in oriental medicine. . It is reported that the stem contains a lot of flavonoid compounds that are effective in lung cancer, colon cancer, skin cancer and uterine cancer. In particular, according to the empirical clinical effects mentioned in herbal milk (本草 拾遺), bark and root bark have been used as folk remedies because they are called baekpipipi to cool the blood and excel in releasing muscles and tendons. .

In the prior art using the kkujimi tree, Korean patent No. 128944, by culturing the hairy roots derived from kkujimi tree plant cells by plant cell tissue culture technology, three times the conventional method of the anti-cancer active material geurikudranin derivatives The company is offering a new method that can produce more than 17 times in volume. However, there is no published report on its antioxidant activity or its effects on cholesterol and related metabolism.

Therefore, the present inventors, while searching for a new hyperlipidemia and atherosclerosis treatment agent with fewer side effects in natural products, it was confirmed that the flavonoid compound isolated from the cooji mulberry has antioxidant activity against LDL and excellent inhibitory activity against ACAT enzyme, and hyperlipidemia The present invention has been completed by confirming that it can be used for the prevention and treatment of cardiovascular diseases such as coronary heart disease, arteriosclerosis and myocardial infarction.

The present invention is to provide a composition for the prevention and treatment of heart circulatory diseases, including flavonoid compounds isolated and purified from Koji.

The present invention provides a composition for the prevention and treatment of cardiovascular diseases comprising a flavonoid compound represented by the following formula (1).

The flavonoid-based compound can be purchased and used commercially available, can be used in the production method known in the art, a method for preparing to separate from the kkujijak according to the present invention or a modified production method thereof.

(In the above formula,

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently H, OH, C ₁ -C ₄ alkoxy or C ₂ -C ₇ alkenyl,

R ₁ and R ₂ may form a pyran substituted with each other with pyran or C ₁ -C ₄ alkyl,

R ₄ and R ₅ may form pyran substituted with each other with pyran or C ₁ -C ₄ alkyl.)

Preferably, the flavonoid compound is

2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6-prenylflavanone;

2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) -3-prenylflavone;

2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -8- (3-hydroxy-3- methylbutyl) flavanone;

2,4,5,7-tetrahydroxy-5,6-diprenyl flavanone; or

2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) flavone.

Each formula is shown in Table 1 below.

compound Chemical formula One

2

3

4

5

The flavonoid compound has an antioxidant activity against LDL and has an excellent effect of inhibiting the activity of ACAT, an enzyme that esterifies cholesterol. Therefore, it can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis, and myocardial infarction, which are known to be induced by the oxidation of LDL or 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 to the flavonoid compound of Formula 1 above.

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, as necessary, including antioxidants, buffers, 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 a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

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 diet, administration time, administration method, excretion rate and severity of disease. The daily dose of the flavonoid compound of Formula 1 according to the present invention is 0.01 to 100 mg / kg, preferably 0.05 to 10 mg / kg, and more preferably administered once daily to several times.

As a result of conducting toxicity experiments by orally administering the flavonoid compound of Formula 1 to the mouse, 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 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 flavonoid compound of the formula 1 of the present invention is used as a food additive, the flavonoid compound of the formula 1 may be added as it is, or may be used together with other food or food ingredients, and may be appropriately used according to a conventional 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, the flavonoid compound of the formula (1) according to the present invention is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw material. 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 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, 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 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 proportion 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 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 and Experimental Examples.

Example 1 Extraction, Separation and Purification of Flavonoid Compounds

1-1: Extraction, Separation and Purification of Flavonoid Compounds from Cucumber Root

2 kg of roots of Kudji mulberry purchased from Hapcheon, Korea were washed and dried. The roots of the dried Cudrania tree were added to 4 L of chloroform, and extracted twice repeatedly while standing at room temperature for 5 days. The extract was filtered through filter paper, and then concentrated under reduced pressure to obtain a chloroform extract (62 g) as a brown oily substance. The mixture was suspended in 1.5 L of a mixed solution of water and methanol (9: 1 volume ratio), and then partitioned into n -hexane and chloroform in order to obtain an oily substance of 21.7 g of soluble extract of n -hexane and 37.2 g of soluble chloroform. .

To 37.2 g of the chloroform soluble extract obtained above, silica gel column chromatography (500 g of silica gel, 70 to 230 mesh (100 g of chloroform) and a mixed solvent of chloroform and acetone (40: 1 to 1: 1 volume ratio) were used as the mobile phase. mesh)) and separated into 11 fractions ( Fr.1-11 ). For the sixth fraction ( Fr.6 , 0.9 g), silica gel column chromatography (100 g, 230 to 400 mesh) was again performed using a mixed solvent of chloroform and acetone (40: 1 to 8: 1 volume ratio) as a mobile phase. 60 fractions ( Fr. 6-1 ~ 60 ) were obtained. The 21-28 fraction ( Fr.6-21-28 ) was concentrated under reduced pressure, and 120 mg of compound 1 was obtained.

Further, Preparative TLC (Preparative TLC) was carried out using a mixed solvent of chloroform and acetone (15: 1 volume ratio) as a mobile phase for the seventh fraction ( Fr.7 , 96 mg) of the chloroform soluble extract to obtain 21 mg of Compound 2 . Separated.

In addition, silica gel column chromatography (8 g, 230-400) using a mixed solvent of chloroform and acetone (30: 1 to 2: 1 by volume) for the eighth fraction ( Fr.8, 2.5 g) of the chloroform soluble extract was used. Mesh) to obtain seven fractions ( Fr.8-1-7 ). Perform silica gel column chromatography (40 g, 230-400 mesh) once again using a mixed solvent of chloroform and acetone (40: 1 to 4: 1 by volume) for the double Fr.8-2 fraction (280 mg) as a mobile phase. It was. 28 mg of Compound 3 was isolated from the obtained Fr.8-2-13-20 fraction.

In addition, silica gel column chromatography (3 g, 230-400 mesh) using chloroform and a mixed solvent of chloroform and acetone (40: 1 to 1: 1 volume ratio) as a mobile phase for the Fr.8-4 fraction (640 mg). ) Were separated into 65 fractions ( Fr.8-4-1 ~ 65 ), and 130 mg of Compound 4 was extracted from the fraction eluted with a mixed solvent of chloroform and acetone (3: 1 or 6: 1 by volume). Separated.

In addition, silica gel column chromatography (2 g, 230-400 mesh) was carried out using a mixed solvent of chloroform and acetone (3: 1 volume ratio) as a mobile phase with respect to the Fr.8-5 fraction (50 mg), and 10 mg of the compound. 5 was separated.

1-2: Structural Analysis of Flavonoid Compounds

The molecular weight and molecular formula of the compound obtained in Example 1-1 were determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima. In addition, ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC (1H-Detected heteronuclear Multiple-Quantum Coherence), HMBC (Heteronuclear Multiple-) through nuclear magnetic resonance (NMR) analysis (Bruker AM 500) Bond Coherence) and DEPT (Distortionless Enhancement by Polarization) spectra were obtained to determine the molecular structure.

The measurement results are shown below.

The results of the comparative analysis of the measurement results with those of the published literature showed that Compound 1 in the compound of Formula 1 was 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6 Prenylflavanone [2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6-prenylflavanone], compound 2 is 2', 4 ', 5-trihydroxy-6 , 7- (2,2-dimethylchromeno) -3-prenylflavone [2 ', 4', 5-Trihydroxy-6,7- (2,2-dimethylchromeno) -3-prenylflavone], compound 3 is 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -8- (3-hydroxy-3-methylbutyl) flavanone [2', 5,7 -trihydroxy-4 ', 5'-(2,2-dimethylchromeno) -8- (3-hydroxy-3-methylbutyl) flavanone], compound 4 is 2,4,5,7-tetrahydroxy-5,6- Diprenyl flavanone [2,4,5,7-tetrahydroxy-5,6-diprenyl flavanone] and compound 5 are 2 ', 4', 5-trihydroxy-6,7- (2,2-dimethyl Chromeno) flavone [2 ', 4', 5-Trihydroxy-6,7- (2,2-dimethylchromeno) flavone].

[Compound 1]: 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6-prenylflavanone

1) Physical property: Pale yellow needles (m.p. 197 ℃)

2) Molecular Weight: 422

3) Molecular formula: C ₂₅ H ₂₆ O

4) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 1.41 (s, 3H, H-4 ″ ′, 5 ″ ′), 1.76 (s, 3H, H-4 ″), 1.81 (s, 3H, H -5 "), 2.87 (dd, J = 17.3, 3.0 Hz, 1H, H-3), 3.13 (dd, J = 17.3, 12.7 Hz, 1H, H-3), 3.35 (2H, br d, J = 7.1 Hz, H-1 "), 5.25 (m, 1H, H-2"), 5.49 (1H, d, J = 9.8 Hz, H-2 "'), 5.56 (1H, dd, J = 12.6, 2.9 Hz, H-2), 6.01 (s, 1H, H-8), 6.25 (1H, d, J = 9.8 Hz, H-1 "'), 6.33 (s, 1H, H-3'), 6.88 ( s, 1H, H-6 '), 12.37 (s, 1H, OH-5).

5) EIMS m / z 422 [M] ⁺ (32), 407 (18), 404 (33), 389 (7), 220 (4), 202 (1), 187 (100), 165 (47).

[Compound 2]: 2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) -3-prenylflavone

1) Physical property: pale yellow powder (m.p. 126 ℃)

2) Molecular Weight: 420

3) Molecular formula: C ₂₃ H ₂₄ O ₆

4) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 1.44 (s, 3H, H-4 ″ ′, 5 ″ ′), 1.52 (s, 3H, H-5 ″), 3.11 (2H, br d, J = 6.7 Hz, H-1 "), 5.11 (m, 1H, H-2"), 5.59 (1H, d, J = 10.0 Hz, H-2 "'), 6.25 (s, 1H, H-8 ), 6.49 (1H, d, J = 0.3 Hz, H-3 '), 6.50 (1H, dd, J = 8.9, 0.3 Hz, H-5'), 6.70 (1H, d, J = 10.0 Hz, H -1 "'), 7.17 (1H, d, J = 8.9 Hz, H-6'), 13.14 (s, 1H, OH-5).

5) EIMS m / z 420 [M] ⁺ (8), 405 (36), 377 (47), 203 (100), 174 (21); HERIMS m / z 420.1553 [M] ⁺ (calcd for C ₂₃ H ₂₄ O ₆ , 420.1571).

[Compound 3]: 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -8- (3-hydroxy-3-methylbutyl) flavanone

1) Physical property: Yellow powder (m.p. 126 ℃)

2) Molecular Weight: 440

3) Molecular formula: C ₂₅ H ₂₈ O ₇

4) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 1.25 (s, 3H, H-4 ″, 5 ″), 1.38 (s, 3H, H-4 ″ ′, 5 ″ ′), 1.64 (m, 2H, H-2 "), 2.61 (m, 2H, H-1"), 2.72 (1H, dd, J = 17.1, 2.7 Hz, H-3), 3.02 (1H, dd, J = 17.1 13.2 Hz, H-3), 5.50 (1H, d, J = 9.8 Hz, H-2 "'), 5.57 (1H, dd, J = 13.2, 2.7 Hz, H-2), 5.97 (s, 1H, H-6 ), 6.25 (s, 1H, H-3 '), 6.30 (1H, d, J = 9.8 Hz, H-1 "'), 7.09 (s, 1H, H-6 ').

5) EIMS m / z 440 [M] ⁺ (6), 422 (29), 404 (47), 387 (5), 361 (14), 349 (71), 213 (64), 187 (100), 165 (60); HERIMS m / z 440.1834 [M] ⁺ (calcd for C ₂₅ H ₂₈ O ₇ , 440.1835).

[Compound 4]: 2,4,5,7-tetrahydroxy-5,6-diprenyl flavanone

1) Physical property: pale yellow powder (m.p. 189 ℃)

2) Molecular Weight: 424

3) Molecular formula: C ₂₅ H ₂₈ O ₆

4) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 1.73 (s, 3H, H-4 ″, 5 ″), 1.74 (s, 3H, H-4 ″ ′), 1.79 (s, 3H, H − 5 "'), 2.83 (1H, dd, J = 17.3, 3.0 Hz, H-3), 3.15 (1H, dd, J = 17.3, 12.8 Hz, H-3), 3.25 (2H, br d, J = 7.1 Hz, H-1 "'), 3.32 (2H, br d, J = 7.1 Hz, H-1"), 5.25 (m, 1H, H-2 "'), 5.54 (1H, dd, J = 12.8 , 3.0 Hz, H-2), 5.59 (s, 1H, H-8), 6.41 (s, 1H, H-3 '), 6.96 (s, 1H, H-6'), 12.30 (s, 1H, OH-5).

5) EIMS m / z 424 [M] ⁺ (48), 406 (100), 363 (45), 351 (25), 221 (10), 220 (4), 205 (9), 189 (12), 165 (58).

[Compound 5]: 2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) flavone

1) Physical property: Yellow needle type (m.p. 194 ℃)

2) Molecular Weight: 352

3) Molecular formula: C ₂₀ H ₁₆ O ₆

4) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 1.34 (s, 3H, H-4 ″, 5 ″), 5.74 (1H, d, J = 10.2 Hz, H-2 ″), 6.45 (s, 1H, H-8), 6.55 (1H, dd, J = 8.4, 2.4 Hz, H-5 '), 6.60 (1H, dd, J = 2.4 Hz, H-3'), 6.65 (1H, d, J = 10.2 Hz, H-1 "), 7.13 (s, 1H, H-3), 7.85 (1H, d, J = 8.4 Hz, H-6 '), 13.56 (s, 1H, OH-5).

5) EIMS m / z 352 [M] ⁺ (28), 337 (100), 203 (25); HERIMS m / z 352.0958 [M] ⁺ (calcd for C ₂₀ H ₁₆ O ₆ , 352.0947).

Experimental Example 1 Antioxidant Activity of Flavonoid Compounds According to the Present Invention by TBARS (Thiobarbituric Acid Reactive Substances) Method

In order to examine the antioxidant activity of low density lipoprotein in the flavonoid-based compound of the present invention, the oxidation of LDL induced by Cu ²⁺ - TBA an oxide of di-aldehyde (dialdehyde) generated by the (Cu ²⁺ mediated LDL oxidation) Antioxidant activity was measured by thiobarbituric acid (Packer, L. Ed. (1994) Methods in Enzymology Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego).

300 ml of human plasma was 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. Next, the specific gravity of the remaining solution was adjusted to 1.063 g / ml, which was centrifuged at 100,000 x g for 24 hours to collect LDL (1.006 < d <1.063 g / ml) suspended in the upper layer again. The collected LDL was dialyzed at 4 ° C. with 10 mM phosphate buffered solution (phosphate-buffered saline, PBS, pH 7.4). The dialyzed LDL was used within 4 weeks with storage at 4 ° C.

20 µl (protein concentration, 50-100 µg / ml) separated as described above was mixed with 210 µl of 10 mM PBS, and 10 µl each of the solution of the flavonoid compound prepared in Example was added. Flavonoid compounds were dissolved in DMSO (dimethylsulfoxide) and diluted to various concentrations before use in experiments. As a negative control, only the solvent DMSO 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. 1 ml of a 0.67% TBA solution dissolved in 0.05 N NaOH solution was added, stirred for 10 seconds, heated at 95 ° C. for 5 minutes to cause a color reaction, and cooled with ice water. This solution was centrifuged at 3000 rpm for 5 minutes to separate the supernatant, and the absorbance at 540 nm was measured by UV-Vis spectroscopy to determine the amount of malondialdehyde (MDA) produced by the color reaction. .

Meanwhile, using a stock solution of tetramethoxypropane malonaldehyde bis (dimethylacetal), 250 μl of PBS standard solution containing 0-10 nmol malondialdehyde was prepared. The standard solution was developed in the same manner as above, and the absorbance at 540 nm was measured to determine the standard curve of malondialdehyde. After treating the flavonoid compound using this standard curve, the amount of malondialdehyde produced was quantified and IC ₅₀ was determined.

The results are shown in Table 2.

compound IC ₅₀ (μM) One 54 2 82 3 58 4 34 5 78

As shown in Table 2, the flavonoid compounds of the present invention exhibited excellent antioxidant activity against LDL due to lower IC ₅₀ values compared to other known flavonoid compounds.

Therefore, it was confirmed that the flavonoid compound according to the present invention can be usefully used for the prevention or treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis, which are known to be caused by oxidizing LDL.

Experimental Example 2: Effect on the ACAT activity of the flavonoid compound according to the present invention

In order to determine the effect on the ACAT activity of the flavonoid compound according to the present invention, the following experiment was performed.

2-1: Preparation of ACAT Enzyme Source

In order to determine the effect on the activity of human ACAT-1 and ACAT-2, the proteins of hACAT-1 and hACAT-2 were obtained using a baculovirus expression system.

Viruses were prepared by inserting cDNAs of hACAT-1 and hACAT-2, respectively, obtained through human liver cDNA library screening into baculovirus delivery vectors, and introducing into insect cells sf9 cells. Next, after separating the recombinant viruses of each of the hACAT-1 and hACAT-2 by plaque purification method, the titer of the storage virus (viral stock) was increased through three amplification processes. Recombinant virus was infected with 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 at 27 ° C for one day. In order to separate microsomal fractions from Hi5 cells overexpressed in cultured hACAT-1 and hACAT-2, cells were collected by centrifugation at 500 × g for 15 minutes and rapidly frozen and thawed in a hypotonic buffer. Cells were broken by the method, and then ultracentrifuged at 100,000 × g for 1 hour. The obtained microsome fractions were suspended in storage buffer so that the protein concentration was 8 mg / ml and stored in a cryocooler until use.

2-2: ACAT Activity Measurement

Determination of ACAT activity was carried out using the method of Brecher & Chan using [1- ¹⁴ C] oleoyl-CoA (56.0 μCi / μmol; Amersham) as substrate. Brecher and C. Chan, Biochim. Biophys. Acta, 617, 458, 1980, was used with some modifications.

10 μl of the flavonoid compound prepared in Example, 4.0 μl of the microsome solution prepared in Experiment 2-1, 20.0 μl of activity assay buffer (0.5 M KH ₂ PO ₄ , 10 mM DTT, pH 7.4; Sigma), Fat-free bovine serum albumin (BSA; bovine serum albumin, stock solution 40 mg / ml; Sigma) 15.0 μl, cholesterol (stock solution 20 mg / ml; Sigma) 2.0 μl, distilled water 41.0 μl Preliminary reaction was carried out for 15 minutes. 8 µl of [1- ¹⁴ C] oleoyl-coei (0.05 µCi, final concentration: 10 µM) was added to the reaction mixture and reacted again at 37 ° C. for 30 minutes, followed by an isopropanol: heptane mixture (4: 1 (v / v)) 1 ml was added to stop the reaction. To this was added 600 μl heptane and 200 μl 0.1 M KH ₂ PO ₄ (pH 7.4), the mixture was vigorously mixed with a vortexer and centrifuged at 300 × g for 5 minutes. 100 μl of the supernatant obtained by centrifugation was placed in a scintillation bottle, and 4 ml of scintillation liquid (Lipoluma) was added thereto. The radiation dose of this mixture was measured with a 1450 Microbeta liquid scintillation counter (Wallacoy).

ACAT activity was calculated from the synthesized cholesteryl oleate picolol (picomol / min / mg protein) 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 3.

compound hACAT-1 IC ₅₀ (μM) hACAT-2 IC ₅₀ (μM) One 103 31 2 120 112 3 75 52 4 74 56 5 125 97

As shown in Table 3, the flavonoid compound of the present invention was found to have excellent inhibitory activity against hACAT-1 and hACAT-2.

Therefore, it was confirmed that the flavonoid compound according to the present invention can be useful for the prevention or treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction caused by the synthesis and accumulation of cholesteryl esters. .

Experimental Example 3: Acute Toxicity of Oral Administration of Flavonoid Compounds According to the Present Invention in Mice

In order to determine the acute toxicity of the flavonoid compound according to the present invention, the following experiment was performed using a mouse.

12 females and 12 males (3 males and 3 females each) were used as specific pathogens free ICR mice at 4 weeks of age in animals with temperature 22 ± 3 ° C, humidity 55 ± 10%, and illumination 12L / 12D. Breeding in the room. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (JeilJedang Co., Ltd., mice and rats) and negative water were supplied after sterilization and free ingestion.

The flavonoid compound prepared in the above example was prepared at a concentration of 50 mg / ml using 0.5% Tween 80 as a solvent, and then 0.04 ml (100 mg / kg) and 0.2 ml (500 mg / kg) per 20 g of mouse body weight. Or 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, and 12 hours after administration, and at least once in the morning and afternoon every day from the day after the administration.

In addition, after dissecting and dissecting the animal on the 7th day of administration, the internal organs were visually inspected, and the weight change of the animal caused by the flavonoid compound was observed by measuring the change in body weight every day from the day of the administration.

As a result of the test, all mice treated with the test substance showed no clinical symptoms, no dead mice, and no toxicity change was observed in weight change, blood test, blood biochemical test, autopsy findings, and the like.

Therefore, the flavonoid compound according to the present invention did not show a toxic change up to 1,000 mg / kg in all mice, and was determined to be a safe substance having a minimum lethal dose (LD ₅₀ ) of at least 1,000 mg / kg or more.

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

Formulation Example 1 Preparation of Pharmaceutical Formulation

1-1: Preparation of Powder

2 g of a flavonoid compound of Formula 1 according to the present invention

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

1-2: Preparation of Tablets

100 mg of the flavonoid compound of Formula 1 according to the present invention

Corn starch 100 mg

Lactose 100 mg

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 Capsule

100 mg of the flavonoid compound of Formula 1 according to the present invention

Corn starch 100 mg

Lactose 100 mg

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 Solution

10 μg / ml of the flavonoid compound of Formula 1 according to the present invention

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the flavonoid compound of Formula 1 according to the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 with dilute hydrochloric acid BP, and adjust the volume with sodium chloride BP for injection. And mixed well. 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

Food containing the flavonoid compound of Formula 1 according to the present invention was prepared as follows.

2-1: Manufacturing Flour Food

0.1 to 10.0 parts by weight of the flavonoid compound of Formula 1 according to the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared in a conventional manner using the mixture to prepare foods for health promotion.

2-2: Preparation of Soups and Gravys

0.1 to 1.0 parts by weight of the flavonoid compound of Formula 1 according to the present invention was added to soups and broth to prepare meat products for health promotion, soups and broths of noodles in a conventional manner.

2-3: Preparation of Ground Beef

10 parts by weight of the flavonoid compound of Formula 1 according to the present invention was added to ground beef to prepare a ground beef for health promotion in a conventional manner.

2-4: Manufacture of Dairy Products

0.1 to 1.0 parts by weight of the flavonoid compound of Formula 1 according to the present invention was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.

2-5: 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.

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 flavonoid compound of Formula 1 according to the present invention was dried under reduced pressure and concentrated in a vacuum concentrator, dried by spraying and a hot air dryer, and ground to a particle size of 60 mesh by a grinder to obtain a dry powder.

Cereals, seeds and the dry powder of the flavonoid compound of Formula 1 according to the present invention were prepared in the following ratio to prepare a conventional method.

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 flavonoid compound of Formula 1 according to the present invention,

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

Formulation Example 3: Preparation of Beverage

A beverage containing a flavonoid compound of Formula 1 according to the present invention was prepared as follows.

3-1: Preparation of Health Beverage

Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight) and the like and flavonoid compounds of the formula 1 according to the present invention 0.1 ~ 2 parts by weight homogeneously blended and then sterilized immediately, and then packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy beverage.

3-2: Preparation of Vegetable Juice

0.5 g of the flavonoid compound of Formula 1 according to the present invention was added to 1,000 ml of a vegetable juice such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.

3-3: Preparation of Fruit Juice

0.1 g of the flavonoid compound of Formula 1 according to the present invention was added to 1,000 ml of fruit such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.

The flavonoid compound according to the present invention, more specifically, the flavonoid compound isolated and purified from Koji mulberry tree not only has excellent antioxidant activity against LDL, but also effectively inhibits the activity of ACAT.

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

Claims (5)

A composition for the prevention and treatment of cardiovascular diseases selected from the group consisting of hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction comprising a flavonoid compound represented by the following formula (1). <Formula 1>

(In the above formula, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently H, OH, C ₁ -C ₄ alkoxy or C ₂ -C ₇ alkenyl, R ₁ and R ₂ may form a pyran substituted with each other pyran or C ₁ ~ C ₄ alkyl, R ₄ and R ₅ may form pyran substituted with each other with pyran or C ₁ -C ₄ alkyl.) According to claim 1, wherein the flavonoid compound 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6-prenylflavanone; 2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) -3-prenylflavone; 2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -8- (3-hydroxy-3- methylbutyl) flavanone; 2,4,5,7-tetrahydroxy-5,6-diprenyl flavanone; And And a 2 ', 4', 5-trihydroxy-6,7- (2,2-dimethylchromeno) flavone. The composition according to claim 1 or 2, wherein the flavonoid compound is separated and purified from Cucumber tree. According to claim 3, wherein the Kudji mulberry is a composition, characterized in that the root of Kudji mulberry. delete