KR100821966B1 - Therapeutic agent comprising biphenol compounds for prevention and treatment of cardiovascular disease - Google Patents

Abstract

The present invention relates to a cardiovascular extract, an acetyl dopamine-based compound or biphenol compound isolated therefrom, as an active ingredient, which relates to the prevention and treatment of cardiac circulatory diseases, specifically, a trademark having excellent antioxidant activity and radical scavenging ability against low-density lipoproteins. The present invention relates to an agent for preventing and treating cardiac circulatory diseases, which comprises an extract, an acetyldopamine compound or a biphenol compound isolated therefrom as an active ingredient. Since the composition of the present invention has the same effect as above, it can be usefully used for the prevention and treatment of atherosclerosis, coronary heart disease, myocardial infarction and cardiac circulatory diseases caused by the above-mentioned low density lipoprotein oxidation and radical production. have.

Description

 Therapeutic agent comprising biphenol compounds for prevention and treatment of cardiovascular disease}

The present invention relates to a cardiovascular extract, an acetyl dopamine compound or biphenol compound isolated therefrom as an active ingredient for cardiovascular disease prevention and treatment, and more particularly, antioxidant activity and radical scavenging activity against low density lipoprotein The present invention relates to an excellent cardiac extract, an acetyl dopamine-based compound or a biphenol 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 causes cerebral pneumoconiosis, and in the case of coronary arteriosclerosis, it is known to cause angina arrhythmias and cause angina pectoris 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 endothelium due to genetic variations, peroxides, hypertension, diabetes, elevated plasma homocysteine levels, and microbial infections. The cell is in a dysfunctional state 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. (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 stimulate the lesion's inflammatory response by inducing the influx and activation of immune cells such as macrophages and 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 complex structure covered with fibres in the necrotic tissue which is MLDL-based (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 resulting 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. 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 (nuclear factor-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, 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, Mantidis ootheca is a medicinal insect used in herbal medicine and folk remedies. The trademark vine is in the shape of a columnar column, about 3-4 cm in length and 2-3 cm in diameter, and its outer surface is yellowish brown or reddish brown with almost no smell. Herbal medicine has been seen since ancient times, fixed urinary efficacy. It is also used in the treatment of oily white turbidity, red white crayfish, urine bins, and pediatric acute pulmonary disease. However, there have been no published antioxidant activities for extracts of medicinal insects, or acetyldopamine-based compounds or biphenol compounds.

Accordingly, the inventors of the present invention, while searching for a natural preventive agent for preventing and treating cardiovascular diseases such as atherosclerosis, coronary heart disease, and myocardial infarction, extracts of medicinal insects, acetyl dopamine compounds, It was confirmed that the biphenol compound has excellent antioxidant activity and DPPH radical scavenging activity against low density lipoprotein, and heart caused by atherosclerosis, coronary heart disease, myocardial infarction and such diseases caused by oxidation and radical production of low density lipoprotein The present invention was completed by confirming that it can be usefully used for the prevention and treatment of circulatory diseases.

The present invention is to provide a prophylactic and therapeutic agent for cardiovascular disease, which comprises the extract of the trademark herb, an acetyldopamine-based compound or a biphenol compound isolated therefrom.

The present invention provides an extract of the trademark herb having an antioxidant activity prepared by extracting the trademark herb with a solvent.

The present invention also provides an antioxidant comprising an acetyldopamine compound and a biphenol compound extracted from the trademark herb.

The present invention also provides a method for producing an acetyldopamine-based compound and a biphenol compound isolated from the trademark herb extract.

In addition, the present invention provides a preventive and therapeutic agent for cardiac circulatory disorders containing an acetyldopamine-based compound and a biphenol compound represented by Chemical Formulas 1 and 2, isolated from the trademark herb extract.

Hereinafter, the present invention will be described in detail.

The present invention provides an extract of the trademark herb having an antioxidant activity prepared by extracting the trademark herb with a solvent.

Specifically, the present inventors washed the trademark herb with water to remove foreign substances, dried in the shade, and then added the appropriate amount of solvent to the dried trademark herb so as to be completely immersed. 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, 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. It was. Antioxidant effect was measured for each soluble extract and it was confirmed that the antioxidant effect was the best in the ethyl acetate soluble extract (see Table 1).

The present invention also provides an antioxidant comprising an acetyldopamine-based compound or a biphenol compound isolated from the trademark herb extract.

Acetyl dopaminergic compound is N -2- (3,4- dihydroxy-phenyl) represented by the formula 1-ethyl acetamide N - [2- (3,4-Dihydroxyphenyl ) -ethyl] -acetamide a biphenol compound 3,5,3 ', 5'-tetra-tet-butyldiphenyl-2,2'-diol (3,5,3', 5'-Tetra- tert -butyl-diphenyl represented by Formula 2 below) -2,2'-diol).

The compounds can be used in the form of pharmaceutically acceptable salts and include all salts, hydrates and solvates prepared by conventional methods.

In addition, the present invention provides a method for preparing the acetyl dopamine-based compound and biphenol compound from the trademark herb extract, which

(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 trademarked herb;

(2) adding water to the crude extract obtained in 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 and a biphenol compound.

Specifically, the ethyl acetate soluble extract obtained above was filtered, followed by silica gel chromatography using chloroform, methanol, and a mixed solvent thereof as a mobile phase solvent, and separated into 13 fractions (fractions 1 to 13). At this time, the antioxidant effect of each fraction was measured to collect the most effective fractions 2 to 5 and subjected to secondary silica gel chromatography to separate into 15 fractions (fractions 1 to 15) (at this time, the mobile phase solvent is chloroform and Methanol mixed solvent, preferably each having a ratio of 10: 1). Antioxidant effect was measured for each fraction to obtain the most effective fractions 2, 9, and 10, and fraction 2 was subjected to tertiary silica gel chromatography to separate 13 fractions (fractions 1 to 13). When the mobile phase solvent is a mixed solvent of nucleic acid and ethyl acetate, preferably a mixed solvent in which each ratio is 1: 1. Subsequently, the active fractions 2 and 3 obtained above were subjected to fourth silica gel chromatography to obtain compound 2. In the secondary silica gel chromatography separation, fractions 9 and 10 having excellent LDL-antioxidant activity were collected and subjected to tertiary silica gel chromatography to separate five fractions (fractions 1 to 5) (in the mobile phase solvents, chloroform and methanol). Mixed solvents, preferably mixed solvents having respective ratios of 1: 1. Antioxidant effect was measured for each fraction to obtain the most effective fractions 4 and 5, and then the reverse phase C-18 (RP-18) column was fixed to the fractions 4 and 5, and a mixed solvent of methanol and water, preferably 7 fractions were obtained by column chromatography using a mixed solvent mixed at 1: 1 (v / v) as a mobile phase. Antioxidant effect was measured for each fraction to give compound 1 from fraction 4.

In addition, the present invention provides a prophylactic and therapeutic agent for cardiac circulatory disorders comprising an acetyldopamine-based compound represented by Formula 1 and 2, and a biphenol compound, which are separated from the trademark herb extract or the same.

Extract of the trademark vinegar of the present invention and N- [2- (3,4-dihydroxyphenyl-ethyl)]-acetamide, 3,5,3 ', 5'-tetra-tersil-butyl-di separated therefrom Antioxidative activity of phenyl-2,2'-diol against low-density lipoproteins was measured by TBARS (Thiobarbituric Acid Reactive Substances) method.The inhibitory effect on the oxidation of low-density lipoproteins and DPPH radical scavenging activity were used as positive controls. It is superior to probucol and therefore has an excellent antioxidant effect (see Table 1). In acute toxicity experiments in mice, there were no deaths and no change in toxicity.

Thus, the trademark herb extract according to the present invention and N- [2- (3,4-dihydroxyphenyl-ethyl)]-acetamide, 3,5,3 ', 5'-tetra-tersil- Butyl-diphenyl-2,2'-diol is useful for the prevention and treatment of atherosclerosis, coronary heart disease, myocardial infarction and cardiovascular diseases caused by such diseases caused by oxidation and radical production of low density lipoproteins Can be used.

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

The therapeutic agent of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the trademark herb extract or acetyldopamine-based compound and biphenol compound separated therefrom.

The therapeutic agent 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 a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The therapeutic agents 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 dose is 10 to 2,000 mg / kg, preferably 50 to 500 mg / kg, for the herbaceous extract. In addition, in the case of the acetyl dopamine-based compound or biphenol compound isolated from the extract of the trademark herb extract is about 0.1 ~ 100 mg / kg, preferably 0.5 ~ 10 mg / kg, it is effective to divide this 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 therapeutic agents of the present invention may 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 therapeutic agent of the present invention may be provided in the form of a health food for the purpose of improving the cardiovascular disease.

When using the trademark herb extract or acetyl dopamine-based compound and biphenol compound isolated from the extract according to the present invention as food additives, the acetyl-dopamine-based compound and biphenol compound isolated from the trademark herb extract or separated therefrom or other food Or may be used together with food ingredients, and may be appropriately used according to conventional methods. 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 manufacture of food or beverage, the trademark vinegar extract of the present invention or the acetyldopamine-based compound and biphenol compound separated therefrom are added in an amount of 1 to 20 parts by weight, preferably 5 to 10 parts by weight based on the raw materials. do. 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, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage of the present invention may contain various flavors or natural carbohydrates and the like as additional ingredients, as in general beverages. 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 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 health food of the present invention.

In addition to the above, the health beverage 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 adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the health beverage 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, the present invention will be described in detail by the following examples and experimental examples. However, the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Crude Herbal Extracts

500 ml of water was added to 200 g of dried trademark vinegar (Yunnan Province, China), and boiled at 90 ° C. for 4 hours, followed by filtration to obtain an extract and a solid residue. The resultant was concentrated under reduced pressure to obtain 3 g of trademarked hot water extract.

In addition, 2000 ml of 95% ethanol (EtOH) was added to 500 g of dried trademark vinegar, and the mixture was left at room temperature for 3 days, and then filtered through a filter paper and concentrated to give 62.3 g of an oily substance, a trademark ethanol extract.

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

Example 2 Preparation of Nonpolar Solvent Soluble Extract of Trademark Herb

200 ml of water was added to the ethanol extract of the trademark herb obtained in Example 1, suspended, and fractionated in the order of n -hexane ( n -hexane), chloroform (CHCl ₃ ) and ethyl acetate (EtOAc), and n-hexane soluble extract. 20.3 g of ( n- hexane-soluble), 6.3 g of chloroform soluble extract (CHCl ₃ -soluble) and 2.7 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 the following examples (Table 1).

Example 3 Separation of Compound of the Invention from Ethyl Acetate Soluble Extract

2.7 g of the ethyl acetate soluble extract obtained in Example 2 were separated in four steps using column chromatography.

First, silica gel column (silica gel: Merk, Art 9385, column size: φ7 × 40 cm) 100% chloroform, chloroform: methanol = 95: 1, 90: 1, 60: 1, 30: 1, 10: 1, Each of 500 ml was developed at 5: 1, 1: 1 (v / v) and 100% of mobile phase solvent conditions of methanol to obtain 13 fractions (Fr. 1 to 13). The antioxidant activity of each fraction was measured in the same manner as in Experimental Example 1, and as a result, 2 to 5 fractions (chloroform: methanol = 90: 1 and 60: 1 solvent conditions, yield 1.18 g) showing excellent antioxidant activity were separated. , A second column chromatography was performed. Second, 400 ml each of silica gel columns (silica gel: Merk, Art 9385, column size: φ4.5 × 30 cm) under mobile phase solvent conditions of chloroform: methanol = 10: 1 (v / v) was developed. Fractions (Fr. 1-15) were obtained. As a result of measuring the antioxidant activity of each fraction by the same method as Experimental Example 1, two fractions (amount yield 331 mg) and 9 to 10 fractions (230 mg) showing excellent antioxidant activity were separated.

The third column was run in two parts. In the first part, fraction 2 was developed on a silica gel column (silica gel: Merk, Art 9385, column size: φ2.5 × 28 cm) with 300 ml of nucleic acid: ethyl acetate = 1: 1 (v / v) each under mobile phase solvent conditions. To obtain 13 fractions (Fr. 1 to 13). As a result of measuring the antioxidant activity of each fraction by the same method as Experimental Example 1, two to three fractions (nucleic acid: ethyl acetate = 1: 1 solvent condition, yield 30.6 mg) showing excellent antioxidant activity were separated. Fractions 2-3 were prepared on a fourth (1-4) silica gel column (silica gel: Merk, Art 9385, column size: φ2.5 × 28 cm) nucleic acid: ethyl acetate = 1: 1 (v / v) mobile phase solvent conditions 100 ml each of them was developed, and 7 fractions (Fr.1-7) were obtained. The antioxidant activity of each fraction was measured by the same method as Experimental Example 1 below, to obtain Compound 2 purified from fraction 1 (nucleic acid: ethyl acetate = 1: 1 solvent condition) showing excellent antioxidant activity, and the yield was 7.6. mg.

Fraction 9-10 obtained through the second silica gel column was subjected to a silica gel column (silica gel: Merk, Art 9385, column size: φ3.2 × 28 cm) under mobile phase solvent conditions of chloroform: methanol = 10: 1 (v / v). 300 ml each was developed, and 5 fractions (Fr. 1-5) were obtained. The antioxidant activity of each fraction was measured by the same method as Experimental Example 1 below, and 4 to 5 fractions (chloroform: methanol = 10: 1 solvent condition, yield 95.3 mg) showing excellent antioxidant activity were separated, and the fourth ( 2-4) Reversed phase C-18 column chromatography (Licroprep RP-18: Merck, Art 13900, column size: φ2.5 × 15 cm) was performed. At this time, 50 ml each of methanol: water = 1: 1 (v / v) was developed under mobile phase solvent conditions to obtain 7 fractions, and the antioxidant activity of each fraction was measured in the same manner as in Experimental Example 1 below. , Purified compound 1 was obtained from fraction 4 having the highest antioxidant activity, and the yield was 19 mg.

Example 4 Structure Analysis of Compounds of the Invention

The following analysis was conducted for structural analysis of the three compounds obtained in Example 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.

As a result of analyzing the results of the above instrumental analysis compared with that of published documents (Naoki, N. et al ., Chem. Pharm. Bull , 48: 1749-1752, 2000), the compound 1 of the present invention is represented by the formula (1), Compound 1 is N- [2- (3,4-dihydroxyphenyl-ethyl)]-acetamide (N- (2- (3,4-Dihydroxy-phenyl) -ethyl) -acetamide), Alexakis et al Compound 2 represented by the above formula (2) with reference to J. Org. Chem., 69; 5660-5667, 2004 is 3,5,3 ', 5'-tetra-tersil-butyl-diphenyl- 2,2'-diol (3,5,3 ', 5'-Tetra-tert-butyl-diphenyl-2,2'-diol), respectively.

(Compound 1)

1) Physical property: reddish brown powder

2) Molecular Weight: 195

3) Molecular Formula: C ₁₀ H ₁₃ NO ₃

4) ¹ H NMR (CD ₃ OD, 500 MHz) δ 1.86 (3H, s, H-4), 2.60 (2H, t, J = 7.0 Hz, H-2), 3.34 (2H, t, J = 7.1 Hz, H-1), 6.70 (1H, d, J = 8.0 Hz, H-5 '), 6.62 (1H, d, J = 1.3 Hz, H-2'), 6.49 (1H, dd, J = 7.9 Hz, J = 1.2 Hz, H-6 ')

6) ESI-MS m / z : 218 (M + Na) ⁺ , 196 (10), 199 (15), 217 (20), 218 (100)

(Compound 2)

1) Physical property: white powder

2) Molecular Weight: 410

3) Molecular Formula: C ₂₈ H ₄₂ O ₂

4) ¹ H NMR (CD ₃ OD, 500 MHz) δ 1.30 (18H, s, 5- t -butyl), 1.43 (18H, s, 3- t -butyl), 5.19 (2H, s, -OH), 7.09 (2H, d, J = 2.1 HZ, H-4), 7.37 (2H, d, J = 2.1 Hz, H-6)

7) EIMS (rel. Int.) M / z : 410 (M) ⁺ , 57 (70), 190 (35), 283 (15), 339 (35), 354 (10), 395 (60), 410 (90).

Experimental Example 1 Measurement of Antioxidant Activity of the Compound of the Present Invention by TBARS Method

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

Antioxidant activity is obtained by TBARS (Thiobarbituric Acid Reactive Substances) method, which is a oxidized product of unsaturated fatty acid 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 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 (Probucol, Sigma-Aldrich Co.) was used. 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, and 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, in order to obtain the standard curve of malondialdehyde, diluting the stock solution (Sigma-Aldrich Co.) of malonaldehyde bis (dimethylacetal) (Sigma-Aldrich Co.) PBS containing 0 ~ 10 nmol malondialdehyde 250 μl of standard solution was prepared and developed 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.

As shown in Table 1, the hydrothermal extract obtained in Example 1 showed an antioxidant effect of 52% at 40 ㎍ / ㎖, while the ethanol extract showed an antioxidant effect of 64% at the same concentration, Confirmed better.

In addition, the ethyl acetate solvent extract of each solvent extract obtained in Example 2 showed an antioxidant effect of 79% against the low density lipoprotein at a concentration of 40 ㎍ / ㎖, the most excellent antioxidant activity compared to the soluble extract of n-hexane, chloroform Confirmed

Further, Compound 1 obtained in Example 3, N- [2- (3,4-dihydroxyphenyl-ethyl)]-acetamide, compound 2 , 3,5,3 ', 5'-tetra-tersil- Antioxidant activity of butyl-diphenyl-2,2'-diol showed IC ₅₀ values of 5.7 μM and 44.7 μM, respectively, and compound 1 was lower than IC ₅₀ of the positive control group, indicating that the antioxidant activity against low density lipoprotein was excellent. .

Experimental Example 2 Measurement of Antioxidant Activity of the Compound of the Present Invention Using DPPH Scavenging Ability

In order to determine the antioxidant activity through the radical scavenging ability of the trademark herb extract, acetyl dopamine-based compound, biphenol compound of the present invention, the following experiment was performed.

DPPH (1,1-Diphenyl-2-picrylhydrasyl) is a very stable free radical, and the radical scavenging ability was measured to the extent of DPPH remaining by the treatment of the compound (Royt, PW et al., Bioorg. Chem. 29: 387-397, 2001).

1 ml of radical DPPH solution (final concentration 100 μM, Sigma-Aldrich Co.) was added to 500 μl of the sample dissolved in MeOH (final concentration 100 μM). As a control, 1 ml of radical DPPH solution and 500 µl of MeOH were used, and only 1.5 ml of MeOH was used as a blank, and the result was measured by a UV-visible spectrometer at 517 nm for 20 minutes. The instrument used for the DPPH method used an 8453 UV-Visible Spectrophotometer (Agilent Technologies Co.).

As a result, 10 minutes after adding the compounds 1 and 2, the remaining radicals were 4.3% and 58.1%, respectively, and the probucol used as a control was 38.7%. Compound 1 showed higher radical scavenging ability than probucol.

Therefore, the extract of the trademark of the present invention, acetyldopamine-based compounds and biphenol compounds separated therefrom have excellent antioxidant activity and radical scavenging ability against low-density lipoproteins. It can be usefully used for the prevention and treatment of atherosclerosis caused by atherosclerosis, coronary heart disease, myocardial infarction and cardiovascular disease caused by such diseases.

Experimental Example 3 Acute Toxicity

In order to determine the acute toxicity of the trademark herb extract, acetyl dopamine-based compounds, biphenol compounds of the present invention, the following experiment was performed.

SPF (specific pathogens free) ICR mice were divided into three 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 Example 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 / kg) per 20 g of mouse body weight. ) And 0.4 ml (1,000 mg / kg). 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 daily intervals 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 therapeutic agents of the present invention are illustrated below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of Powder

2 g of acetyl dopamine-based compound or biphenol compound isolated from the trademark extract according to the present invention

1g lactose

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

<1-2> Preparation of Tablet

100 mg of acetyldopamine-based compounds or biphenol compounds isolated from the trademark extract according to the present invention

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 Capsule

100 mg of acetyldopamine-based compounds or biphenol compounds isolated from the trademark extract according to the present invention

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 Solution

10 g / ㎖ of the extract of the trademark according to the present invention or acetyl dopamine-based compound, biphenol compound isolated therefrom

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

In a suitable volume of sodium chloride BP for injection, each of the trademark extract according to the present invention or the acetyldopamine-based compound and biphenol compound isolated therefrom is dissolved, and the pH of the resulting solution is adjusted to pH 3.5 using dilute hydrochloric acid BP. The volume was adjusted 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.

Preparation Example 2 Preparation of Food

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

<2-1> Preparation of Cooking Seasoning

The trademark vinegar extract according to the present invention, or acetyl dopamine-based compounds isolated from the biphenol compound, 0.2 to 10 parts by weight, respectively, was prepared for health promotion cooking seasoning.

<2-2> Preparation of Tomato Ketchup and Sauce

Health care tomato ketchup or sauce was prepared by adding 0.2 to 1.0 parts by weight of each of the trademark herb extract or acetyldopamine-based compound and biphenol compound separated from tomato ketchup or sauce according to the present invention.

<2-3> Preparation of flour food

0.1 ~ 5.0 parts by weight of each of the extract of the trademark according to the present invention or the acetyldopamine-based compound and biphenol compound separated therefrom are added to flour, and the bread, cake, cookies, crackers and noodles are prepared using the mixture to improve health. Dragon food was prepared.

<2-4> Preparation of Soups and Gravys

0.1 to 1.0 parts by weight of each of the trademark herb extract or acetyldopamine-based compound and biphenol compound separated from the extract 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

10 parts by weight of each of the trademark herb extract or acetyldopamine-based compound and biphenol compound isolated from the extract was added to ground beef to prepare ground beef for health promotion.

<2-6> Manufacture of Dairy Products

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

<2-7> Preparation 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 dried powder obtained by label extract according to the present invention or acetyl dopamine-based compound and biphenol compound separated therefrom under reduced pressure and concentrated in a vacuum concentrator, dried by spraying and hot air dryer, was pulverized to 60 mesh particle size with a grinder to dry powder. Got it.

The grains, seeds and the vinegar extract according to the present invention or the acetyldopamine-based compound and biphenol compound dried powder prepared in the above ratio were prepared in the following ratio.

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),

Extract of the trademark herb according to the present invention or acetyl dopamine-based compounds isolated therefrom, dry powder (1 parts by weight), ganoderma lucidum (0.5 parts by weight), sulfuric acid (0.5 parts by weight)

Preparation Example 3 Preparation of Beverage

<3-1> Preparation of Carbonated Drink

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 Sterilize at 20 ℃ for 180 seconds and mix with cooling water at a ratio of 1: 4, and then inject 0.5 to 0.82% of carbon dioxide gas to contain acetyldopamine-based compounds and biphenol compounds separated therefrom. Carbonated beverages were prepared.

<3-2> Preparation of health drink

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and acetylchopamine compounds isolated from the trademark herb extract according to the present invention , Biphenol compound was homogeneously blended and then sterilized immediately and then packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

<3-3> Preparation of Vegetable Juice

Health promotion vegetable juice was prepared by adding 0.5 g of acetyl dopamine-based compound or biphenol compound separated from the trademark herb extract according to the present invention to 1,000 ml of tomato or carrot juice.

<3-4> Preparation of fruit juice

Health promotion fruit juice was prepared by adding 0.1 g of acetyldopamine-based compound or biphenol compound separated from the trademark herb extract according to the present invention to 1,000 ml of apple or grape juice.

Since the extract of the trademark of the present invention, the acetyl dopamine-based compound and the biphenol compound separated therefrom have excellent antioxidant activity and radical scavenging ability against the low density lipoprotein, the composition comprising the same is induced by oxidation and radical production of the low density lipoprotein. It can be usefully used for the prevention and treatment of atherosclerosis, coronary heart disease, myocardial infarction and cardiovascular diseases resulting from such diseases.

Claims (5)

 Coronary heart disease, arteriosclerosis, and myocardium containing 3,5,3 ', 5'-tetra-tersil-butyl-diphenyl-2,2'-diol, which is a biphenol compound represented by the following formula (2), as an active ingredient Pharmaceutical compositions for the prevention and treatment of cardiovascular diseases selected from the group consisting of infarctions: <Formula 2>

. The pharmaceutical composition according to claim 1, wherein the biphenol compound is separated from the trademark herb extract. From the group consisting of coronary heart disease, arteriosclerosis and myocardial infarction containing 3,5,3 ', 5'-tetra-tersil-butyl-diphenyl-2,2'-diol as an active ingredient according to claim 1 Health functional food for improvement of selected cardiovascular disease. (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 trademarked herb; (2) adding water to the crude extract obtained in step 1 and suspending it, and sequentially dividing with n-hexane, chloroform and ethyl acetate to obtain an ethyl acetate soluble extract; And (3) 3,5,3 ', 5'-tetra-tersil-butyl according to claim 1, comprising the step of separating and purifying the ethyl acetate soluble extract obtained in step 2 to obtain a biphenol compound. Method for preparing diphenyl-2,2'-diol. 5. The method of claim 4, wherein the chromatography of step 3 is silica gel chromatography.