KR20060073751A - Agent comprising terpenoid compounds for prevention and treatment of cardiovascular disease - Google Patents

Abstract

The present invention relates to a prophylactic and therapeutic agent for cardiovascular diseases comprising a terpenoid compound as an active ingredient.

The terpenoid compounds according to the present invention can be effectively used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters by effectively inhibiting the activity against ACAT.

Description

Agent comprising terpenoid compounds for prevention and treatment of cardiovascular disease

The present invention relates to a prophylactic and therapeutic agent for cardiovascular diseases comprising a terpenoid compound as an active ingredient.

Recently, as well as an increase in adult disease, vascular disorders such as arteriosclerosis have been greatly increased. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral arteriosclerosis, headache, dizziness, and mental disorders are indicated and cause encephalopathy, and in the case of coronary arteriosclerosis, pain and arrhythmia in the heart cause angina and myocardial infarction. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading cause of death in modern society, especially among men in their 50s and 60s.

Cardiac circulatory disorders occur when blood cholesterol levels are high. To reduce blood cholesterol levels, it is necessary to suppress the absorption of cholesterol by dieting to reduce cholesterol and fat intake or by inhibiting enzymes related to lipid metabolism. Therefore, studies have been actively conducted to develop drugs that can lower the total cholesterol concentration in the blood for the purpose of preventing cardiovascular diseases. As a result, a number of substances that inhibit the biosynthesis of cholesterol in the human body have been developed and commercialized. However, in order to improve the side effects reported by these drugs, research has recently been focused on finding compounds that can only selectively regulate blood cholesterol, and one of them is related to ACAT inhibitors.

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

First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol 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 point of view. Since the proliferation of foam cells in the vessel wall is directly related to the increase in ACAT activity, the development of ACAT inhibitors as a potent anti-arterial 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 in the liver to reduce blood cholesterol levels. Third, it is expected to prevent arteriosclerosis by preventing cholesterol from accumulating in blood vessel wall cells.

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

Human ACAT includes human ACAT-1 and human ACAT-2. Human ACAT-1 (50 kDa) acts mainly on the liver, adrenal glands, macrophages and kidneys of adults. Human ACAT-2 (46 kDa) It acts in 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 Medicine 6, 1341-1347, 2000).

On the other hand, the visa ( Torreya nucifera ) is an evergreen conifer tree belonging to the family Taxaxaceae and is distributed only in Korea and Japan worldwide. Viburnum is used for edible, ornamental, industrial and medicinal purposes. Seed is eaten or squeezed oil. In addition, in oriental medicine and private medicine, fruit is used as a medicine for insect repellent, hair growth, health, landscaping, intestinal bleeding, and wood is used for building materials, furniture materials, and marine materials. Ⅰ, p40, Seoul National University Press, 1996; Yuk Chang-soo, Asian Herbal Medicine Book, p23, Book Publishing Kyungwon, 1997). Sesquiterpenoids (sesquiterpenoids, Sakai, T. et al., Bull. Chem. Soc. Japan , 38: 381, 1965), labdane family And abietane type diterpenoids, Sayama, Y. et al, Agric. Bio. Chem ., 35: 1068, 1971; Harrison, L. and Asakawa, Y., Phytochemistry , 26: 1211, 1987 ) And flavonoids (flavonoids, Kariyone, T. and Sawaka, T., Ykugaku Zasshi, 78: 1010, 1958).

Thus, the inventors of the present invention, while finding a new hyperlipidemia and atherosclerosis treatment agent having fewer side effects in natural products, confirmed that the terpenoid-based compound separated from non-tree extracts had excellent inhibitory activity against ACAT enzyme and completed the present invention.

The present invention aims to provide a prophylactic and therapeutic agent for cardiovascular diseases comprising terpenoid compounds as an active ingredient.

The present invention provides a prophylactic and therapeutic agent for cardiovascular diseases comprising a terpenoid compound as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a prophylactic and therapeutic agent for cardiac circulatory diseases, wherein any one or more compounds of the terpenoid compounds represented by the following Chemical Formulas 1 to 4 are active ingredients.

Among the terpenoid compounds represented by Chemical Formulas 1 to 4, the compounds of Chemical Formulas 1 and 2 are isopimaric acid and dehydroabietinol, which are abiethane diterpenoid compounds, and Chemical Formula 3 The compound of is a carboxyl diterpenoid-based compound (kayadiol), and the compound of formula 4 is a sesquiterpenoid-based compound delta-cardinol (δ-cadinol). The compounds can be used in the form of pharmaceutically acceptable salts and include all salts, hydrates and solvates prepared by conventional methods.

Terpenoid compounds used in the present invention can be obtained by any conventional method, and commercially available reagents can be used. In the present invention, the terpenoid-based compound can be used by extraction, separation and purification from non-trees.

Extraction, separation and purification methods of terpenoid compounds according to the present invention are as follows.

1 kg of dried non-tree leaves were placed in methanol, left at room temperature for 3 weeks, filtered through a filter paper, and then mixed with filtrate and stirred at room temperature for 12 hours. The solution is filtered and concentrated under reduced pressure to give a yellow oily substance. Water was added and suspended therein, and filtered using a filter paper. The upper layer obtained is dissolved in ethyl acetate and concentrated to give a yellow oily substance. The concentrated solution obtained above was dissolved in dichloromethane, n-hexane was added slowly to recrystallization, filtered using a filter glass, and the liquid phase was concentrated to obtain an oily substance.

At this time, the oil fraction obtained above was measured for inhibition of human acyl coei: cholesteryl acyltransferases 1 and 2, resulting in an inhibitory effect.

The oil fraction obtained above is separated by silica gel column chromatography using a mixed solvent of ethyl acetate and n-hexane as a mobile phase. N-hexane: ethyl acetate = 10: 1, 5: 1, 3: 1, 1: 1 (v / v) and EtOAc in 11 fractions (fractions 1 to 11) under mobile phase solvent conditions of 100%. Separate.

Fraction 5 having strong double inhibitory activity was separated by silica gel column chromatography using a mixed solvent of n-hexane and ethyl acetate as a mobile phase. At this time, it was developed under mobile phase solvent conditions of n-hexane: ethyl acetate = 7: 1 (v / v), and separated into seven fractions (fractions 5-1 to 5-7). Fraction 5-4 having excellent ACAT inhibitory activity was separated by silica gel column chromatography. At this time, four fractions (fractions 5-4-1 to 5-4-4) were separated under mobile phase solvent conditions of 100% chloroform. In fraction 5-4-1, a compound of formula 1 (isopimarinic acid, 76 mg), which is a pure active substance, is obtained.

Fraction 10 obtained in the first column was subjected to silica gel column chromatography under mobile phase solvent conditions of methylene chloride: methanol = 50: 1 (v / v) to obtain 14 fractions (fractions 10-1 to 10-14). To separate. At this time, the active fraction 10-4 was separated into 11 fractions (fractions 10-4-1 to 10-4-11) by C18 reverse phase column chromatography under a methanol: water = 15: 1 mobile phase solvent. Double-inhibitory fraction 10-4-5 was n-hexane: ethyl acetate = 50: 1, 30: 1, 10: 1, 1: 1 (v / v) and 100% EtOAc in silica gel column under mobile phase solvent conditions Chromatography is performed to separate the five fractions (10-4-5-1 to 10-4-5-5). In fraction 10-4-5-2, a compound of formula 2 (dehydroaviethanol, 25 mg), which is a pure active substance, is obtained.

In addition, the fraction 11 obtained in the first column is used as a recrystallized solvent n-hexane: ethyl acetate = 5: 1 (v / v) to obtain a compound of formula 3 (kayadiol, 40 mg) as a pure active substance.

In the second column, fraction 10-6 , which is the active fraction of 14 fractions, was subjected to C18 reverse phase column chromatography using methanol: water = 10: 1 (v / v) as a mobile phase solvent. 10-6-1 to 10-6-12). N-hexane: ethyl acetate = 15: 1, 10: 1, 5: 1, 1: 1 (v / v) and 100% EtOAc in silica gel column as mobile phase solvent Chromatography is separated into five fractions (fractions 10-6-3-1 to 10-6-3-5). In fraction 10-6-3-1, a compound of formula 4 (delta-cardinol, 15 mg) is obtained which is a purely active substance.

Terpenoid compounds according to the present invention effectively inhibit the activity against ACAT in hACAT-1 and hACAT-2.

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

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the terpenoid-based compound.

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

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 dosage of the terpenoid-based compound according to the present invention is about 0.1-100 mg / kg, preferably 0.5-10 mg / kg, and more preferably administered once to several times a day.

As a result of the toxicity experiment by orally administering the terpenoid-based compound of the present invention, 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is determined 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 can be added to health foods for the purpose of improving cardiovascular disease. When the terpenoid-based compound of the present invention is used as a food additive, the terpenoid-based compound may be added as it is or used 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 terpenoid-based compound of the present invention is added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight, based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

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

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

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

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

Example  : Extraction, Separation and Purification of Terpenoid Compounds from Non-trees

1. Extraction, separation and purification from non-tree

1 kg of non-tree leaves purchased from Jeju Island, Korea were washed and dried. The dried non-tree leaves were placed in 4 l of 100% methanol and left at room temperature for 3 weeks, filtered through a filter paper. Then, complex call (100 g) was added to the filtrate and stirred at room temperature for 12 hours. The solution was filtered and concentrated under reduced pressure to give a yellow oily substance. 200 ml of water was added to this, and it suspended and filtered using filter paper. The upper layer obtained above was dissolved in ethyl acetate and concentrated to give 40 g of a yellow oily substance. The concentrated solution obtained above was dissolved in dichloromethane, n-hexane was added slowly to recrystallization, filtered using a filter glass, and the liquid phase was concentrated to give 30 g of an oily substance.

In this case, the oil fraction obtained above was measured for inhibition of human acyl COA: cholesterol acyl transfer enzymes 1 and 2, and showed an inhibitory effect.

Some 16 g of the oil fraction obtained above was separated by silica gel column chromatography (silica gel: Merk, Art 9385, column size: φ7 × 40 cm) using a mixed solvent of ethyl acetate and n-hexane as a mobile phase. At this time, n-hexane: ethyl acetate = 10: 1, 5: 1, 3: 1, 1: 1 (v / v) and 100 ml of mobile phase solvent under 100% of mobile phase solvent was developed, each of 11 fractions (fractions) 1 ~ 11), and ACAT inhibitory activity of each fraction was observed.

Fraction 5 (6 g) having strong double inhibitory activity was separated by silica gel column chromatography (column size: φ4 × 20 cm) using a mixed solvent of n-hexane and ethyl acetate as a mobile phase. At this time, it was developed under mobile phase solvent conditions of n-hexane: ethyl acetate = 7: 1 (v / v), and separated into seven fractions (fractions 5-1 to 5-7) of 250 ml each. Fraction 5-4 (100 mg) having excellent ACAT inhibitory activity was separated by silica gel column chromatography (column size: φ2.5 × 10 cm). At this time, it was developed under mobile phase solvent conditions of 100% of chloroform, and separated into four fractions (fractions 5-4-1 to 5-4-4) of 50 ml each. In this case, the compound of formula 1 (25 mg) was obtained as a pure active substance in fraction 5-4-1.

Fraction 10 (1.34 g) obtained in the first column carried out above was subjected to silica gel column chromatography (column size: φ1.5 × 20 cm) while developing under mobile phase solvent conditions of methylene chloride: methanol = 50: 1 (v / v). As a result, each of 50 fractions were separated into 14 fractions (fractions 10-1 to 10-14). At this time, the active fraction 10-4 (100 mg) was subjected to C18 reverse phase column chromatography (column size: φ1.5 × 10 cm) while developing methanol: water = 15: 1 under mobile phase solvent conditions. Fractions (fractions 10-4-1 to 10-4-11) were separated, and the ACAT inhibitory activity of each fraction was observed. Double inhibitory fraction 10-4-5 (72 mg) was extracted with n-hexane: ethyl acetate = 50: 1, 30: 1, 10: 1, 1: 1 (v / v) and 100% EtOAc in mobile phase solvent. Silica gel column chromatography (column size: φ 1 × 8 cm) was carried out under the conditions, and each fraction was separated into five fractions (10-4-5-1 to 10-4-5-5) at 30 ml. Compound 10 (76 mg) was obtained as a pure active substance in fraction 10-4-5-2.

Also, Compound 11 (40 mg) was obtained using fraction 11 (149 mg) obtained in the first column using n-hexane: ethyl acetate = 5: 1 (v / v) as a recrystallization solvent.

C18 reverse phase column chromatography using column 10-6 (85 mg), which is the active fraction of 14 fractions, in a second column, using methanol: water = 10: 1 (v / v) as the mobile phase solvent (column size: φ1.5 × 13 cm) was separated into 12 fractions (fractions 10-6-1 to 10-6-12), each of 45 ml, of which the active fraction 10-6-3 (40 mg) was separated from the mobile phase solvent. N-hexane: ethyl acetate = 15: 1, 10: 1, 5: 1, 1: 1 (v / v) and 100% EtOAc developed under mobile phase solvent conditions, and silica gel column chromatography (column size: φ1 × 7 Cm) and separated into 5 fractions (fractions 10-6-3-1 to 10-6-3-5), each 30 ml. In this case, the compound of Formula 4 (15 mg) was obtained as a pure active substance in fraction 10-6-3-1.

2. Structural Analysis of Terpenoid Compounds

The material obtained in 1 was determined by using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima, and the molecular weight and molecular formula were determined by using a polarizer (Jasco DIP-181 digital). polarimeter). Nuclear Magnetic Resonance (NMR) analysis (Bruker AMX 300, 500) also revealed ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC ( ¹ H-Detected heteronuclear Multiple-Quantum Coherence), HMBC Multiple-Bond Coherence (DEP) and Distortionless Enhancement by Polarization (DEPT) spectra were obtained and the molecular structure was determined.

The measurement results are as follows. As a result of the comparative analysis with that of the published literature, Compound 1 isopimaric acid [Y.-H. Kuo and W.-C. Chen, J. Chin. Chem. Soc ., 1999, 46 , 819], compound 2 is dehydroabietinol [HL Ziegler et al., Planta Med. , 2002, 68 , 547], compound 3 is kayadiol [JDP Teresa et al., Argic. Biol. Chem., 1971, 35 , 1068] and compound 4 are delta-cardinol [ Bull, Chem, Soc, Jpn. 1963, 37 , 1053.

[Compound 1]: isofimaric acid

1) Physical property: Colorless prism, Melting point (M.P.) = 185 ~ 187 ℃

2) Luminous intensity: [α] _D ²⁵ + 10.5 ° (c = 0.42, EtOH)

3) Molecular weight: 302

4) Molecular formula: C ₂₀ H ₃₀ O ₂

5) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 0.86 (s, 3H, H-19), 0.91 (s, 3H, H-20), 1.12 (m, 1H), 1.27 (s, 3H, H -17), 1.37 (m, 2H), 1.48 (m, 1H), 1.55 (m, 3H), 1.67-2.03 (m, 9H), 4.87 (dd, J = 1.7, 10.8 Hz, 1H, H-16ab , H-16ax), 4.93 (dd, J = 0.6, 17.5 ㎐, 1H, H-16ba, H-16bx), 5.32 (d like, J = 4.1 ㎐, 1H, H-7), 5.80 (dd, J = 10.8, 17.6 μs, H-15ax, H-15bx), 12.1 (brs, 1H, -COOH).

6) ¹³ C-NMR (CDCl ₃ , 125 MHz) δ 15.3 (C-20), 17.1 (C-19), 17.9 (C-2), 20.0 (C-17), 25.2 (C-6), 35.0 (C-10), 36.1 (C-12), 36.8 (C-3), 37.0 (C-13), 38.8 (C-1), 45.0 (C-5), 46.1 (C-4), 46.3 ( C-14), 52.0 (C-9), 109.3 (C-16), 121.0 (C-7), 135.7 (C-8), 150.3 (C-15), 185.6 (C-18).

7) EIMS (rel. Int.) M / z [M] ⁺ 105 (40.7), 187 (36.9), 241 (47.8), 257 (30.9), 273 (26.9), 287 (47.1), 302 (100.0) .

[Compound 2]: dehydroaviethanol

1) Physical property: viscous oil

2) Luminous intensity: [α] _D ²⁵ + 50 ° (c = 0.24, CHCl ₃ )

3) Molecular Weight: 286

4) Molecular formula: C ₂₀ H ₃₀ O

5) ¹ H-NMR (CDCl ₃ , 300 MHz) δ 0.90 (s, 3H, H-19), 1.22 (s, 3H, H-20), 1.23 (t like, J = 3.6 Hz, 6H, H- 16, 17), 1.34-1.50 (m, 3H, H-1α, H-3), 1.63-1.82 (m, 5H, H-2, 5, 6), 2.29 (d like, J = 12.6 ㎐, 1H , H-1β), 2.79-2.92 (m, 3H, H-7, 15), 3.24 (d, J = 11.1 ㎐, 1H, H-18α), 3.48 (d, J = 11.1 ㎐, 1H, H- 18β), 6.89 (s, 1H, H-14), 6.99 (d, J = 8.1 μs, 1H, H-12), 7.19 (d, J = 7.8 μs, 1H, H-11).

6) ¹³ C-NMR (CDCl ₃ , 75 MHz) δ 17.4 (C-19), 18.6 (C-2), 18.8 (C-6), 24.0 (C-16, 17), 25.3 (C-20) , 30.1 (C-7), 33.4 (C-15), 35.1 (C-3), 37.3 (C-10), 37.8 (C-4), 38.4 (C-1), 43.9 (C-5), 72.2 (C-18), 123.8 (C-12), 124.2 (C-11), 126.8 (C-14), 134.7 (C-8), 145.5 (C-13), 147.3 (C-9).

7) EIMS (rel. Int.) M / z [M] ⁺ 159 (47.5), 173 (60.9), 185 (27.0), 253 (100), 271 (87.2), 286 (32.6).

[Compound 3]: Kayadiol

1) Physical property: amorphous powder

2) Luminous intensity: [α] _D ²⁵ + 18.4 ° (c = 0.3, CHCl ₃ )

3) Molecular Weight: 306

4) Molecular formula: C ₂₀ H ₃₄ O ₂

5) ¹ H-NMR (CDCl ₃ , 500 MHz) δ 0.72 (s, 3H, H-20), 0.75 (s, 3H, H-18), 1.02 (dt, J = 4.2, 12.7 Hz, 1H), 1.28 (m, 1H), 1.35 (dt, J = 4.2, 12.7 ㎐, 1H), 1.37-1.48 (m, 4H), 1.55-1.65 (m, 5H), 1.67 (s, 3H, H-16), 1.76-1.84 (m, 2H), 2.00 (dt, J = 4.6, 12.6 ㎐, 1H), 2.38 (m, 1H), 3.10 (d, J = 10.9 ㎐, 1H, H-19a), 3.42 (d, J = 10.9 ㎐, 1H, H-19b), 4.15 (d, J = 6.7 ㎐, 2H, H-15), 4.52 (s, 1H, H-17a), 4.84 (s, 1H, H-17b), 5.39 (t, J = 6.6 Hz, 1H, H-14).

6) ¹³ C-NMR (CDCl ₃ , 125 MHz) δ 15.0 (C-20), 16.4 (C-16), 17.6 (C-2), 18.7 (C-11), 21.8 (C-6), 24.2 (C-19), 35.4 (C-3), 38.0 (C-1), 38.1 (C-12), 38.4 (C-10), 38.6 (C-7), 39.5 (C-4), 48.5 ( C-5), 56.2 (C-9), 59.4 (C-15), 72.0 (C-18), 105.5 (C-17), 123.1 (C-14), 140.5 (C-13), 148.3 (C -8).

[Compound 4]: Delta-cardinol

1) Physical property: white powder, melting point (M.P.) = 135.5 ~ 136 ℃

2) Luminous intensity: [α] _D ²⁵ -100 ° (c = 0.24, CHCl ₃ )

3) Molecular Weight: 222

4) Molecular formula: C ₁₅ H ₂₆ O

5) ¹ H-NMR (CDCl ₃ , 300 MHz) δ 0.79 (d, J = 6.9 Hz, 3H, H-12 or H-13), 0.87 (d, J = 6.6 Hz, 3H, H-13 or H -12), 1.09 (m, 1H), 1.28 (m, 1H), 1.27 (s, 3H, H-15), 1.41-1.60 (m, 6H), 1.63 (s, 3H, H-14), 1.85 -2.01 (m, 5H), 5.51 (d like, J = 4.2 μs, 1H, H-7).

6) ¹³ C-NMR (CDCl ₃ , 75 MHz) δ 15.3, 18.5, 21.5, 21.7, 23.6, 26.4, 27.9, 31.1, 35.3, 36.8, 44.1, 45.4, 72.5 (C-2), 124.6 (C-7 ), 134.3 (C-6).

Experimental Example 1  : Effect on ACAT Activity of Terpenoid Compounds of the Present Invention

In order to determine the effect on the ACAT activity of the terpenoid-based compound of the present invention, the following experiment was performed.

1. Preparation of ACAT Enzyme Source

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

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

The cultured hACAT-1 and hACAT-2 were collected by centrifugation at 500 xg for 15 minutes to separate microsomal fractions from overexpressed Hi5 cells, respectively, by rapid freezing and thawing in hypotonic buffer. The cells were broken and ultracentrifuged at 100,000 × g for 1 hour.

The obtained microsomal fractions were suspended in a storage buffer such that the protein concentration was 8-10 mg / ml and stored in a cold freezer until use.

2. ACAT activity measurement

Determination of ACAT activity was carried out using Brecher &Chan's method [1. ¹⁴ C] oleoyl-CoA as substrate. Brecher and C. Chan, Biochim. Biophys. Acta 1980 , 617 , 458, with some modifications.

10 μl of sample (terpenoid compound prepared in Example), 4.0 μl of microsome solution obtained in 1, 20.0 μl of activity assay buffer (0.5 M KH ₂ PO ₄ , 10 mM DTT, pH 7.4), Bovine serum albumin (BSA; bovine serum albumin, stock solution 40 mg / ml), 2.0 μl cholesterol (stock solution 20 mg / ml) and 41.0 μl H ₂ O Preliminary reaction was carried out for 15 minutes at. 8 μl of [1- ¹⁴ C] oleoyl-coei (0.05 μCi, final concentration: 10 μM) was added to the reaction solution and reacted again at 37 ° C. for 30 minutes, followed by isopropanol: heptane mixture (4: 1; v). / v) 1 ml was added to stop the reaction, then 600 μl heptane and 200 μl 0.1 M KH ₂ PO ₄ (pH 7.4) were added and the mixture was vigorously mixed with vortex and then at 300 × g. Centrifuge for 5 minutes.

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

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

The results are shown in Table 1.

compound IC ₅₀ (μM) hACAT-1 hACAT-2 One Dehydroaviethanol 41 60 2 Isofimaric acid 229 263 3 Kayadiol 120 155 4 Delta-cardinol 79 -

As shown in Table 1, it can be seen that the terpenoid compounds of the present invention have very excellent inhibitory activity against acyl coei: cholesterol acyltransferase enzymes in hACAT-1 and hACAT-2.

Therefore, the terpenoid compounds according to the present invention can be usefully used for the prevention or treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters.

Experimental Example 2  : Acute Toxicity in Oral Administration in Mice

In order to determine the acute toxicity of the terpenoid-based compound according to the present invention, the following experiment was performed.

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 an animal room of temperature 22 ± 3 ° C, humidity 55 ± 10%, and illumination 12L / 12D. Breeding in. 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 terpenoid compounds prepared in the above examples (isopimaric acid, dehydroaviertinol, kayadiol, delta-cardinol) were prepared at a concentration of 50 mg / ml using 0.5% Tween 80 as a solvent, and then the mouse body weight 20 g 0.04 ml (100 mg / kg), 0.2 ml (500 mg / kg) and 0.4 ml (1,000 mg / kg) were orally administered. 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 terpenoid compounds.

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

Therefore, the terpenoid-based compound according to the present invention did not show toxicity change up to 1,000 mg / kg in all mice, and was determined to be a safe substance having a minimum oral 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 Formulations

Pharmaceutical formulations comprising any one of the terpenoid compounds represented by Formulas 1 to 4 were prepared as follows.

1. Preparation of powder

Terpenoid Compound 2g

1g lactose

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

2. Preparation of Tablets

Terpenoid Compound 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.

3. Preparation of Capsule

Terpenoid Compound 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.

4. Preparation of Injection Solution

10 ㎍ / ml terpenoid compound

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

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

Formulation Example 2  : Manufacture of food

Food containing a compound of any one of the terpenoid-based compound represented by Formula 1 to Formula 4 was prepared as follows.

1. Preparation of Cooking Seasonings

0.2 to 10% by weight of terpenoid compounds were prepared for cooking spices for health promotion.

2. Preparation of Tomato Ketchup and Sauce

0.2 to 1.0 wt% of the terpenoid compound was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.

3. Manufacturing of Flour Foods

0.1 to 5.0% by weight of the terpenoid-based compound was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for promoting health.

4. Preparation of soups and gravy

0.1-1.0 wt% of terpenoid compounds were added to soups and broths to prepare meat products for health promotion, soups of noodles and broths.

5. Preparation of Ground Beef

10% by weight of the terpenoid-based compound was added to the ground beef to prepare a ground beef for health promotion.

6. Manufacture of Dairy Products

0.1 to 1.0% by weight of the terpenoid-based compound was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, 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 terpenoid compound was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The dry powders of the grains, seeds and terpenoid compounds prepared above were prepared by blending in the following ratios.

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

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

Dry powder (1% by weight) of the terpenoid compound,

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3  : Preparation of Beverages

A beverage containing any one compound of the terpenoid-based compounds represented by Formulas 1 to 4 was prepared as follows.

1. Preparation of Carbonated Drinks

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

2. Manufacture of health drinks

Immediately sterilize by homogeneously mixing terpenoid compounds with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Healthy drinks were prepared by packing them in small packaging containers such as bottles and plastic bottles.

3. Preparation of Vegetable Juice

0.5 g of terpenoid compounds were added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

0.1 g of the terpenoid-based compound was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

The terpenoid compounds according to the present invention can be effectively used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters by effectively inhibiting the activity against ACAT.

Claims (3)

A prophylactic and therapeutic agent for cardiac circulatory diseases comprising any one or more compounds of the terpenoid compounds represented by the following Chemical Formulas 1 to 4 as an active ingredient. <Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

The method of claim 1, wherein the terpenoid-based compound is obtained by extracting, separating and purifying from a non-tree. The method of claim 1 or 2, wherein the cardiovascular disease is hyperlipidemia and atherosclerosis.