KR19980037405A - Process for producing acyl-CoA: cholesterol acyltransferase (ACAT) activity inhibitor from ginseng and composition containing the same - Google Patents

Abstract

The present invention relates to a method for producing a polyacetylene compound specifically inhibiting the activity of Acyl-CoA: Cholesterol Acyltransferase (ACAT) from Panax ginseng CA Myers and a composition containing the same , Polyacetylene compounds having the following formula (1), (2) or (3) obtained by extracting and extracting from ginseng can be usefully used for the prevention and treatment of cardiovascular diseases caused by hypercholesterolemia:

or

(Wherein R, R ₁ and R ₂ are each OH or O, which may be the same or different, and R ₃ is OH or OOH, provided that when R ₁ and R ₂ are all O, an epoxy group is formed) .

Description

Process for producing acyl-CoA: cholesterol acyltransferase (ACAT) activity inhibitor from ginseng and composition containing the same

Figure 1 shows the hydrogen nuclear magnetic resonance (NMR) spectrum of phenoxynol.

Figure 2 shows the hydrogen NMR spectrum of the paracrystalline.

Figure 3 shows the hydrogen NMR spectrum of paraxyldiol.

4 shows a hydrogen NMR spectrum of peracid peroxide.

Figure 5 shows the hydrogen NMR spectrum of paraxyltriol.

FIG. 6 shows a hydrogen NMR spectrum of paraxaxide oxide.

[Object of the invention]

The present invention relates to a method for producing an acyl-CoA: cholesterol acyltransferase (hereinafter referred to as ACAT) activity inhibitor from Korean ginseng ( Panax ginseng CA Myers ) and a composition containing the same. More particularly, the present invention relates to a method for extracting a polyacetylene compound having ACAT inhibitory activity from ginseng root of ginseng which is used as a treatment and prevention agent for various adult diseases, and a method for synthesizing a derivative thereof and a method for inhibiting blood cholesterol lowering and cholesterol absorption inhibiting these polyacetylene compounds ≪ / RTI >

[TECHNICAL FIELD OF THE INVENTION AND RELATED ART OF THE SAME]

Coronary artery heart disease is a cardiovascular disease that accounts for one-third of all deaths in the world. It is the leading cause of mortality in most developed countries such as the US and Europe. In Korea, the incidence of coronary artery disease is increasing On the other hand, the incidence rate is very low in Asia such as China and Southeast Asia. This is due to the difference in dietary life and lifestyle, and research on arterial sclerosis, which is the main cause of coronary artery heart disease, has become important as Korean food culture gradually becomes westernized (Brown Goldstein, Ann. Rev. Biochem. , 52, 223-261 (1983)).

Atherosclerosis is an irreversible denaturation of the arterial walls that thicken and thicken as the artery wall thickens and becomes thickened due to the deposition of cholesterol on the inner wall of the artery. This causes mainly blood flow to the coronary artery, Resulting in coronary heart disease such as angina and myocardial infarction. Hypercholesterolemia, smoking, hypertension, diabetes and obesity are known to be causative factors of atherosclerosis. In particular, hypercholesterolemia is a major factor in intensifying atherosclerosis, presumably because cholesterol is converted to low-density lipoprotein (LDL) and deposited in the blood vessel wall.

Exogenous and endogenous cholesterol is the source of LDL. Exogenous cholesterol, which is caused by food intake, is absorbed by the ACAT in the form of cholesterol ester (CE) in the small intestine, and absorbed triglyceride, And cholomicron, which is a type of lipoprotein, is secreted into the lymphatic system and then transferred to the blood, and along the bloodstream, the capillaries of the adipose tissue and the muscle tissue It binds to the endothelial cell wall. These chylomicrons are degraded by the lipoprotein lipase present in the endothelial cell wall of each tissue, resulting in a small size particle called residual chylomicron remnant consisting of residual triglyceride, apoprotein and CE And the fatty acid produced by the triglyceride degradation is used or stored as an energy source in fat and muscle tissue cells. The apolipoprotein portion of the remaining chylomicron binds to the hepatocyte receptor, enters the cell, is degraded in the lysosome, released into the CE, and transformed into cholesterol by the cholesterol ester hydrolase (CEH). Some of the released cholesterol is used in the synthesis of bile acids and secreted into the intestines by bile acids or used for cell membrane production. The remaining cholesterol is stored in the hepatocytes in the form of CE by ACAT again, or very low-density lipoprotein (VLDL ) In the form of intermediate-density lipoprotein (IDL) through the form of LDL becomes.

In addition, the endogenous cholesterol produced by in vivo synthesis in the liver is converted to CE by the ACAT enzyme in the hepatocyte, secreted in the form of VLDL particles surrounded by apoprotein with CE, high concentration of triglyceride, , The triglyceride is removed by the action of lipoprotein lipoprotein in the endothelium and the endothelium is converted into IDL with a large amount of CE at the center. IDL then undergoes two types of metabolic pathways: the remaining half of the total IDL is removed by the pathway taken into the hepatocyte, such as the remaining chylomicrons, and the remaining 50% is removed from the blood by the residual triglycerides in the blood, There is a pathway that leads to LDL present at high concentration. That is, LDL contains a high concentration of cholesterol and has a half-life of 1.5 days and stays in the blood for a long time, so that most of the cholesterol (60 to 70%) in human blood is in the form of LDL. In the liver and other tissues, blood LDL is absorbed by binding to apoptoprotein B-100 receptor. Normally, when cholesterol is required for the production of specific substances in certain cells, the synthesis of LDL receptor is increased, Mediated intracellular uptake is increased, and cholesterol transported into cells is used for cell membrane formation, bile acid production, or steroid hormone production (Brown Goldstein, Scientific American, 251, 52-60 (1984)). On the other hand, in the presence of excess cholesterol, LDL receptor synthesis is inhibited, and a large amount of CE-containing LDL is increased in blood vessels. When LDL is increased in the blood, LDL is absorbed into the endothelial cells of the arterial blood vessels, and the phospholipid of LDL is oxidized by heavy metal. The oxidized phospholipid oxidizes the lysine of apoprotein B-100 protein, Change the polarity. The oxidized apoprotein B-100 protein binds to the scavenger receptors of macrophages to form foam cells. On the other hand, oxidized LDL is cytotoxic and damages the endothelial cells of the arterial blood vessels, and platelets are deposited on the injured endothelial cells. Platelet-derived growth factor (PDGF) and other growth factors are secreted into the deposited platelets to promote cell division, resulting in proliferation of the inner wall of the blood vessel, resulting in arteriosclerosis. Thus, LDL is directly related to the concentration of cholesterol in the blood, and it can be seen that a high concentration of LDL is a critical factor in causing atherosclerosis.

Unlike VLDL or LDL, which is liberated by exogenous or endogenous cholesterol, cholesterol liberated by cell necrosis or cell turnover is absorbed by high-density lipoprotein (HDL) To 25%. Cholesterol in HDL particles is converted to CE by the action of lecithin: cholesterol acyltransferase (LCAT). HDL is converted to LDL via IDL, and it functions to remove cholesterol from peripheral tissue cells by hepatocyte, and its plasma concentration is inversely related to coronary heart disease or atherosclerosis.

As a result of examining the migration pathway of exogenous and endogenous cholesterol, when the cholesterol is biosynthesized in the body such as endogenous cholesterol, the concentration of the exogenous cholesterol is very precisely controlled and balanced. However, in the case of exogenous cholesterol, The change in LDL concentration can be said to depend on the amount of exogenous cholesterol absorbed.

As a mechanism to prevent the induction of hypercholesterolemia, there is a method of inhibiting the biosynthetic process of cholesterol or inhibiting the absorption of cholesterol by CE in order to suppress the increase of the total cholesterol level due to excessive exogenous cholesterol absorption. The currently known therapeutic agent for hypercholesterolemia is HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A reductase), an enzyme that regulates the step of converting HMG CoA into mevalonate in the cholesterol biosynthesis pathway , Inhibitors for the synthesis and secretion of VLDL, fibric acids, cholestyramine resin and colestipol, nicotinic acids, probucol, ), Neomycin, dextrothyroxine, β-sitosterol, which interferes with the absorption of cholesterol in the small intestine and liver due to its similar structure to that of cholesterol. Among them, therapeutic agents other than inhibitors for HMG-CoA reductase are not known precisely. HMG-CoA reductase inhibitors include mevastatin ( penicillium sp .; originally referred to as compactin), lovastatin ( Aspergillus and Mona-scus sp .; originally Monaco Colin K, (Called monacolin K, mevinolin), and the semisynthetic compounds simvastatin and pravastatin have been commercialized and used clinically. However, they have been shown to be involved in coenzyme Q (ubiquinone), dolichol, haem A, farnesylated proteins (Ras, lamin B), which are produced in the midst stage of cholesterol biosynthesis during long- And cholesterol-producing steroid hormones, vitamin D, bile acids and lipoproteins. In particular, studies have shown that continuous use may reduce the synthesis of coenzyme cue, which plays an important role in cardiac function and immune function, thereby adversely affecting atherosclerosis and heart disease (Willis, et al., PNAS, 87, 8928-8930 (1990)).

ACAT is expected to be promising as a new form of treatment that can overcome the problems of existing hypercholesterolemia and atherosclerosis treatment. ACAT is involved in the formation of LDL by forming VLDL in the liver by converting cholesterol into CE form to promote absorption of cholesterol into the small intestine, and accumulation of CE in peripheral cells of the vascular wall wound (JC Goldstein, et al. , Proc. Natl. Acad. Sci. USA, 93, 3178 (1978)). In addition, it has been found that when more CE is present than necessary, it accumulates in the blood vessel wall or increases the viscosity of blood and causes various cardiovascular diseases (D. Steinburg, N. Engl. J. Med. 320, 915 The amount of cholesterol in the intestine can be reduced by lowering the amount of CE in the blood, thereby lowering the viscosity of the blood and increasing the amount of cholesterol in the liver, thereby reducing the amount of cholesterol absorbed in the intestine. It seems to be possible.

Thus, many researchers have attempted to synthesize or extract ACAT inhibitors from natural sources (K. Matsuda, Med. Res. Rev., 14, 271 (1994)). For example, synthetic ACAT inhibitors include urea derivatives (DR Sliskovic, et al., J. Med. Chem., 37, 560 (1994)) or imidazole derivatives (RG Wilde, et al., Bioorg. ( 5), 167 (1995)). Pyripyropenes derivatives produced from Aspergillus fumigatus (S. Omura, et al J. Antibiotics, 48, 751 (1995)), ssdosporium spp . ( J. Antibiotics, 46, 116-119 (1993); Kwon et al., Tetrahedron Lett. , 35, (K. Kuroda et al., J. Antibiotics, 46, 1196-1202 (1993)), which is produced from Scedosporium sp., And obovatol Obovatol, Magnolol and Honokiol (Korean Patent Application No. 95-41207) and lignan compounds isolated from Omija (Korean Patent Application No. 95-41207) have been developed.

Accordingly, the inventors of the present invention have continued to develop an ACAT inhibitor capable of lowering the blood cholesterol concentration from natural herbal medicines which have been used for a long period of time, and as a result, they have found that they are contained in Korean ginseng Low-polar lipid compounds lower blood total cholesterol (AA Qureshi, ZZ Din, et al., Atherosclerosis, 48, 81-94 (1983) The polyacetylene compound capable of inhibiting the reaction can be separated, thereby completing the present invention.

[Technical Problem]

It is an object of the present invention to provide a method for producing a novel ACAT activity inhibitor from ginseng and a composition for lowering blood cholesterol comprising the same.

[Structure and operation of the invention]

According to the above object, the present invention provides a process for producing a polyacetylene compound having the following general formula (1), (2) or (3), which comprises extracting and purifying ginseng with an organic solvent and a process for producing a polyacetylene compound having the general formula A composition for preventing and treating cardiovascular diseases caused by hypercholesterolemia, comprising:

[Chemical Formula 1]

(2)

or

(3)

(Wherein R, R ₁ and R ₂ are each OH or O, which may be the same or different, and R ₃ is OH or OOH, provided that when R ₁ and R ₂ are all O, an epoxy group is formed) .

The polyacetylene compound of formula (1), (2) or (3) is obtained by extracting ginseng with methanol and concentrating, separating the concentrate by adding ethyl acetate and water, separating the organic layer, concentrating, and concentrating the concentrate by silica gel chromatography And the like.

As the polyacetylene compound obtained according to the present invention, compounds represented by the following formulas (1), (4), (5), (6), (7)

[Chemical Formula 1]

or

The compound of the formula (1) is a compound of the formula (1), the compound of the formula (4) is panaxydol, the compound of formula (5) is panaxydiol, the compound of formula (6) is panaxyperoxid, The compound of formula 7 was found to be panaxytriol and the compound of formula 8 to be panaxyoxide (K. Hirakura, et al., Phytochemisty, 31, 899-903 (1992); . Hirakura, et al, Phytochemisty, 30, 3327-3333 (1991);.... BM Kwon, et al, Chem Pharm Bull, 44, 444-445 (1996)).

The polyacetylene compound isolated from ginseng according to the present invention has an IC ₅₀ of 16 to 50 μg / ml as a result of measuring ACAT inhibitory activity. These compounds, which inhibit the action of ACAT, reduce cholesterol levels in the blood because they inhibit the absorption of cholesterol in the intestines and reduce the accumulation of cholesterol, and thus can be used for the treatment and prevention of various cardiovascular diseases caused by hyperlipidemia . Furthermore, it is believed that ginseng has long been used as a herbal medicine and edible medicine, and that the compounds of the present invention, which have been extracted and isolated from them, are also free from toxicity and side effects.

The polyacetylene compound separated and purified according to the present invention can be formulated into a unit dosage form or a multiple dose formulation such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by a conventional method to inhibit cholesterol absorption, Prevention and treatment of hyperlipemia, and prevention and treatment of various cardiovascular diseases caused by hypercholesterolemia.

The pharmaceutical composition containing the derivatives isolated in the present invention as an active ingredient can be administered parenterally or orally according to the purpose. The phenoxynol of formula (1) is administered in an amount of 15 to 20 mg per kg of body weight per day, Panaxyldiol can be administered in an amount of 8 to 15 mg divided into 1 to several times. The dosage level for a particular patient can be varied depending on the type of the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration and excretion, and drug combination and severity of the disease.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

EXAMPLES Example 1: Separation and Purification of Polyacetylene Compound

The ginseng used in the present invention was obtained by purchasing ginseng grown in Geumsan, Chungcheongnam-do, Korea. 1 kg of completely dry ginseng root was finely pulverized, 2 L of methanol was added, and the mixture was allowed to stand at room temperature for 3 days. The mixture was stirred and separated using a filter paper. The filtrate was collected, concentrated under reduced pressure, and then 1 liter of ethyl acetate and 1 liter of water were added to separate the water layer and the organic solvent layer. The water layer was extracted three times with 1 liter of ethyl acetate to collect the portion which was dissolved in the organic solvent.

The degree of inhibition of enzyme activity was measured by separating the organic solvent layer and the water layer. As a result, it was confirmed that the active material was contained in the organic solvent layer. The organic solvent layer was concentrated to dryness under reduced pressure, and the resulting liquid substance was dissolved in 50 ml of methanol, adsorbed on 500 ml of C18, eluted from methanol / water (70/30) to methanol / water (100/0) ≪ / RTI > The eluate was concentrated under reduced pressure to give a yellowish brown liquid material. After filling a column (4.5 x 40 cm) with silica gel (Merck, product name: 9385) using a hexane / ethyl acetate (9/1) solution, the yellow oily oily substance was dissolved in methylene chloride and adsorbed onto silica gel, : Ethyl acetate was changed from 9.5: 0.5 to 7: 3, the active fraction was separated by silica gel column chromatography twice. Finally, four pure polyacetylene compounds were purified using preparative TLC. It was obtained at a yield of 80 mg of pure phenoxynol finally separated per kg of ginseng root, 210 mg of pancoxidol, 40 mg of panaxdialol and 30 mg of peroxyneperoxide.

EXAMPLES Example 2: Polyacetylene derivatives Preparation of paraxitriol having formula (7)

Panaxyltriol was prepared by hydrolysis of the epoxide group in the presence of sulfuric acid in the following manner. That is, 50 mg of pancuricolide was dissolved in 10 ml of a 2: 1 mixture of THF: water (containing 2% sulfuric acid), stirred at room temperature for 30 hours, and then confirmed by thin layer chromatography Respectively. The reaction solution was added to a mixed solution of 50 ml of saturated aqueous sodium chloride and 100 ml of ethyl acetate to quench the reaction. The organic solvent layer was separated and pure purified by preparative TLC to obtain pure paraxyltriol (11 mg ).

EXAMPLES Example 3: Polyacetylene derivatives Preparation of paraxyxide having formula (8)

The paraxaxyl oxide was prepared by reacting the panaxidone having the formula (4) with manganese dioxide in the following manner. That is, 50 mg of paraxylylide was dissolved in 50 mg of methylene chloride, 300 mg of manganese dioxide was added, and the mixture was stirred at room temperature for 10 hours. The reaction was confirmed by thin layer chromatography. The reaction solution was filtered to remove manganese dioxide, the organic solvent layer was dried under reduced pressure, and the filtrate was purified by preparative TLC to obtain pure paroxetized oxide (32 mg).

Example 4: Device analysis and structure determination

The structure of the ACAT inhibitory active substance isolated and purified in Example 1 was analyzed as follows.

Ultraviolet (UV) Visible Absorbance Analysis: The active substance finally separated and purified by HPLC was dissolved in 100% methanol and the absorption wavelength was analyzed using an ultraviolet-visible light spectrometer (Shimazu, UV-265) , And the results are summarized in Table 1.

Infrared (IR) absorbance analysis: 1 mg of the active substance sample was dissolved in chloroform, applied to an AgBr window, dried, and analyzed with a ratio recording infrared spectroscope (Bio-Rad Digilab Division, FTS-80). An absorption band characteristic of the triple bond of these compounds was confirmed at 2240cm ^-1, it showed an absorption peak indicating the presence of OH groups at 3500cm ^-1. The CO group of paraxaxite showed an absorption peak at 1660 cm ^-1 .

Molecular weight analysis: Molecular weight was measured by a high-resolution electron chemical ionization (HRCI) -MS method using a VG70-VSEQ mass spectrometer, and the results are shown in Tables 1 and 2.

Nuclear magnetic resonance (NMR) analysis: After the complete drying of the active substance dissolved in CDCl ₃ was determined by NMR analysis with Varian UNITY 300 aircraft placed in 5mm tubes ¹ H-NMR are as 299.949 MH _Z, ¹³ C-NMR is 75.430 MH _Z .

The ¹ H-spectrum of the compounds of the present invention is shown in Figures 1-6.

The physicochemical properties of these compounds are summarized in Table 1 below.

Physicochemical properties of polyacetylene compounds Panoxynol Pavement stone Paraxanthiol Peracid peroxide Appearance Light yellow liquid Light yellow liquid Light yellow liquid Light yellow liquid Molecular formula C ₁₇ H ₂₄ O C ₁₇ H ₂₄ O ₂ C ₁₇ H ₂₄ O ₂ C ₁₇ H ₂₄ O ₃ Molecular Weight 224 260 260 276 UV (UV) nm 207, 230, 242, 255 204, 229, 240, 255 215, 253, 269, 284 207, 254270, 286 Availability Organic solvent Organic solvent Organic solvent Organic solvent Insoluble H ₂ O H ₂ O H ₂ O H ₂ O

Example 5: ACAT inhibition assay of polyacetylene compound

The activity of ACAT was determined by the method of Tabas et al., J. Biol. Chem., 261, 3147-3154 (1986) , using [1- ¹⁴ C] oleoyl- .

10 μl of fermentation supernatant, 4.0 μl of rat liver tissue microsomal enzyme, 20.0 μl of assay buffer (0.5 M KH ₂ PO ₄ , 10 mM DTT, pH 7.4), 15.0 μl of 40 mg / ml bovine serum albumin / Ml of cholesterol and 41.0 물 of water were mixed and preliminary reaction was carried out at 37 캜 for 15 minutes. To this reaction solution, 8 占 퐇 of [1- ¹⁴ C] oleoyl-CoA (0.02 占 폚, final concentration 10 占)) was added and reacted again at 37 占 폚 for 15 minutes. Isopropanol-heptane, 4: 1, v / v) was added to stop the reaction, and 0.6 ml of heptane and 0.4 ml of assay buffer diluted 5-fold were added and centrifuged. After adding 5 ml of cocktail to 100 쨉 l of the supernatant obtained by centrifugation, the enzyme activity was measured using a liquid scintillation counter. The ACAT inhibitory activity is calculated as follows:

[Equation 1]

The blank test was performed at 0 ° C and chlorpromazine was used as a positive control.

The ACAT inhibition of these compounds was found to be 15 to 50 μg / ml IC ₅₀ . Table 2 below shows the results of measuring the enzyme inhibitory activity of the active compounds of the present invention.

ACAT inhibition activity (IC ₅₀ value) of polyacetylene compounds compound IC ₅₀ ([mu] g / ml) Panoxynol 38 Pavement stone 42 Paraxanthiol 15 Peracid peroxide 25 Panacytriol 50 Paraxial oxide 34

[Effects of the Invention]

The polyacetylenic compounds obtained by extracting and purifying from ginseng, which is a herbal medicine with stability over a long period of time, are effective in lowering the cholesterol concentration in the blood due to its ACAT inhibitory activity. Therefore, various kinds of cardiovascular diseases such as hyperlipidemia and arteriosclerosis Can be usefully used as agents for the prevention and treatment of the related diseases.

Claims (3)

A process for producing a polyacetylene compound having the following general formula (1), (2) or (3), which comprises extracting ginseng with an organic solvent and purifying it: [Chemical Formula 1] (2) or (3) (Wherein R, R ₁ and R ₂ are each OH or O and may be the same or different, and R ₃ is OH or OOH, provided that when R ₁ and R ₂ are all O, an epoxy group is formed). The method according to claim 1, Extracting ginseng with methanol, concentrating, separating the layers by adding ethyl acetate and water to the concentrate, separating and concentrating the organic layer, and subjecting the concentrate to silica gel chromatography. A composition for preventing and treating cardiovascular diseases caused by hypercholesterolemia comprising a polyacetylene compound having the following formula 1, 2 or 3: [Chemical Formula 1] (2) or (3) (Wherein R, R ₁ and R ₂ are each OH or O and may be the same or different, and R ₃ is OH or OOH, provided that when R ₁ and R ₂ are all O, an epoxy group is formed).