KR20060021674A - Composition comprising acyclic triterpenoid compounds with acyl-coa cholesterol acyltransferase inhibitory activity - Google Patents

Abstract

The present invention relates to an acyclic triterpenoid compound having an acyl-CoA: cholesterol transferase inhibitory activity and a pharmaceutical composition containing the same, and more particularly, to an acyclic triterpenoid of Formula 1 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (2,3,22,23) -tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene), or compounds thereof, by confirming that they effectively inhibit the activity of acyl-CoA: cholesterol transferase The present invention relates to a composition or food for preventing and treating various cardiovascular diseases such as hyperlipidemia and arteriosclerosis due to hypercholesterolemia containing a pharmaceutically acceptable salt as an active ingredient.

Acyl-Coay: cholesterol transferase, 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene, cardiovascular Relationship disease

Description

Composition comprising acyclic triterpenoid compounds with acyl-CoAːcholesterol acyltransferase inhibitory activity}             

1 shows a hydrogen-nuclear magnetic resonance spectrum (CDCl ₃ , 500.13 MHz) of the compound represented by Chemical Formula 1 according to the present invention.

Figure 2 shows the carbon-nuclear magnetic resonance spectrum (CDCl ₃ , 125.75 MHz) of the compound represented by Formula 1 according to the present invention.

Figure 3 shows the mass spectrometry spectrum (ESI-Mass) of the compound represented by Formula 1 according to the present invention.

Figure 4 is a diagram showing the ACAT (acyl-CoA: cholesterol acyltransferase) inhibitory activity of the compound represented by the formula (1) according to the present invention.

The present invention relates to an acyclic triterpenoid compound having an acyl-CoA: cholesterol transferase inhibitory activity and a pharmaceutical composition containing the same, more specifically 2,3,22,23-tetrahydroxy-2 , 6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl 6,10,14,18-tetracosatetraene) has been shown to effectively inhibit the activity of acyl-CoA: cholesterol transferase, thereby preventing hypercholesterolemia containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a composition or food for preventing and treating various cardiovascular diseases such as hyperlipidemia and atherosclerosis.

Cardiovascular disease is mainly caused by hyperlipidemia, and the mortality rate of this disease ranks high among all mortality rates.

The cholesterol required by the human body is known to be endogenous cholesterol synthesized in the liver and exogenous cholesterol from food intake. However, hyperlipidemia due to excessive inflow of triglycerides and cholesterol into the blood is excessively high cholesterol and triglycerides in the blood and become a major factor causing atherosclerosis. These symptoms are caused by abnormal metabolism of lipoproteins due to abnormalities during the formation, transport and degradation of lipoproteins.

According to epidemiologic studies, most of the ischemic heart diseases are predominantly coronary atherosclerosis, and the rise of blood cholesterol is known to be an important factor in the development and progression of the disease. Therefore, methods for promoting cholesterol inhibition in the small intestine, inhibition of biosynthesis of cholesterol in the liver, and excretion of bile acids have been proposed to lower blood cholesterol.

Drugs currently used to lower blood cholesterol levels are those that inhibit the synthesis of biosynthetic cholesterol in the liver, and are manufactured by Sankyo, Japan, and Merck, USA, and are known as pravastatin and simvastatin, which are biologically modified derivatives of compactin. (simvastatin) has the highest market share and elongation. The mechanism of action of these drugs is to inhibit 3-hydroxy-3-methyl glutaryl Co-A reductase (hereinafter referred to as "HMG Co-A"), which is involved in the intermediate stages of cholesterol biosynthesis in the liver. . However, long-term use of HMG Co-A reductase inhibitors on the mechanism of action requires ubiquinone, dolichol, dolichol, which require the human body to be produced in the secondary pathways of cholesterol synthesis following mevalonate. It has been reported to affect the production of steroid hormones, vitamin D, bile acids and lipoproteins produced in heem A, farnesylated protein and cholesterol [Grunler J., J. Ericsson and G. Dalloner 1994. Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins: Biochim. Biophys, Acta 1212, 259-277]. Indeed, continuous use of HMG Co-A reductase inhibitors has been shown to reduce the synthesis of coenzyme cues, which play an important role in cardiac and immune function, and may be adversely affected in patients with atherosclerosis or heart disease. [Willis RA, K., Folkers. J.L. Tucker, C.Q. Ye,, L.J. Xia, and H Tamagawa. Lovastatin decreases coenzyme Q levels in rats: Proc. Natl. Acad. Sci. USA, 87, 8928-8930.

Currently used antilipidemic drugs are inhibitors that inhibit the biosynthesis of cholesterol synthesized in the liver, and anion exchangers that bind to bile acids secreted by the liver to digest food and are reabsorbed in the large intestine. In addition, there is a need for the development of new hyperlipidemia drugs with no restrictions on their use, certain mechanisms of action, and fewer side effects. Sliscovik's research has shown that ACAT (acyl-CoA: cholesterol acyltransferase) inhibitors are effective in the prevention and treatment of hyperlipidemia [Sliskovic DR and AD White 1991. Therapeutic potential of ACAT inhibitors as lipid lowering and antiatherosclerotic agents. Trends in Pharmacol. Sci. 12: 194-199], in particular, the development of ACAT inhibitors as a part of the development of a therapeutic agent for hyperlipidemia with a new mechanism of action that is directly related to the mechanism of atherosclerosis.

ACAT is known as an enzyme involved in the acylation of cholesterol, the absorption of cholesterol in the small intestine, the synthesis of very low density lipoprotein (VLDL) in the liver, and the accumulation of storage-type cholesterol in adipocytes and blood vessel walls.

The ACAT inhibitors studied so far are mainly chemical compounds, and urea-, amide- and phenol-based synthetic compounds are predominant in Warner Lambert, Pfizer, and Yamanouchi. [Matsuda K. 1994. ACAT inhibitors as antiatherosclerosis agent compound and mechanisms. 14, John Wiley & Son, Inc., 271-305. In addition, exploratory studies are underway on microbial resources to develop ACAT inhibitors with a new structure, and epik from Sankyo, Japan, begins with the discovery of the structure of purpactin at Kitasato Research Institute in Japan. -Pi-cohliquinone A (Japanese Patent Laid-Open No. 4-334383, 1992), acatelin, helmintosporol, helmintosporol, lateritin, straw Gypsetin, enanthins from the Kitasato Institute of Japan, gnisoprenins, pyripyropenes, terpendols, AS-183 from Kyowa Hakko, Japan, AS-186, GERI-BP-001 and GERI-BP-002 of the Korea Research Institute of Bioscience and Biotechnology are reported. In particular, ACAT inhibitor avasimibe, which Pfizer is developing recently, has attracted attention as a new drug for the treatment of hyperlipidemia in Phase III clinical trials [Heinonen TM., 2002. Acyl coenzyme A: cholesterol acyltransferase inhibition: potential atherosclerosis therapy or springboard for other discoveries: Expert Opin Investig Drugs. 11: 1519-1527.

Alpinia katsumadai used in the present invention is a herb belonging to the family Zingiberaceae, and it is known that it has effects such as health and soil . Chodugu is distributed in southern China, Taiwan and Hong Kong, and is cultivated in the tropics and is known for its detoxification, constitution improvement, oral hygiene and anti-inflammatory effects.

Recently, the pendulum extract inhibits the activity of pancreatic cholesterol esterase (pancreatic cholesteryl ester hydrolase (pCEH), so that the pendulum extract has been patented for the prevention and treatment of atherosclerosis [Domestic Patent Registration No. 307667] ]. However, only the active fractions of turmeric were obtained, and the structure of the specific substance was not revealed. In addition, when the drug is prepared in fractions, various compounds are contained together in the fractions, and thus may exhibit activities that are not related to the desired drug efficacy.

On the other hand, 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene which is an acyclic triterpenoid compound No pharmacological activity of (2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene) has been reported.

Therefore, the inventors of the present invention, while studying cholesterol metabolism inhibitors from natural products, found that the active ingredient from acephalycoa is an acyclic triterpenoid-based compound of Formula 1, and the compound specifically inhibits ACAT activity. The present invention was completed by confirming for the first time that there is an action.

Accordingly, an object of the present invention is to provide a composition for the prevention and treatment of cardiovascular diseases comprising the acyclic triterpenoid compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, an object of the present invention is to provide a food having an acyclic triterpenoid compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The present invention is characterized by the composition for the prevention and treatment of cardiovascular diseases comprising the acyclic triterpenoid compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention includes a food having an acyclic triterpenoid-based compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The present invention will be described in more detail as follows.

The present invention relates to 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14, which are acyclic triterpenoid compounds of Formula 1 , 18-tetracosatetraene (2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene) acyl-coei: cholesterol transfer The composition or food for preventing and treating various cardiovascular diseases such as hyperlipidemia and atherosclerosis, which is caused by hypercholesterolemia containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient by confirming effective inhibition of enzyme activity. It is about.

First, a method for separating the compound represented by Chemical Formula 1 from the scalp sphere according to the present invention is as follows.

The green bean curd is washed with water, dried in the shade and then powdered with a grinder. Three times of ethanol (relative to the weight of dried caudal gourd) was added to the powdered cedar, followed by extraction at room temperature for 7 days, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a crude extract.

The crude extract was separated using chloroform, ethyl acetate and water, and ACAT activity was measured on the obtained chloroform layer, ethyl acetate layer and water layer. As a result, ACAT activity was strongly inhibited in the chloroform layer.

The chloroform layer is separated by silica gel column chromatography using a mixed solvent of chloroform and methanol. At this time, it is preferable that chloroform and methanol use the solvent which is 100-0: 0-100 (v / v).

The ACAT inhibitory activity of the separated active fractions was measured, and the fractions having the highest ACAT inhibitory activity were collected and separated by reverse phase column chromatography using 70%, 80%, 90%, and 100% methanol as the elution solvent.

In the final fractions obtained, the fractions having high ACAT inhibitory activity were collected, and the final compound was separated by HPLC while flowing at 5 ml / min using 90% methanol.

As described above, the compound of Formula 1 may be extracted and separated from the cephalosphere or synthesized by organic synthesis.

In addition, the compound represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt, the acid addition salt formed by the pharmaceutically acceptable free acid is useful as a salt. The compound of Formula 1 may form an acid addition salt which is pharmaceutically acceptable according to a conventional method in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

The compound of formula (I) isolated from the extract of Cultivation of the present invention showed an IC ₅₀ value of 24 μg / ml for rat liver ACAT and effectively inhibited ACAT activity in a concentration-dependent manner.

Accordingly, the present invention includes compositions for preventing and treating various cardiovascular diseases, such as hyperlipidemia and arteriosclerosis, which are caused by hypercholesterolemia containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the compound of Formula 1.

The composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, are used. It is prepared by. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. The daily dosage is about 10 to 50 mg / kg of the compound of formula 1, preferably 15 to 20 mg / kg, and more preferably administered once to several times a day.

As a result of toxicity experiments by orally administering the compound represented by Formula 1 of the present invention to rats, 50% lethal dose (LD ₅₀ ) of the compound of Formula 1 is at least 0.1 g / kg, which is considered a safe substance.

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 compound represented by Formula 1 of the present invention is used as a food additive, the compound of Formula 1 may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of foods or beverages, the compound of formula 1 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 the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

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

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, 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 composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of the Compound of Formula 1

1) Extraction from Cultivation

The pseudonym was washed with water and dried in the shade, and then powdered with a grinder. Three times of ethanol (relative to the dried caudal weight) was added to 100 g of powdered crustacean, extracted at room temperature for 7 days, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude extract.

In order to isolate and purify the active substance from the crude extract, chloroform, ethyl acetate and water were separated into their respective fractions, and the ACAT inhibitory activity was measured by the following Experimental Example 1. As a result, it showed the best ACAT inhibitory activity in the chloroform layer.

2) Separation and Purification of Chloroform Layer

The chloroform layer obtained in 1) was concentrated under reduced pressure (7.35 g), and column chromatography of silica gel was performed using a step gradient solvent system consisting of chloroform: methanol = (100/0 to 0/100). The active fractions were separated.

The ACAT inhibitory activity of the separated active fractions was measured, and the fractions with the highest ACAT inhibitory activity were collected, and reversed-phase column chromatography was performed using 70%, 80%, 90%, and 100% methanol as the elution solvent. column fractionation, ODS gel). Among them, the fractions having high ACAT inhibitory activity were collected and finally, 90% methanol was flowed at 6 ml / min as an elution solvent, followed by high performance liquid chromatography (HPLC, YMC Jsphere ODS H-80 (250 × 20 mm). )) To give a pure compound (50.8 mg). The active substance was detected at UV 210 nm, and the ACAT inhibitory substance was eluted at 25 minutes.

3) Structure analysis of ACAT inhibitory active substance

The completely isolated and purified compound was a dark, colorless oil. [M + Na] + was m / z 501 and the molecular formula was estimated to be C ₃₀ H ₅₄ O ₄ in high resolution ESI-MS. Ultraviolet absorbance of the compound was measured and the maximum absorption was found at 210 nm. During the NMR test to determine the structure of the compound, 15 carbons, equivalent to 30 half carbons estimated in the mass spectrometry, were observed in ¹³ C-NMR, indicating that the compound had a symmetrical structure. gave. Four olefinic protons (δ 5.13, 5.18) were observed in the ¹ H-NMR spectrum, four methyl protones (δ 1.59, 1.61), and six methylene protons (δ) linked to the olefin group. 1.99, 2.08, 2.01) were observed. In addition, four methylene protons (δ 1.40, 1.58, 2.06 and 2.23), two methine protones (δ 3.36) and four terminal methyl protons (δ 1.15, 1.19) bound to hydroxyl groups Was observed (FIGS. 1, 2 and 3).

Comparative analysis of the compound with data of published documents (Nishiyama Y, Moriyasu M, Ichimaru M, Tachibana Y, Kato A, Mathenge SG et al. Acyclic triterpenoids from Ekebergia capensis. Phytochemistry 1996; 42 (3): 803-807) As a result, 2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (2,3,22,23 -tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene), and the structural formula of the compound is represented by the following Chemical Formula 1.

[Formula 1]

Experimental Example 1: Confirmation of the ACAT activity inhibitory effect of the compound represented by the formula (1)

In order to determine the effect on the ACAT activity of the compound represented by the formula (1) isolated from the scalp according to the present invention, the following experiment was performed.

1) Preparation of ACAT Enzyme Source

Liver of rats (Male Sprague-Dawley rats, 250-300 g) was isolated with ACAT enzyme source and washed with microsome buffer A (0.25M sucrose, 1 mM EDTA, 0.01M tris-hydrochloric acid, pH 7.4), Shears were cut to 1 to 2 cm 3 in size and homogenized using a homogenizer made of Teflon-glass. The homogenate was centrifuged at 14,000 xg for 15 minutes to collect the supernatant and again centrifuged at 100,000 xg for 1 hour.

To separate the microsomes containing ACAT, centrifuged precipitate was added to microsome buffer B (0.25M sucrose, 0.01M Tris-hydrochloric acid, pH 7.4) and centrifuged again at 100,000 xg for 1 hour. It was.

In order to homogenize the protein concentration of the enzyme source during the test, the microsomal buffer B is appropriately dissolved in the centrifuged precipitate, and the protein is prepared by the Lowry method using bovine serum albumin as the protein standard. After the concentration was determined, the protein concentration was adjusted to 10 mg / ml by dilution with microsomal buffer B, which was dispensed into 1 ml vials and stored at -70 ° C for use in experiments.

2) ACAT activity measurement

ACAT activity was measured by using [1- ¹⁴ C] oleoyl-coei and cholesterol as a substrate and by modifying some of the following known methods [Kim Y. K, HW Lee, K. H Son, B. M Kwon] , T. S Jeong, D. H Lee, JH Shin, Y W. Seo, SU Kim, SH Bok 1996.GERI-BP002-A, Novel inhibitors of acyl-CoA: cholesterol acyltransferase produced by Aspergillus fumigatus F93: J. Antibiotics 49: 31-36.

As the reaction solution, 10.0 μl of sample solution dissolved in methanol, 4.0 μl of liver microsomal enzyme from liver tissue, rat standard buffer solution (0.5 M KH ₂ PO ₄ , 10 mM DTT) (Dithiothreitol), pH 7.4] 20.0 μl, bovine serum albumin, 40 mg / ml prepared by expressing essential fatty acid free to prevent the fatty acid from entering the system, 15.0 µl, 2.0 µl of 20 mg / ml cholesterol and 41.0 µl of distilled water were added thereto, mixed well, and the preliminary reaction was performed while shaking at 37 ° C. for 20 minutes.

8.0 μl of [1- ¹⁴ C] oleoyl-coei (0.05 μCi, final concentration of 10 μM) was added to the reaction solution and reacted again at 37 ° C. for 25 minutes. v / v)) was added to stop the enzyme reaction. Centrifugation was performed by adding 0.6 ml of heptane and 0.4 ml of standard buffer diluted five-fold.

To measure the ACAT activity, 100 µl of the supernatant obtained by centrifugation was added to a scintillation bottle, and 3 ml of scintillation cocktail (Lipoluma, Lumac Co.) was added, followed by radioactivity using a liquid scintillation counter. radioactivity was measured

Inhibition of ACAT activity was measured by using a radiometric measuring apparatus by adding a test sample to a substrate and an enzyme labeled with radioactivity, and calculating the inhibition of ACAT activity using the following equation (1).

CPM (T): CPM (counter per minute) when sample and enzyme are added

CPM (C1): No sample added, enzyme added CPM

CPM (C2): CPM when sample was added but no enzyme was added

CPM (B): CPM without sample and enzyme

At this time, all the basic manipulations were carried out on an ice bath, and obovatol was used as a positive control.

As a result of measuring the ACAT inhibitory activity using Equation 1, the IC ₅₀ value of obovatol, which is a positive control, was 44 μM, and the IC ₅₀ value of the compound represented by Formula 1 of the present invention was 24 μg / ml. Appeared.

In addition, as shown in Figure 4, the compound of Formula 1 of the present invention effectively inhibited ACAT activity with increasing concentration.

Therefore, the compound of Formula 1, which exhibits concentration-dependent activity, can be usefully used for the prevention and treatment of various cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by hypercholesterolemia.

Experimental Example 2 Effect of the Compound of Formula 1 on Cholesterol Acylation in HepGe Cells

In order to determine the effect on the cholesterol acylation of the compound of formula (I) isolated from the scalp according to the present invention, it was measured by modifying some known methods [Namatame I, H. Tomoda, H. Arai, K. Inoue, S. Omura 1999. Complete inhibition of mouse macrophage-derived foam cell formation by triacsin C: J Biochem 125: 319-327].

Human liver-derived HepG2 cell line used in this experiment was cultured using D-MEM medium (Dulbecco's Modified Eagle Medium) to which 10% bovine serum was added.

Incubate HepG2 cells in semi-saturated cells in each well of a 48 well plate and mix with 2.5 μl of sample and 7 mg / ml bovine serum albumin [ ¹⁴ C] olenic acid (0.5 μCi, 33.4 μM). ) Was added and reacted at 37 ° C for 4 hours.

The reaction solution was removed, the cells were washed three times with standard buffer solution (PBS), and 0.5 ml of 0.1% (wt / vol) sodium dodecyl sulfate (SDS) solution mixed in the PBS solution was added to lyse the cells. 1 ml of isopropanol-hexane (2: 3 (v / v)) was added to extract the intracellular lipid components.

The extracted organic solvent fraction was concentrated completely using nitrogen gas, and then separated by TLC plate using petroleum ether: ether: acetic acid (90: 10: 1, v / v / v) as a developing solvent. It was. [ ¹⁴ C] Oleic acid-acylated cholesterol radioactivity was measured by a bio-imaging analyzer (BAS1500, Fuji).

Cholesterol acylation inhibitory activity in HepG2 cells, the compounds of formula (I) is the IC ₅₀ value was significantly lower than the IC ₅₀ value of ACAT enzyme inhibitory concentration to 16.28 ㎍ / ㎖.

As shown in Table 1, the compound represented by Formula 1 of the present invention effectively inhibited cholesterol acylation with increasing concentration.

Therefore, the compound of Formula 1 can be usefully used for the prevention and treatment of various cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by hypercholesterolemia.

Experimental Example 3: Acute Toxicity Test

In order to determine the acute toxicity of the compound represented by Formula 1 of the present invention, an acute toxicity test was conducted in the following manner.

6-week-old SPF rats were used as experimental animals and divided into 5 groups per group. The compound of Formula 1 obtained in the above example was suspended in 0.5% methylcellulose solution and administered orally at a dose of 0.1 g / kg.

After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

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

As a result, the compound did not show a change in toxicity in rats up to 0.1 g / kg, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of at least 0.1 g / kg or more.

Formulation Example 1 Preparation of Pharmaceuticals

1) tablet manufacturing

Tablets containing 10 mg of the compound of formula 1 of the present invention as an active ingredient were prepared by the following method.

1.0 g of the compound of Formula 1 was mixed with 7.0 g of lactose, 1.5 g of crystalline cellulose, and 0.5 g of magnesium stearate, and then a tablet was prepared by a direct tableting method.

The total amount of each tablet was 100 mg, of which the active ingredient content was 10 mg.

2) Manufacture of capsule

A capsule containing 10 mg of the compound represented by Formula 1 of the present invention as an active ingredient was prepared by the following method.

1.0 g of the compound of Formula 1 was mixed with 5.0 g of corn starch and 4.0 g of carboxy cellulose to prepare a powder. 100 mg of the powder was put in a hard capsule to prepare a capsule.

Formulation Example 2: Food Preparation

Food containing a compound represented by the formula (1) of the present invention was prepared as follows.

1) Preparation of Cooking Seasonings

20 to 95% by weight of the compound of formula 1 was added to general spices such as soybean paste and soy sauce to prepare a cooking seasoning for health promotion.

2) Preparation of Tomato Ketchup and Sauce

0.2 to 1.0% by weight of the compound of formula 1 was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.

3) Preparation of flour food

0.5 to 5.0% by weight of the compound of Formula 1 was added to the flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles to prepare health promoting food.

4) Preparation of soup and gravy

0.1 to 5.0% by weight of the compound of formula 1 was added to soups and broth to prepare meat products for health promotion, soup of noodles and broth.

5) Preparation of Ground Beef

10% by weight of the compound of Formula 1 was added to the ground beef to prepare a ground beef for health promotion.

6) Manufacture of dairy products

5 to 10% by weight of the compound of formula 1 was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

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.

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 compound of formula 1 of the present invention was decompressed and concentrated in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds and the dry powder of the compound of formula (1) prepared above were formulated in the following ratio.

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

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

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

Ganoderma lucidum (0.5% by weight),

Sulfur (0.5 wt%)

8) Preparation of Tea

0.1 to 10% by weight of the compound of formula 1 and liquid ingredients such as fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), water (75%) Prepared.

Formulation Example 3 Preparation of Beverage

1) Preparation of Carbonated Drinks

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

2) Preparation of Health Drinks

Instant sterilization by homogeneously combining the compound of formula 1 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

5 g of the compound of Formula 1 was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4) Preparation of fruit juice

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

As described above, the compound of Formula 1 according to the present invention effectively inhibits the activity of acyl-CoA: cholesterol transferase, thereby preventing and treating various cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by hypercholesterolemia. This can be useful for.

Claims (4)

A composition for preventing and treating cardiovascular diseases, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

According to claim 1, wherein the compound is a composition for the prevention and treatment of cardiovascular diseases, characterized in that isolated from Alpinia katsumadai . A food comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

4. A food according to claim 3, wherein the compound is isolated from Alpinia katsumadai .

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