KR980008237A - Method for producing acyl-CoA: cholesterol acyltransferase (ACAT) activity inhibitor from Omija and composition containing same - Google Patents

Abstract

The present invention relates to a process for producing a rarein-based compound which specifically inhibits the activity of acyl-CoA: cholesterol acyltransferase (ACAT) from Schizandra chinensis Baillon and a composition containing the same The lignan compound having the following formula 1 obtained by extracting and purifying omiza with an organic solvent can be usefully used for the prevention and treatment of cardiovascular diseases caused by hypercholesterolemia.

(Wherein R is a hydrogen, hydroxyl, alkyl or aryl group, and R < ₁ > is hydrogen or a hydroxyl group.

Description

METHOD FOR PREPARING ACYLCOE: CHOLESTEROL ACACY TRANSFERASE (ACAT) ACTIVITY INHIBITOR FROM OMIZA AND COMPOSITION CONTAINING THE SAME

1 shows a hydrogen nuclear magnetic resonance (NMR) spectrum of hyperinsin N. Fig.

Fig. 2 shows a carbon NMR spectrum of hyperinsin N. Fig.

Fig. 3 shows the ultraviolet absorbance spectrum of hyperinsin N. Fig.

Fig. 4 shows a hydrogen NMR spectrum of hyperinsin A. Fig.

5 shows a hydrogen NMR spectrum of hyperinsin K3.

Fig. 6 shows a hydrogen NMR spectrum of hyperinsin.

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

The present invention relates to a method for producing an acyl-CoA: cholesterol acyltransferase (hereinafter referred to as ACAT) activity inhibitor from Schizandra chinensis Baillon which is naturally occurring in Korea and a composition containing the same . More particularly, the present invention relates to a method for producing a lignan compound having an ACAT inhibitory activity from an omega which is used for herbal medicine and food, and a composition for lowering blood cholesterol and inhibiting cholesterol absorption, including the lignan compound.

Hypercholesterolemia, which is caused by an increase in blood cholesterol levels, is a cause of arteriosclerosis, heart attack, angina pectoris, and various types of stroke, and has become one of the most important causes of death in developed countries (WB Kannel, et al., N. J. Med., 311, 1144 (1984)). Accordingly, various studies have been made to reduce the amount of cholesterol in the blood, and in particular, efforts have been made to suppress the absorption of cholesterol taken from foods in connection with excessive high-fat diets.

The advanced civilization is focusing on the development of therapies in this area because of the major causes of death such as cardiovascular disease (hypertension, arteriosclerosis, hypercholesterolemia and hyperlipidemia) and cancer. For example, Merck & Co., Inc. (USA) has introduced 3-hydroxy-3-merhyl glutaryl-CoA reduction, an enzyme necessary for cholesterol biosynthetic metabolism An enzyme inhibitor has been developed as a blood cholesterol lowering agent.

ACAT has been shown to be an enzyme that promotes the esterification of intracellular cholesterol (J. C. Goldstein, et al., Proc. Natl. Acad. Sci. USA, 93, 3178 (1978)). The cholesterol absorbed or biosynthesized in the liver is esterified

It is then transported by lipoprotein to the tissues via the blood vessels. It is known that when cholesterol ester is present excessively, it accumulates in the blood vessel wall or increases the viscosity of blood and becomes a cause of various cardiovascular diseases (D. Steinburg, N. Engl. J. Med., 320, 915 )). Therefore, when the activity of ACAT is inhibited, the amount of cholesterol ester in the blood of the animal is lowered, the viscosity of the blood is lowered, and the amount of cholesterol is increased in the liver, thereby reducing the synthesis thereof and reducing the amount of cholesterol absorbed in the intestine (K. Matsuda, Med. Res. Rev., 14, 271 (1994)), since it seems that ultimately various cardiovascular treatment or prevention is feasible, an ACAT inhibitor has been attempted to be synthesized or extracted from natural products. . For example, synthetic ACAT inhibitors include urea derivatives (DR Sliscovic, et al., J. Med. Chem., 37, 560 (1994)) or imidazole derivatives (RGWiilde, et al., Bioorg. And P. pyripyropenes (Aspergillus fumigatus) (S. Omura, et al., J. Immunol. Lett., 5, 167 (1995)) and those isolated from microbial metabolites include Aspergillus fumigatus J. Antibiotics, 46, 116-119 (1993); Kwon, et al., Tetrahedron Lett., (1994) J. Antibiotics, 48, (1995)), Scedosporium sp. (Obo vatol, Magnolol, and the like) isolated from natural herbaceous preparations, and AS-183 (K. Kuroda et al., J. Antibitics, 46, 1196-1202 ) And Honokiol (Korean Patent Application No. 95-41207) have been developed.

Accordingly, the present inventors have continued to develop an ACAT inhibitor capable of lowering blood cholesterol from natural herbal medicines which have been used for a long time, and as a result, they have found that they can inhibit ACAT activity The present invention has been completed.

[Technical Problem]

It is an object of the present invention to provide a method for producing an ACAT activity inhibitor from Omija 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 preparing a lignan compound having the following general formula (1), comprising the step of extracting and purifying Schizandra chinensis Baillon with a solvent, and a process for producing a lignan compound represented by the general formula There is provided a composition for preventing and treating cardiovascular diseases caused by high cholesterol blood.

(Wherein R is a hydrogen, hydroxyl, alkyl or aryl group, and R < ₁ > is hydrogen or a hydroxyl group.

The lignan compound having ACAT inhibitory activity according to the present invention is obtained by extracting omiza with methanol, concentrating the concentrate, separating the concentrate into water and organic solvent layers by adding hexane and water, separating the organic layer containing the active ingredient Concentrated and the concentrate can be separated and purified by silica gel chromatography and HPLC.

As the lignan compound obtained according to the present invention, compounds represented by the following general formula (2), (3), (4) or (5)

It was confirmed that the compound of Formula 2 is Gomisin N, the compound of Formula 3 is hypermisin A, the compound of Formula 4 is hyperimin K3, and the compound of Formula 5 is hyperimin J (Y. Ikeya , et al., Chem. Pharm. Bull., 27, 2695 (1979)).

According to the present invention, the lignan compound isolated and purified from Omiza has an IC ₅₀ of 10 to 50 / / ml, in particular, a high microsin N of 10 쨉 l / ㎖, a high microsin A of 50 / / 15 μl / ml of high myosin K3, and 20 μl / ml of hypericin J. Since these compounds are isolated from medicinal materials used for herbal medicine and food, there are no problems such as toxicity, and lignan compounds prevent hepatotoxicity (H. Hikino, et al., Planta Medica, 213 (1984) ) There is no problem of hepatotoxicity that most medicines have. Therefore, the compounds isolated in the present invention can reduce the amount of cholesterol in blood through inhibition of ACAT activity, and can be used for the prevention and treatment of various cardiovascular diseases caused by hypercholesterolemia.

The lignan compound according to the present invention may be formulated into a unit dosage form or a multiple dose formulation such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by conventional methods to prevent cholesterol absorption, inhibition , Prevention and treatment of hyperlipidemia, and preparations for the 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 hypermethylated N is administered in an amount of 5 to 10 mg per 1 kg of body weight per day, 25 to 50 mg of hypericin A, 8 to 15 mg of hypericin K3 and 10 to 20 mg of hypericin J may be administered in 1 to several doses. 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 for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example 1]

Isolation and purification of lignan compounds

The Schizandra chinensis Baillon, which is native to the Republic of Korea, was purchased for the present invention. To 1 kg of fully dried Omija, 2 L of methanol was left for 3 days at room temperature, and then separated into a liquid phase and a solid phase using a filter paper. The liquid phase was collected, concentrated under reduced pressure, and then 1 liter of hexane 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 hexane to collect an organic solvent.

The organic solvent layer and water layer were separated and the degree of inhibition of enzyme activity was measured. 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 200 ml of methanol, adsorbed on 500 ml of C18, and then eluted from methanol / water (80/20) to methanol / water (95/5) . The eluate was concentrated under reduced pressure to give a yellowish brown liquid material. The active ingredient fractions were adsorbed onto a silica gel through a column (4.5 x 30 cm) packed with silica gel (Merck, trade name: 9385), and the silica gel column was washed twice with a silica gel column Chromatography was performed to isolate the active fractions. Ultimately, four pure lignan compounds were purified using HPLC. YMC-PackC8 SH-243-5 (250 x 21.2 mm) manufactured by YMC was used as an HPLC column. When hyperinsin N and A were eluted with methanol / water (85/15) as a solvent, they were separated as a single component at 30 minutes and 20 minutes respectively, and hyperinsin K3 was eluted with methanol / water (80/20) And the most polar hypermycin J was purified by eluting with methanol / water (70/30). Among the finally separated pure compounds, hyperinsin N was 0.1%, and hyperinsin A, K3 and J were obtained in a yield of 0.03 to 0.5%.

[Example 2]

Device analysis and structure determination

In order to determine the structure of the ACAT inhibitory active substance isolated and purified in Example 1, the following instrumental analysis was performed.

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). At 1605cml < ^-1 >, the aromatic compound showed a characteristic absorption peak, and the superimin A, K3 and J showed an absorption peak indicating the presence of 3500cml ^- 1OH group.

Molecular weight analysis: The molecular weight was measured by HRC (High-Resolution Electron Chemical Ionization) -MS method using a VG70-VSEQ mass spectrometer. The results are shown in Table 1.

Nuclear magnetic resonance (NMR) analysis: The active substance fully dried was dissolved in CDCl ₃ was determined by NMR analysis with Varian UNITY 300 models placed in a 5mm tube ¹ H-NMR is 299.949MH _Z, ¹³ C-NMR is 75.430MH _Z Respectively

¹ H-spectrum and ¹³ C-spectrum of the compounds of the present invention are shown in FIGS. 1, 2 and 3 respectively show the hydrogen NMR spectrum, the carbon NMR spectrum and the ultraviolet absorbance spectrum of hyperinsin N, and FIGS. 4, 5 and 6 show the hydrogen NMR spectra of hyperinsin A, K3 and J, respectively.

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

Table 1 Physicochemical properties of hypermics N, A, K3 and J

[Example 3]

ACAT inhibition activity of lignan compounds

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㎕ fermentation supernatants, tissue microsomal liver enzymes in mice 4.0㎕, assay buffer _{(0.5M KH 2 PO 4, 10mM} DTT, pH 7.4) 20.0㎕, 40mg / ml bovine serum albumin (fatty acid member) 15.0㎕, 20mg / ml Of cholesterol and 41.0 占 퐇 of water were mixed and pre-reacted at 37 占 폚 for 15 minutes. 8 μl of [1- ¹⁴ C] oleoyl-CoA (0.02 μCi, final concentration 10 μM) was added to the reaction mixture and reacted at 37 ° C. 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 solution obtained by centrifugation, enzyme activity was measured using a liquid scintillation counter. ACAT inhibitory activity is calculated as follows.

CPM (sample) -CPM (blank test)

% Inhibition = 100 × [1 - --------- --------- --------- -------]

CPM (control) - CPM (blank test)

The blank test was carried out at 0 ° C and chlorpromazine was used as a control.

The ACAT inhibition of these compounds was determined and the IC ₅₀ was found to be _10-50 μg / ml.

The following Table 2 shows the results of measuring the enzyme inhibitory activity of the active compounds hyperinsin N, A, K3 and J of the present invention.

Table 2 ACAT inhibition activity (IC50 value) of lignan compounds

[Example 4]

Acute Toxicity Experiments of Hypericin N, A, K3 and J

As a result of subcutaneous injection of high myosin N, A, K3 and J into mice (female) weighing 23 to 25 g each at a dose of 500 mg / kg, no abnormal abnormal symptoms were observed and they did not die until 4 days after administration. The acute toxicity effect of the compound was not observed.

[Effects of the Invention]

According to the present invention, the lignan compound obtained by extracting and purifying from Omija, a herbal medicine whose stability has been ensured by long-term use, has ACAT inhibitory activity and is effective for lowering the blood cholesterol concentration, And can be usefully used as a preparation for the prevention and treatment of various cardiovascular diseases such as cardiovascular diseases.

Claims (5)

A method for producing a lignan compound having the following formula (1), comprising the step of extracting Schizandra chinensis Baillon with an organic solvent and purifying: (Wherein R ¹ is hydrogen, hydroxyl, alkyl, or allyl group, and R ¹ is hydrogen or a hydrosyl group.) The method of claim 1, wherein the lignan compound is a compound having the formula: The method according to claim 1, wherein the omis is extracted with methanol, concentrated, and the concentrate is subjected to layer separation by adding hexane and water, and then the organic layer is concentrated, and the concentrate is purified by silica gel chromatography and HPLC. 4. The method according to any one of claims 1 to 3, wherein the lignan compound is a compound having an acyl-CoA: cholesterol inhibitory activity. A composition for preventing and treating cardiovascular diseases caused by hypercholesterolemia comprising a lignan compound represented by the following formula (1): ※ Note: It is disclosed by the contents of the first application.