KR20030075390A - Inhibitor of HMG-CoA reductase activity containing isoflavone - Google Patents

Abstract

PURPOSE: An inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activities is provided which inhibits cholesterol synthesis and accumulation in body tissues. Therefore, it is used in the prevention and treatment of cardiovascular disease. CONSTITUTION: An inhibitor of HMG-CoA reductase contains isoflavones as an effective ingredient. The isoflavones are separated from fermented soybean paste(toenjang). The isoflavones are selected from the group consisting of genistein, daidzein, glycitein, daidzin, genistin, glycitin, 6'-0-acetyl-genistin, 6'-0-acetyl-daidzin, 6'-0-acetyl-glycitin, 6'-0-malonyl-genistin, 6'-0-malonyl-daidzin and 6'-0-malonyl-glycitin. Preferably daily dosage of isoflavones is in the range of 1 and 25mg/day.

Description

Inhibitor of HMG-CoA reductase activity containing isoflavone}

The present invention relates to an inhibitor of HMG-CoA reductase activity, and more particularly, to inhibit the activity of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase), an enzyme involved in cholesterol biosynthesis. It relates to an HMG-CoA reductase activity inhibitor comprising an isoflavone having an active ingredient.

Cardiovascular disease is known as the leading cause of death in the United States and Europe, and is one of the diseases that frequently occurs in Koreans. Factors causing cardiovascular disease include hereditary predisposition, sex, smoking, lifestyle and age. In particular, hyperlipemia and hypercholesterolemia caused by low density lipoprotein cholesterol (hereinafter referred to as 'LDL-cholesterol') and total cholesterol are major causes of cardiovascular disease. Thus, lowering blood LDL-cholesterol and total cholesterol levels can reduce the incidence of cardiovascular diseases such as atherosclerosis and myocardial infarction.

Cholesterol lowering agents used to lower LDL-cholesterol and total cholesterol levels in the blood, including nicotinic acid, cholestyramine, clofibrate (CPIB), neomycin, and plant sterols. ), Drugs such as triparanol (MER-29), D-thyroxine and estrogen have been used. However, many problems have been reported with these drugs. In other words, nicotinic acid is known to cause side effects such as skin vasodilation, rash, gastrointestinal abnormalities, uric acidemia, hyperglycemia, liver dysfunction. In addition, cholestyramine, although effective for many patients, causes discomfort from overdose. Clofibrate is a cholesterol lowering agent that is typically used, but its effect is minimal and no specific action at the biochemical stage has yet been identified. Neomycin has side effects such as nausea, diarrhea, phytosterol disrupts the absorption of cholesterol in the intestinal tract, triparanol has cataracts, D-thyroxine has arrhythmias, angina pectoris, and complex infarction, and the estrogen hormone is used in men In this case, there is a problem that causes a feminization phenomenon has been limited use.

Therefore, recently, inhibitors that inhibit the activity of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) such as lovastatin have been developed and used as cholesterol lowering agents (Hunninghake, Curr. Opin. Lipidol , 3: 22-28, 1992; Brown et al ., N. Engl. J. Med ., 323: 1289-1298, 1990).

HMG-CoA reductase is a glycoprotein with a molecular weight of 97 kDa and is an enzyme that plays an important role in cholesterol biosynthesis (Rodwell et al ., Adv. Lipid Res ., 14: 1-74, 1976). . During cholesterol biosynthesis, acetyl-CoA and acetoacetyl-CoA are converted to HMG-CoA by HMG synthase, which in turn converts HMG-CoA reductase. After conversion to mevalonate by means of several steps, the final cholesterol is synthesized (Goldstein and Brown, J. Lipid Res ., 25: 1450-1461, 1984). At this time, the conversion of HMG-CoA to mevalonate by HMG-CoA reductase is known as a rate-limiting step of the cholesterol synthesis process, and thus can inhibit the action of HMG-CoA reductase. If so, it can effectively inhibit the synthesis of cholesterol.

However, conventional inhibitors of HMG-CoA reductase activity, such as lovastatin, have a strong inhibitory effect, despite myopathy, rhabdomyolysis, suicidal tendency, neurosis. It is known to cause side effects such as (Corpier et al ., JAMA , 260: 239-241, 1988; Pierce et al ., JAMA , 264: 71-75, 1990; Reaven and Witztum, Ann.Intern. Med ., 109: 597-598, 1988; Duits and Bos, Lancet , 341: 114, 1993; Engelberg, Lancet , 339: 727-729,1992).

Therefore, to overcome this problem, Korean Patent Application No. 1998-37962 discloses a composition comprising a substance called diosmin extracted from citrus fruits, and Korean Patent Application No. 1997-55581 discloses vitamin C, hesperidin and naringin. Including inhibitors of HMG-CoA reductase activity has been disclosed. However, there is still a need for the development of HMG-CoA reductase activity inhibitors and cholesterol inhibitors using the same while effectively lowering blood cholesterol levels and having no side effects on the human body.

On the other hand, isoflavone (isoflavone) is a kind of plant hormone that is separated from the plant is effective in the treatment of osteoporosis (Korean Patent Application No. 2000-714485), and is known to have effects such as antioxidant activity, anticancer action. It has also been found that by reducing blood levels of LDL-cholesterol and total cholesterol, it inhibits or delays the development of atherosclerosis. That is, Korean Patent Application No. 1997-6053 discloses a method for inhibiting cholesterol biosynthesis using isoflavones, genistein, and Korean Patent Application No. 2000-21186, which has an effect of lowering cholesterol concentration, genistein and die A composition comprising isoflavones such as zein, glycine, and the like has been disclosed. However, there is no research on how this isoflavone inhibits cholesterol biosynthesis.

Therefore, while the present inventors are studying a method that can effectively inhibit cholesterol production without side effects in the human body, the activity of HMG-CoA reductase in which isoflavones isolated from soybean fermented soybeans act on cholesterol biosynthesis metabolism The present invention was completed by revealing that it can suppress and, as a result, lower the amount of cholesterol in the human body.

The technical problem to be achieved by the present invention is to provide an inhibitor of HMG-CoA reductase activity having a function of inhibiting the activity of HMG-CoA reductase and consequently inhibiting cholesterol synthesis.

Figure 1 is a graph (Lineweaver-Burk plot) for measuring the inhibitory activity of Genistein HMG-CoA reductase, Figure 1a using HMG-CoA as a substrate, Figure 1b is used as a substrate NADPH.

Figure 2 is a graph (Lineweaver-Burk plot) for measuring the inhibitory activity of Dyzein against HMG-CoA reductase, Figure 2a is used as a substrate HMG-CoA, Figure 2b is used as a substrate NADPH .

3 is a graph for measuring the inhibitory activity of glycine HMG-CoA reductase (Lineweaver-Burk plot), Figure 3a is used as a substrate HMG-CoA, Figure 3b is used as a substrate NADPH .

Figure 4 is a graph (Lineweaver-Burk plot) for measuring the inhibitory activity of Genistin HMG-CoA reductase, Figure 4a is a substrate using HMG-CoA, Figure 4b is a substrate using NADPH.

5 is a microscopic observation of the inhibitory effect of HMG-CoA reductase activity by isoflavones in vero cells, FIG. 5A shows cells treated with 2 μg of genistein, and FIG. 5B simultaneously shows genistein and mevalonate. Treated cells, FIG. 5C is a photograph of observation of control cells treated with nothing.

In order to achieve the above object, the present invention provides an HMG-CoA reductase activity inhibitor comprising isoflavones as an active ingredient.

In addition, the present invention provides a cardiovascular disease treatment agent comprising the HMG-CoA reductase activity inhibitor.

Hereinafter, the present invention will be described in detail.

Isoflavone is a flavonoid family of soybeans and is called phyto estrogen because it has a similar chemical structure to the female hormone estrogen. The present invention is characterized by providing an HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) activity inhibitor comprising the isoflavone as an active ingredient.

In general, isoflavones exist in glycoside form in nature, and are converted into nonglycoside forms by β-glucosidase, which is difficult to absorb in the human body. However, in the case of fermented foods, most of the glycosides of isoflavones are transformed into non-glycoside forms during fermentation, and even when ingesting isoflavones in the form of glycosides, many of them are converted into non-glycoside forms during digestion and absorption. Thus, the same effect as the isoflavone nonglycoside form can be obtained. Therefore, as an inhibitor of HMG-CoA reductase activity according to the present invention, both isoflavones in the form of glycosides and isoflavones in the form of glycosides can be used.

The isoflavone in the non-glycoside form is preferably selected from the group consisting of genistein, diedzein, and glycidin.

In addition, the isoflavone in the glycoside form is genistin, didzin, glycidin, 6'-O-acetyl-genistin, 6'- 6'-O-acetyl-daidzin, 6'-O-acetyl-glycitin, 6'-O-malonylzenistin (6'-O) group consisting of -malonyl-genistin, 6'-O-malonyl-daidzin, and 6'-O-malonylglycitin (6'-O-malonyl-glycitin) It is preferred to be selected from.

The isoflavones may be isolated from natural plants or chemically synthesized by known methods. Specifically, the isoflavones are preferably separated from soybeans, most preferably from fermented forms of soybeans such as soybean paste. Separation of isoflavones from the doenjang can be separated by chromatography methods which are known in the art.

Although isoflavone may be used directly as an inhibitor of HMG-CoA reductase activity according to the present invention, a material processed and manufactured by adding a pharmaceutically acceptable carrier may be used according to a pharmaceutical preparation method known in the art.

Examples of the carrier in the above, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose And polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the HMG-CoA reductase activity inhibitor according to the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

HMG-CoA reductase activity inhibitors according to the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

In addition, the HMG-CoA reductase activity inhibitor according to the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous or intramuscular.

The daily dose of isoflavone used as the inhibitor of HMG-CoA reductase activity according to the present invention is preferably in the range of 1 to 25 mg / kg. However, the actual dosage should be determined in consideration of several relevant factors such as the route of administration, the age, sex, weight of the patient and the severity of the patient and the like does not limit the scope of the invention in any aspect.

On the other hand, the present invention provides a cardiovascular disease treatment comprising the HMG-CoA activity inhibitor containing the isoflavones.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Isolation and Purification of Isoflavones from Doenjang

Isoflavones were isolated and purified from the doenjang by chromatography, a method known in the art.

First, lyophilized doenjang was crushed finely, and then powdered by filtering with a 35 mesh (mesh). 30 g of the powdered doenjang was mixed at a ratio of 1:30 (w / v) with a mixture of methanol and dichloroform (1: 1, v / v) and homogenized at 10,000 rpm for 5 minutes. The homogenized mixture was shaken for 18 hours in a Rotary Separatory Funnel, filtered using Whatman paper, and concentrated using a rotary evaporator to obtain miso extract.

The concentrated extract was adsorbed onto silica gel and subjected to VLC (vaccum liquid chromatography). VLC is a sintered glass funnel using thin layer chromatography grade packing (Silica gel 60H, Merck, Germany), Iwaki glass, Japan, 25G4, porosity: 5-10㎛, diameter: 90mm, column height: 50mm). The concentrate adsorbed on silica gel was developed in a thin layer chromatography (TLC) plate with a developing solvent of chloroform-methanol-acetic acid (85: 10: 5, v / v), and the color development was confirmed using a UV lamp. The fractions showing the same UV absorbance were combined and then subjected to VLC (vaccum liquid chromatography). After performing VLC twice, the obtained materials were dissolved in methanol, and chromatography was performed using a Lipophilic Sepadex LH-20, 20 × 400 mm, Sigma Chemical Co., St. Louis, MO. Was performed.

Each of the components obtained above was purified using HPLC (High performance liquid chromatography), NMR (Nuclear Magnetic Resonance) was performed to determine the type of isoflavones contained in the purified material. As a result, each component isolated and purified from isoflavones is genistein, dyedzein, glycidine, dydazine, genistin, glycidine, 6'-O-acetylidazine, 6'-O-acetylgeninistin , 6'-O-acetylglycithin, 6'-O-malonyldide, 6'-O-malonylgenistin and 6'-O-malonylglycitin was confirmed.

<Example 2>

Inhibitory Effect of HMG-CoA Reductase Activity by Isoflavones

The inhibitory effect of isoflavones on Syrian Hamster HMG-CoA reductase (fromprofessor Rodwell in Department of Biochemistry, Purdue University, USA) was verified.

HMG-CoA (Sigma Co., USA) and NADPH (ICN Biochemicals, Inc., USA) were used as substrates of the enzyme, and genistein, dydane, glycine in isoflavones isolated as in Example 1 above as inhibitors. Genistin, a glycoside of cysteine and genistein, was used. Each isoflavones were dissolved in alkaline state using 0.1 N NaOH and used in experiments with different concentrations.

Non-linear regression method based on the formula of Duggleby and Leonard, DNRPEASY, a data analysis computer program (1992) to determine the inhibitory activity characteristics (V _max , K _m , K _i ) regression method) was used, and the reaction rate was measured according to the change of the concentration of the substrate, and represented by a double-reverse plot (Lineweaver-Burk's plot).

First, as a result of measuring the characteristics of the enzyme HMG-CoA reductase used, V _max value (μmol NADPH / min / mg) was 15 ± 5, K _m value was 141 ± 12 μM for NADPH, HMG-CoA Were measured at 32 ± 5 μM, respectively.

In order to observe the inhibitory effect of isoflavones on HMG-CoA, the substrate of HMG-CoA reductase, the NADPH concentration was fixed at 200 μM and the HMG-CoA concentration was changed to 9-50 μM. In addition, to observe the inhibitory effect of isoflavones on NADPH, HMG-CoA concentration was fixed at 200 μM and NADPH concentration was changed to 28-150 μM.

As a result, as shown in Figures 1 to 4, it was confirmed that each isoflavones act as a competitive inhibitor against HMG-CoA, and a non-competitive inhibitor against NADPH. In addition, as a result of calculating each K _i value, Genistein is 27.7 ± 4.3 μM (Fig. 1), Dyzedein is 49.5 ± 4.5 μM (Fig. 2), Glycsteine is 94.7 ± 11.9 μM (Fig. 3), Genistin was confirmed to be 40.0 ± 3.4 μM (FIG. 4). That is, the isoflavones were found to have a competitive inhibitory activity against HMG-CoA, a substrate of HMG-CoA reductase, and as a result, it was confirmed that the cholesterol biosynthesis process could be inhibited.

<Example 3>

Isoflavone-induced HMG-CoA Reductase Inhibitory Activity in Animal Cells

Animal cells were used to measure HMG-CoA reductase inhibitory effect of isoflavones.

Inhibition of HMG-CoA reductase activity by isoflavones was confirmed using Vero cells (KCLB 10081), a representative cell line used to search for HMG-CoA reductase activity inhibitors. Vero cells were seeded in 96-well plates containing 100 μl of Eagle's minimal basal medium (2% Calf serum-minimal eagles's media; CS-MEM) at a concentration of 3 × 10 ⁴ cells each. . After 1 hour of incubation, two pairs of paper disks containing various concentrations of xenstein dissolved in methanol were administered. At each concentration, a pair of 1 mg of mevalonate, a product produced by HMG-CoA reductase, was simultaneously administered to recover the cell morphological changes caused by HMG-CoA reductase inhibition of isoflavones. . As a control, only paper discs were administered and compared.

As a result, as shown in Figure 5a, when the administration of 2μg of the non-glycoside form of isoflavone genistein in each well changes the intracellular metabolic system to change the shape of the cell growth control (see Figure 5c) It was confirmed that the inhibition compared with the. In addition, as shown in Figure 5b, it was confirmed that the morphology of the cells is restored by the addition of mevalonate, a result produced by the catalysis of HMG-CoA reductase. That is, the cholesterol biosynthesis metabolism was restored by the addition of mevalonate to confirm that the growth of the cells was recovered.

These results show that isoflavones act as inhibitors of HMG-CoA reductase in the cellular environment.

The HMG-CoA reductase activity inhibitor containing isoflavones according to the present invention acts as a competitive inhibitor for HMG-CoA reductase, which plays a major role in cholesterol biosynthesis. Therefore, by using the HMG-CoA reductase activity inhibitor of the present invention, blood cholesterol content can be lowered, and as a result, cardiovascular diseases can be prevented and treated.

Claims (4)

HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) activity inhibitor comprising isoflavone as an active ingredient. The method of claim 1, wherein the isoflavone is genistein, diedzein, glycidin, diidzin, genistin, glycidin, 6 '. 6'-O-acetyl-genistin, 6'-O-acetylidazine, 6'-O-acetyl-glycithin (6'-O) -acetyl-glycitin), 6'-O-malonyl-genistin, 6'-O-malonylidezin (6'-O-malonyl-daidzin), and 6'-O -HMG-CoA reductase activity inhibitor, characterized in that selected from the group consisting of malonyl glycidin (6'-O-malonyl-glycitin). The HMG-CoA reductase activity inhibitor according to claim 1, wherein the isoflavone is isolated from doenjang. A cardiovascular disease treatment comprising the HMG-CoA reductase activity inhibitor according to claim 1.