KR19990069246A - Inhibitory Effects of Soybean Sprout Extracts on Residual Oxidase, Aldose Reductase and Squalene Synthase - Google Patents

Abstract

The present invention relates to inhibitors of residual oxidation oxidase, aldose reductase, and squalene synthetase, and invented that a substance extracted from bean sprouts has inhibitory activity. This substance is useful for the treatment of gout caused by the abnormality of purine acid in association with xanthine oxidase, and is used as an inhibitor of aldose reductase to prevent and improve complications caused by diabetes. In addition, by inhibiting cholesterol biosynthesis, the rate of synthesis of LDL-recepto may be increased, thereby lowering plasma cholesterol levels, which may be used for the prevention and improvement of cardiovascular diseases.

Description

Inhibitory Effect of Soybean Sprout Extract on Residual Oxidase, Aldose Coenzyme and Squalene Synthase

1 is a diagram of the growth stages of bean sprouts.

2 is a graph showing the sugar content of each stage of sprout sprout growth.

3 is a graph showing the inhibitory effect of the residual oxidase, aldose reductase, squalene synthetase of day 3 sprouts extract.

(1) Description of residue oxidase inhibitors

The present invention relates to a residual oxidase inhibitor that can be used for the treatment of gout. Xanthine oxidase is distributed in various species of organisms, from bacteria to humans. In mammals, it is an enzyme distributed in various tissues such as liver and small intestine mucosa. The enzyme is involved in the oxidation of various substrates, such as various purine-based substances in the cell and ethanol in the extracellular cells, but is known as rate-limiting in the final oxidation of all purines. Gout caused by abnormal purine metabolism associated with xanthine oxidase is associated with an increase in uric acid in the blood. The final product of human purine metabolism, uric acid, is in the monosodium (monosodium) state in the body, due to its low solubility in the blood concentrations of the crystals to form precipitates or deposits in the kidney tissue causing kidney failure. Gout is usually caused by a long period of hyperuricemia, which leads to the precipitation of uric acid, which develops into acute inflammation. The development of uric acid hyperemia, which has long been considered the most important and dangerous factor in the beginning of gout, is caused by the overproduction of uric acid, decreased excretion of uric acid in the kidneys, or a combination of both. Gout treatment with xanthine oxidase inhibitors effectively reduces the amount of uric acid in the blood and urine, so that the accumulation of hypoxanthine and xanthine is again used in the production of nucleotides by the salvage pathway, and also by IMP, GMP and The accumulation of AMP feed back inhibition of purine biosynthesis, resulting in no accumulation of debris, resulting in reduced blood uric acid without significantly affecting purine metabolism. However, the present inhibitors have many side effects such as nephrotoxicity in the body or compete with substrates for enzymes, and also do not inhibit the production of oxygen radicals of residual oxidase. Screening revealed that the bean sprout extract showed very strong activity.

(2) Description of aldose reductase inhibitor

Two enzymes, aldose reductase and polyol dehydrogenase, are known to be involved in the sorbitol synthesis process. These enzymes are present in the tissues of the body, including the peripheral nerves, retina, cornea, iris, lens, kidney, and red blood cells. Aldose reductase is an enzyme that causes the first reaction of sorbitol synthesis. It is a rate-limiting enzyme that converts glucose to sorbitol using NADPH as a coenzyme. And, once sorbitol is produced, sorbitol is converted to fructose by polyol dihydrogenase. Under physiological conditions, the flow of synthesis by these two enzymes is in equilibrium with most tissues. The accumulation of sorbitol is almost negligible in most tissues, and the affinity of aldose reductase for glucose is lower than that of hexokinase. Therefore, since most intracellular glucose is phosphorylated, the rate of sorbitol formation is lowered. Thus, in the case of diabetics, the further increase in the concentration of glucose in the blood causes sorbitol and fructose accumulation due to this increased activity. At this time, the produced polyol does not easily penetrate the cell membrane, so the concentration of this metabolite in the cell increases, resulting in an increase in the molar osmolarity. Under these conditions, the amount of myoinositol in the nervous tissue is reduced. The resulting deficiency of miyoinositol causes various metabolic changes, including the reduction of sodium-potassium adenosine triphosphatase (ATPase), electrophysiological changes, reduced energy consumption in nerve tissues, and changes in nerve tissues. The result of this change is a decrease in cell membrane stability, leading to diabetic complications such as neuropathy, nephropathy, retinopathy and cataract, which eventually lead to cell death. Aldose reductase inhibitors bind to aldose reductase and thus inhibit the conversion of glucose to sorbitol. This prevents the reduction of miyoinositol and damage of sodium-potassium ATPase. Aldogenases and compounds cause noncompetitive inhibition of aldose reductase. Inhibitors that have been discovered so far include cannons such as sorbinil, alrestatin, epalrestat, ponalestat, tolrestat, and many other inhibitors have been developed or synthesized. But so far, these aldose reductase inhibitors have had short-term stability for neuropathy in diabetics or a slight improvement in neurological function, but not enough for long-term effects. There are still side effects in treating diabetic complications in the long term, so the search and development of new inhibitors are still ongoing.

(3) Description of squalene synthase inhibitor

The most common cause of death in Korea, as well as in the United States, is cardiovascular disease, which is known to be closely related to plasma cholesterol levels. Plasma cholesterol levels in Koreans are still lower than those of Westerners, but have risen more than 10 mg / dl over the last 20-30 years, especially in low age groups. As a result, in recent years, the number of patients with hypertension, cerebrovascular disease, and heart disease has been increasing year by year, and the age of myocardial infarction patients is gradually decreasing. This change may be attributable to changes in the diet of Koreans with a significant increase in animal fat and protein intake. Cholesterol, on the other hand, is an insoluble steroid compound with 27 carbon atoms, which is not only an essential membrane component of animal cells but also progestins, corticosteroids, and sex hormones, androgens. It acts as a precursor to estrogens, forming bile acids necessary for the absorption of fat or fat-soluble vitamins from the intestine, and ultra-low-density lipoproteins (VLDL) that are formed in the liver to transport fat from the liver to peripheral tissues. It is also used to form low-density lipoproteins. Cholesterol must be transported from the synthesized or absorbed tissue to its required tissue for its accumulation or utilization and, like triglyceride, is transported and metabolized in the form of lipoproteins in plasma. The transport and metabolism of these lipoproteins shows that cholesterol and triglycerides absorbed by the intestine through foods are made in the form of chylomicrons, and along the bloodstream go to capillaries of fat and muscle tissues Under the action of lipoprotein lipase, triglycerides are broken down into residual chylomicron remnants. It is ingested and broken down by the hepatocytes and released into cholesterol and used to form cell membranes, bile acids and steroid hormones. In addition, the liver secretes cholesterol and triglycerides mainly in the form of ultra low density lipoproteins, which are transformed into intermediate-density lipoproteins (IDLs) as triglycerides are removed by the action of lipoproteins in the blood vessel walls. Density lipoproteins are converted to low-density lipoproteins (LDL) by removing residual triglycerides.Low-density lipoproteins remain in the blood for a long time and most of the plasma cholesterol (60-70%) is in the form of low-density lipoproteins. . Low density lipoproteins in the blood are ingested again by the liver and peripheral tissues. Excess cholesterol in the terminal tissue is transported back to the liver via a reverse cholesterol transport system, secreted into the intestine in the form of bile salts and excreted.

LDL in the blood is transported into the cell through the LDL-receptor of the cell membrane. When the concentration of intracellular cholesterol is high, the synthesis rate of the LDL-receptor is decreased and the absorption of cholesterol from the blood is slowed. High levels of cholesterol in the blood can lead to serious illness. When the amount of cholesterol from biosynthesis and food intake is greater than the amount necessary for the synthesis of cell membranes, bile acids, and steroid hormones, blood vessels accumulate cholesterol and Narrowing can interfere with blood flow, and in severe cases, blockage of blood vessels (coronary arteriosclerosis) occurs, stopping blood flow and causing tissue death. At present, the main risk factors for cardiovascular disease include an increase in plasma cholesterol level and an increase in LDL-cholesterol. Therefore, maintaining the normal level is aimed at preventing cardiovascular disease. In humans, approximately 300 to 500 mg of cholesterol is consumed in the diet and 700 to 900 mg is synthesized in the body. Since about two thirds of the total cholesterol in the body is directly synthesized in the body, it inhibits cholesterol biosynthesis. Lowering plasma cholesterol levels by increasing the rate of synthesis of LDL-receptors is believed to be effective.

The therapeutic agents for cardiovascular diseases thus far can be classified into three types: agents that lower plasma cholesterol, agents that alleviate and alleviate arteriosclerosis, and agents that block or reverse the progress of cardiovascular diseases. Among them, plasma cholesterol lowering agents are the most widely used and mainly act to lower the level of LDL-cholesterol or total cholesterol in the blood. The agent lowers plasma cholesterol levels by promoting catabolism of intracellular cholesterol, depending on the mechanism of action, such as bile acid sequestrant, derivatives of fibric acid, nicotinic acid. ) And probucol, which inhibit cholesterol cholesterol synthesis enzyme HMG-CoA reductase (3-hydroxy-methyl-glutaryl coenzyme A reductase), which increases the rate of synthesis of LDL-receptors to lower plasma cholesterol levels. Formulations, for example, lovastatin (lovastatin), provastatin, simvastatin (comppactin) derivatives, and the like and inhibit the activity of the cholesterol transfer enzyme acyl-Co A: cholesterol acyltransferase (ACAT) Agents that lower plasma cholesterol levels by inhibiting the absorption of cholesterol, such as, for example, perpactin and epicory, which are mainly developed in Japan. Epicohliquinone, acaterin, helminthosporols, lacteritin, gypsetin, enniatin, glisoprenins, pyripyrophene (pyripyropenes), terpendoles (terpendoles) and the like.

In addition, attempts to prevent atherosclerosis by inhibiting hyperlipidemia by inhibiting cholesterol ester transfer protein (CETP), an enzyme that transfers cholesterol in HDL to LDL, have been conducted by many pharmaceutical companies, including Upjohn Co. and Merck Co. Is going on. In addition, a recent study to search for inhibitors of squalene synthase (SQS), an important enzyme involved in cholesterol synthesis, has recently begun, such as Merago's zaragozic acid and Glaxo's squalestatin. It has been reported that new substances are separated from microorganisms.

The content of the present invention will be described in detail as follows.

The supernatant obtained by boiling water sprouts of each growth stage provided by Pulmuone Co., Ltd. was extracted with ethyl acetate in order to prevent denaturation by hydrogen ion concentration or temperature.

Hereinafter, the present invention will be described in more detail with reference to Examples.

(Example 1)

Preparation of Residual Oxidase Coenzyme Solution

Pig livers were washed with 0.25 M glucose solution and then homogenized with a double volume of 0.1 M potassium phosphate buffer (pH 7.8). The homogenized liver was centrifuged at 15,000 x g for 20 minutes to discard the precipitate and the supernatant was precipitated with 35% ammonium sulfate and centrifuged at 15,000 x g for 20 minutes. Again, this supernatant was precipitated with 65% ammonium sulfate and centrifuged at 15,000 x g for 20 minutes. The precipitate was dissolved again in 0.1 M potassium phosphate buffer (pH 7.8) and dialyzed with the same buffer before use in the experiment.

(Example 2)

Preparation of aldose oxidase coenzyme solution

200 g of pig kidney was placed in the same amount of 0.01 M sodium phosphate buffer (pH 7.0), homogenized and centrifuged at 20,000 x g for 30 minutes to remove insoluble matters. The supernatant was taken and precipitated with 20.8g / 100ml ammonium sulfate, and then left for 5 hours to remove the precipitate by centrifugation. Slowly add 23.6 g / 100 ml ammonium sulfate to this supernatant. After standing for 5 hours, the precipitates were collected by centrifugation, and the precipitates were dissolved in 0.01 M sodium phosphate buffer (pH 7.0) and stored at −70 ° C. and used for experiments.

(Example 3)

SQS production from pork liver

It was chopped pork liver 100g to prepare the SQS sliced fully gearing homogenization was added to homogenize the solution (0.3M sucrose _{/ 5mM DTT / 10mM MgCl 2 /} 0.1M phosphate buffer pH 7.4 containing 50mM KCl) of 200ml, and then, 4,000 × g The supernatant was collected by centrifugation for 15 minutes at, followed by centrifugation at 15,000 × g for 30 minutes to obtain a supernatant. Subsequently, the obtained supernatant was ultracentrifuged at 105,000 × g for 1 hour to precipitate microsomes, washed by addition of 50 ml of the homogenization solution, and then homogenized sufficiently, followed by ultracentrifugation at 105,000 × g for 30 minutes. The moths were precipitated. The precipitated microsomes were suspended in 2 ml of homogenization solution and used as an enzyme source of SQS.

(Example 4)

Preparation of Bean Sprout Extract

On day 3, 300 g of bean sprouts were put in an appropriate amount of water, boiled for 3 hours, and extracted with hot water. Distilled water was added to the hot water extract to adjust the total volume to 300 ml, and then the solution was adjusted to pH 3 and extracted with the same amount of ethyl acetate to prevent denaturation by hydrogen ion concentration or temperature. 300 µl of ethyl acetate, which was equivalent to the boiling water extraction molar, was concentrated under reduced pressure and used to measure the inhibition of enzymatic activity.

(Example 5)

Residual Coral Enzyme Inhibitory Activity

The activity of the enzyme was measured at 292 nm by the increase in absorbance due to the uric acid produced by reacting for 30 minutes at 37 ℃ using a spectrophotometer. The reaction solution used for the activity measurement was 3 ml in total volume and contained 0.1 M potassium phosphate buffer (pH 7.8), 100 μM residue, and an appropriate amount of coenzyme solution. Inhibitor activity was measured by adding an inhibitor to a reaction solution containing no substrate and reacting the inhibitor with an enzyme at 37 ° C. for 30 minutes.

(Example 6)

Aldose Reductase Inhibitory Activity

Coenzyme solution was prepared from the porcine kidney by 35-70% ammonium sulfate precipitation and DEAE sepharose anion exchange resin column chromatography. The activity of the enzyme was measured as the amount of decrease in absorbance of NADPH which occurred for 5 minutes at 37 ° C. and 340 nm using spectrophotometry UV1201, SHIMADZU, JAPAN. 0.2 M sodium phosphate buffer (pH 7.0), 5 mM DL-glyceraldehyde, 0.125 mM NADPH, and an appropriate amount of coenzyme solution were added to 1 ml of total volume.

(Example 7)

Inhibition of SQS activity

To measure the activity of SQS, 100 μl phosphate buffer solution containing 50 μl of microsomes (5 mg protein ml), the enzyme source of SQS prepared in Example 1, and a reaction solution (5 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 2 mM NADPH) pH 7.4) 150 μl was mixed and left at 37 ° C. for 10 minutes, and then 20 μl of [ ³ H] -panesyl pyrophosphate (0.05 μCi) was added thereto, followed by reaction for 30 minutes at 37 ° C. Then, 200 μl The reaction was stopped by adding cold ethanol, and 900 µl of n-hexane was added and mixed vigorously for 3 minutes, followed by centrifugation at 8,000 rpm for 10 minutes to obtain a hexane layer. The enzymatic activity was measured by mixing the cocktail solution and scintillation counter, and as a result, the cpm value of the positive control group was about 20,000 without adding the inhibitor to the reaction solution, and 50 μl of the reaction solution was added instead of the microsome. Cpm of control Was 400 degree.

The degree of inhibition of SQS activity was determined by the cpm value of the sample reacted with 50 µl of the microsome and 20 µl of the inhibitor of Example 3 described below in 150 µl of the reaction solution. Prior to this, farnesyl pyrophosphate is a water-soluble substance with a strong polarity, and squalene synthesized from the farnesyl pyrophosphate by the enzymatic action of SQS is a low-polar fat-soluble substance. Of course, the amount of squalene extracted in n-hexane also increases naturally, resulting in a higher cpm value. On the contrary, when SQS enzyme activity is inhibited and the amount of squalene synthesized decreases, the amount of squalene extracted in the n-hexane layer decreases, thereby lowering the cpm value.

Accordingly, inhibition rate (%) was calculated by the following equation.

Claims (4)

Inhibitory Effect of Soybean Sprout Extract. The method according to claim 1, wherein the bean sprout extract is used to treat gout by inhibiting residual oxidase involved in gout. The method of claim 1, wherein the bean sprout extract is used to inhibit aldose reductase involved in the complications of diabetes mellitus and to treat diabetes complications. The method of inhibiting cholesterol biosynthesis according to claim 1, wherein the bean sprout extract is used to inhibit the activity of squalene synthase.