KR100862610B1 - Composition for preventing or treating cardiovascular diseases comprising an extract of rubus coreanum fermentation as active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of cardiovascular diseases comprising bokbunja fermented extract as an active ingredient, more specifically in the perfusion model of the adrenal glands extracted from Sprague-Dowely rats with normal blood pressure Fermented extract isolated from Bokbunjaju significantly inhibits catecholamine (CA) secretion of adrenal gland by various secretagogues such as acetylcholine, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazone acid By revealing that, the composition comprising the bokbunja fermented extract according to the present invention as an active ingredient can be usefully used for the prevention or treatment of cardiovascular diseases.

Bokbunja fermentation, extract, adrenal gland, catecholamines, cardiovascular disease

Description

COMPOSITION FOR PREVENTING OR TREATING CARDIOVASCULAR DISEASES COMPRISING AN EXTRACT OF RUBUS COREANUM FERMENTATION AS ACTIVE INGREDIENT}

1 is a schematic diagram showing a method for separating and separating adrenal gland (left) and perfusion of adrenal gland (right) from Sprague-Dowely rats.

FIG. 2 is a flowchart illustrating a process of separating polyphenolic compound of fermented liquor of Rubus coreanum ( PCRC ).

Figure 3 is a graph showing the time and concentration-dependent effect of the bokbunja fermentation extract on catecholamine (CA) secretion by acetylcholine (ACh) in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats.

X axis: Perfusion liquid collection time (min) after polyphenol compound (PCRC) treatment, an extract obtained from Bokbunjaju.

Y-axis: amount of catecholamines secreted from the adrenal glands for 4 minutes (100% of control (1371 ± 20 ng / 4 min)).

Figure 4 is a graph showing the time and concentration-dependent effects of bokbunja fermentation extract on catecholamines (CA) secretion by high potassium in the perfusion model of the adrenal glands isolated from the sprag-dolly rats.

FIG. 5 is a graph showing time and concentration-dependent effects of bokbunja fermentation extract on catecholamine (CA) secretion by DMPP in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats.

FIG. 6 is a graph showing time and concentration-dependent effects of bokbunja fermentation extracts on catecholamine (CA) secretion by McN-A-343 in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats.

7 is a graph showing the effect of bokbunja fermentation extract on catecholamine (CA) secretion by Bay-K-8644 in the perfusion model of the adrenal gland isolated from the sprag-dolly rats.

8 is a graph showing the effect of bokbunja fermentation extract on catecholamine (CA) secretion by cyclopiazonic acid in the perfusion model of the adrenal gland isolated from the sprag-dolly rats.

Figure 9 is a catecholamine (ACH) by acetylcholine (ACH) in bokbunja fermentation extract + L-NAME (N-ω-nitro-l-arginine methyl ester hydrochloride) in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats CA) is a graph showing the effect on secretion.

10 is a graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by high potassium in the perfusion model of the adrenal glands isolated from the sprag-dolly rats.

FIG. 11 is a graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by DMPP in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats.

12 is a graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by McN-A-343 in the perfusion model of the adrenal glands extracted from the sprag-dolly rats.

FIG. 13 is a graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by Bay-K-8644 in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats.

FIG. 14 is a graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by cyclopiazonic acid in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats.

The present invention relates to a composition for the prevention or treatment of cardiovascular diseases comprising bokbunja fermented extract as an active ingredient.

In vivo, blood is always balanced by a complex and sophisticated control system, so its flow is not interrupted by bleeding or blood clots under normal conditions. However, when this equilibrium is broken by various factors, the flow of blood vessels is not smooth and cardiovascular disease occurs. Atherosclerosis is the most representative of cardiovascular diseases, which is a very dangerous disease causing myocardial infarction or cerebral infarction by causing ischemic conditions in important organs such as heart and brain.

When atherosclerosis occurs, the function of vascular endothelial cells in the vascular endothelium decreases or is impaired. As a result, cell adhesion substances are highly expressed and monocytes, T-lymphocytes, and platelets adhere to the vascular lining. They pass between vascular endothelial cells and infiltrate into the vessel wall. Monocytes entering the blood vessel wall are phagocytic lipoproteins, transformed into macrophages, proliferate and activate smooth muscle cells in the blood vessel walls to produce an extratracelluar matrix (ECM), which further accumulates fat and causes atherosclerosis. Will occur. When atherosclerosis occurs, the function of blood vessels is weakened and the flexibility is reduced, so that they can be easily ruptured even by weak irritation.When the extracellular matrix such as collagen in the inner wall of blood vessels is exposed, platelets in the blood adhere, activate, and aggregate, and the coagulation system Activate to form a rapid blood clot. This phenomenon is commonly observed in sudden death due to myocardial infarction. If the coronary artery of a deceased patient is examined under a microscope, the narrowing of blood vessels due to atherosclerosis and the clot of blood vessels can be seen. The result is an instantaneous interruption of the blood supply to the heart that passes through the ischemic state and causes tissue necrosis. Therefore, in order to effectively prevent cardiovascular disease, a decrease in blood cholesterol, platelet activity, inhibition of blood coagulation activity, and inhibition of proliferation of vascular smooth muscle cells causing vascular restenosis are required.

Recently, due to the high incidence and mortality of vascular diseases, research is being actively conducted with a lot of medical attention. In the prevention and treatment of thrombotic diseases, thrombotic release, antiplatelet agents and anticoagulants are commonly used (Jeong Kwang Ho. Research and development of new antithrombotic agents. Korean Society for Hemothrombosis, 3: 1-12, 1996). Thrombolytic agents, such as urokinase or tissue-plasminogen activator (t-PA), are used as effective treatments to help blood flow by dissolving already formed blood clots.Aspirin, an antiplatelet agent, has excellent efficacy but does not affect gastrointestinal disorders. It has been reported to cause side effects (Miwa, K., et al., Am. Heart J., 105: 262-273, 1983), and anticoagulant Coumadin has side effects such as bleeding due to its strong activity. There is a limit to use as a long-term primary preventive. The administration of such effective drugs is a prescription for the second prevention for patients who have already developed cardiovascular disease to the last, and fundamentally it has not been developed as a primary prevention agent for prevention.

Bokbunja ( Rubus coreamum Miquel) has been used in Chinese medicine to treat senile diseases, premature ejaculation (oil well) and erectile dysfunction (sonic). These bokbunja are known to contain several polyphenolic compounds, such as (-)-epicatechhin, (+)-catechin, and proanthocyanidine, but there have been no reports on their effects on cardiovascular diseases.

The present invention aims to develop an effective primary preventive agent that can reduce the incidence of cardiovascular disease by identifying and ingesting an active ingredient related to the prevention and treatment of cardiovascular disease using natural products, preferably bokbunja, and thereby The purpose is to prevent or treat tea.

Accordingly, the present inventors remarkably suppressed catecholamine (CA) secretion of the adrenal gland induced by various secretagogues in the perfusion model of the adrenal gland in which the polyphenolic compound, a bokbunja fermentation extract, was isolated from the Sprague-Dawley rat with normal blood pressure. The present invention was completed by suggesting that it can be used as a prophylactic or therapeutic agent for cardiovascular disease after confirming that it can be inhibited.

An object of the present invention is to provide a composition for the prevention or treatment of cardiovascular diseases comprising bokbunja fermented extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of cardiovascular diseases, including the bokbunja fermented extract as an active ingredient. In this case, the cardiovascular disease may be hypertension, stroke, angina pectoris, heart failure, myocardial infarction, or arteriosclerosis.

Preferably, the bokbunja fermented liquor extract extracts the bokbunja fermented liquor with water, alcohol having 1 to 4 carbon atoms, or a mixture thereof, and the ethanol by loading the extract on a styrene-divinylbenzene type adsorption column And it may be an ethanol fraction obtained by eluting with a mixture of water, more preferably, the ethanol fraction is a polyphenol extract.

In addition, the composition may further comprise one or more additives selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders and lubricants in addition to the active ingredient, wherein the type of the additives is not particularly limited.

In addition, the composition may be used orally, for example, it may be prepared as a warning agent, granules, powders, tablets, capsules and the like.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention or treatment of cardiovascular diseases, including the bokbunja fermented extract as an active ingredient.

At this time, the extract is made of a process for obtaining the extract of fermented wine after preparing the bokbunja fermented wine.

Specifically, the process of manufacturing the bokbunja wine, for example, by crushing the bokbunja, pectinase treatment, and the control of sugar can be prepared by inoculating and incubating the fermentation yeast.

In one embodiment of the present invention, the method for producing the bokbunja wine is enzymatic treatment by adding a pectinase to bokbunja crushed bokbunja juice. The concentration of the pectinase added to the bokbunja grinding fluid is 200-1000 ppm, preferably 200 ppm, the treatment temperature is 40-50 ° C, preferably 50 ° C, and the treatment time is 30-60 minutes, preferably Can be processed for 50 minutes.

Potassium metabisulfite (K ₂ S ₂ O ₅ ) is added to the enzyme-treated juice, followed by filtration, and sugars are added to adjust the sugar content. Potassium metabisulfite (K ₂ S ₂ O ₅ ) may be added at a concentration of 100-300 ppm per bokbunja raw material, preferably 200 ppm per 10 L of bokbunja raw material. The sugar is not particularly limited, but the sugar can be adjusted to 10-40 Brix, preferably 25 Brix by adding any one selected from sugar, glucose and fructose.

Fermented yeast is incubated in pretreated bokbunja juice. The pre-treated bokbunja juice and yeast starter can be inoculated at a rate of 1-3% (v / v), preferably 2% (v / v) can be incubated at 23-26 ℃ for 10 days. Fermented yeast may be commercially available or may be prepared by direct culture. The yeast is not particularly limited, but may be prepared by inoculating a liquid malt medium by inoculating a liquid yeast culture medium separated from yeast, an extract of yeast, and a separated yeast, which are purely separated from traditional medicines, melange, and meju. . For example, the starter can be prepared for each yeast by inoculating purely isolated yeast isolated yeast into liquid malt medium.

The step of separating the extract from the bokbunja wine may be an ethanol fraction obtained by extracting the bokbunja fermented wine with the extraction solvent, performing the adsorption column chromatography using a mixture of water and alcohol as eluent. Preferred extracts of the bokbunja wine of the present invention can be extracted from the bokbunja fermented liquor as an extraction solvent for adsorbing the polyphenol component as a polyphenol component. An example of the column may include a Daion HP series, and the eluate may be a solution obtained by mixing ethanol and water in the same amount. In addition, the process of obtaining the extract may include an additional concentration process, it may be dried in a conventional drying method, for example, spray drying, freeze drying or simple drying to prepare a powder form.

Although it does not restrict | limit especially as said extraction solvent, It is preferable to add distilled water, an organic solvent, or a mixture thereof to a bokbunja fermentation product, and to elute. In addition, the organic solvent is not particularly limited, but may be an alcohol having 1 to 5 carbon atoms, hexane, chloroform or ethyl acetate. Examples of the alcohol may be selected from the group consisting of ethanol, methanol, butanol, isopropanol and propanol. It is possible to use, preferably ethanol.

Specific examples of the bokbunja wine extract manufacturing process of the present invention, for example, extracting the bokbunja fermented liquor with distilled water and ethanol mixture and evaporated to remove ethanol; 2) separating the extract component by chromatography using a Diaion column of the eluate; And 3) extracting the solution with ethanol, vacuum filtration, and evaporating and lyophilizing the same.

In addition, the effective content of the extract in the composition for the prevention or treatment of cardiovascular diseases is not particularly limited, but may be 0.1 to 99.9% by weight, preferably 10 to 60% by weight. This is because, when the content of the extract is less than 0.1% by weight, it is necessary to administer a large amount due to the insufficient treatment or prevention effect of cardiovascular diseases such as hypertension and arteriosclerosis caused by the extract. The effective amount of the extract in the composition may be appropriately adjusted according to the method of use and / or the purpose of treating the cardiovascular disease.

In addition, the composition of the present invention may further comprise at least one additive selected from the group consisting of pharmacologically acceptable conventional excipients such as disintegrants, binders and lubricants, in addition to the active ingredient, the kind of the additive is limited thereto. It is not possible. In addition, the composition may further include one or more additives selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders and lubricants in addition to the active ingredient, wherein the type of the additives is not particularly limited. In addition, the composition may be used orally, for example, it may be prepared as a warning agent, granules, powders, tablets, capsules and the like. At this time, the dosage of the composition is preferably adjusted according to the use, purpose of use, the condition of the patient, age, sex, weight, prescription drugs and diseases. For example, when administered orally to an adult it may be administered in a dose of 80 to 180 mg.

The bokbunja fermentation extract (20-180ug / ml) according to the present invention is acetylcholine (5.32mM), high potassium (56mM, membrane depolarizing agent), DMPP (100uM, selectivity) for 90 minutes at the time of perfusion into the adrenal vein. Nicotine receptor agonist) and McN-A-343 (100 uM, selective muscarinic receptor agonist) could significantly inhibit the amount of catecholamines released (see FIGS. 3-6). In addition, in the presence of bokbunja fermented extract (60ug / ml), catecholamine secretion by Bay-K-8644 (L-type calcium channel activator) and cyclopiazone acid (Ca ²⁺ -ATPase inhibitor in the cytoplasm) was also significantly inhibited ( 7 and 8). On the other hand, when bokbunja fermented extract (60ug / ml) and L-NAME (100uM) were simultaneously perfused into the adrenal vein for 90 minutes, acetylcholine, high potassium, DMPP, McN-A-343, Bay-K-8644 treated alone And the inhibitory effect of the bokbunja fermentation extract in the increase in catecholamine secretion by cyclopiazone acid was relatively reduced, it can be seen that the catecholamine secretion recovered to the control level (see FIGS. 9 to 14).

Therefore, the bokbunja fermentation extract of the present invention in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats with normal blood pressure significantly inhibits catecholamine secretion by cholinergic (nicotine and muscarinic receptor) excitatory action and membrane depolarization. And it was found. The inhibitory effect of the bokbunja fermentation extract increases NO production by the activity of NO Synthase in the adrenal medulla isolated from Sprague-Dawley rats, inhibits calcium influx into chromofriendly cells and inhibits calcium free from intracellular calcium storage. It is believed that this is at least due to the interaction with the nicotine receptor itself.

In addition, pheochromocytoma is a tumor derived between the sympathetic nerve and the adrenal medulla tissue, which has been reported to produce catecholamines (Bravo, EL, et al., N. Engl. J. Med., 301: 682-686). , 1979). The clinical characteristics of the tumors are hypertension and tachycardia, which are caused by catecholamines secreted into the circulating blood, and are generally known to be caused by an increase in plasma or urinary excretion of catecholamines and metabolites at diagnosis (Engelman). , K. Clin. Endocrinol. Metab., 6 (3): 769-797, 1977; Maiter, D. Acta. Clin.Blg., 59 (4): 209-219, 2004). That is, in patients with pheochromocytoma, the blood concentration of catecholamine is significantly increased. Norepinephrine and Epinephrine have some advantages in measuring blood levels of these patients (Engelman, K. Clin. Endocrinol. Metab., 6 (3): 769-797, 1977). Differential diagnosis of hypertensive patients and pheochromocytoma patients can be easily performed by examining urinary catecholamines or their metabolites. In addition, catecholamine concentrations have been reported to be significantly higher in about half of hypertensive patients compared to those of normal blood pressure and hypertensive patients (Kopin, IJ, John Wiley and Sons. 267-276, 1979; Kopin, IJ, et al., Clin.Exp. Hypertens., 2 (3-4): 379-394, 1980a; Kopin, IJ, et al., Springer New York, 1980b; Goldatein, DS, et al., Life Sci., 28 (5): 467-475, 19811: Goldatein, DS., Clin.Endocrinol. Metab., 6 (3): 769-797, 1983), spontaneously hypertensive rats ( It has been reported that the amount of catecholamines secreted in the perfusion adrenal gland of SHRs is significantly higher in the adrenal glands of normal blood rats (Lim, DY, et al., Auton. Autacoid. Pharmacol., 22 (4): 225-232, 2002).

From the above results, the polyphenol compound, which is an active ingredient present in the bokbunja fermentation extract according to the present invention, can be used to prevent or treat cardiovascular diseases such as hypertension, stroke, angina pectoris, heart failure, myocardial infarction, or arteriosclerosis. It offers potential as a useful resource.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

Example 1 Separation of Bokbunja Fermented Extract

<1-1> Bokbunja Fermented Wine

Pectinase (Pectinex 100L) was added to 10 L of bokbunja grinding solution at 500 ppm, and the enzyme was treated at 50 ° C. for 50 minutes. In order to suppress the harmful bacterial breeding of fruit juice, potassium metabisulfate (K ₂ S ₂ O ₅ ) was added and filtered at a concentration of 200 ppm per 10 L of bokbunja raw material, and sugar was added to adjust the sugar to 25 Brix. After diluting traditional medicines, muru and meju with excellent aroma and taste in sterile water, smear 0.1mL in medium and incubate at 25 ℃ to isolate the characteristic yeast colony, and contain 0.1% of Tween 80 again. After diluting with the diluted solution and repeatedly incubated 3-4 times, pure water was separated. Yakju isolated yeast obtained from the above was inoculated in liquid malt medium to produce a starter (starter) for each yeast. The starter was inoculated at 2% (v / v) in the bokbunja juice obtained above, and cultured at 25 ° C. for 10 days to prepare a bokbunja wine.

<1-2> Extract Separation

Fresh bokbunjaju was purchased from Bokbunja Research Institute (Gochang-gun, Jeollabuk-do, Korea) and separated by the process of FIG. 2 according to the method of Caderni et al. (Carcinogenesis, 21, 1965-1969, 2000) (FIG. 2). Specifically, bokbunjaju was extracted with distilled water and ethanol, ethanol was removed by distillation, and the remaining solution was passed through the Diaion HP Column (Mitsubish Chemical Corporation, Japan). After washing with distilled water to remove sugars and organic acids, Bokbunjaju extract was extracted with 100% ethanol, vacuum filtered and evaporated and sprayed and then freeze-dried using Coldvac-80 (Hanil R & D, South Korea). Finally, 2.9 g of extract (bokbunja fermentation extract) per 1 L of bokbunja strain was obtained, and the bokbunja fermentation extract was diluted with 0.9% physiological saline on the day of the experiment and filtered after administration in the present invention.

Example 2 Bokbunja Fermented Extract of Catecholamine by Acetylcholine (ACh), High Potassium, DMPP, McN-A-343, Bay-K-8644 and Cyclopiazonic Acid in Sprague-Dawley Rat Perfusion Adrenal (CA) Efficacy on Secretion

Type of drug

Drugs used in the present invention, acetylcholine (ACh), norepinephrine bitartrate, KCl, L-NAME (N-ω-nitro-l-arginine methyl ester hydrochloride), DMPP (1,1 -dimethyl-4-phenyl piperazinium iodide), McN-A-343 [3- (m-cholro-phenyl-carbamoyl-oxy) -2-butynyltrimethyl ammonium chloride, RBI, USA] and cyclopiazonic acid, Sigma, USA) were all diluted with distilled water and added to Krebs-bicarbonate solution. Bay-K-8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4- (2-trifluoromethyl-phenyl ) -pyridine-5-carboxylate] was dissolved in 99.5% ethanol and properly diluted with Krebs-bicarbonate solution (alcohol final concentration was 0.1% or less). The concentrations of all the drugs were used in molar concentrations, norepinephrine was dissolved in 0.9% acid saline (pH = 4.0).

Adrenal Extraction Separation

Sprague-Dowley rats weighing 200-250 g were used as experimental animals. Anesthesia was administered by intraperitoneal administration of 50 mg thiopental-Na per kg body weight. The anesthetized rat was fixed in the coordination of the animal stator and then cut along the abdominal midline and the surrounding organs were fixed with a three-hook retractor to expose the adrenal gland to prevent blood clotting. The fluid (400 IU / ml) was injected ascending from the vena cava just below the renal vein. As shown in Figure 1, ligation of all blood vessels around the adrenal gland for perfusion of the adrenal gland was fixed by inserting a thin polyethylene tube. A small slit is made in the adrenal cortex opposite the entrance to the adrenal vein and immediately begins perfusion to check for leaks from other parts of the adrenal gland, and then slowly and carefully separated along the ligated vessel and the cannula and fixed on the leucite chamber. Perfusion was continued with the next Krebs-bicarbonate solution.

Perfusion of the adrenal glands

The isolated adrenal glands were perfused at a rate of 0.31 ml / min using a peristaltic pump (Isco, USA), and Krebs-bicarbonate solution was used as the perfusate. At this time, the composition of the perfusate was prepared by mixing 118.4 mM NaCl, 4.7 mM NaCl, 2.5 mM CaCl _2, 1.18 mM MgSO _4, 25 mM NaHCO _3, 1.2 mM KH ₂ PO _4, and 11.7 mM Glucose. Krebs-bicarbonate solution was supplied with 95% O ₂ and 5% CO ₂ until the end of the experiment and the final pH was adjusted to 7.4-7.5. In addition, disodium EDTA (10 µg / ml) and ascorbic acid (100 µg / ml) were added to prevent oxidation of catecholamine (CA). Perfusion experiments were performed at 37 ± 1 ° C. 1 is a schematic diagram showing a method for separating and separating adrenal gland (left) and perfusion method (right) of the adrenal gland from a sprag-dolly rat.

drug injection

Acetylcholine (ACh) and the like were injected directly into the perfusate through a fixed three-way stopcock during drug administration. The dose was adjusted to 0.05 ml. In addition, other drugs were administered by diluting the solution with Krebs-bicarbonate and perfusion. Through preliminary experiments, it was confirmed that the catecholamine secretion effect by one administration of the secretagogue was restored to the pre-administration state after approximately 4 to 8 minutes, and all adrenal perfusates were collected for 4 to 5 minutes after administration of the drug. . After the experimental procedure was completed, perfusion was performed with Krebs-bicarbonate solution for about 1 hour, and acetylcholine was administered to confirm the secretory function from the adrenal medulla. The collection of perfusate from the adrenal gland was collected in an ice bath to minimize degradation of catecholamines.

Perfusate Collection

Perfusion solution was collected for 4 minutes to measure basal catecholamine secretion before stimulation with various secretagogues. Immediately after collecting the control sample, the perfusion solution was collected from the time point when the perfusate containing the secretagogue reached the adrenal gland. Perfusion samples stimulated with secretagogues were collected for 4-8 minutes. The catecholamine content in the sample was expressed in all figures by converting the catecholamine secretion secreted by the secretory stimulant minus the catecholamine secretion of the control group to the pure catecholamine secretion.

In order to investigate the effect of bokbunja fermentation extract on the catecholamine secretion by the control and secretion accelerator, perfusate samples stimulated with the control and secretagogue for a period of time while collecting the adrenal glands for 90 minutes through the bokbunja fermentation extract was collected. .

Catecholamine (CA) secretion measurement

The catecholamine content in the adrenal perfusate was measured using a fluorospectrophotometer (Kontron, Italy) without intermediate purification on alumina according to the methods of Anton and Sayre (J. Pharmacol. Exp. Ther., 138, 360-375, 1962). It was measured directly. At this time, 0.2 ml of the collected perfusate was collected and used to measure the catecholamine. The calculation of the catecholamine content in the perfusate was compared with the standard norepinephrine (converted to base) value of the known concentration and expressed as a total amount. All data in this experiment were reported as (mean ± standard error). Statistical differences were compared using Student's paired “t” test or ANOVA test, and tested using computer programs of Tallarida and Murray (1987).

First, in order to investigate the efficacy of the bokbunja fermentation extract itself on catecholamine secretion in the adrenal perfusion model of Sprague-Dowley rats, oxygenated Krebs-bicarbonate solution was used as a Sprague-Dowley rat. ) Extraction of Rats After perfusion for 1 hour to the perfusion adrenal gland, the free amount of catecholamine was measured, and it was confirmed that 20 ± 2 ng (n = 12) for 2 minutes. This means that the bokbunja fermented extract of the present invention (20-180 ㎍ / ㎖) itself does not have any effect on the existing catecholamine secretion in the adrenal perfusion model of Sprague-Dowley rats. Therefore, in this Example, the effect of bokbunja fermentation extracts on catecholamine secretion by choline receptor excitatory action and membrane depolarization was investigated, for this purpose, administration of several secretagogues at 15-20 minute intervals and the control free amount of the secretagogues After establishment, the bokbunja fermentation extract was perfused for 90 minutes.

Specifically, when 0.05 ml of acetylcholine (5.32 × 10 ^-3 M) was injected into the perfusion solution, the catecholamine secretion amount was 1371 ± 120 ng for 4 minutes, whereas acetylcholine was perfused for 20 minutes at 20-80 ug / ml concentration. It was confirmed that catecholamine secretion by choline was inhibited at concentration and time-dependently. 3 is a graph showing the time and concentration-dependent effect of bokbunja fermentation extract on catecholamine (CA) secretion by acetylcholine (ACh) in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats. As described above, the bokbunja fermentation extract was administered to the adrenal vein at 15 min intervals by 0.5 ml of acetylcholine (5.32 × 10 ⁻³ M) while perfused for 90 minutes at concentrations of 20, 60, and 180 ug / ml. Numbers in parentheses indicate the number of rat adrenal glands and each bar graph represents a standard error (SEM). Statistical difference was calculated by comparing the catecholamine secretion corresponding to each concentration of the bokbunja fermentation extract with the catecholamine secretion of the control (CONT) (perfusate with acetylcholine collected for 4 minutes; *: P <0.05, **: P <0.01, ns: no statistical significance).

X axis: perfusion liquid collection time (min) after polyphenol compound (PCRK) treatment.

Y-axis: The amount of catecholamines secreted by the adrenal gland for 4 minutes (expressed in% relative to the control (1371 ± 20 ng / 4 min) secretion).

As shown in Figure 3, it was confirmed that the free amount of catecholamine by acetylcholine in the presence of bokbunja fermentation extract is suppressed to 71% of the control free amount (100%) (Fig. 3).

In addition, depolarizing agents such as KCl significantly promoted catecholamine secretion (746 ± 3 ng / 0-4 min). FIG. 4 is a graph showing time and concentration-dependent effects of bokbunja fermentation extracts on catecholamine (CA) secretion by high potassium in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats. 5.6 × 10 ^-2 M) is shown in the adrenal vein at 15 minute intervals. As shown in FIG. 4, after pretreatment with 20 ug / ml bokbunja fermentation extract, the catecholamine secretion by high potassium (5.6 × 10 ⁻³ M) was not affected after 60 minutes after treatment compared with the control secretion. After lapse, catecholamine secretion by high potassium was suppressed, and when treated with 60-180 ug / ml high concentration bokbunja fermented extract, it was observed that catecholamine secretion was effectively inhibited after 60 minutes (FIG. 4).

Nicotine receptor agonist agent DPP (10 ^-4 M) was also significantly promote the catecholamine secretion (1182 ± 53ng / 0-8 minutes). 5 is a Sprague-Dawley in perfusion model was separated from the rat adrenal extraction bokbunja fermentation extract is time to effect on catecholamine (CA) secretion by DPP and concentration-dependent manner as shown by the graph, the DPP 0.05ml (10 ^{- 4} M) shows the results of adrenal vein administration for 2 minutes at 20 minute intervals. As shown in FIG. 5, the pretreatment of the bokbunja fermented extract showed that the catecholamine secretion by DPP was suppressed by 84% compared to the control group (FIG. 5).

In addition, the selective muscarinic M ₁ receptor agonist agent ^{McN-A-343 (10 -4} M) for the catecholamine secretion was increased during perfusion 4 minutes into the adrenal veins according to the procedure of FIG. 1 (578 ± 26ng / 0-4 minutes) . FIG. 6 is a graph showing the time and concentration-dependent effects of bokbunja fermentation extracts on catecholamine (CA) secretion by McN-A-343 in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats. After administration, McN-A-343 (10 ^-4 M) was administered to the adrenal vein for 4 minutes at 15 minute intervals. As shown in FIG. 6, catecholamine secretion by McN-A-343 was suppressed to 71% of the control secretion amount (100%) in the presence of bokbunja fermented extract (FIG. 6).

Meanwhile, Bay-K-8644, a calcium channel activator, is used for basal calcium intake (Garcia, AG, et al., Nature, 309, 66-71, 1984) and catecholamine free (Lim, DY, et al., Arch. Pharm. Res., 28, 914-922, 1992), is useful for investigating the efficacy of bokbunja fermented extracts on the change of catecholamine secretion by Bay-K-8644 in Sprague-Dawley rat isolated perfusion adrenal gland. Thus, catecholamine secretion by Bay-K-8644 (10 ⁻⁵ M) was measured in the perfusion model of the adrenal gland extracted from the rat. 7 is a time-dependent graph showing the effect of Bokbunja fermentation extract on catecholamine (CA) secretion by Bay-K-8644 in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats. Bay-K-8644 (10 ^-5 M) was injected into the adrenal vein at 15 minute intervals for 4 minutes. As shown in FIG. 7, catecholamine secretion by Bay-K-8644 in the presence of bokbunja fermentation extract was inhibited to 69% of the control (100%) except for the first 30 minutes when compared to the control secretion (FIG. 7). .

Cyclopiazone acid, a mycotoxin isolated from Aspergillus and Penicillium , is also known as a selective inhibitor of Ca ²⁺ -ATPase in skeletal muscle myoplasmic reticulum (SR). Goeger, DE, et al., Biochem.Pharmacol., 38, 3995-4003, 1989; and Seidler, NW, et al., J. Biol. Chem., 264, 17816-17823, 1989). Therefore, the inhibitory effect of bokbunja fermentation extract on the catecholamine secretion of cyclopiazone acid in the perfusion model of the adrenal gland extracted from the rat was investigated. 8 is a time-dependent graph showing the effect of bokbunja fermentation extracts on catecholamine (CA) secretion by cyclopiazone acid in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats. Zonic acid (10 ^-5 M) is the result of administration of the adrenal vein for 4 minutes at 15 minute intervals. As shown in FIG. 8, catecholamine secretion by cyclopiazone acid in the presence of bokbunja fermented extract was suppressed to 70% of the control secretion (427 ± 18ng / 0-4min, 100%) (FIG. 8).

Therefore, it was found that the bokbunja fermented extract of the present invention effectively inhibits catecholamine secretion due to cholinergic excitation and membrane depolarization in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats.

<Example 3> (Bokbunja fermentation extract + L-NAME) is acetylcholine (ACh), high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazone acid in the Sprague-Dawley rat perfusion adrenal gland Efficacy of Catecholamine (CA) Secretion by Cyclopiazonic Acid)

In order to investigate the correlation between the inhibitory action of NO and bokbunja fermentation extract in catecholamine secretion by cholinergic excitation and membrane depolarization in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats, Inject the bokbunja fermented extract and NO dornor L-NAME in the same manner and then to acetylcholine (ACh), high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid Catecholamine (CA) secretion change was measured.

First, in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats, the catecholamine secretion by acetylcholine (5.32 mM) was 1050 ± 50 ng (0-4 min) before perfusion of the bokbunja fermented extract + L-NAME. . 9 is a graph showing the time-dependent effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by acetylcholine in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats. As shown, the result of administration of 0.05 ml of acetylcholine (5.32 × 10 ^-3 M) to the adrenal vein at 15 minute intervals was once perfused for 60 minutes at 60ug / ml bokbunja fermented extract and 30uM L-NAME. As shown in FIG. 9, when bokbunja fermented extract (60ug / ml) and L-NAME (30uM) were simultaneously perfused for 90 minutes, catecholamine secretion by acetylcholine was not affected until 0-34 minutes, but 90-94 minutes. Up to 85% of the control secretion (1050 ± 50ng, 0-4 minutes) in (Fig. 9).

In the perfusion model of the adrenal glands isolated from the sprag-dolly rats, catecholamine secretion by high potassium (56 mM) was zero in the presence of bokbunja fermented extract (60ug / ml) and L-NAME (30uM) which had been perfused for 90 minutes. There was no change until -64 minutes, but up to 82% of the control secretion (717 ± 51 ng, 0-8 minutes) in the 90-94 minutes (Fig. 10). 10 is a time-dependent graph showing the effect of (bokbunja fermentation extract + L-NAME) on the secretion of catecholamines (CA) by high potassium in the perfusion model of the adrenal glands extracted from the sprag-dolly rats. As indicated by the arrow, 0.05ug of high potassium (5.6 × 10 ^-2 M) was injected into the adrenal vein at 15 minute intervals while simultaneously perforating 60ug / ml bokbunja fermented extract and 30uM L-NAME for 90 minutes. will be.

In addition, in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats, the catecholamine secretion of DMPP was 1126 ± 57 ng (0-8 min) before perfusion of the bokbunja fermentation extract and L-NAME. FIG. 11 is a time-dependent graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by DPP in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats, indicated by arrows. As shown, the result of administration of 0.05 ml of DPP (10 ^-4 M) to the adrenal vein for 20 minutes at 20 minutes while perfused for 60 minutes at 60ug / ml bokbunja fermented extract and 30uM L-NAME at the same time. As shown in FIG. 11, when bokbunja fermented extract (60ug / ml) and L-NAME (30uM) were simultaneously perfused for 90 minutes, catecholamine secretion by DPP was not affected until 0-48 minutes, but at 80-88 minutes. Up to 91% of control secretion (1126 ± 57 ng, 0-4 min) (FIG. 11).

In the perfusion model of the adrenal glands isolated from the sprag-dolly rats, the amount of catecholamines secreted by McN-A-343 was 0-in the presence of bokbunja fermented extract (60ug / ml) and L-NAME (30uM) which had been perfused for 90 minutes. There was no change until 79 minutes, but it was suppressed by 81% of the control secretion (512 ± 34 ng, 0-4 minutes) in the 90-94 minutes (Fig. 12). 12 shows time-dependent effects of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by McN-A-343 in the perfusion model of the adrenal glands isolated from Sprague-Dawley rats. As a graph, McN-A-343 (10 ^-4 M) was administered to the adrenal vein at 15 minute intervals for 15 minutes while simultaneously perfusion of 60 ug / ml bokbunja fermented extract and 30 uM L-NAME as indicated by the arrow. The results are shown.

In the perfusion model of the adrenal gland isolated from Sprague-Dawley rats, catecholamine secretion by Bay-K-8644 in the presence of bokbunja fermented extract (60ug / ml) and L-NAME (30uM) was 0- There was no effect until 64 minutes, but up to 79% of control secretion (448 ± 25 ng, 0-4 minutes) in the 90-94 minutes (FIG. 13). FIG. 13 is a time-dependent graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by Bay-K-8644 in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats. As indicated by the arrow, Bay-K-8644 (10 ^-4 M) was injected into the adrenal vein for 15 weeks at a time of 60 minutes perfusion for 30 minutes at 60 μg / ml bokbunja fermented extract and 30 μM of L-NAME. It is shown.

In addition, in the perfusion model of the adrenal gland isolated from Sprague-Dawley rats, the catecholamine content of cyclopiazone acid (10 ^-5 M) before the perfusion of L-NAME was 435 ± 21 ng (0-4 min) However, while bokbunja fermented extract and L-NAME were perfused for 90 minutes, the catecholamine secretion by cyclopiazone acid had no effect until 0-64 minutes, but in the 90-94 minutes control secretion (435 ± 21, 0-4 minutes) It was confirmed that up to 77% of (FIG. 14). 14 is a time-dependent graph showing the effect of (bokbunja fermentation extract + L-NAME) on catecholamine (CA) secretion by cyclopiazone acid in the perfusion model of the adrenal gland isolated from Sprague-Dawley rat As shown by the arrow, cyclopiazone acid (10 ^-4 M) was administered to the adrenal vein for 4 weeks at intervals of 15 minutes while permeating 60ug / ml bokbunja fermented extract and 30uM L-NAME at the same time for 90 minutes. .

As described above, the composition comprising the bokbunja fermented extract according to the present invention as an active ingredient can be usefully used for the prevention or treatment of cardiovascular diseases.

Claims (6)

A composition for preventing or treating hypertension, comprising an ethanol fraction obtained by removing the fraction eluted with water on a Diaion HP column and eluting with ethanol again as an active ingredient. delete delete delete The composition for preventing or treating hypertension according to claim 1, wherein the composition further comprises one or more additives selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders and lubricants. The composition for preventing or treating hypertension according to claim 1, wherein the composition is an oral composition having a formulation of tablets, capsules, solutions, suspensions, or syrups.

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