KR20110112783A - Composition comprising stilbene derivative compounds for preventing or treating of vascular disease - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of vascular diseases comprising a stilvin derivative compound as an active ingredient, and more particularly, the present invention relates to piaceanol-3'-O-beta-, which is a stilbene derivative compound. Piceatannol-3'-O-beta-D-glucopyranoside, raponticin, rapontigenin, isorhapontin, deoxyrhapontigenin, Resveratrol compounds increase the production of nitric oxide (NO) through the action of inhibiting the activity of arginase and at the same time inhibit the production of free radicals, thereby improving dysfunction of vascular endothelial cells. The composition containing can be usefully used as a pharmaceutical or nutraceutical for preventing and treating vascular diseases.

Description

Composition comprising stilbene derivative compounds for preventing or treating of vascular disease

The present invention relates to a novel use of stilbin derivative compounds, and more particularly to a composition for the prevention or treatment of vascular diseases comprising a stilbin derivative compound as an active ingredient.

In vivo, blood is always balanced by a complex and sophisticated control system so that flow is not interrupted by bleeding or blood clots under normal conditions. However, if this equilibrium is broken due to various factors, blood vessels may not flow smoothly, resulting in vascular diseases. Atherosclerosis is the most representative of vascular diseases, and is one of the very dangerous diseases causing myocardial infarction or cerebral infarction by causing ischemic condition in important organs such as heart and brain.

On the other hand, the heart and blood vessels are the organs that supply oxygen and nutrients to the whole body and can be said to be essential for humans to maintain life. Therefore, various causes of the cardiovascular system is abnormal, if the blood and oxygen supply to each tissue and organs is not smooth, local tissue necrosis, as well as severe life-threatening dangerous diseases. Cardiovascular disease is more frequently caused by aging of the body, especially as it ages. For this reason, prevention and treatment of cardiovascular disease is becoming very important.

To date, cardiovascular diseases such as Schwarz's ISMO, such as nitrate-based drugs, have been introduced, but these drugs are effective, but they have side effects such as positional hypotension and palpitations, such as hypotension and glaucoma. A beta-blocker is used as a beta-blocker, Astra's beta-lock (BETALOC), etc. This drug is also effective, but a sudden cardiac arrest, such as congestive heart failure should be used carefully.

On the other hand, normal production of endothelial nitric oxide is important for the maintenance of vascular function. Increased nitric oxide release of endothelial cells results in reduced elasticity and reduced vascular resistance of smooth muscle cells. However, under conditions of hypertension and hyperlipidemia, endothelial dysfunction causes a marked decrease in nitric oxide release, resulting in increased vasoconstriction and vasospasm, enlarged monocytes and LDL infiltration, proliferation of vascular smooth muscle cells, It leads to an increase in oxidative stress and an increase in platelet aggregation. Therefore, the substance that restores endothelial function and nitric oxide metabolism can be said to be effective in the treatment of vascular diseases including coronary artery disease and heart dysfunction.

Most cells that synthesize nitric oxide also have an enzyme called arginase, which catalyzes the hydrolysis of L-arginine to release L-ornithine and urea. It is a group of enzymes that produces. Arginase I is constitutively expressed in cells, and arginase II is known to be induced by endotoxin. The enzymes also play a very important role in altering the production of nitric oxide in inflammatory conditions, but also play an important role in the constant synthesis of nitric oxide.

Arginase, on the other hand, also competitively inhibits NOS through the use of a common substrate, L-arginine (Berkowitz et al., 2003; Holowatz et al., 2006; Morris et al. , 1998; Peyton et al., 2009; Simon et al., 2003; Steppan et al., 2006). The expression and function of arginase I in macrophages, hepatocytes and vascular smooth muscle cells is lipopolysaccharide (LPS), IL-13, altered oxygen partial pressure and coronary arteries. Stimulated by balloon dilatation (Chicoine et al., 2004; Klasen et al., 2001; Louis et al., 1998; Modolell et al., 1995; Morris et al., 2004; Nelin et al ., 2007; Que et al., 1998; Ryoo et al., 2006), and also the activity and expression of arginase II are diverse including OxLDL, LPS, TNF-a, IFN-, 8-bromo-cGMP and hypoxia. May be induced by vascular factors.

Recently reported data suggest that inhibition of arginase actively increases nitric oxide production and is associated with vascular dysfunction in normal heart function and atherosclerosis, aging, erectile dysfunction and sickle cell disease. It has been found to produce useful effects (Berkowitz et al., 2003; Bivalacqua et al., 2001; Bivalacqua et al., 2007; Hsu et al., 2007; Morris et al., 2004; Steppan et al., 2006; White et al., 2006; Xu et al., 2007).

Therefore, it is predicted that a substance that inhibits the activity of arginase may be useful for improving and treating vascular dysfunction. However, in the case of a conventionally developed arginase inhibitor, there is a side effect and the effect is insignificant. The development of arginase inhibitors is urgent.

The present inventors have described stilvin derivative compounds, in particular, piceatanol-3'-O-beta-D-glucopyranoside, raponticin, la Rhapontigenin, isorhapontin, deoxyrhapontigenin, and resveratrol compounds inhibit arginase activity in vascular endothelial cells, increase nitric oxide production and simultaneously increase ROS The present invention was completed by confirming that vascular disease can be prevented or treated through an activity of inhibiting production.

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of vascular diseases, which contains a stilvin derivative compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, another object of the present invention is to provide a health functional food for the prevention or improvement of vascular diseases containing the composition according to the present invention as an active ingredient.

In order to achieve the above object, the present invention is a stilvin derivative compound piaceanol-3'-O-beta-D-glucopyranoside (piceatannol-3'-O-beta-D-glucopyranoside), Lafonti A compound selected from the group consisting of Rhaponticin, Rhapontigenin, isorhapontin, isorhapontin, deoxyrhapontigenin, Resveratrol, and pharmaceutically acceptable salts thereof is an active ingredient. It provides a composition for the prevention or treatment of vascular diseases comprising.

In one embodiment of the invention, the stilbin derivative compound may be derived from rhubarb (rhubarb).

In one embodiment of the present invention, the stilbin derivative compound may have an effect of inhibiting the activity of arginase, increasing the production of nitric oxide or inhibiting the production of reactive oxygen species (ROS).

In one embodiment of the present invention, the arginase may be arginase I or arginase II.

In one embodiment of the present invention, the vascular disease is hypertension, arteriosclerosis, heart attack, stroke, cancer, aging, erectile dysfunction, sickle cell disease, diabetes, angina pectoris, myocardial infarction, arthritis, diabetic It may be selected from the group consisting of retinopathy, psoriasis, neovascular glaucoma, hemangioma and ischemic disease.

In one embodiment of the present invention, the stilbin derivative compound may be included in a concentration of 0.1 ~ 150 uM relative to the total volume of the composition.

In addition, the present invention provides a health functional food for the prevention or improvement of the vascular disease containing the composition for the prevention or treatment of vascular diseases according to the present invention as an active ingredient.

The stilbin derivative compound according to the present invention can improve the production of nitric oxide (NO) through the action of inhibiting the activity of arginase and at the same time can improve the dysfunction of vascular endothelial cells by inhibiting the production of free radicals The composition containing this as an active ingredient may be usefully used as a medicine or health functional food for the prevention and treatment of vascular diseases.

1 is a mouse in order to confirm the effect of reducing the activity of arginase I and II by piceanol-3'-O-beta-D-glucopyranoside (PG) in the stilbin derivative compounds according to the present invention After preparing cell lysates from the liver and kidney, Western blots were shown, and (A), (b) and (c) show the degree of inhibition of arginase activity according to concentration of PG.
Figure 2 shows the results of confirming the protein levels of arginase activity inhibition (A), NOx production (B) and arginase II and eNOS after treatment with PG at each concentration in vascular endothelial cells. will be.
Figure 3 shows the inhibition of arginase activity after treatment with the PG compound according to the present invention in the mouse aorta compared to the control group (A), the control group not treated with the fluorescence intensity of DAF according to the PG treatment time And graphs comparing the group treated with PG and L-NAME (B and C).
Figure 4 shows the result of measuring the degree of inhibition of arginase I and II activity after treatment with different concentrations of PG.
5 is arginase of stilbin derivative compounds according to the present invention, raponticin (Rhaponticin), rapontigenin (Rhapontigenin), isorhapontin, deoxyrhapontigenin and resveratrol compounds The degree of inhibition of I and II activity was measured in kidney and liver tissues of mice.

The present invention identifies a novel pharmaceutical use of a stilbene derivative compound, and more specifically, the present invention relates to piceanol-3'-O-beta-D-glucopyranoside (piceatannol-3'-). O-beta-D-glucopyranoside, Raponticin, Rapontigenin, Rhapontigenin, Isorhapontin, Deoxyrhapontigenin, Resveratrol and their pharmaceutically acceptable It is characterized by providing a composition for preventing or treating vascular diseases containing at least one compound selected from the group consisting of salts as an active ingredient.

The stilbin derivative compound according to the present invention is a derivative compound of the stilbin compound consisting of aromatic hydrocarbons having the formula C ₁₄ H _12. Preferably, piceanol-3'-O-beta-D-glucopyranoside (piceatannol) -3'-O-beta-D-glucopyranoside, Raponticin, Rhapontigenin, Rhapontigenin, Isorhapontin, Deoxyrhapontigenin and Resveratrol.

The piceatanol-3'-O-beta-D-glucopyranoside of the stilvin derivative compound of the present invention is represented by the following formula (1).

 <Formula 1>

Piaceanol -3'-O- beta -D- Of glucopyranoside  constitutional formula

In addition, the raponticin (Rhaponticin) compound is named as 3 ', 5-dihydroxy-4'-methoxystilbene 3-O-beta-D-glucopyranoside, is a compound represented by the following formula (2).

 <Formula 2>

Lafonticin ( Rhaponticin Structural formula of the compound

The rappontigenin is a compound represented by the following formula (3).

<Formula 3>

Lafontigenin ( Rhapontigenin Structural formula of the compound

In addition, the isolapontin (isorhapontin) is a compound represented by the following formula (4), deoxyrhapontigenin (deoxyrhapontigenin) is a compound represented by the formula (5).

<Formula 4>

Isolapontin ( isorhapontin Structural formula of the compound

<Formula 5>

Deoxylafontigenin ( deoxyrhapontigenin Structural formula of the compound

In addition, the resveratrol compound is a compound represented by the following formula (6).

<Formula 6>

Resveratrol ( Resveratrol Structural formula of the compound

Furthermore, the stilbin derivative compounds according to the present invention may be used in the form of salts, preferably pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

Stilbin derivative compounds according to the present invention can be isolated from nature or prepared by chemical synthesis methods known in the art.

Preferably, the pisteanol-3'-O-beta-D-glucopyranoside, stilvin derivative compound of the present invention (piceatannol-3'-O-beta-D-glucopyranoside), raponticin (Rhaponticin), Rapontigenin, isorhapontin, deoxyrhapontigenin and resveratrol compounds can be obtained from nature using methods for extracting and separating conventional materials, The compounds according to the present invention can be extracted and separated from rhubarb using extraction and purification methods known to those skilled in the art.

In one embodiment of the present invention, stilbin derivative compounds isolated and purified from Rheum palmatum were obtained from Catholic University and used in experiments. Rhubarb is known to be extremely cold in nature, tasteless and poisonous. Efficacy in bleeding blood and menstruation is not effective, it helps to pass urine well and cool down, menstrual abnormalities, hematuria, acute peritonitis, harvesting, bleeding, edema, food obesity, bacterial dissociation, degenerative arthritis, fever, fever with fever It is known to be used for symptoms, each, swelling, and burns. In addition, herbal medicine has been used as an anti-inflammatory laxative since BC and has been used in various prescriptions.

As a suitable solvent for obtaining an extract containing the compound according to the present invention from the rhubarb, water or an organic solvent may be used. Preferably, purified water, methanol, ethanol, propanol, isopropanol (isopropanol), butanol, acetone, ether, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Various solvents such as (cyclohexane) may be used alone or in combination, and the extraction method may be any method used in the art such as hot water extraction, reflux cooling extraction, and cold needle extraction.

In addition, the separation and purification of the stilbin derivative compounds of the present invention from rhubarb include column chromatography, high-performance liquid chromatography (HPLC), etc., which are filled with various synthetic resins such as silica gel and activated alumina. May be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

Thus, in the present invention, still-derived derivatives derived from rhubarb, more specifically, piceanol-3'-O-beta-D-glucopyranoside (piceatannol-3'-O-beta-D-glucopyranoside), Raponticin, Rhapontigenin, isorhapontin, isorhapontin, deoxyrhapontigenin, and Resveratrol compounds inhibit the production of nitric oxide through the inhibition of arginase activity. For the first time, it has been found to be effective in preventing or treating vascular diseases by increasing and inhibiting the generation of reactive oxygen species (ROS).

Genetic factors, lifestyle, and complications of diabetes mellitus are widely known as major factors for developing cardiovascular diseases, but from the modern medical point of view, reactive oxygen species (ROS), increased vascular oxidative stress, and endothelial cells Nitrogen oxide is known to be due to a decrease in the activity of endothelial type Nitric Oxide Synthase (eNOS).

In particular, nitric oxide produced by endothelial nitric oxide synthase is a potent vasorelaxant and inhibits platelet aggregation, vascular muscle cell proliferation, monocyte deposition, and atherosclerosis related protein expression to regulate overall cardiovascular homeostasis. (Forstermann et al., Circulation, 113: 1708-1714, 2006), active oxygen species also regulate adhesion molecule expression, proliferation of vascular smooth muscle cells (VSMC). It is known to cause cardiovascular disease by causing stimulation of migration and regulation of oxidative lipoprotein.

On the other hand, the present invention has been characterized for the first time that the stilbin derivative compound has the activity of inhibiting the activity of arginase, the activity inhibition of the arginase according to the recently reported data production of nitric oxide It has been found to have a beneficial effect on vascular dysfunction by increasing the.

Accordingly, the present inventors could expect that the stilbin derivative compounds according to the present invention can prevent and treat vascular diseases through the inhibition of arginase activity.

More specifically, in accordance with an embodiment of the present invention, to determine the inhibitory action of arginase activity of the piceanol-3'-O-beta-D-glucopyranoside compound in the stilbin derivative compound, After each tissue was separated from the liver and kidney, the tissue lysate was treated with the piaceanol-3'-O-beta-D-glucopyranoside compound, respectively, and the activity of arginase was measured. In the group treated with the compound, arginase activity was decreased in the group treated with the compound, especially arginase I in the liver tissue and arginase II in the kidney tissue. This was shown to be suppressed (see FIG. 1 and Example 2).

In addition, according to another embodiment, after treatment with the physethanol-3'-O-beta-D- glucopyranoside compound of the present invention to vascular endothelial cells, intracellular nitric oxide through the inhibition of arginase activity It was confirmed that the production of was increased. In the group treated with Piaceanol-3'-O-beta-D-glucopyranoside compound in HIVEC cells, the activity of arginase was significantly higher than that of the untreated control group. On the other hand, the amount of nitric oxide produced was found to increase, and the extent of the increase was found to be proportional to the concentration of the compound of the present invention treated (see FIG. 2 and Example 3). In contrast, the expression level of nitric oxide synthase was not significantly changed in the group treated with the compound of the present invention and the group not treated.

Therefore, through the above results, the present inventors have found that the action of piaceanol-3'-O-beta-D-glucopyranoside compound according to the present invention increases the production of nitric oxide in vascular endothelial cells through arginase inhibitory activity. It can be seen that through the prevention and treatment of vascular disease, the inhibitory activity of arginase to the compound of the present invention is slightly higher than the boronic acid derivatives, but a new role of inhibiting arginase activity By clarifying the compounds of the present invention can contribute to the development of new drugs that can be used as a therapeutic agent for vascular diseases.

Furthermore, the inventors of the present invention confirmed that the piaceanol-3'-O-beta-D-glucopyranoside compound also has an activity of reducing the production of free radicals, namely, The endothelial cells were treated with the physatanol-3'-O-beta-D-glucopyranoside compound and analyzed by measuring the fluorescence intensity of DHE, an oxygen sensitive fluorescence reagent. It was shown to be significantly reduced in proportion to the treatment concentration of the tanol-3'-O-beta-D-glucopyranoside compound, and through these results, the present inventors found that the piacetanol-3'-O- It can be seen that the beta-D-glucopyranoside compound has an activity of inhibiting the generation of reactive oxygen (see FIG. 3 and Example 4).

As well as the present inventors, other stilvin derivative compounds in addition to the phyceanol-3'-O-beta-D-glucopyranoside compound are raponticin, rapontigenin, isolahapontin, Deoxyrhapontigenin and resveratrol compounds were also examined for arginase inhibitory activity. To this end, each tissue was isolated from the liver and kidney of the mouse, and then the stilbin derivative compound in the tissue lysate. After treatment with Arginase activity, Arginase I and II were found to be treated with raponticin (Rhaponticin), isorhapontin, deoxyrhapontigenin and resveratrol. It was shown that there is activity to inhibit both arginase I and II, and in the case of Rapontigenin, there was an inhibitory activity of arginase I.

Therefore, through these results, the present invention can provide a composition for the prevention or treatment of vascular diseases containing stilvin derivative compounds or salts thereof according to the present invention as an active ingredient, the compounds are the inhibitory activity of arginase, Increasing the production of nitric oxide or inhibiting the production of reactive oxygen species (ROS) can prevent and treat vascular diseases.

In the above, the arginase, in which the compound of the present invention may inhibit its activity, may be arginase I or arginase II, and particularly, in the present invention, piaceanol-3'-O-beta-D-glucopy The ranoside compound exhibited an IC ₅₀ at about 11 uM level for arginase I and II activity.

In the present invention, the vascular disease is not limited thereto, but hypertension, arteriosclerosis, heart attack, stroke, cancer, aging, erectile dysfunction, sickle cell disease, diabetes, angina pectoris, myocardial infarction, arthritis, diabetic It may be selected from the group consisting of retinopathy, psoriasis, neovascular glaucoma, hemangioma and ischemic disease.

Therefore, the composition for preventing or treating vascular diseases according to the present invention can be used as a pharmaceutical composition that can treat vascular diseases.

In addition, the stilbin derivative compound in the present invention may be included in a concentration of 0.1 ~ 150 uM relative to the total volume of the composition.

The composition for preventing or treating vascular disease according to the present invention may include a pharmaceutically effective amount of stilvin derivative compound alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent, ameliorate, and treat vascular disease. The pharmaceutically effective amount of the stilbin derivative compound according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of symptoms of vascular disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease, preferably 1 to 10000 mg / weight kg / day, more preferably 10 to 1000 mg / weight kg / day It may be administered several times a day at an effective dose of. In addition, the composition for the prevention or treatment of vascular diseases according to the present invention may be administered in parallel with a known compound having the effect of preventing, improving or treating vascular diseases.

Accordingly, the present invention may provide a medicament for preventing and treating vascular diseases, including a composition containing a stilvin derivative compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the composition for the prevention or treatment of vascular diseases according to the present invention contains stilbin derivative compounds isolated and purified from nature, so there is no toxicity and side effects for drugs and can be used safely even for long-term use. The composition is characterized by very stable to the body.

Therefore, the composition for preventing or treating vascular disease of the present invention can be added to food for the purpose of preventing and improving vascular disease symptoms, the composition of the present invention can be used as a composition for food for the prevention and improvement of vascular disease symptoms Can be.

Therefore, the composition for food for the prevention and improvement of the symptoms of vascular disease of the present invention can be easily utilized as foods, such as the main ingredients, side ingredients, food additives, functional foods or beverages that are effective in preventing and improving the symptoms of vascular diseases. have.

As used herein, the food means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step. It includes all food additives, functional foods and beverages.

Foods to which the composition for preventing and improving vascular disease symptoms according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the functional food is a biological defense rhythm control, disease prevention and recovery of a food group or a food composition that has added value to the food by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body regulatory function related to the living body, specifically, it may be a health functional food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, in the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy a taste and includes a functional drink. The beverage contains, as an essential ingredient, a composition for the prevention and amelioration of the symptoms of vascular disease as an essential ingredient, and there are no particular restrictions on the other ingredients, and it contains various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. can do.

Furthermore, in addition to the above-described foods containing a composition for the prevention and improvement of symptoms of the vascular disease of the present invention, a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, colorants and fillers ( Cheeses, chocolates, and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The components can be used independently or in combination.

In the food containing the composition for the prevention and improvement of the symptoms of vascular disease of the present invention, the amount of the composition according to the present invention may comprise from 0.001% to 90% by weight of the total food weight, preferably 0.1% by weight To 40% by weight, and in the case of a beverage, it may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, for health and hygiene purposes or for health control purposes. In the case of long-term intake to be less than the above range, since the active ingredient has no problem in terms of safety can be used in an amount above the above range is not limited to the above range.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

The experimental materials and experimental methods used in the Examples performed in the present invention are as described below.

<1-1> Experimental material

Arginase lysates were prepared from liver and kidney of anesthetized mouse C57BL / 6, MnTBAP (Mn (III) Tetra (4-benzoic acid) porphyrin chloride), L-NAME (-nitro-L-arginine methyl ester) was used from Calbiochem. Hereinafter, all other reagents were purchased from Sigma, and Piceatannol-3-O-beta-D-glucopyranoside (PG), a compound according to the present invention, was used from Catholic University.

<1-2> Cell culture

HUVECs cells were purchased from Cascade biologics and cultured and stored in medium supplemented with Medium230 plus low-serum growth supplement (LSGS) under 5% carbon dioxide and 37 ° C according to the supplier's protocol.

<1-3> Arginase ( arginase Activity measurement

For measuring the activity of arginase, the cells were first homogenized at 4 ° C., then centrifuged at 14,000 × g for 20 minutes, lysis buffer (50 mM Tris-HCl, pH7.5, 0.1 mM EDTA and Lysates were prepared by lysing the cells with protease inhibitors, and arginase activity assays were performed using supernatants of the lysates (Ryoo et al., 2006). In addition, the activity assay was analyzed by measuring the activity of arginase in the presence of a known arginase inhibitor (ABH, 2 (S) -amino-6-boronohexanoic acid, 20 uM).

<1-4> NOx  Measure

The Nitric Oxide Assay Kit (Calbiochem) was used to determine NO by determining the ratio of nitrate / nitrite (NOx) by the Griess reaction after reduction of nitrate to nitrite by nitrate reductase. The content was measured and the NOx concentration in HUVECs was expressed as uMol / mg protein.

<1-5> Weston Blot  analysis

Liver or kidney of C57BL / 6 mice (week 10) was buffered (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 ug / ml leupeptin, 1 ug / ml pepstatin, 1 ug) / ml aprotinin, 1 mM phenylmethylsulfonylflouride, 1 mM sodium orthovanadate and 1 mM NaF) and homogenized and centrifuged at 14,000 xg for 30 minutes. Then, the amount of protein in the supernatant was measured by Bradford method, the protein (100 ug) was electrophoresed in 10% SDS-PAGE, and transferred to the nitrocellulose membrane (Bio-Rad). Subsequently, a monoclonal anti-arginase I (Santa Cruz), arginase II (Santa Cruz), anti-endothelial nitric oxide synthase (eNOS, BD Bioscience) or anti-beta tubulin (BD bioscience) antibody was added to the membrane. After addition and reaction, the reaction was performed with a secondary antibody (Amersham). The signal analysis was then detected with an X-ray film using a chemiluminescence detection reagent.

<1-6> In isolated mouse aorta DAF - FM  or DHE Using NO  Occurrence measurement

Dulbeccos modified Eagles isolated from mouse aortic rings and added 2% FBS and antibiotic (1X) in the presence of picaetannol-3-O-beta-D-glucopyranoside (50 uMol / L) compound according to the present invention Incubated overnight in medium (DMEM) medium at 37 ° C. and 5% carbon dioxide (White et al., 2006). The aorta was then cut longitudinally and filled with HEPES buffer (NaCl 120 mM, KH 2 PO 4 2.6 mM, KCl 4 mM, CaCl 2 2 mM, MgCl 2 0.6 mM, HEPES 25 mM, glucose 14 mM, pH 7.4). The bottom of the chamber (silgard coated chamber) (endothelial layer) was fixed. The chamber was balanced with a heating stage at 37 ° C. for 30 minutes, and the chamber was maintained in static bath conditions during fluorescence measurements. Afterwards, Olympus 10x objective, intensified camera (Luca 658M-TL) and custom with optimized excitation and emission wavelengths (DAF-FM, 470/525 nm; or DHE, 470/580) An image acquisition program (Cell software, Olympus) was used to measure the fluorescence of tissue background and DAF-FM (4-Amino-5-methylamino-2 ', 7'-difluorofluorescein) Diacetate or dihydroethidine (DHE). Following initial equilibrium, background fluorescence was recorded and the aorta was allowed to room temperature for 15 minutes. The aorta was then loaded into 5 uM DAF-FM or 5 uM molecular probes (DHE) for 45 minutes in HEPES buffer, washed with DAF-FM or DHE, and then equilibrated at 37 ° C. for 20 minutes. . Fluorescence intensities were averaged from the full field of view (5 frames, 2 × 2 binning) and recorded by acquisition program. After re-treatment of L-NAME (mol / L) or MnTBAP (mol / L), washing with DAF-FM or DHE to set baseline changes in intensity, then recording changes in DAF-FM or DHE fluorescence It was. The slope of fluorescence change over time was measured by linear regression in Origin (version 7.5, OrignLab Corp, Northampton, Mass.) And used for statistical comparison.

<1-7> Statistical processing

All data are expressed as mean ± SD of at least four independent experiments. The unpaired Student's t-test between the two groups was used to assess the significance of the deviation. P <0.05 was accepted as significant.

< Example  2>

Piaceanol -3'-O-beta-D- On glucopyranoside  Arginase Activity Assay

The inventors of the present invention, first to investigate whether there is an inhibitory activity for the arginase of picetanol-3'-O-beta-D-glucopyranoside (PG) in the stilbin derivative compounds according to the present invention, A lysate of the tissues was obtained according to the method of <1-1> from liver and kidney of Pseathanol-3'-O-beta-D-glucopyranoside compound according to the present invention. Prior to treatment, the presence of an isomer of arginase was confirmed by the Western method described in <1-5> using antibodies of arginase I and arginase II, and then physethanol- was dissolved in the lysate. After treating the 3'-O-beta-D-glucopyranoside compound with each concentration (1, 3 and 10 uM), respectively, the activity of arginase was measured according to the method of <1-3>.

As a result, as shown in FIG. 1, it was confirmed that a large amount of arginase I was present in liver tissue, while in the kidney, arginase II was present. In addition, as a result of culturing PG in the concentration of the liver lysate, arginase I activity was significantly reduced compared to the control group not treated with PG (75u5% activity for 1uM, 72u for 3uM). Activity of ± 7%, 62 ± 1% at 10 uM). In addition, when treated with 1, 3 and 10 uM of PG for kidney lysate, the remaining arginase activity was found to be 75 ± 6, 74 ± 5 and 53 ± 8%. The inventors have found that for the PG compounds according to the present invention there is no particular selectivity for the isotype of arginase, and it can be seen that it has strong and specific inhibitory activity against both arginase I and II. there was.

< Example  3>

Piaceanol -3'-O-beta-D- On glucopyranoside  by NOx  Generate increase activity analysis

Furthermore, the present inventors have found that nitric oxide is inhibited by intracellular HUVEC cells through the inhibition of arginase activity in the treatment of the piaceanol-3'-O-beta-D-glucopyranoside compound according to the present invention. It was investigated whether it could increase production. That is, for this purpose, after culturing the vascular endothelial cells as in <1-1>, and then treated with the piaceanol-3'-O-beta-D-glucopyranoside compound according to the method of <1-4>. The production of NOx was investigated by measuring the content of nitrate / nitrite (NOx).

As a result, as shown in Figure 2, after treatment with PG in HUVEC cells and incubated for 12 hours, the activity of arginase was found to decrease very much, and the degree of reduction was proportional to the concentration of treated PG. On the other hand, NOx production was found to increase.

Therefore, the present inventors have found that the inhibition of arginase was found in rat aortic endothelial cells (Berkowitz et al., 2003), bovine lung endothelial cells (Chicoine et al., 2004), and porcine coronary artery model (Zhang et al., In 2001, the results showed that the results of the study were consistent with the previous studies, whereas the PG according to the present invention had little effect on the expression level of arginase II and eNOS in the cells.

Therefore, the above results according to the present invention showed that the production of NOx was increased by treatment with the PG compound of the present invention, and it was found that the increase of NOx is dependent on the increase of L-arginine through the inhibition of arginase activity.

< Example  4>

According to the invention PG  Inhibiting arginase activity of the compound, NO  Generate increase and ROS Decreased activity measurement of the

In order to determine whether increased NOx production in HUVECs is altered by redox response-regulation of aortic tissue endothelial cells, we treated the cells with PG at different time intervals and then measured the intensity of DAF-AM and DHE fluorescence. Measured. The measurement was carried out according to the method described in <1-6>.

As a result, as shown in FIG. 3, when treated with PG compounds overnight in HUVEC cells, arginase activity in the aorta isolated from mice was decreased (704 for the control group and 445 for the PG treatment). ± 14 pmol Urea / mg protein / minute, p <0.01). In addition, DAF-FM diacetate, a NO-sensitive fluorescence staining reagent, was used to investigate whether PG-dependent arginase inhibition increased NO production, and the average slope of DAF fluorescence was significantly increased by PG treatment. On the other hand, the treatment of N ^G -Nitro-L-arginine methyl ester (L-NAME) dramatically reduced the fluorescence gradient of DAF.

Furthermore, the present inventors further investigated whether the inhibition of arginase activity by the treatment of the PG compound according to the present invention increases the production of NO while simultaneously reducing the production of ROS. generation was measured ^- O2 using a sensitive sample of DHE ^- - O2 in the cell.

As a result, the DHE fluorescence intensity was significantly decreased compared to the control group in a time and concentration-dependent manner of the treated PG compound, and the DHE signal was completely suppressed upon MnTBAP treatment.

In addition, the results are consistent with the new concept that arginase can regulate NO production by NOS by limiting the L-arginine substrate as a general mechanism for the regulation of NOS. Therefore, it was found that inhibition of arginase could increase the amount of L-arginine and further inhibit eNOS from segregation with ROS production in reduced endothelial cells.

< Example  5>

Concentration dependent PG Inhibition of Arginase Activity by

Through the above examples, the present inventors confirmed that the PG compound according to the present invention inhibits the activity of arginase, and furthermore, liver and mouse from the mouse according to the method described in the above example to more clearly prove these results. After preparing the lysates of kidneys, the PG compounds according to the present invention were treated at different concentrations (25, 50, 75, 100 and 125 uM), and then arginase activity in each tissue was specified. The activity was analyzed by measuring IC ₅₀ (Inhibitory concentration of 50%) using Graphpad prizm 4.0 software.

As a result, as shown in Figure 4, the PG compound according to the present invention was shown to effectively inhibit the activity of arginase in liver and kidney tissues of mice, in particular IC ₅₀ in liver tissues The value was found to be 11.22 uM, and for kidneys, 11.06 uM, and the inhibition of arginase activity was found to increase in a concentration dependent manner of the treated PG compound.

< Example  6>

Steel bean  Of derivative compounds Arginase  Activity analysis

Furthermore, the inventors of the present invention, in addition to the piceatannol-3'-O-beta-D-glucopyranoside compound, raponticin, which is another stilbin derivative compound, The following experiments were performed to determine whether arginase inhibitory activity was observed for Rhapontigenin, isorhapontin, deoxyrhapontigenin, and resveratrol compounds. First, a lysate of the respective tissues was obtained from the liver and kidney of the mouse according to the method of Example <1-1>, and arginase I and argina were treated before the stilbini derivative compound described above was treated to the lysate. The presence of an isomer of arginase was confirmed by the Western method described in <1-5> using an antibody of ase II, and the activity of arginase was measured according to the method of <1-3>.

As a result, as shown in FIG. 5, it was found that the stilbin derivative compound of the present invention has an activity of effectively inhibiting arginase in liver and kidney tissues of mice, and particularly, raponticin and isola. Isorhapontin, deoxyrhapontigenin, and resveratrol have been shown to have activity inhibiting both arginase I and II, and resveratrol has been shown to contain arginase I, laponti God has been shown to have a superior activity in inhibiting arginase II.

Above, the present inventors through the experimental results as described above, the present inventors are the stilbin derivative compounds according to the present invention (Rhaponticin), rapontigenin (Rhapontigenin), isorhapontin (isorhapontin), deoxyrhapontigenin (deoxyrhapontigenin) and Resveratrol and Piaceanol-3'-O-beta-D-glucopyranoside compounds are excellent in inhibiting the activity of arginase, while at the same time promoting the production of nitric oxide It can be seen that the action of inhibiting the production of vascular diseases, particularly cardiovascular diseases and cancer can be prevented and treated.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (7)

Picetanol-3'-O-beta-D-glucopyranoside, stilbene derivative compound, raponticin, rapontigenin (Rhapontigenin), isorhapontin, deoxyrhapontigenin, resveratrol, and pharmaceutically acceptable salts thereof. Therapeutic composition. The method of claim 1,
The stilbin derivative compound is a composition for the prevention or treatment of vascular diseases, characterized in that derived from rhubarb (rhubarb). The method of claim 1,
The stilbin derivative compound is a composition for preventing or treating vascular disease, characterized in that it has the effect of inhibiting the activity of arginase, increase the production of nitric oxide or inhibit the production of reactive oxygen species (ROS). The method of claim 3,
The arginase is a composition for preventing or treating vascular diseases, characterized in that the arginase I or arginase II. The method of claim 1,
The vascular diseases include hypertension, arteriosclerosis, heart attack, stroke, cancer, aging, erectile dysfunction, sickle cell disease, diabetes, angina, myocardial infarction, arthritis, diabetic retinopathy, psoriasis, neovascular glaucoma, hemangioma And ischemic diseases. The composition for the prevention or treatment of vascular diseases, characterized in that selected from the group consisting of. The method of claim 1,
The stilvin derivative compound is a composition for the prevention or treatment of vascular diseases, characterized in that it comprises a concentration of 0.1 ~ 150 uM relative to the total volume of the composition. Health functional food for the prevention or improvement of vascular diseases containing the composition of any one of claims 1 to 6.

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