KR20150118470A - Composition for preventing or treating cardiovascular diseases comprising serine as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating cardiovascular diseases and, more specifically, to a pharmaceutical composition for preventing or treating cardiovascular diseases, containing serine which is a kind of amino acid, as an active ingredient, and a food composition and a health functional food for improving cardiovascular diseases. The serine according to the present invention may effectively reduce a concentration level of homocystein corresponding to a cardiovascular toxicant and drastically inhibit apoptosis and endoplasmic stress in a cell due to the homocystein. Therefore, the serine according to the present invention can be used as a functional supplement food as well as a medicine like a pharmacological composition as a material which can prevent or treat the cardiovascular diseases, so being expected that the value is very high in the case of being applied in a medical industry and a health functional food industry. Particularly, the serine is low-priced and intrinsic amino acid so almost having no toxicity to be safe even if being used for a long time.

Description

[0001] The present invention relates to a composition for preventing or treating cardiovascular diseases comprising serine as an active ingredient,

The present invention relates to a composition for preventing or treating cardiovascular diseases, and more particularly to a pharmaceutical composition for prevention or treatment of cardiovascular diseases including serine which is one kind of amino acid as an active ingredient, A food composition for improving disease, and a health functional food.

According to the statistics of the World Health Organization (WHO), the death rate of the world population due to cardiovascular disease in 1999 was more than 30%, and in 2010 it was expected to be the number one disease in advanced countries. . This is presumably due to an increase in risk factors for coronary artery disease due to changes in access to an aging society and eating habits.

Endothelial dysfunction has been associated with hypertension, atherosclerosis, hyperlipidemia, and hypertension since 1990, when non-ideal vessel relaxation was found in patients with hypertension (Panza JA et al., New England Journal of Medicines, 323: , Diabetes, and obesity (Brunner H. et al., J. Hypertens, 2005, 23: 233-246). Endothelial cells are epithelial cells lined up along the coronary arteries of the heart, blood vessels, and lymphatics. The main function is to regulate both vascular tone and structure by producing vasodilating and vasoconstrictor neural mediators.

BACKGROUND ART Cardiovascular disease is a disease characterized by an initial endothelial dysfunction and a heart and vascular system abnormality which has been conventionally known and has been known as atherosclerosis, hypertension, hyperlipemia, coronary artery disease (heart attack), cerebrovascular disease (stroke, , Cardiac arrhythmia, heart failure, congestive heart disease, myocardial disease, and the like, but are not limited thereto.

The main factors of cardiovascular disease manifestation are genetic factors, lifestyle, and diabetic complications. However, from the viewpoint of modern medicine, reactive oxygen species (ROS) and vascular endothelial growth factor Is known to be due to an increase in oxidative stress and a decrease in nitric oxide due to decreased activity of endothelial type Nitric Oxide Synthase (eNOS). Nitric oxide produced by endothelial nitric oxide synthase is a potent vasodilator and inhibits platelet aggregation, vascular myocyte proliferation, vascular deposition of mononuclear cells, and arteriosclerosis-related protein expression, resulting in an overall control of cardiovascular homeostasis (Forstermann et al., Circulation, 113: 1708-1714, 2006). However, the production of NADPH oxidase, which is responsible for the production of reactive oxygen species in the blood vessel due to various factors, decreases the production of nitrogen oxides. Also, the produced reactive oxygen species regulate the expression of adhesion molecules [Lo et al. Am. J. Physiol., 264: L406-412, 1993], proliferation and migration stimulation of VSMC, vascular smooth muscle cells [Griendling and Ushio-Fukai, J. Lab. Clin. (Lynch and Frei, J. Lipid Res., 34: 1745-1753, 1993), which causes oxidative lipoprotein regulation and the like. In addition, the production of reactive oxygen species increased by NAD (P) H 2 O oxidase is associated with clinical risk factors of atherosclerosis and nitric oxide (NO) function of injured endothelial cells in patients with coronary artery disease And it causes the contraction of blood vessels in origin.

Therefore, reduction of active oxygen species in blood vessels, inhibition of the generation of active oxygen species downstream and induction of activity of endothelial nitric oxide synthase are recognized as important targets in the prevention of cardiovascular diseases every week.

In addition, myocardial infarction accounts for 75% of these cardiovascular diseases. The myocardial infarction is the most common cause of death with cancer. Methods for inhibiting the onset of myocardial infarction include a method of preventing the occlusion of blood vessels by treating hypertension and hyperlipemia before the occlusion of the blood vessels, or a method of suppressing the death of myocardial tissue due to a shortage of blood flow after occlusion of blood vessels And so on. Even though the most preferable method is to prevent the occlusion of the blood vessel, since the myocardial tissue is killed after 3 to 6 hours after the occlusion of the blood vessel, the result is irreversible, Research to develop a method for inhibiting the death of tissue is being actively pursued. Although it is practically impossible for all patients to undergo reperfusion treatment at an appropriate time point, the most preferable method for inhibiting the death of myocardial tissue after occlusion of the blood vessel is to be delivered to the hospital immediately after the onset of vascular occlusion, Studies are underway to develop a method to retard myocardial tissue death after occlusion. In particular, since death of myocardial tissue has mainly been reported to be due to apoptosis, studies have been actively conducted to develop a method of inhibiting apoptosis and improving cell viability.

Although the mechanism of apoptosis has not yet been clearly elucidated, it is generally recognized that the production of ATP is decreased by the deficiency of oxygen and glucose supply, and edema occurs, or due to accumulation of excessive glutamic acid in the extracellular environment It has been reported that it proceeds by intracellular inflow of glutamic acid and a rapid increase of intracellular calcium concentration or by an increase of intracellular radicals due to sudden supply of oxygen by reperfusion of occluded blood vessels and DNA damage by the radicals . These mechanisms of apoptosis are known to be applied individually or in combination depending on the type of each tissue and the external conditions given to each cell. In the case of myocardial infarction, the production of ATP due to the shortage of oxygen and glucose, It is known that cell suicide is induced by the occurrence of edema.

Efforts to develop a therapeutic agent capable of treating a disease causing cell apoptosis by effectively suppressing cell apoptosis based on the action mechanism of cell apoptosis are continuing. For example, minocycline, a tetracycline-based antibiotic, enhances cell viability by suppressing cell suicide, and is useful for the treatment of ischemic diseases such as cerebral infarction, myocardial infarction and ischemic acute renal failure as well as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis amyotrophic lateral sclerosis, Huntington's disease, and spinal cord injury (Korean Patent No. 0404134, US Patent No. 6,716,822 and No. 6,818,625). In addition, aminoglycoside and quinolone antibiotics, like minocycline, improved the survival of cells in ischemic conditions. Among them, aminoglycoside G418 (geneticin) was effective in treating myocardial infarction (US Patent No. 6,716,822). In the subsequent experiments, G418 inhibited apoptosis in ischemic conditions and showed therapeutic effects in not only myocardial infarction but also cerebral infarction. As a result, the samples exhibiting the same cell survival improvement effect as is G418 in ischemic conditions ultimately have an effect on the treatment of ischemic diseases such as myocardial infarction, and also prevent and treat diseases caused by apoptosis such as degenerative brain diseases The results of this study showed that the extracts of Ganoderma lucidum, including wheat, showed the effect of improving the survival of the cells. Based on the results, the wheat extract was applied to the animal models of ischemic diseases and degenerative brain diseases, And ischemic diseases such as myocardial infarction and degenerative brain diseases such as Alzheimer's disease (Korea Patent No. 0723950 and International Patent Publication No. WO 2006/073265).

On the other hand, homocysteine is a sulfur-containing amino acid, an important intermediate for the metabolism of the essential amino acid methionine. Homocysteine is maintained at a very low concentration in cells, and extra homocysteine is released extracellularly, that is, into the blood. Thirty years ago, it was reported by Dr. McCully that this homocysteine is responsible for vascular conditions such as atherosclerosis, myocardial infarction (Am J Pathol 1969; 56: 111-128). Subsequent clinical studies have demonstrated that homocysteine levels are high in patients with atherosclerosis in the peripheral blood vessels and cerebral blood vessels (N Eng J Med 1985; 313: 709-715). It has also been reported that in most subsequent clinical trials, there is a correlation between elevated homocysteine levels and cerebral infarction (Stroke. 1984; 15: 1012-1016, Eur J Cli Invest. 1992; 22: 214-221 , Stroke 1990; 21: 572-576, Lancet 1995; 346: 1395-1398, Stroke 1994; 25: 1924-1930, Stroke 1998: 29: 2478-2483). In large trials, a 25% increase in serum homocysteine (3 μM as absolute) has been reported to increase the risk of coronary disease by 10% and the risk of cerebral infarction by 20% (JAMA 2002; 288: 2015-22 ), Suggesting that they are now independent risk factors.

At present, as a method of lowering homocysteine, only the supplement of the metabolic enzyme, that is, vitamin B6, vitamin B12, and folic acid (folic acid) has been attempted. It is known that the level of homocysteine in the blood is reduced to some extent by the intake of these vitamins (J Nutr 1996; 126 (suppl) 1276S-1280S). In addition, improvement of vascular endothelial function by vitamins and reduction of carotid plaques have been reported (Circulation 1999; 99: 1156-1160; Eur J Clin Invest 1995; 25: 176-181; Lancet 1998;

However, in the large-scale study (Vitamin Intervention for Stroke Prevention (VISP)) that examined the effect of the vitamin 3 method on the recurrence of cerebral infarction and myocardial infarction, no recurrence prevention effect was observed. (JAMA 2004; 291: 565-575) suggests that vitamin therapies have not shown a sufficient decrease in homocysteine. In this report, it has been found that if the baseline homocysteine level is reduced by 3 μm, the risk of recurrence of cerebral infarction can be reduced by 10% and the risk of myocardial infarction by 26%.

Therefore, a substance that can effectively lower homocysteine value without exhibiting toxicity or side effects upon long-term administration can be effectively used as a drug substance for treating cardiovascular diseases such as atherosclerosis, myocardial infarction, and the like.

Accordingly, the inventors of the present invention have made efforts to search for a substance which is easy to ingest, safe for human body and can treat cardiovascular diseases, and as a result, it has been found that certain amino acids effectively reduce the level of intracellular homocysteine, Thereby completing the invention.

Korean Patent Publication No. 10-2013-0062809 Korean Patent Laid-Open No. 10-2012-0115963 Korean Patent Laid-Open No. 10-2009-0079584

Accordingly, an object of the present invention is to provide a pharmaceutical composition which is stable to human body and can effectively prevent or treat cardiovascular diseases even after long-term use.

Another object of the present invention is to provide a health functional food which is stable to human body and can effectively improve cardiovascular diseases even after long-term use.

In order to achieve the above-mentioned object of the present invention,

The present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases comprising L-serine as an active ingredient.

In one embodiment of the present invention, the L-serine may comprise the compound itself or a pharmaceutically acceptable salt and solvate.

In one embodiment of the present invention, the L-serine may be included in the composition at a concentration of 1 to 1000 μM.

In one embodiment of the present invention, the L-serine may have a therapeutic effect on cardiovascular diseases through activity of decreasing the concentration of intracellular homocysteine.

In one embodiment of the present invention, the L-serine may have an effect of inhibiting apoptosis and endoplasmic reticulum of endothelial cells induced by homocysteine.

In one embodiment of the invention, the cardiovascular disease is selected from the group consisting of myocardial infarction, angina pectoris, hypertension, heart failure, cardiomyopathy, primary cardiac arrest, congestive heart disease, ischemic heart failure, arteriosclerosis, arrhythmia, coronary artery disease, stroke and peripheral vascular disease ≪ / RTI >

The present invention also provides a health functional food for improving cardiovascular diseases comprising L-serine as an active ingredient.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foodstuff, confectionery, pizza, ramen, other noodles, rice cake, gum, candy, ice cream, alcoholic beverage, ≪ / RTI >

The serine according to the present invention can effectively lower the concentration level of homocysteine corresponding to a vascular toxic substance and is remarkably effective in suppressing homocysteine-induced apoptosis and endoplasmic reticulum stress. Therefore, the serine of the present invention can be used as a functional food or the like as well as a medicine such as a pharmacological composition as a substance that can prevent or treat a cardiovascular disease, and is highly valuable when applied to the pharmaceutical industry and the health functional food industry . In particular, serine is an inexpensive, endogenous amino acid that has little toxicity and is safe for long-term use.

FIG. 1A shows the cell viability of human vascular endothelial cells after treatment with homocysteine (5 mM) and L-serine (1, 5, and 10 mM) at different concentrations (MTT assay).
FIG. 1B shows the results of treatment of homocysteine (5 mM) with L-serine at concentrations of 1, 5, and 10 mM in human vascular endothelial cells, and the degree of apoptosis was measured using a flow cytometer Results.
FIG. 2 a shows the results of treatment with homocysteine (5 mM) and L-serine (1, 5, and 10 mM) on human vascular endothelial cells after 24 hours of treatment with Grp78, which is a representative index of endoplasmic reticulum stress The amount of protein expression in CHOP was determined by Western blotting.
FIG. 2B is a graph showing the effect of L-serine on human endothelial cells treated with homocysteine (5 mM) and L-serine (1, 5 and 10 mM) The results of PCR confirmed the mRNA levels of CHOP and spliced-XBP1 (XBP1 splicing form).
FIG. 3 is a graph showing changes in intracellular homocysteine concentration after 24 hours of treatment with homocysteine (5 mM) and L-serine (1, 5 and 10 mM) in human vascular endothelial cells using a Homocysteine EIA Reagent kit Respectively.

The present invention is characterized by providing a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising serine as an active ingredient.

The serine defined in the present invention is characterized by having the following formula (1).

In the present invention, the serine includes an L-type or D-type isomer, preferably L-serine. D-serine is synthesized from L-form in the brain and acts as a neurotransmitter, so it is desirable to use pure L-form to avoid unexpected action. The L-serine of the present invention includes the compound itself or a pharmaceutically acceptable salt and solvate thereof.

The serine according to the present invention can be appropriately selected and used either separately or commercially purchased by methods well known in the technical field of the present invention, and the method or substance is not particularly limited.

The L-serine compounds of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be filtered by suction.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid glutamic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid may be used as the acidic compound, such as glutaric acid, glutamic acid, glutaric acid, glucuronic acid,

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate (Salicylate) salts, which are known in the art and which are known in the art, such as, for example, ≪ / RTI >

The L-serine of the present invention can effectively lower the concentration level of homocysteine, which is a vascular toxic substance, and has an effect of inhibiting apoptosis and endoplasmic reticulum by homocysteine in cells, Or as a material for treatment.

Homocysteine is a sulfur-containing amino acid, an important intermediate for the metabolism of the essential amino acid methionine. Homocysteine is maintained at a very low concentration in cells, and extra homocysteine is released extracellularly, that is, into the blood. Thirty years ago, it was reported by Dr. McCully that this homocysteine is responsible for vascular conditions such as atherosclerosis, myocardial infarction (Am J Pathol 1969; 56: 111-128). Subsequently, clinical studies have shown that homocysteine levels are high in patients with atherosclerosis in the peripheral blood vessels and cerebral blood vessels.

Therefore, the present inventors have tried to find a substance which is easy to ingest, safe for human body and can treat cardiovascular diseases, and as a result, it is firstly experimented that L-serine can effectively reduce the level of homocysteine in vascular endothelial cells .

More specifically, in Example 1 below, the effect of L-serine on homocysteine-induced apoptosis of vascular endothelial cells was examined, and as a result, the survival rate of vascular cells reduced by homocysteine was significantly higher than that of serine Dependent manner, and it was confirmed that apoptosis was effectively reduced in the group treated with L-serine (see FIGS. 1A and 1B).

In addition, in Example 2, the effect of L-serine on homocysteine-induced vascular endothelial cell stress was investigated. As a result, the factors of the stress of the endoplasmic reticulum (Grp78, CHOP, spliced- XBP1) and gene transcription levels were greatly decreased when L-serine was treated together (see FIGS. 2A and 2B).

In addition, in Example 3 below, it was confirmed that L-serine could actually reduce the level of homocysteine present in the cells. As a result, it was confirmed that L- Homocysteine concentration was significantly reduced (see FIG. 3).

From the above results, it was experimentally proved that L-serine of the present invention has an activity of decreasing the concentration of intracellular homocysteine and also inhibits homocysteine-induced vascular endothelial cell apoptosis and endoplasmic reticulum stress .

Therefore, the composition of the present invention comprising L-serine as an active ingredient can effectively prevent or treat cardiovascular diseases.

The composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients as the pharmaceutical composition comprising the L-serine as an active ingredient. Examples of the adjuvants include excipients, , A sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the L-serine of the present invention may be contained in the composition at a concentration of 1 to 1000 μM, and the L-serine of the present invention may be contained in an amount of 0.1 to 50% .

Examples of diseases for which the pharmaceutical composition of the present invention can exhibit a therapeutic effect include cardiovascular diseases such as myocardial infarction, angina pectoris, hypertension, heart failure, cardiomyopathy, primary cardiac arrest, congestive heart failure, ischemic heart failure, arteriosclerosis, arrhythmia, Diseases, stroke, peripheral vascular disease, and the like.

The pharmaceutical composition of the present invention may further comprise, as an active ingredient, a substance having an additional therapeutic effect on cardiovascular diseases other than L-serine.

The present invention also provides the use of a composition comprising L-serine as an active ingredient for the manufacture of a medicament for the prevention or treatment of cardiovascular diseases. The composition of the present invention comprising L-serine as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of cardiovascular diseases.

The present invention also provides a method of preventing or treating cardiovascular diseases comprising administering L-serine to a mammal.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

The composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The composition according to the present invention can be used alone or in combination with the methods of using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention and treatment of fatty liver disease.

Since L-serine according to the present invention has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The present invention also provides a food composition comprising L-serine as an active ingredient.

Such a food composition may contain, as an additional ingredient, various flavors or natural carbohydrates such as an ordinary food composition besides L-serine as an active ingredient.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition to the L-serine as an active ingredient, the above-mentioned food composition may also contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate, etc.) Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

Since L-serine, an active ingredient of the present invention, is a natural substance with little toxicity and side effects, it can be safely used for long-term administration for the purpose of improving cardiovascular diseases.

The present invention also provides a health functional food for preventing or improving cardiovascular diseases comprising L-serine as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of prevention and improvement of cardiovascular diseases.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in tablet form may be prepared by granulating a mixture of L-serine, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives by a conventional method, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a normal hard capsule with a mixture of L-serine, an active ingredient of the present invention, with an excipient such as an excipient. The soft capsule may contain L-serine as an excipient And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of L-serine, an active ingredient of the present invention, excipient, binder, disintegrant, and the like by a conventionally known method. If necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of L-serine, an active ingredient of the present invention, excipient, binder, disintegrant, etc., into granules by a known method, And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Inhibitory effect of serine on homocysteine-induced cell death

In order to investigate the effect of serine on homocysteine-induced vascular endothelial cell death, cells were treated with homocysteine alone or with serine, and cell viability was measured by MTT (3- [4,5-dimethylthiazol- 2-yl] -2,5-diphenyltetra sodium bromide). In order to avoid unexpected behavior, all experiments of the present invention were conducted using L type.

Specifically, homocysteine (5 mM) and L-serine (1 mM, 5 mM, 10 mM) were treated in the same manner as in Example 1, after culturing 7 × 10 ³ human blood vessel cell line EA.hy926 cells (ATCC: CRL- . After 24 hours, the medium was removed and cultured in MTT 1 mg / mL solution for 1-2 hours. The resulting purple formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and absorbance was measured at 540 nM. L-serine and homocysteine used in this experiment were purchased from Sigma chemical. Amino acid was directly dissolved in the medium, and 5 mM homocysteine was treated on the cells, or homocysteine and 1 mM, 5 mM, and 10 mM L-serine were treated for 24 hours.

As a result, as shown in Fig. 1A, it was confirmed that the viability of vascular cells reduced by homocysteine was restored depending on the treatment concentration of L-serine.

In addition, a flow cytometer was used to measure the extent of apoptosis. For this, EA.hy926 cells treated with homocysteine and L-serine at different concentrations were stained with Annexin V and PI (propodium iodide), and stained cells were quantified using a flow cytometer .

Specifically, 5.0 × 10 ³ human blood vessel cell line EA.hy926 cells were cultured in a 100 mm dish, homocysteine (5 mM) and L-serine (1 mM, 5 mM, 10 mM) were treated for 24 hours together, After washing twice with cold PBS and re-dispersing in 500 μl of Annexin binding buffer (0.1 M Hepes / NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl 2), Annexin V-FITC and propidium iodide iodide) were added to each well and stained for 15 minutes. The stained cells were analyzed using a FACScalibur flow cytometer (BD, USA) equipped with a 488 nm argon laser.

In the early stage of apoptosis, cell membranes are confused by the formation of phospholipids, and phosphatidylserine is exposed to the outside of the cell membrane. Annexin V is a Ca2 + -dependent phospholipid binding protein specifically binding to phosphatidylserine , It is currently used mainly for detecting phosphatidylserine on the surface of apoptotic cells. PI (propodium iodide) can also be used to stain damaged DNA. Therefore, we tried to detect definite apoptosis stage by the degree of binding of annexin V to phosphatidylserine and staining of PI.

As a result, as shown in FIG. 1B, it was confirmed that the cell survival rate was significantly increased (32.6%) and the apoptosis was effectively reduced by 21.4% in the group treated with L-serine.

< Example  2>

Inhibitory effect of serine on homocysteine-induced ER stress

In the present invention, to examine the effect of L-serine on endothelial cell endothelial cell stress induced by homocysteine, cells were treated with homocysteine alone or with L-serine, The amount of expression of Grp78 and CHOP was confirmed by Western blotting, and mRNA levels of Grp78, CHOP and XBP1 genes were measured by PCR.

For reference, ER stress refers to the infiltration of immature proteins into the endoplasmic reticulum beyond the ability of the endoplasmic reticulum to be treated by the physiological or pathological environment, or depletion of calcium in the endoplasmic reticulum, It means that the folding capacity is exceeded and the ER protein folding load is exceeded. As the ER stress related genes, IRE1, ATF6, PERK, elF2, XBP1, CHOP or GRP 78 are known.

<2-1> Western Blasting

First, 2.5 × 10 ⁴ EA.hy926 cells were divided into 60 mm dishes and stabilized. Homostin (5 mM) and L-serine (1 mM, 5 mM, 10 mM) were treated together and 24 hours later, Protein concentration was measured by Bio-Rad Protein Assay Reagent (Bio-Rad, USA). The obtained protein was added in the same amount to SDS sample buffer (2.5 ml of 1 M Tris (pH 6.8), 5 ml of 80% glycerol, 5 ml of 20% SDS, 0.2 ml of 1% bromophenol blue, For 5 minutes, and then electrophoresed on a 10% SDS-polyacrylamide (SDS-PAGE) gel. The SDS-PAGE gel was transferred to a PVDF membrane (polyvinyl difluoride membrane) and diluted with a tris-buffered saline tween (TBS-T; 20 mM tris, 137 mM sodium chloride, 0.1%) containing 5% skim milk % tween-20, pH 8.0) for 1 hour at room temperature to prevent non-specific binding. The primary antibody reaction was reacted with anti-GRP78, anti-CHOP at 4 DEG C for 18 hours. Secondary antibody reaction was carried out using 5% skim milk (anti-rabbit IgG (Cell signaling, CA, USA) antibody labeled with horseradish peroxidase and anti-mouse IgG (Calbiochem, San Diego, TBS-T containing skim milk was diluted 2,000 times and reacted at room temperature for 1 hour. Then, the protein was expressed using the ECL plus chemiluminescence system (Amersham, USA).

As a result, as shown in Fig. 2a, the expression level of Grp78 and CHOP protein was increased in the test group treated with homocysteine alone, whereas in the test group treated with homocysteine and L-serine, Grp78 And the amount of CHOP protein expression was markedly decreased. In other words, ER stress indexes increased by homocysteine were significantly decreased when serine was treated together.

<2-2> RT - PCR

For this experiment, total RNA was extracted using Easy-Blue ™ Total RNA Extraction Kit (Intron Biotechnology, Korea). RNA concentration was measured by absorbance at 260 nm and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo, USA) and 300-500 ng / μL of extracted RNA was obtained. CDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen, USA). The cDNA was amplified by incubating at 42 ° C for 2 minutes, 42 ° C for 15 minutes, and 95 ° C for 3 minutes. RT-PCR was performed using Maxime PCR PreMix Kit (Intron Biotechnology, Korea). The cDNA was diluted 1/5 with distilled water and mixed with each primer (forward and reverse). Gene expression was performed according to the instructions of the gene expression protocol as set forth in Eco Real-Timc PCR (Illumina Inc.). GAPDH was used as a control for mRNA levels as a house keeping gene. Table 4 below shows the set of primers used in RT-PCR.

Primer set gene Primer sequence Grp78 Forward 5 'CTG GGT ACA TTT GAT CTG ACT GG 3' Reverse 5 'GCA TCC TGG TGG CTT TCC AGC CAT TC 3' CHOP Forward 5 'CCA CTC TTG ACC CTG CTT 3' Reverse 5 'GAG TCG CCT CTA CTT CCC 3' XBP1 Forward 5 'AAA CAG AGT AGC AGC TCA GAC TGC 3' Reverse 5 'TCC TTC TGG GTA GAC CTC TGG GAG 3' GAPDH Forward 5 'AACGGATTTGGTCGTATTG 3' Reverse 5 'GCTCCTGGAAGATGGTGAT 3'

As a result, as shown in FIG. 2B, the levels of Grp78, CHOP mRNA and spliced-XBP1 (spliced form of XBP1) were increased in the group treated with homocysteine alone, while those treated with homocysteine and L-serine In the experimental group, Grp78 and CHOP mRNA levels and spliced-XBP1 levels were significantly decreased depending on L-serine treatment concentration. These results are consistent with the embodiment <2-1>.

< Example  3>

Reduction effect of serine on homocysteine

In the present invention, in order to confirm whether L-serine can actually reduce the level of homocysteine present in cells, homocysteine is treated with homocysteine alone or with L-serine in human vascular endothelial cells, .

Specifically, 5.0 × 10 ⁴ EA.hy926 cells were divided into 100 mm dishes and stabilized. Homocysteine alone (5 mM) or homocysteine and L-serine (1, 5, 10 mM) were used in combination and reacted for 24 hours. Then, the cells were washed three times with PBS, and the cells were scraped off and re-dispersed by adding 0.3 ml of PBS. Half were used for protein quantification, and half were used for homocysteine determination. To dissolve homocysteine from cells, perchloric acid (1: 3) was added in triplicate in PBS. The cell debris was removed by centrifugation at 14000 rpm for 15 minutes, and the concentration of homocysteine contained in the supernatant was measured using a Homocysteine EIA Reagent kit (Axis-Shield) according to the manufacturer's instructions. Protein concentration was measured by Bio-Rad Protein Assay Reagent (Bio-Rad, USA) and the amount of homocysteine was corrected.

As a result, as shown in Fig. 3, it was confirmed that the concentration of homocysteine in the cell was significantly decreased depending on the serine concentration in the experimental group treated with L-serine (control group: 0.34 ± 0.31 mmol / mg protein, homocysteine group: 36.03 Homocysteine + serine 1 mM: 27.37 ± 0.65 mmol / mg protein, homocysteine + serine 5 mM: 18.76 ± 2.26 mmol / mg protein, homocysteine + serine 10 mM: 9.51 ± 1.22 mmol / mg protein).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Hcy: homocysteine
Ser: serine

Claims (8)

A pharmaceutical composition for preventing or treating cardiovascular diseases comprising L-serine as an active ingredient. The method according to claim 1,
Wherein said composition comprises L-serine itself or a pharmaceutically acceptable salt and solvate thereof. The method according to claim 1,
Wherein the L-serine is contained in the composition at a concentration of 1 to 1000 μM. The method according to claim 1,
Wherein the L-serine has a therapeutic effect on cardiovascular diseases through an activity of decreasing the concentration of intracellular homocysteine. The method according to claim 1,
Wherein said L-serine has an effect of inhibiting apoptosis and endoplasmic reticulum of endothelial cells induced by homocysteine. 6. The method according to any one of claims 1 to 5,
Wherein said cardiovascular disease is selected from the group consisting of myocardial infarction, angina pectoris, hypertension, heart failure, cardiomyopathy, primary cardiac arrest, congestive heart disease, ischemic heart failure, arteriosclerosis, arrhythmia, coronary artery disease, stroke and peripheral vascular disease &Lt; / RTI &gt; A health functional food for improving cardiovascular diseases comprising L-serine as an active ingredient. 8. The method of claim 7,
Wherein the food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, rice cakes, gums, candy, ice cream, alcoholic beverages, alcoholic beverages, Health functional foods for the prevention or improvement of degenerative neurological diseases.

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