KR101290749B1 - Composition for preventing or treating the brain ischemia disease containing extract of codonopsis lanceolata - Google Patents

Abstract

The present invention relates to a composition for preventing or improving ischemic cerebrovascular disease comprising the deodeok extract as an active ingredient, more specifically, the deodeok extract is excellent in neuronal cell protection and neuronal cell death of the brain hippocampal tissue, and glial cells It has been confirmed that the activity of inhibiting the activity of the cerebral hippocampal tissue CA1 region known to be sensitive to cerebral ischemia is effectively prevented, as well as harmless to the human body, preventing ischemic cerebrovascular disease comprising the deodeok extract as an active ingredient or The composition for improvement is expected to be applied in various ways for the treatment, prevention or improvement of diseases caused by cerebral ischemia.

Description

Composition for preventing or ameliorating ischemic cerebrovascular disease comprising deodeok extracts {COMPOSITION FOR PREVENTING OR TREATING THE BRAIN ISCHEMIA DISEASE CONTAINING EXTRACT OF CODONOPSIS LANCEOLATA}

The present invention relates to a composition for preventing or improving ischemic cerebrovascular disease comprising a deodeok extract as an active ingredient.

According to the latest data released by the National Statistical Office, Korea also accounted for 7.2% of the population aged 65 and over in 2000, and the average life expectancy of the population was 77.5 years in 2003. As of 2005, the proportion of the elderly aged 65 or older accounted for 9.1% of the total population as of 2005, and the proportion of the younger population aged 14 or younger is gradually decreasing, and Korea has already entered the aging society. Moreover, in 2019, the proportion of the elderly aged 65 or older out of the total population will exceed 14%, and by 2020, this ratio will reach 20%, making it the next aging country next to Japan.

In addition, according to a 2003 UN announcement, the world's population will grow to 10 billion people by 2050, with two billion older people aged 60 or older. As described above, as the aging population progresses rapidly, interest in elderly welfare such as housing, health, culture, and leisure of the elderly is increasing.

In addition, Korea's GNI per capita announced by the National Statistical Office was $ 14,162 in 2004, and it is increasing by more than 10% every year.

As a result of recent changes in the living environment and dietary patterns due to the improvement of quality of life, modern people are becoming more interested in degenerative neurological diseases including aging of biological tissues. In addition, as the aging issue becomes a social issue, the public's interest in the characteristics of the elderly population, the elderly, such as housing, health, culture, leisure, etc. is increasing, and the demand for statistics is increasing.

With these changes in social conditions, chronic degenerative diseases are becoming more serious problems than acute infectious diseases, which have been the leading cause of death for the last 50 years. More specifically, the number of deaths from infectious diseases, which are the major cause of death in the past, is rapidly decreasing, while the number of deaths from degenerative diseases such as cancer, cerebrovascular disease, and diabetes is steadily increasing. In particular, with the rapid growth of the elderly population, degenerative neurological diseases including brain, spine or peripheral nerve damage continue to increase, and death of cerebrovascular disease among degenerative diseases is 2% of mortality caused by single disease. It has become a very important disease to record the stomach.

The cerebrovascular disease, which is the second largest cause of death in Korea, is often referred to as stroke, and can be classified into two types. One is a hemorrhagic brain disease such as cerebral hemorrhage, which is mainly caused by a blood vessel in the brain exploded by an external shock, such as a traffic accident. The other is a disease frequently occurring in elderly people. Disease.

The cerebral infarction is also referred to as ischemic cerebrovascular disease, it means a disease caused by brain cells die because the blood vessels are blocked by the blood vessels of the brain, the brain hemorrhagic is also called hemorrhagic cerebrovascular disease, It means blood flow and stiffness, resulting in brain damage. When cerebral infarction or cerebral hemorrhage causes loss of brain function, physical symptoms such as apparent paraplegia, speech and consciousness disorders may appear, and in severe cases, death.

Among these, ischemic cerebrovascular disease refers to a disease caused by delayed neuronal death in the hippocampus CA1 region due to a decrease in blood flow in the brain and the supply of oxygen and glucose. In addition, the ischemic brain disease is known to be caused by not supplying blood to the brain in pathological situations such as stroke or heart attack.

Until now, two known neuronal cell death mechanisms by cerebral ischemia are known.

One is the excitatory neuronal death mechanism in which excess glutamate accumulates outside the cell by cerebral ischemia, and this glutamate enters the cell and eventually causes neuronal death due to the accumulation of excess intracellular calcium. Oxidative neuronal cell death mechanism is caused by the sudden oxygen supply during reperfusion, the radical in vivo is increased, and the increase of the radical in vivo is caused by damage to DNA and cytoplasm.

The cerebral ischemia and reperfusion have been reported to induce delayed neuronal death in the hippocampal CA1 region. The cause of the delayed neuronal death is known to be the influx of calcium into the cell, free radical-related neuronal damage or glutamate-receptor mediated neuronal damage.

More specifically, when cerebral ischemia occurs, the supply of oxygen and glucose is blocked so that neurotransmitters cause inversion of active transport due to a decrease in Adenosine Triphosphate (ATP), resulting in depolarization of neurons, and glutamate of synapses. Is liberated to increase the concentration of extracellular glutamate. Accumulation of the extracellular glutamate includes N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4 -isoxazole-propionic acid (AMPA), and metabotropic glutamate receptor. ) Induces continuous activity of calcium, resulting in excessive cellular influx of calcium. Intracellular influx of calcium triggers the activity of calcium-dependent proteases, lipases and modulators, and in turn leads to the generation of cytotoxic molecules, including free radicals, that break down cellular structures such as DNA and cell membranes, causing neuronal death. . Since neuronal cell death occurs after a significant time after brain ischemia, this is called delayed neuronal death.

The delayed neuronal cell death can be examined through an experiment using a transient forebrain ischemic model using a Mongolian gerbil, and in the Mongolian gerbil model, four days after induction of cerebral ischemia for 5 minutes, the hippocampus (hippcampus) Neuronal cell death has been reported in the CA1 region (Kirino T., Sano K., Acta Neuropathol., 62, pp 201-208 (1984)).

When irreversible nerve cell damage occurs by the cerebral ischemia, it is known to cause a decrease in motor performance, a decrease in sexual ability, and a decrease in memory. In addition, in relation to diseases caused when brain tissue damage occurs due to the brain ischemia, various cerebral nervous system diseases, including senile dementia, Alzheimer's disease, Huntington's disease, and Parkinson's disease, are reported according to parts of the brain tissue damaged by the brain ischemia. It is becoming.

In order to treat the diseases caused by cerebral ischemia, cerebral nervous system disease or neurodegenerative diseases, various treatments such as cell transplantation and surgical surgery have been proposed, but most of them show risk factors and side effects, and the nerves are substantially damaged due to the complexity of the damage mechanism. Therapies for protecting cell damage have not been developed.

More specifically, many studies have been conducted on the basis of these mechanisms to search for substances that effectively inhibit neuronal cell death in cerebral ischemia or to reveal the mechanism of the substances. However, there are few substances that effectively inhibit neuronal cell death caused by cerebral ischemia.

Tissue plasminogen activator (Tissue plasminogen activator) is currently the only FDA-certified therapeutic agent for cerebral ischemia, and it is a substance that induces rapid supply of oxygen and glucose by melting blood clots that cause cerebral ischemia with thrombolytics. Therefore, it is not necessary to directly protect nerve cells, so it is necessary to use them quickly. Due to the characteristics of thrombolytics, excessive use or frequent use may cause thinning of the blood vessel walls and eventually cause hemorrhagic cerebrovascular disease.

In addition, a clinical test was performed for MK-801, a calcium channel blocker for inhibiting glutamate-induced cytotoxicity by effectively inhibiting early cellular influx of calcium, but the drug was discarded due to its side effects. There is a bar.

On the other hand, in addition to a lot of substances are conducting a clinical test, rather than the therapeutic effect on stroke rather than side effects due to the administration of the drug is very serious, it is reported that it is difficult to use as a real drug.

In addition, although it has recently been reported that regeneration of neurons is possible, the regeneration of these neurons is expected to be not easy considering the histological characteristics.

Therefore, in consideration of the characteristics of cerebrovascular disease, the disease caused by cerebral ischemia or cerebral nervous system tends to emphasize the prevention rather than the treatment, and cerebrovascular disease by continuously ingesting substances that can protect the brain cells The need to prevent this is newly recognized.

In this respect, it has been argued that diseases caused by cerebral ischemia, in particular ischemic cerebrovascular disease, should be given more emphasis on preventing than treating the disease.

In the case of drug administration for the prevention of the disease, as in the case of drugs for general diseases, natural complexes are preferred to a single compound that can be a side effect problem. In accordance with this trend, with the increasing interest and demand for health foods, a number of natural substances in Korea have been marketed as health foods that are effective in preventing stroke.

However, most of them are not scientifically verified, and they are often social problems because they cause health food abuse.

Therefore, there is an urgent need for an effort to objectively verify natural resources that have been used as foods for a long time and to prove their safety, and to develop natural materials having the effect of treating and preventing brain diseases.

The present invention effectively prevents or improves ischemic cerebrovascular disease, inhibits brain damage caused by cerebral ischemia, as well as a composition for preventing or improving ischemic cerebrovascular disease comprising an extract of a natural plant harmless to the human body and the composition. An object of the present invention is to provide a dietary supplement for preventing or improving ischemic cerebrovascular disease.

In order to achieve the above object, the present invention provides a composition for treating or preventing ischemic cerebrovascular disease comprising a deodeok extract as an active ingredient.

In addition, in order to achieve the above object, the present invention provides a health functional food composition for preventing or improving ischemic cerebrovascular disease comprising a deodeok extract or the composition for preventing or improving ischemic cerebrovascular disease.

In addition, in order to achieve the above object, the present invention provides a neuronal protective composition for protecting the deodeok extract as an active ingredient. The neuronal protective composition is for protecting or preventing damage to nerve cells caused by cerebral ischemia, specifically nerve cells of brain tissue, more specifically nerve cells of the brain hippocampal tissue CA1 region.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention, while studying the composition for preventing and improving cerebrovascular disease from natural plants, especially those that have been used as a food for a long time, is known that deodeok extract or steamed duck extract is sensitive to cerebral ischemia In the brain hippocampal tissue CA1 region of the brain has the effect of preventing damage, in particular, the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed deodeok extract was found to be remarkably excellent in the neuronal effect of the neurons, based on this The invention was completed.

In the present invention, neuronal protection means inhibiting the death of nerve cells of the brain or central nervous system, for example, the brain or spinal cord, or protecting them from damage of nerve cells. More specifically, neuronal protection may be to inhibit and protect the death of neurons in the brain, for example, the hippocampus, caused by cerebral ischemia.

In the present invention, ischemia means that the blood supply to the tissue is reduced or the blood supply is abolished. If ischemia results in a combined deficiency of oxygen and nutrients in the tissue, it can lead to cell death (necrosis) in the tissue where the ischemia occurs. When ischemia occurs, damage to brain tissue induced by ischemia can be divided into two stages. The first stage is the injury caused by a lack of oxygen, which causes cell metabolism to stop, resulting in the death of some cells and tissues within minutes, and the second stage, after an early induced ischemic condition is improved, apoptosis ( A series of cascades, such as apoptosis, start up and last up to 12 hours. The tissue damaged by this second stage is known to be fatal and more dangerous to the subject than the damage caused by the first stage.

In the present invention, the patient is meant to include both humans and animals.

In the present invention, a composition is meant to include a product comprising the specified components in the specified amounts, as well as products resulting directly or indirectly from the combination of the specified components in the specified amounts.

In the present invention, an effective amount or therapeutically effective amount means an amount of an extract or composition of the present invention effective to produce the desired therapeutic, alleviation, inhibitory or prophylactic effect.

The present invention relates to a composition for the prevention or improvement of ischemic cerebrovascular disease comprising the deodeok extract as an active ingredient.

Codonopsis lanceolata ) is a perennial vine plant of the family Campanula, also called samsam or white ginseng. The deodeok is a perennial herb that is wild in the mountains or forests of Korea, Japan or China and is a food material that has been commonly used for general food. In Korea, roots are edible in the mountains in spring. It is large, light in shape and tastes sweet and slightly bitter with a unique scent. The young leaves of the deodeok is boiled and eaten or eaten with ssam, the roots are eaten raw, pressed or roasted, eaten with red pepper paste or soaked with intestines or liquors.

The roots of Deodeok are similar to bellflower or ginseng, and the vine stem extends over 2m in length. Purple broad bell-shaped flowers bloom from August to October, with corollas splitting into five, with the tip fold outwards, with a flowery greenish green on the outside and dark purple arongjin spots on the inside.

The duodeok is not particularly limited in form, but may preferably be a dude, that is, raw or steamed duodeok. The raw deodeok means that the collected deodeok as it is or to add the washing process to the collected deodeok. The steamed duck means that steaming or steaming is carried out with hot steaming or the washing process and / or drying process are additionally performed before and after the steaming process, and the enzyme is inactivated in the cell. It is also called blanching, which is a high temperature treatment carried out for the purpose. The term “steaming” or “steaming process” is a concept that is distinguished from boiling in boiling water in that it is cooked with seaweed. The steaming duck, for example, may be a steaming process for 5 minutes to 3 hours with hot steam at a temperature of about 80 ℃ to 105 ℃, the steaming process may be performed repeatedly. In addition, the deodeok may be performed by further performing a washing step, drying step and / or grinding process.

The extract of the present invention may be a solvent extract extracted with an extraction solvent, a fraction obtained by adding a fractional solvent to an extract prepared by extracting with an extraction solvent, or a purified product obtained by performing chromatography on the fraction.

The extraction solvent may be any one selected from the group consisting of water, an organic solvent or a mixed solvent thereof, preferably water, an alcohol having 1 to 5 carbon atoms, and a mixture thereof, which can be used for extracting natural products. The organic solvent may be a linear or branched alcohol having 1 to 5 carbon atoms such as methanol or ethanol, a polar solvent such as ethyl acetate or acetone, a nonpolar solvent such as hexane or dichloromethane, or a neutral solvent such as ketone having 3 to 5 carbon atoms. It may be a mixed solvent, preferably a straight-chain or branched alcohol solution of 1 to 5 carbon atoms, such as 30 to 90% ethanol, or a ketone having 3 to 5 carbon atoms of 30 to 90%, more preferably 40 to 80% ethanol Aqueous solution or 40-80% acetone aqueous solution, even more preferably 50-70% ethanol aqueous solution.

The fractional solvent may be water, alkane having 5 to 7 carbon atoms, linear or branched alcohol having 1 to 5 carbon atoms, including butanol, ethyl acetate, methylene chloride, chloroform, hexane, or a mixture thereof. May be any one selected from the group consisting of ethyl acetate, hexane, methylene chloride and water, and more preferably ethyl acetate. More specifically, when the deodeok is raw, the fractional solvent may be any one selected from the group consisting of ethyl acetate, hexane, methylene chloride and water, preferably ethyl acetate or hexane, more preferably ethyl Acetate, when the deodeok is steamed, the fractional solvent may be any one selected from the group consisting of ethyl acetate, methylene chloride, hexane and water, preferably ethyl acetate, methylene chloride or water, more Preferably it may be ethyl acetate or methylene chloride, even more preferably may be ethyl acetate.

Deodeok extract of the present invention may be prepared according to the conventional method for producing a plant extract. More specifically, it can be carried out by adding an extraction solvent in addition to the removal of impurities and performing the extraction process. The extraction process may be cold extraction, hot extraction, pressure extraction, reflux extraction or ultrasonic grinding extraction.

In addition, the preparation of the fraction of the deodeok extract is added to the extract prepared by the extraction method, that is, the crude extract is a fraction solvent different in polarity from the extraction solvent, for example, hexane, chloroform, methylene chloride, ethyl acetate, butanol and water, More preferably hexane, methylene chloride, ethyl acetate, butanol and water, more preferably hexane, methylene chloride, ethyl acetate, butanol and water, followed by layered hexane fractions, chloroform fractions, ethyl acetate fractions, butanol fractions and water. This can be done by the method of obtaining fractions.

The fractionation method by layer separation may be a method of obtaining a fraction obtained for each application by applying the nonpolar order of the solvent, for example, hexane obtained by fractionating the hexane layer separated by adding hexane to the ethanol aqueous solution extract. Fractions; Methylene chloride fraction obtained by separating the hexane fraction and separating the methylene chloride layer separated by adding methylene chloride to the remaining aqueous layer; Separation of the methylene chloride fraction and adding ethyl acetate to the remaining aqueous layer may be a method of obtaining the ethyl acetate fraction obtained by fractionating the ethyl acetate layer separated from the ethyl acetate fraction and the water fraction, which is the remaining layer after separating the ethyl acetate fraction. .

Chromatography for the purification of the fractions can be carried out using various chromatographies such as silica gel column chromatography, thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) .

Further, the extract may be subjected to extraction, fractionation or purification, followed by vacuum filtration or further concentration and / or lyophilization to concentrate or remove the solvent. Therefore, the deodeok extract in the present invention is used as a meaning including the dried product of the extract dried in a conventional manner and the concentrate of the extract concentrated in a conventional manner, the dilution of the extract, dried or concentrate. The obtained deodeok extract can be stored in a deep freezer until use.

Ischemic cerebrovascular disease in the present invention is a generic term for a disease that occurs because the blood vessels of the brain is blocked for some reason, the blood is not supplied to the brain, also referred to as cerebral ischemic disease.

The cerebral ischemia is most commonly seen clinically in cardiac arrest or stroke, resulting in intractable neuronal cell damage, which can lead to disability or coma and, in severe cases, death do.

When the blood flow of the brain is reduced by the cerebral ischemia and oxygen and glucose supply is not normally performed, oxidative phosphorylation is reduced, and consequently, anaerobic glycolysis is also stopped and used as a cell energy source. Adenosine triphosphate (ATP) is depleted. By depletion of the energy source, cell functions essential for life maintenance are inhibited, which causes various degenerative processes, resulting in cell death. The apoptosis occurs not only during the ischemic period but also during reperfusion by free radicals generated by oxygen supplied when the blood flow at the ischemic site is restored and reperfusion occurs.

In particular, apoptosis occurs in neurons in the CA1 region of the hippocampal tissue known to be sensitive to cerebral ischemia among brain cells, and ischemic diseases caused by death of the neurons, ie neuronal death, in particular, delayed neuronal death. It is called cerebrovascular disease.

Examples of the ischemic cerebrovascular disease include cerebral infarction (ischemic stroke), cerebral hemorrhage, subarachnoid hemorrhage, cerebral nerve disease, cerebral ischemia, ischemic nerve disease, degenerative neurological disease, stroke, dementia, senile dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease , Creutzfeldt-Jakob disease, epilepsy, Lou Gehrig's disease, memory loss, thrombosis, thromboembolism, microinfarction or white matter dysfunction, etc., preferably cerebral infarction, cerebral hemorrhage, senile dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease and thromboembolism It may be one or more selected from the crowd consisting of.

Deodeok extract of the present invention is anesthetized by jeolbil after administration for a certain period of time to induce cerebral ischemia by ligation of the whole neck artery, and the brain hippocampal tissue and brain cells in the experiment to measure the efficacy by staining neurons in the CA1 region of the algae tissue It was confirmed that the neuronal cell death inhibitory effect, especially the extract of the deodeok extract, specifically, the ethyl acetate fraction of the raw deodeok extract and the ethyl acetate fraction of the steamed deodeok extract crude extract confirmed to have superior neuronal cell death inhibition effect compared to the control group Or it was confirmed to have a remarkably excellent neuronal protective effect compared to the other fractions.

Deodeok extract of the present invention has the same effect as above, it may be included as an active ingredient of the composition for the prevention or improvement of ischemic cerebrovascular disease.

In this aspect, the present invention relates to a composition for the prevention or improvement of ischemic cerebrovascular disease comprising a deodeok extract as an active ingredient.

The composition for preventing or improving the ischemic cerebrovascular disease may include 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight of the deodeok extract based on the total weight of the composition.

In addition to the deodeok extracts, the composition includes nutrients, vitamins, electrolytes, flavors, colorants, neutralizers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, It may further contain a carbonation agent used in the carbonated beverage. The components may be added to the composition independently or in combination. The content of the additional ingredient may be added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the deodeok extract.

In this aspect, the composition for preventing or improving ischemic cerebrovascular disease may be a pharmaceutical composition for preventing, improving or treating ischemic cerebrovascular disease.

The composition containing the deodeok extract as an active ingredient may further comprise a suitable carrier, excipients and diluents commonly used in the preparation thereof.

 Carriers, excipients and diluents which may be included in the composition comprising the extract of the present invention as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol And liquid paraffin may be at least one selected from the group consisting of, but is not limited thereto, conventional carriers, excipients or diluents can be used. The ingredients may be added independently or in combination with the active ingredient Deodeok extract.

The composition containing the deodeok extract of the present invention as an active ingredient may be used in oral formulations, such as powders, granules, tablets, suspensions, emulsions, syrups, etc. according to conventional methods.

The composition comprising the deodeok extract as an active ingredient is 0.001% to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1% to 50% by weight of the deodeok extract relative to the total weight of the composition It may include. In addition, the content of the additional component is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the deodeok extract.

In addition, the composition comprising the deodeok extract as an active ingredient, when formulated, may further comprise a conventional filler, extender, binder, disintegrant, surfactant, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier or preservative It can be used orally or parenterally.

Particularly, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have.

In addition, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, and in particular, by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated. Examples of the specific formulations may be granules, powders, syrups, liquid preparations, suspensions, premixes, infusions, tablets, suppositories, injections, main preparations, capsules, pills, soft or hard gelatine capsules and the like. Furthermore, the therapeutic compositions of the present invention can be prepared using methods known in the art or as described in Remington ' s Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. 18th, 1990 And the like.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. . Preferably, the pharmaceutical composition of the present invention is administered at 0.0001 to 1000 mg / kg per day based on the amount of the deodeok extract, 0.01 to 100 mg / kg to be more effective. The administration may be carried out once a day or divided into several times. The dose and the number of administrations do not limit the scope of the present invention in any aspect.

In addition, in order to achieve the above object, in another aspect, the present invention provides a food composition for the prevention or improvement of ischemic cerebrovascular disease comprising a deodeok extract as an active ingredient.

In the present invention, the term "food" means a natural product or a processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, and a conventional meaning As intended, it is intended to include all dietary supplements, beverages, food additives and beverage additives.

The food of the present invention includes, for example, various foods, beverages, gums, tea, a vitamin complex, a health functional food, and the like. In addition, the food in the present invention includes special nutritional products (e.g., crude oil, infant food, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods (E.g., soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages (Eg, fruit, vegetable drinks, soy milk, fermented beverages, etc.) and natural seasonings (eg, ramen soup, etc.), but are not limited thereto. The food, the health functional food, the beverage, the food additive and the beverage additive can be produced by a usual production method.

In the present invention, a health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition which has added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to fully express the gymnastics function on recovery.

The health functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include a health functional beverage. The beverage is not particularly limited to other ingredients other than including the deodeok extract as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and Xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention can be used for the production of natural fruit juice, fruit juice drink, Can also be added.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 20 parts by weight per 100 parts by weight of the deodeok extract of the present invention.

In the present invention, the health functional beverage includes a beverage group to which added value is imparted so that the function of the beverage functions to a specific purpose using physical, biochemical, biotechnological techniques, etc., Means a beverage which is processed by being designed so that the body control function related to recovery and the like is sufficiently expressed to a living body.

The health functional beverage is not particularly limited to other ingredients except the deodeok extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol , Sorbitol, and erythritol. Natural flavorings (tau martin), stevia extracts (for example, rebaudioside A and glycyrrhizin), and synthetic flavors (saccharin, aspartame, etc.) may be used as flavors other than those described above. The proportion of said natural carbohydrates is generally about 1-20 g, preferably 5-12 g per 100 ml of the composition of the present invention.

In addition, in the food composition for the purpose of preventing or improving the ischemic cerebrovascular disease, the amount of the deodeok extract may be appropriately determined according to the purpose of use in the manufacture of food or beverage, for example, The extract may comprise 0.01 to 15% by weight of the total food weight, in the case of a beverage composition may be included in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml of the total food weight.

The composition and food comprising the deodeok extract of the present invention as an active ingredient has an excellent neuronal cell protective effect of inhibiting neuronal cell death in the hippocampus region of male Mongolian gerbil (Harlan, USA) In particular, the ethyl acetate fraction of the deodeok extract was found to be remarkably superior neuroprotective effect as compared to the control by the deodeok extract or other solvent fractions recognized as well as neuroprotective and neuronal cell death.

As described above, the deodeok extract of the present invention can effectively prevent neuronal damage caused by cerebral ischemia by protecting neurons, and can inhibit neuronal cell death caused by cerebral ischemia, thereby preventing, treating or improving ischemic cerebrovascular disease. Not only can be usefully used in the pharmaceutical composition or nutraceutical composition because it does not cause side effects to the human body.

1 is a picture taken by staining the hippocampus tissue of experimental animals 4 days after the induction of cerebral ischemia with cresyl violet in order to determine the protective effect of the deodeok extract according to an embodiment of the present invention In one picture, A is a picture of the whole hippocampus of the normal group (Sham) that does not cause cerebral ischemia, B is a picture of the CA1 region, and C is a control group that induced cerebral ischemia after administration of a solvent only ( Vehicle is a picture of the whole hippocampus, D is a picture of the CA1 area, E is a picture of the whole hippocampus of the Raw CL Crude, F is a picture of the CA1 area, The G is a photograph of the hippocampus of the ethyl acetate fraction of the deodeok extract, H is a photograph of the CA1 region, the I is a photograph of the walrus fraction of the hexane fraction of the deodeok extract, J is a CA1 region Shooting The photograph is a photograph of the seahorse of the methylene chloride fraction of the deodeok extract, K is a photograph taken, L is a photograph of the CA1 region, the M is a photograph of the hippocampus of the water fraction of the deodeok extract, N Is a photograph of the CA1 region, O is a photograph of the whole hippocampus of Steamed CL Crude, P is a photograph of the CA1 region, Q is the ethyl acetate fraction of the steamed duck extract The whole hippocampus is photographed, R is a photograph of the CA1 region, the S is a photograph of the whole hippocampus of the hexane fraction of steamed duck extract, T is a photograph of the CA1 region, the U is steamed A photograph of the hippocampus of the methylene chloride fraction of the deodeok extract is taken, V is a photograph of the CA1 region, W is a photograph of the hippocampus of the water fraction of the deodeok extract, X is a CA1 region It is a photograph of English.
2 is a graph showing the survival rate of neurons in 4 days after the induction of cerebral ischemia in relative numbers with respect to the normal group in order to determine the protective effect of the deodeok extract according to an embodiment of the present invention to be. In the graph, Sham means normal group, Vehicle means control group, Raw CL means raw duck, Steamed CL means steamed duck, Crude means extract, EtoAC means ethyl acetate fraction. Hexane means hexane fraction, DCM means methylene chloride fraction, Water means water fraction.
Figure 3 in order to determine the ischemic cerebrovascular disease protective effect of the deodeok extract, more specifically the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of steamed deodeok extract according to an embodiment of the present invention, 4 days after the induction of cerebral ischemia The CA1 region of the hippocampal tissue of the experimental animal was observed by immunohistochemistry using mouse anti-NeuN (NeuN), an antibody against neurons, and Fluoro-Jade B (F- It is a fluorescent stained picture using JB). In the picture, Sham means the normal group (A, a), Vehicle means the control group (B, b), ERC (C, c) means the ethyl acetate fraction of the raw extract, ESC (D, d) means the ethyl acetate fraction of steamed duck extract.
Figure 4 is to determine the ischemic cerebrovascular disease protective effect of the deodeok extract, more specifically, the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the deodeok extract according to an embodiment of the present invention, Figure 4a is cerebral ischemia-induced 4 The number of surviving cells after the day is a graph showing the relative value with respect to the normal group, Figure 4b is a graph showing the number of cells killed after 4 days after the cerebral ischemia induction relative to the control group. In the graph, Sham means the normal group, Vehicle means the control group, Raw Codonopsis means the ethyl acetate fraction of the raw deodeok extract, Steamed Codonopsis means the ethyl acetate fraction of steamed duck extract.
5 is a marker of microprogenitor glial cells in order to determine the ischemic cerebrovascular disease protective effect of the deodeok extract, more specifically the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of steamed deodeok extract according to an embodiment of the present invention The photograph was observed by immunohistochemistry using an anti-IB4 antibody (IB4). In the above picture, Sham means normal group (A), Vehicle means control group (B), ERC means ethyl acetate fraction (C) of raw deodeok extract, ESC means ethyl acetate fraction of steamed duck extract ( D) means.
6 is stained with IB4 4 days after cerebral ischemia-induced cerebral ischemia in order to examine the protective effect of the deodeok extract, more specifically the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed deodeok extract according to the embodiment of the present invention In the graph, Sham means normal group, Vehicle means control group, Raw Codonopsis means ethyl acetate fraction of Saengdeok extract, and Steamed Codonopsis shows Ethyl acetate fraction of steamed duck extract.

Hereinafter, preferred embodiments of the present invention will be described. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  1. Deodeok Extract and Fraction  Produce

Steamed deodeok was prepared using deodeok purchased fresh in the traditional market in Chuncheon, South Korea, and deodeok extracts were prepared using the deodeok and steamed deokdeok respectively.

More specifically, the steamed duck was performed in the following manner. The fresh raw water was washed with water, and dried at 60 ° C. for 3 hours using a hot air dryer, and then steamed at 105 ° C. for 3 hours using an autoclave. The process was repeated two more times, followed by further drying at 60 ° C. for 3 hours using a hot air dryer to finally prepare steamed duck.

Extracts were prepared by the solvent extraction method for the deodeok and steamed deodeok, and further fractions were prepared.

More specifically, after the addition of 30 L 70% ethanol to each of the deodeok and each steamed deodeok, the extraction process was carried out for 2 hours at 70 ℃. The extract obtained by repeating the above procedure two more times was filtered using Whatman filter No2. (Whatman, UK) to obtain a filtrate. The solvent of the obtained filtrate was removed under a reduced pressure concentrator (Buchi Rotavapor R-220, Switzerland) and freeze-dried at -20 ° C to give a final deodorant 70% ethanol extract (1,334.81 g, 22.25%) and steamed decanter 70% ethanol extract. (2,557.75 g, 42.63%) was obtained. The obtained extract was stored frozen until used in the experiment.

In order to fractionate the obtained deodeok 70% ethanol extract and steamed duck 70% ethanol extract according to the polarity, a systematic fraction using an organic solvent was performed.

More specifically, after suspending by adding a certain amount of water to the ethanol extract, n-hexane, methylene chloride, ethyl acetate, ethyl-butanol and water Addition afforded fractions.

First, water was added to the ethanol extract to suspend, and after addition of normal-hexane, the mixture was sufficiently shaken and left at room temperature for 5 hours, followed by separation. The process was repeated three times to obtain a hexane fractionation layer, filtered, concentrated under reduced pressure, and lyophilized to obtain a final hexane fraction. After the hexane fractionation layer was removed, methylene chloride was added and methylene chloride fractions were obtained in the same manner as above. After the methylene chloride fractions were removed, ethylene acetate was added and ethylene acetate fractions were obtained in the same manner as above. After removing the ethylene acetate fraction, n-butanol was added, and a butanol fraction was obtained in the same manner as above. The butanol fraction was removed and the remaining water layer was lyophilized to obtain a water fraction.

Deodeokane hexane fraction (26.8 g, yield: 22.25%), deodeok methylene chloride fraction (15.62 g, yield: 1.17%), deodeok ethyl acetate fraction (3.39 g, yield: 0.25%), deodeok butanol fraction (by the fractionation process) 107.95 g, yield: 8.09%), deodeok water fraction (691.09 g, yield: 51.77%), steamed duck hexane fraction (21.64 g, yield: 0.85%), steamed methylene chloride fraction (60.26 g, yield: 2.36%) And steamed ethyl acetate fractions (24.67 g, yield: 0.96%) were obtained.

Example  2. Deodeok Extract of Experimental Animals Brain ischemia  Optic nerve cell protection effect Measurement 1

Neurodegenerative effect of the deodeok extract, deodeok fraction, steamed duck extract and steamed duck extract prepared in Example 1 anesthetized male Mongolian gerbil (Mongolian gerbil, Harlan, USA) of 65g to 75g body weight (common carotid) artery) was exposed after ligation to induce cerebral ischemia, and was confirmed by staining and monitoring the hippocampal tissue of Mongolian gerbils.

Example  2-1. Breeding of experimental animals

40 male Mongolian gerbil (Meriones unguiculatus, Halan, USA), weighing between 65 and 75 grams, with a constant contrast cycle of light from 7 am to 7 pm, temperature from 21 ° C. to 25 ° C. and 45% to 65% It was raised under the condition of relative humidity of. The experimental animals were fed with pellet dried feed (Korean Experimental Animals, Korea), and feed and water were constantly consumed.

Example  2-2. Cerebral ischemia  Measurement of nerve cell protection efficacy

Experimental animal bred in Example 2-1, 350 mg each of the deodeok extract, deodeok fraction, steamed duck extract and steamed duck extract prepared in Example 1 dissolved in 50 ml of distilled water from a week ago every day using an esophageal needle The extract or fractions were orally administered to 50 mg per 1 kg body weight of the experimental animal.

After injecting the extracts for one week, the experimental animals were anaesthetized using gas mixed with 3% isoflurane (isoflurane, Baxtor, USA) in a gas mixture of nitrogen and oxygen 7: 3. The animals were operated with the gas mixture of nitrogen and oxygen mixed with 2.5% isoflurane while maintaining the anesthetic state of the experimental animals.

The anesthesia was performed by shaving and disinfecting the neck of the neck, exposing both coronary arteries, and aneurysm clip (Staelting, USA) for 5 minutes to induce cerebral ischemia. And then reperfused. At this time, ophthalmoscope was used to observe the blood circulation of the central artery of the retina to confirm the complete occlusion of the common coronary artery.

 During the induction of cerebral ischemia, the body temperature was measured by inserting a rectal temperature thermometer, and the body temperature was kept constant at a normal body temperature of 36.7 to 37.3 ° C using a thermostatic pad which was automatically controlled according to the temperature of the test animal.

The experimental group was anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight after 4 days after induction of cerebral ischemia, and then at 4 ° C. containing 1,000 IU per 1,000 ml. Physiological saline was injected into the left ventricle and washed perfusion. The perfusion-cleaned animals were perfused with 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4, pH 7.4) at 4 ° C.

Using a bone cutter, the brain was extracted by opening the head bone space of the experimental animals after the perfusion fixation. The brains of the extracted experimental animals were post-fixed for 4 hours using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4). After the post-fixation, the brain was immersed in a 30% sucrose solution (in 0.1 M phosphate buffer) until it was submerged on the bottom, and then transferred to a slide microtome (Reichert-Jung, Germany) The tissue was cut into a thickness of 30 mu m to prepare a tissue section. The tissue sections were stored in 6-well plates containing a storage solution and stored at 4 ° C. until staining was performed.

From the tissue sections, tissues with well-formed hippocampal formation were selected, and washed three times with 0.01 M PBS for 10 minutes to remove the preservatives from the tissue sections. The washed tissue sections were plated on gelatinized slides and dried sufficiently at 37 ° C. After sufficiently drying the tissue sections in distilled water, the tissue sections were stained for 1 minute in 2% cresyl violet acetate (Sigma, USA) solution.

The stained tissue sections were sufficiently washed with running water to remove excess dye from the slides. The tissue sections from which the excess dye was removed were briefly immersed in distilled water, and then sequentially treated with 50%, 70%, 80%, 90%, 95%, and 100% ethanol solution to perform dehydration and excess cresyl violet washing. After confirming that the tissue section was visible in the Nissle body (Nissle body), it was immersed in xylene (Junsei, Japan), and then transparent and encapsulated in Canada Balsam (Kanto, Japan).

The control group was prepared in the same manner as above except that only the distilled water used to dissolve the extract was orally administered, and the normal group was not administered any substance including the deodeok extract, and no ischemia-reperfusion surgery was performed. Tissue sections of the normal group were prepared in the same manner as above.

AxioM1 microscope (Carl Zeiss, Germany) with a digital camera (Axiocam, Cal Zeiss, Germany) was used to enlarge the hippocampus area by 25 times and CA1 area by 200 times. . Figure 1 shows the results of photographing the tissue sections of the experimental group, the control group and the normal group administered the deodeok extract and fractions. In order to verify the significance, FIG. 1 is a photograph taken by selecting the most general part of each group.

In addition, using the image analyzer (Optimas 6.5, USA) program for the photographed pictures were counted neurons of the tissue section stained purple. One-way ANOVA test was performed to verify the significance of each group. The result of the counting of the neurons in the measured tissue sections was shown in FIG. 2, where the relative number of neurons in the normal group (ie, the neuronal cell count result of the normal group) was 100%.

As can be seen in Figure 1, the normal group (Sham) was observed that the neurons are deeply stained purple in the entire area of the hippocampus, especially in the CA1 region it was observed that the neurons are concentrated in the pyramid layer. However, in the case of the control group which induced cerebral ischemia without the extract, neuronal staining was hardly observed because neuronal cell death occurred in the CA1 region of the hippocampus due to delayed neuronal cell death caused by cerebral ischemia. It was confirmed that most neurons were lost in the pyramid layer of the region.

In addition, it was confirmed that the neuronal cell death occurred in E-N, which was administered 50 mg / kg of Deodeok extract or a fraction of Deodeok extract, the Deodeok extract, methylene chloride fraction and water fraction, and confirmed the CA1 region of the hippocampus In the results, it was confirmed that the deodeok extract, the methylene chloride fraction, and the water fraction had a slight loss of neurons in the pyramidal layer in the CA1 region. As a result of treatment of the ethyl acetate fraction and the hexane fraction, it was observed that the neurons were deeply stained purple in the entire hippocampus region as in the normal group, and the neurons were concentrated in the pyramidal layer of the CA1 region. From the above results, it was confirmed that the deodeok extract, the ethyl acetate fraction of the deodeok extract and the hexane fraction of the deodeok extract is significantly superior neuronal protective effect than the other extract or fraction.

As can be seen in Figure 2, the control group was observed about 12% of the number of CA1 region neurons of the hippocampus compared to the normal group, in the case of the deodeok extract was confirmed that about 25% of the neurons survived, Deodeok extract It has been confirmed that the neuron protective effect. In addition, about 25% of the deodeok extract, about 25% of the methylene chloride fraction, and about 35% of the water fraction, about 35% of the neurons survived, whereas the hexane fraction of about 53% The neurons survived, and about 75% of the neurons survived in the ethyl acetate fraction, and the protective effect of the ethyl acetate fraction and the hexane fraction, especially the ethyl acetate fraction, was significantly superior to the other extracts. It became.

In addition, as can be seen in Figure 1, in the O to X administered a fraction of 50 mg / kg steamed duck extract or steamed duck extract, in the case of steamed duck extract, hexane fraction and water fraction neuronal cell death occurred As a result of confirming the CA1 region of the hippocampus, it was confirmed that in the pyramidal layer of the CA1 region, nerve cells were somewhat lost in the steamed duck extract, hexane fraction and water fraction. As a result of treatment of the ethyl acetate fraction and the methylene chloride fraction, as in the normal group, the neurons were darkly colored in purple throughout the hippocampus, and the neurons were concentrated in the pyramidal layer of the CA1 region. From the above results, it was confirmed that in the case of steamed duck extract, the ethyl acetate fraction of steamed duck extract and the methylene chloride fraction of steamed duck extract were significantly superior in neuronal cell protection effect than other extracts or fractions.

In addition, as can be seen in Figure 2, in the case of steamed duck extract, about 20% of the neurons were found to survive, compared to the control group of about 12%, even in the case of steamed duck extract has a neuronal protective effect This was confirmed. In addition, it was confirmed that about 20% of the nerve cells survived about 20% of the extract and steamed hexane fraction. In addition, about 40% of the neurons survived in the water fraction, and about 300% of the neurons in the control group were identified. About 57% of the neurons survived in the hexane fraction, and about 500% of the neurons in the control group were identified. In the ethyl acetate fraction, about 85% of the neurons survived, and the ethyl acetate fraction of steamed duck extract showed the best neuroprotective effect among the extracts of deodeok extract, deodeok extract and steamed duck extract and steamed duck extract. It was confirmed.

Ethyl acetate fraction of the deodeok extract or ethyl acetate fraction of the steamed deodeok extract is counted 75% and 85% of the neurons compared to the normal group, respectively, even when cerebral ischemia occurs, neurons of a similar degree to the normal group is maintained Was confirmed. Therefore, in the case of ethyl acetate fraction, it is possible to suppress the death of nerve cells in a part or the whole of the brain, and even in the event of cerebral ischemia, the nerve cell damage of related parts such as movement, language, vision or smell may be suppressed to the maximum. In addition, the ethyl acetate fraction is evaluated as having excellent neuroprotective effect because it can prevent or improve cerebral dysfunction such as ischemic cerebrovascular disease or exercise loss, cerebral palsy, speech disorder, visual and olfactory disorders caused by cerebral ischemia. Can be.

Example  3. Deodeok Extract of Experimental Animals Brain ischemia  Optic nerve cell protection effect Measurement 2

In order to further confirm the neuroprotective effect of the deodeok extract, more specifically, the anti-apoptotic effect of neurons by cerebral ischemia, ethyl acetate fraction and steamed deodeok extract of the deodeok extract, which was found to have the best neuronal protective effect in Example 2 For the administration group to which the ethyl acetate fraction of the extract was administered, immunohistochemistry staining was performed using an antibody against a neuronal specific nuclear protein (NEUN), which is a marker of neurons, and a marker for killed neurons. Tissue fluorescence was performed using Fluoro-Jade B.

More specifically, the tissue sections of the tissue sections prepared in Example 2 were selected for the hippocampus complex and used in the experiment. The selected tissue sections were 0.3 to remove endogenous peroxidase present in the tissue. Reaction was performed with% H ₂ O ₂ (in 0.001 M PBS) for 30 minutes. The tissue sections were incubated with 3% normal goat serum for 30 minutes to prevent nonspecific immune responses of the tissue sections reacted with 0.3% H ₂ O ₂ (in 0.001 M PBS).

For staining of neurons, tissue sections reacted with the 3% normal goat serum and mouse anti-NeuN (mouse anti-NeuN, Chemicon, USA), diluted 1: 800, were used at 4 ° C. The reaction was time. After the reaction, the tissue sections were reacted with anti-mouse IgG (Vector), a secondary antibody diluted at 1: 200, at room temperature for 2 hours, and then ABC solution, a tertiary antibody diluted at 1: 200, for 2 hours. The reaction was carried out with (Vector) for 2 hours, and 3,3'-DAB (diaminobezidine) was developed as a substrate. In performing each step of the reaction with the antibody, the tissue sections were washed three times for 10 minutes using 0.01M PBS for each step.

After completion of the reaction using the tertiary antibody, the tissue sections were plated on a gelatin-coated slide glass, and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration process. After performing the reaction with the NeuN antibody, the enclosed tissue sections were observed using a microscope, and the photographs of the observation results were taken and shown in FIG. 3.

In addition, the stained nerve cells were counted using the image analyzer program as in Example 2-2, and the result of the counting is shown in FIG. 4 as a relative value with respect to the normal group (Sham). .

As shown in the right column of FIG. 3, in the normal group (Sham), a large number of cells immunostained with the mouse anti-NeuN antibody, which is the primary antibody, were observed throughout the CA1 region of the hippocampus. However, in the case of the control group (Vehicle) in which only the solvent and not the extract was induced and induced the cerebral ischemia, the cells immunostained by the mouse anti-NeuN antibody, which is the primary antibody in the CA1 region, were markedly expressed in the CA1 region. It was confirmed that the reduction. In addition, 50 mg / kg of deodeok ethyl acetate fraction and steamed deodeok ethyl acetate fractions were treated, and as a normal group, a large number of cells immunostained by the mouse anti-NeuN antibody, the primary antibody, throughout the CA1 region of the hippocampus. Was observed. From the above results, it was reconfirmed that the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract had a very good neuronal protective effect.

In addition, as can be seen in Figure 4a, the number of neurons stained with the mouse anti-NeuN antibody of the primary antibody of the control group was measured to be only about 14% of the normal group, but the derivative of ethyl acetate fraction In this case, about 72% of the neurons of the normal group were found to survive, and about 68% of the normal groups of the ethyl acetate fraction were found to have survived. The protective effect was found to be remarkably superior.

In addition, tissue fluorescence staining was performed using Fluoro-Jade B (F-J B), a marker of degenerative neurons, for staining of dead neurons. The tissue sections were washed three times for 5 minutes with distilled water and then immersed in 0.06% potassium permanganet solution for 15 minutes. Thereafter, the mixture was washed for 2 minutes with distilled water and dipped in 0.001% F-J B (Histochem, Jefferson, AR, USA) solution containing 0.1% acetic acid for 30 minutes. The stained tissue was washed with distilled water three times for 1 minute, dried in a slide warmer at 50 ° C for 20 minutes, immersed in xylen, and sealed with DPX (Sigma, USA). The encapsulated tissue was observed using a fluorescence microscope (Ex: 385 nm, Em: 425 nm), and the photograph taken was taken and shown in FIG.

In addition, the stained neurons were counted using the image analyzer program as in Example 2-2, and the counted results are shown in FIG. 4 as a relative value with respect to the control (Vehicle).

As shown in the left column of FIG. 3, the normal group (Sham) is hardly stained by Fluoro-Jade B, which is a marker for the killed or dying neurons in the entire CA1 region of the hippocampus, while the control group (Vehicle) ) Was darkly fluorescently stained throughout the CA1 region of the hippocampus, indicating that many neurons are dying or dying. In addition, as a result of treatment of 50 mg / kg deodeok ethyl acetate fraction and steamed deodeok ethyl acetate fraction, Fluoro-Jade B, which is a marker for the killed or dying neurons throughout the CA1 region of the hippocampus, as in the normal group Few stained cells were observed. From the above results, it was confirmed that the degree of neuronal cell death was significantly reduced by the ischemia when the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract were treated.

In addition, as can be seen in Figure 4b, in the experimental group treated with the deodeok ethyl acetate fraction, the number of neurons stained with Fluoro-Jade B, which is a marker for killed or dying neurons is normal group In the experimental group treated with the ethyl acetate fraction, the number of neurons stained with Fluoro-Jade B, a marker for killed or dying neurons, was about 16% of the normal group. In spite of about 23%, the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract can be suppressed the death of nerve cells due to the ischemia, the ethyl acetate fraction and the steamed duck extract of the deodeok extract Ethyl acetate fraction was found to be remarkably excellent neuroprotective effect.

Example  4. Deodeok Extract of Experimental Animals Brain ischemia  Optic nerve cell protection effect Measurement 3 (Measurement of Glue Cell Activity Change)

In order to further confirm the neuroprotective effect of the deodeok extract, more specifically, the anti-apoptotic effect of neurons by cerebral ischemia, ethyl acetate fraction and steamed deodeok extract of the deodeok extract, which was found to have the best neuronal protective effect in Example 2 In addition to the administration group to which the ethyl acetate fraction of the extract was administered, immunohistochemistry staining was performed to change the activity of the glial cells, specifically, the microglia.

More specifically, the tissue sections of the tissue sections prepared in Example 2 was selected for the hippocampus complex was used in the experiment, the selected tissue sections were subjected to immunohistochemical staining by applying the method described in Example 3. .

In the immunohistochemical staining process, anti-IB4 (rabbit anti-IB4, Sigma, USA) diluted 1: 1000 was used as a primary antibody to confirm the change of microprogenitor cells. After performing the immunohistochemical staining process by applying the method described in Example 3 using the primary antibody, the encapsulated tissue sections were observed using an electron microscope, and the photographs of the observation results were taken. Shown in

As shown in FIG. 5, in the normal group (Sham), little cells were immunostained by the mouse anti-IB4 antibody, which is an antibody against activated microglia. On the other hand, in the case of the control vehicle (Vehicle), a large number of cells immunostained with the mouse anti-IB4 antibody was observed, it was confirmed that a large number of IB4 expression, which is a marker of the microglia cells. On the other hand, in the experimental group administered the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed deodeok extract at 50 mg / kg, respectively, the expression level of IB4, which is known as a marker of activated microprotuberant glial cells, was lower than that of the control group. Expression of glial cells was found to be significantly suppressed.

In addition, as can be seen in Figure 6, the microsolipids confirmed to be activated by confirming the immune response to the cells immunostained by IB4, that is, the primary antibody against the activated microlithium cells, IB4 Cells were identified as about 9,000% compared to the normal group (Sham). In addition, in the experimental group administered the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract at 50 mg / kg, respectively, the expression level of IB4, which is known as a marker of activated microprogenitor cells, was 600% compared to the normal group, respectively. And 640%. As a result, in the control group, the microglia were abnormally activated by ischemia, but the microglia were activated in the experimental group administered with the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract. Although it was possible to suppress the abnormal activation as compared to the control group, the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract was remarkably excellent in the neuronal protective effect.

As described above, the deodeok extract and the steamed duck extract, in particular, the ethyl acetate fraction of the deodeok extract and the ethyl acetate fraction of the steamed duck extract were observed in the neuronal cells, especially the hippocampal region, in which the hippocampus was stained even when ischemia was induced. Inhibition of neuronal cell death in the whole area of, particularly the CA1 region of the hippocampus, was confirmed by FJ B, an anti-NeuN antibody to neuronal cells and an indicator for killed neurons. It was confirmed that the inhibition, and through the anti-IB4 antibody was confirmed that the very excellent inhibition of the activation of the microglia glial cells, the deodeok extract not only has an excellent effect on neuronal cell protection but also activates the glial cell activation The inhibitory effect was also evaluated to be excellent.

Therefore, the deodeok extract can significantly inhibit the delayed neuronal cell death caused by ischemia, and even when cerebral ischemia occurs, it inhibits the neuronal cell damage in the relevant areas such as movement, language, vision or smell to the maximum, cerebral ischemia. It is possible to prevent or improve cerebral dysfunction such as ischemic cerebrovascular disease or loss of motion, numbness of the senses, speech impairment, visual and olfactory disorders.

Manufacturing example : Formulation using Deodeok Extract

The following preparation example is to specifically explain the preparation of the composition for preventing or improving ischemic cerebrovascular disease according to the present invention using the deodeok extract prepared in Example 1, the content of the present invention is limited by the following description no.

Manufacturing example  One. Sanje  Produce

Deodeok Extract Powder 30 mg

Lactose 100 mg

Talc 10 mg

According to the preparation method of powder in the Pharmacopoeia General Regulations, the above ingredients were mixed per bag and filled in an airtight cloth to prepare a powder.

Manufacturing example  2. Preparation of tablets

Deodeok Extract Powder 20 mg

Anhydrous calcium hydrogen phosphate 750 mg

Silicon dioxide 2 mg

20 mg sodium starch glycolate

Magnesium Stearate 3 mg

Titanium oxide appropriate

Diethylphthalate Proper

Aluminum Lake Proper

Lecithin

Indigocarmine aluminum ray

According to the preparation method of tablets in the Pharmacopoeia General Formulation, the above ingredients were mixed and tableted per tablet to prepare a tablet.

Manufacturing example  3. Preparation of Capsule

Deodeok Extract Powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

The capsules were prepared by mixing the above components per capsule according to the preparation method of capsules in the pharmacopeia formulation and filling into gelatin capsules.

Manufacturing example  4. Liquid  Produce

Deodeok Extract Powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Microcrystalline cellulose

Potassium Dihydrogen Phosphate

Purified water

According to the preparation method of liquid formulation in the Pharmacopoeia General Regulations, each component was added to and dissolved in purified water, and lemon flavor was added appropriately. After mixing the above components, the total volume was adjusted to 100 ml by adding purified water and filled into a brown bottle. The solution was prepared by sterilization.

Manufacturing example  5. Preparation of dietary supplements

Deodeok Extract Powder 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. The granules can then be prepared and used in the manufacture of nutraceuticals in accordance with conventional methods.

The composition ratio of the drug and the functional food is one of the component ratios relatively suitable for the manufacture of the drug or functional food, the compounding ratio may be arbitrarily modified according to the preference of the consumer, such as the country of demand, the demand hierarchy and the intended use.

Claims (8)

Codonopsis lanceolata ) composition for preventing or improving ischemic cerebrovascular disease comprising an extract as an active ingredient. delete The method of claim 1,
The deodeok is a deodeok or steamed deodeok ischemic cerebrovascular disease prevention or improvement composition. The method of claim 1,
The deodeok extract is a composition for preventing or improving ischemic cerebrovascular disease is a solvent extract extracted by using any one selected from the group consisting of water, alcohol having 1 to 5 carbon atoms and mixtures thereof. 5. The method of claim 4,
The deodeok extract is a composition for preventing or improving ischemic cerebrovascular disease that is fractionated in the deodeok solvent extract with one or more fractions solvent selected from the group consisting of ethyl acetate, hexane, methylene chloride, butanol and water. The method of claim 3,
The deodeok extract is a composition for preventing or improving ischemic cerebrovascular disease which is a fraction obtained by fractionation of ethyl acetate or hexane as a fraction solvent in a deodeok ethanol extract or a deodeok ethanol aqueous solution extract. The method of claim 3,
The deodeok extract is a composition for preventing or improving ischemic cerebrovascular disease is a fraction fractionated by one or more fractions solvent selected from the group consisting of ethyl acetate, methylene chloride and water in steamed deodeok ethanol extract or steamed deodeok ethanol aqueous extract. Codonopsis lanceolata ) Food composition for preventing or improving ischemic cerebrovascular disease comprising an extract as an active ingredient.

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