KR100756357B1 - Composition for preventing or improving the ischemia damage containing extract of onion - Google Patents

Abstract

A composition for preventing or improving ischemic cerebrovascular disease comprising the extract of onion(Allium cepa) is provided to prevent damage of neuronal cells in the brain hippocampus tissue CA1 region and inhibit apoptosis of neuronal cells without harmful effects on human. A composition for preventing or improving ischemic cerebrovascular disease comprises 0.1-99 wt.% of the extract of onion which is prepared by extracting onion with water or 50-100% C1-C4 lower alcohol, wherein the ischemic cerebrovascular disease includes Alzheimer's disease, vascular dementia and Huntington's disease. A food composition for preventing or improving ischemic cerebrovascular disease comprises 0.01-15 wt.% of the composition and is food, food additive or drink.

Description

Composition for preventing or improving ischemic cerebrovascular disease, including onion extract {COMPOSITION FOR PREVENTING OR IMPROVING THE ISCHEMIA DAMAGE CONTAINING EXTRACT OF ONION}

1 is a schematic diagram showing a process of obtaining an onion extract according to an embodiment of the present invention,

Figure 2 is a graph measuring the amount of lactate dehydrogenase secretion in a hypoxic environment in PC12 culture cells according to the administration of the onion extract in order to determine the neuronal cell death effect of the onion extract according to an embodiment of the present invention Is,

3 is a photograph of staining hippocampus tissue of experimental animals 4 days after the induction of cerebral ischemia with cresyl violet in order to determine the neuronal cell death prevention effect of the onion extract according to an embodiment of the present invention. ,

4 is a graph measuring the number of surviving cells after 4 days of cerebral ischemia, the survival rate of neurons in order to determine the neuronal cell death prevention effect of the onion extract according to an embodiment of the present invention,

5 is a GFAP, oligodendrocytes, markers of glial cells of hippocampal tissues of experimental animals 4 days after the induction of cerebral ischemia, in order to determine the neuronal cell death prevention effects of onion extracts according to an embodiment of the present invention. It is a photograph observed by the immunohistochemistry method using the marker Rip and Iba-1, the glial glia.

[Industrial use]

The present invention relates to a composition comprising an onion extract having a neuronal protective effect, and more particularly to a composition for the prevention and improvement of cerebral ischemic disease comprising the onion extract.

[Private Technology]

Of the total population released in 2005, the National Statistical Office recently made up 9.1% of the population aged 65 and over, and the proportion of young people aged 14 and younger is decreasing. The proportion of the population aged 65 and over is 7.2% in 2000. It is estimated that our society has already entered an aging society. In addition, as of 2003, the average life expectancy of Korean people is 77.5 years old, and the elderly population is increasing rapidly. Meanwhile, GNI per capita announced by the National Statistical Office was $ 14,162 in 2004, and it is increasing by more than 10% every year. The quality is getting better. Due to such changes in social conditions, the number of deaths from infectious diseases, which are the major causes of death, has been drastically decreasing, while the number of deaths from degenerative diseases such as cancer, cerebrovascular disease, and diabetes is steadily increasing.

Cerebrovascular disease, which is the second leading cause of death in Korea, is often called stroke, and is caused by clogging or bursting blood vessels in the brain. It can be divided into two types: cerebral infarction and cerebral hemorrhage.

The cerebral infarction is also referred to as ischemic cerebrovascular disease, and it is a disease caused by brain cells dying because the brain is blocked from receiving blood and oxygen. Brain hemorrhage is also called hemorrhagic cerebrovascular disease. This is the case when the brain is damaged due to flow. 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 is a disease caused by delayed neuronal death in the hippocampus CA1 region due to a decrease in blood flow in the brain and supply of oxygen and glucose (Kirino, Brain Res., 239, pp 57-69, 1982; Petito et al., Neurology, 37, pp 1281-1286, 1987; Pulsinelli et al., Ann. Neurol., 11, pp 491-498, 1982). In addition, the ischemic brain disease is known to be caused by the lack of blood supply to the brain in pathological conditions such as stroke or heart attack (Nedergaard, Acta. Neurol. Scand., 77, pp81-101, 1988; White et. al., Neurology, 43, pp 1656-1665, 1993).

The cause of delayed neuronal death in the hippocampal CA1 region caused by transient cerebral ischemia and reperfusion is caused by the influx of calcium into cells (Hara et al., Brain Res. Bull., 29, pp659-665, 1992; Nedergaard , Acta Neurol. Scand., 77, pp81-101, 1988), known as free radical related neuronal damage and glutamate-receptor mediated neuronal damage (Won et al., Neurosci. Lett., 301, pp 139-142, 2001). That is, when cerebral ischemia occurs, depolarization of neurons occurs, and glutamate of synapses is released to increase the concentration of extracellular glutamate.

In addition, the reversal of active transport caused by the reduction of Adenosine Triphosphate (ATP) accelerates the accumulation of extracellular glutamate. Accumulation of extracellular glutamate is characterized by N-methyl-D-aspartic acid (NMDA), ??-amino-3-hydroxy-5-methyl-4 -isoxazole-propionic acid (AMPA), and metabotropic glutamate receptor. ), Resulting in an over-flow of calcium into cells (Olney et al., Science, 244, pp1360-1362, 1989). 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. .

Stroke treatment includes acute treatment and preventive treatment for prevention of recurrence, and the treatment effect can be seen only if you go to the hospital within 3 hours after the stroke and receive acute stroke treatment. That is, once cerebrovascular disease is due to irreversible nerve cell damage, it is known to cause a decrease in motor skills, sexual performance, memory loss.

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

Considering the characteristics of cerebrovascular disease, cerebrovascular disease tends to be prevented rather than treated, and the necessity of preventing the cerebrovascular disease by continually ingesting a substance that can protect the nerve cells is newly introduced. It is recognized.

In line with this need, efforts are underway to find active ingredients from natural plant-derived substances that are effective in protecting neuronal cells.

The present invention effectively prevents neuronal cell damage caused by cerebral ischemia, prevents neuronal cell death caused by cerebral ischemia, and ischemic cerebrovascular disease comprising a composition containing onion extract as an active ingredient and onion extract as an active ingredient. It is an object to provide a food composition for the prevention or improvement of the.

In order to achieve the above object, the present invention comprises an onion extract as an active ingredient and provides a composition for preventing or preventing ischemic cerebrovascular disease having neuroprotective effect.

In addition, in order to achieve the above object, the present invention provides a food composition for preventing or preventing ischemic cerebrovascular disease comprising onion extract as an active ingredient and having neuroprotective effect. Examples of the food composition of the present invention include food, food additives or beverages.

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

The inventors of the present invention, while studying the composition for preventing and preventing cerebrovascular disease that ensures human safety from natural plant-derived material, can effectively prevent the damage of nerve cells in the CA1 region of the brain hippocampal tissue known that onion extract is sensitive to cerebral ischemia It was confirmed, and based on this, the present invention was completed.

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

Onium ( Allium cepa ) is a vegetable belonging to the family Liliaceae , originating from the west coast of Asia or the Mediterranean. One. The scales vary slightly depending on the variety, but the flat round or round shape has a thin filmy purplish brown shell on the outside, and the scales on the inside are thick, layered and spicy. The leaves are hollow cylinders, and the flowers bloom white in September, forming a scented inflorescence at the end of the stalks between the leaves. There are six stamens, three of which have small bumps on both sides under the operating table, with one pistil.

Onions are mainly used for edible scales, and are used for salads, soups, and meat dishes. The scales contain compounds such as propyl disulfide and allyl sulfide, which are the causes of unique odors, which physiologically promote the secretion of digestive fluids and have effects such as excitement, sweating, and diuresis. In addition, the scales contain minerals such as calcium and phosphoric acid together with various vitamins to remove harmful substances in the blood. Onion leaves contain about 5,000 IU of vitamin A, about 45 mg of vitamin C, about 80 mg of calcium, about 24 mg of magnesium, and about 220 mg of potassium.

Onions have long been used as a folk remedy in the treatment of various diseases. Onion extracts contain arachidonic acid, which inhibits cyclooxygenase and lipooxygenase. It has the action of, and the pigment called guelchin in the tan shell is known to be good for the prevention of hypertension, arteriosclerosis, blood clots, etc., because it acts to strengthen the capillaries of the brain.

The solvent of the extract of the present invention may be water or an organic solvent such as a polar solvent such as lower alcohol having 1 to 4 carbon atoms such as methanol or ethanol, ethyl acetate, and a nonpolar solvent of hexane or dichloromethane, or a mixed solvent thereof. Preferably it is C1-4 lower alcohol, such as 50-100% of methanol, ethanol, More preferably, it is 70-100% ethanol.

Onion extract of the present invention may be prepared according to a conventional method for producing a plant extract. Preferably, distilled water is added to the onion from which the impurities are removed from the outer surface, which is then pulverized while heating. The residue is removed to obtain crude onion extract, the crude extract is adsorbed, and the compound adsorbed with ethanol is desorbed to prepare an extract. can do.

At this time, the amount of distilled water added is added 1 to 5 times the weight of the onion, preferably 1 to 3 times the weight of distilled water, the heating temperature is 75 ℃ to 140 ℃, preferably 90 ℃ to 120 ℃, more preferably 95 ℃ to 110 ℃, the heating time is 5 minutes to 75 minutes, preferably 10 minutes to 60 minutes more preferably 15 minutes to 40 minutes most preferably 20 minutes, the grinding May be pulverized by a conventional method, and preferably pulverized so as not to be seen with a hand crusher, and the crude extract may be obtained by removing the residue through a filter net after the heat treatment and pulverization, and adsorption of the extract The adsorbent used in the present invention uses a conventional adsorbent, preferably a synthetic adsorbent, more preferably a synthetic adsorbent (Diaion HP-20), and the amount of the adsorbent is usually used for extraction of plant extracts. The amount is used, preferably 3L to 12L, more preferably 5L to 10L, most preferably 7L to 9L, the desorption is first washed with distilled water and then desorbed using alcohol, The amount is a commonly used amount, preferably 15L to 30L, more preferably 18L to 27L, most preferably 21L to 25L, the alcohol is preferably a low alcohol or acetone having 1 to 4 carbon atoms, more preferably Ethanol is used, and the desorption compound may be preferably a phenolic compound of onion.

The onion extract is an extract of the above-mentioned extract and the onion residue removed in the process of obtaining the crude extract using an alcohol having a low carbon of 1 to 4 carbon atoms, preferably ethanol, preferably extracted by cold or hot extraction method Combined, it may be concentrated under reduced pressure.

The onion extract obtained may be stored in a deep freezer until use.

In addition, the onion extract may be one from which the moisture is completely removed by concentrating and lyophilizing the obtained extract.

In the present invention, the cerebrovascular disease refers to a term that refers to various diseases caused by abnormalities of blood vessels of the brain, and cerebrovascular diseases are largely classified into hemorrhagic cerebrovascular disease and ischemic cerebrovascular disease.

The ischemic cerebrovascular disease is a disease caused by delayed neuronal death in neurons of the CA1 region of the hippocampus known to be sensitive to cerebral ischemia because brain blood flow is decreased and oxygen and glucose are not normally supplied. Ischemic cerebrovascular disease may include Alzheimer's disease, dementia or Huntington's disease.

Onion extract of the present invention is exposed to the hypoxic environment of PC12 cells of the adrenal derived neuronal cell line by measuring the lactate dehydrogenase concentration released from the cells to measure the efficacy and anesthesia by ligation of the cervical artery to cerebral ischemia After inducing the neuronal staining, neuronal cell death was significantly inhibited in all experiments measuring the efficacy of the neurons, and showed an excellent neuronal protective effect.

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

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

The present invention provides a food composition or pharmaceutical composition comprising onion extract as an active ingredient for the prevention or improvement of the cerebral ischemic disease.

Food composition or pharmaceutical composition comprising the onion extract as an active ingredient may further comprise a suitable carrier, excipients and diluents commonly used in the preparation thereof.

Carriers, excipients and diluents that may be included in the composition comprising the onion 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, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising the onion extract of the present invention as an active ingredient may be used in oral formulations of powders, granules, tablets, suspensions, emulsions, syrups and the like according to conventional methods, respectively.

The oral formulation is meant to include a solid preparation and a liquid preparation for oral administration, the solid preparation for oral administration may include tablets, pills, powders, granules, capsules, etc., such solid preparation is the extract At least one excipient, for example, may be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and serums.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

The preferred dosage of the composition containing the onion extract of the present invention depends on the condition and weight, the extent 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 composition containing the onion extract of the present invention is 0.0001 to 1000mg / kg per day based on the amount of the onion composition, in order to be more effective it is preferable to administer in 0.01 to 100mg / kg. Administration may be administered once a day or may be divided several times. The dosage and frequency of administration are not intended to limit the scope of the invention in any aspect.

The composition of the present invention can be used as food. In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages.

Examples of the food to which the onion extract of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, 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, fruits, vegetable drinks, soy milk, fermented beverages, etc.) and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additives may be prepared by a conventional manufacturing method.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastics function related to the living body. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In the present invention, the drink is a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional drink. The beverage is not particularly limited to other ingredients other than including the onion extract as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in the usual beverage. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate may generally be about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. It may further contain a pulp for.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, 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 extract of the present invention.

Functional beverage in the present invention is a biological defense rhythm control, disease prevention and the like having a beverage group or a beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the beverage to a specific purpose Means a beverage that is designed and processed to fully express the gymnastics function related to recovery.

The functional beverage is not particularly limited to other ingredients other than containing the onion 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 glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.

In addition, in the food composition aimed at the effect of preventing or improving cerebral ischemic disease, the amount of the extract may include 0.01 to 15% by weight of the total food weight, the beverage composition is 0.02 to 5 based on 100 ml g, preferably 0.3 to 1 g.

Compositions and foods comprising onion extract of the present invention as an active ingredient have a neuroprotective effect that significantly inhibits cell death of PC12 cells, which are cell lines derived from adrenal medulla, exposed to a hypoxic environment, and induces ischemia, a male Mongolian gerbil (Mongolian) gerbil, Harlan, USA) has been shown to have an excellent neuronal protective effect that significantly inhibits neuronal cell death in the hippocampus region.

Hereinafter, preferred embodiments of the present invention are described. The following examples are only for illustrating the present invention and the present invention is not limited by the following examples.

EXAMPLE

Example 1: Preparation of Onion Extract

10 kg of freshly purchased onion was removed from impurities on the surface, and 20 kg of distilled water was added thereto, followed by heat treatment at 100 ° C. for 20 minutes, and crushed until the dryness was not seen with a hand grinder. The residue was removed from the pulverized pulverized product through a filter net to obtain a water extract of onion. The water extract was adsorbed using a synthetic adsorbent (Diaion HP-20, 8L), washed with 24 L of distilled water, and the phenolic compound of onion was desorbed with alcohol (ethanol). The residue was extracted by cold extraction using ethanol (ethanol), and then combined with phenolic fractions desorbed from a synthetic adsorbent, concentrated under reduced pressure, and lyophilized.

Example 2 Measurement of Neuronal Protective Effects of Onion Extracts in Cerebral Ischemia

Method of measuring the concentration of lactate dehydrogenase released from the adrenal PC12 cell line derived from the adrenal gland in a hypoxic environment and then released into the neurons to prevent neuronal cell death and improvement effect of the onion extract of Example 1 and male Mongolian weighing 65g to 75g After anesthesia was used to anesthetize gerbils (Mongolian gerbil, Harlan, USA) to expose the common carotid artery and induce cerebral ischemia, staining was observed in Mongolian gerbil tissue.

2-1: Determination of Neuroprotective Effect of Onion Extracts in Brain Ischemia by Lactate Dehydrogenase (in vitro)

Lactate dehydrogenase is obtained by culturing PC12 cells in the adrenal glands of rats and incubating them with CoCl ₂ to induce neuronal damage through a hypoxic environment. By measuring the concentration, the presence of damage to the neurons was confirmed. PC12 cells were treated at 37 ° C with 6% CO ₂ concentration in culture medium containing 7% fetal calf serum and 7% horse serum in Dulbecco's Modified Eagle Medium (DMEM, Mediatech, USA). The lactate dehydrogenase was measured by taking a cell culture solution at 20 hours when almost secretion was completed from damaged and destroyed cells.

The onion extract obtained in Example 1 for the PC12 cell line culture medium was treated at a concentration of 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 5 mg / ml, respectively, and then 30 After minutes treated with 150 ?? M CoCl ₂ . After incubating the PC12 cells treated with CoCl ₂ for 24 hours at 37 ° C., a culture solution of PC12 cells was obtained, and the concentration of lactate dehydrogenase secreted from the cells was determined using Zhong Sheng Biotech's standard reagent. Enzymatic mechanical method using a Beckman DU-640 absorbance photometer (Hou et al. J. Neurosci. Res., 74, pp123-133, 2003; Yamakawa et al. Neurol. Res., 23, pp522-530 , 2001), and the results are shown in FIG.

As shown in Figure 2, the onion extract treated group, treated only with the solvent, compared to the control group that caused a low oxygen environment, the lactate dehydrogenase secretion rate was relatively low. In the case of the onion extract treated group, the lactate dehydrogenase secretion decreased by the concentration of lactate dehydrogenase until the treated concentration of the onion extract was 1 mg / ml. This was observed to remain unabated. In the case of the onion extract treated cells at a concentration of 1 mg / ml, it was confirmed that the secretion of lactate dehydrogenase was reduced by 60% compared to the cells administered with the solvent only.

As a result of the above results, it was confirmed that the onion extract has a neuronal protective effect and an improvement effect, and the neuronal protective effect and the improvement effect was the greatest at a concentration of 1 mg / ml.

2-2. Determination of Neuroprotective Effect of Onion Extracts in Cerebral Ischemia by Animal Experiment

Animal experiment using gerbil confirmed the neuronal protective effect of onion extract.

2-2-1. 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. For the feed of the experimental animals, general pellet dry feed (Korean experimental animals, Korea) was used, and the feed and water were always available.

2-2-2. Efficacy Test of Onion Extracts for Neuronal Cell Protection in Cerebral Ischemia

Dissolving the 700 mg of the onion extract of Example 1 in 50 ml of distilled water in the experimental animal was orally administered to 100 mg of the onion extract per 1 kg body weight of the experimental animal using an esophageal needle every day from a week ago. After the onion extract was added for one week, the experimental animal was anaesthetized using a gas containing 3% isoflurane (isoflurane, Baxtor, USA) in a gas mixed with nitrogen and oxygen 7: 3. Surgery was performed on the experimental animals while maintaining anesthesia of the experimental animals by using the gas of 2.5% isoflurane mixed with the nitrogen and oxygen mixed gas. Surgery for the experimental animals was performed by cutting and disinfecting the hair of the neck and making an incision to expose both warm arteries and ligation for 5 minutes using an aneurysm clip (Aneurysm clip, Staelting, USA). Removal and reperfusion were carried out. At this time, each experimental group was checked for blood circulation in the central artery of retina by using an ophthalmoscope to confirm whether there was complete obstruction of the entire artery. The body temperature was measured by inserting a rectal thermometer during the induction of cerebral ischemia, and the body temperature was constantly maintained at a normal body temperature of 36.7 ° C. to 37.3 ° C. using a thermal pad automatically adjusted according to the temperature of the experimental animal.

The experimental group was anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight four 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. Perfusion fixation was performed using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer (PB, 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 6 hours using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4). After the post-fixed brain was placed in a 30% sucrose solution (in 0.1 M phosphate buffer) and settled to the bottom, the brain tissue was slid with a sliding microtome (Reichert-Jung, Germany). Tissue sections were cut to thickness. The tissue sections were stored in 6-well plates containing a storage solution and stored at 4 ° C. until staining was performed.

Among 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 immersing the dried tissue sections in distilled water for a while, the tissue sections were stained for 1 minute in a 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 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 onion extract was prepared. The normal group did not perform ischemia-reperfusion surgery as well as any substance including onion extract.

AxioM1 microscope (Carl Zeiss, Germany) with a digital camera (Axiocam, Cal Zeiss, Germany) was used to photograph each tissue section with 40 times the total hippocampus area and 200 times the CA1 area. . Figure 3 shows the results of photographing the control group, the normal group and the experimental group administered with 100 mg / kg onion extract.

A and C of Figure 3 represent the total hippocampal region of the normal group and the control group, respectively, B and D represent the CA1 region of the hippocampus of the normal group and the control group, respectively. In addition, E and F of Figure 3 shows the total hippocampal region and CA1 region of the hippocampus of the experimental group administered 100mg / kg onion extract.

In order to verify the significance, Figure 3 was taken to pick the most general part of each group.

As can be seen in Figure 3, the normal group was able to observe the deep staining of nerve cells throughout the area of the hippocampus. However, it was observed that a large portion of nerve cells were not stained by neuronal cell death due to cerebral ischemia in the control group that was not administered onion extract. On the other hand, in the experimental group administered 100 mg / kg onion extract was confirmed that the cells are deeply stained in the whole area of the hippocampus, it was confirmed that the onion extract significantly inhibits neuronal cell death.

In addition, neurons of the tissue sections stained in purple were counted using an image analyzer (Optimas 6.5, USA) program. One-way ANOVA test was performed to verify the significance of each group, and the data were expressed as mean and standard deviation.

The result of counting the nerve cells of the tissue sections is shown in Figure 4 by the relative number. As can be seen in Figure 4, the control group was observed about 11.7% of the number of CA1 region cells of the hippocampus compared to the normal group, 78.4% of cells survived compared to the control group when administered 100 mg / kg onion extract Since the cell viability was observed, it was confirmed that the onion extract has a very excellent effect on neuronal cell protection.

Example 3. Changes of Glue Cell Activity According to Administration of Onion Extract

Animal experiments using gerbils confirmed changes in glial cell activity of onion extracts by immunohistochemistry.

Breeding, ischemic induction, brain extraction and tissue treatment of the experimental animals were performed in the same manner as in Example 2-2-2, divided into control, normal and experimental groups.

Tissue sections showing hippocampus complexes were selected and used in the experiment. The selected tissue sections were treated with 0.3% H ₂ O ₂ (in PBS) for 30 minutes to remove endogenous peroxidase. Reacted. In order to prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum for 30 minutes. In order to confirm the change of astrocytic cells, the tissue sections reacted with 3% normal goat serum were reacted with rabbit anti-GFAP (Chemicon International, USA), a primary antibody diluted 1: 1000, and rare oligodendrocytes. In order to confirm the change of glial cells, the tissue sections were reacted with a mouse anti-Rip (Hybidoma bank, USA), a primary antibody diluted 1: 200, and the tissue sections were confirmed in order to confirm the change of microprogenitor cells. Was reacted with rabbit anti-Iba-1 (Wako, USA), a primary antibody diluted 1: 1000, at 4 ° C. for 48 hours. After completion of the reaction, the tissue sections were reacted for 2 hours with anti-rabbit IgG (Vector) or anti-mouse IgG (Vector), which were diluted 1: 200, respectively. The solution was reacted for 2 hours, and 3,3'-diaminobezidine (DAB) was developed as a substrate.

Each step was washed three times for 10 minutes using 0.01 M PBS.

After tertiary reaction, the tissue sections were plated on gelatin-coated slide glass and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration and transparency process.

Immunostaining photographs of the tissue sections that completed the reaction with the GFAP, Rip, and Iba-1 are shown in FIG. As shown in FIG. 5, in the experimental group administered with the onion extract, the expression of the glial cells was significantly reduced compared to the control group in which the solvent was administered. In particular, the expression of GFAP and the microglial cells, known as markers of astrocytic cells, were confirmed. It was confirmed that the expression of Iba-1, known as a marker, was significantly reduced. Therefore, it was confirmed that the onion extract not only has a very excellent effect on neuronal cell protection, but also inhibits the expression of microglia and astroglia.

Example 4. Acute Toxicity Test Using Laboratory Rats

4-1. Toxicity test by oral administration to Mongolian gerbil

After dissolving the onion extract of Example 1 in 50 ml of 4 mg, 28 mg, 70 mg, 140 mg and 280 mg distilled water to the experimental animal of Example 2 daily 0.5 weeks orally using an esophageal needle for a week Tissue sections of the brains of the extracted experimental animals were stained with Cresyl Biolet Acetate solution and observed using an Axiom1 microscope equipped with a digital camera of Example 2.

As a result of observing the entire hippocampal region and CA1 region of the hippocampus, the neurons almost survived the entire hippocampal CA1 region, and no side effects were observed.

4-2. Acute Toxicity Test by Oral Administration in Rats

In order to confirm the acute toxicity of onion extract, the onion extract obtained in Example 1 was dissolved in distilled water and orally injected into 6-week-old specific pathogen-free (SPF) SD rats (Sprague-Dawley, Harlan, USA). Was administered in the manner of. After the oral administration of 14 mg, 28 mg, 70 mg, 140 mg, and 280 mg of onion extract in 1 ml of distilled water, the mortality, clinical symptoms, and weight changes of the animals were observed. Blood biochemical tests were performed, and the experimental rats were necropsied and visually observed for abnormal abdominal and thoracic organs.

As a result, there were no clinical symptoms or dead animals in all experimental animals treated with onion extract, and no toxic change was observed in weight change, blood test, blood biochemical test and autopsy findings. According to the above results, the onion extract of the present invention did not show a change in toxicity even in rats up to 280 mg, respectively, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 280 mg or more.

Preparation Example 5 Formulation Using Onion Extract

Preparation Example 1 Preparation of Powder

Onion Extract Powder 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Preparation of Tablet

Onion Extract Powder 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional manufacturing method of the tablet.

Preparation Example 3 Preparation of Capsule

Onion Extract Powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 4 Preparation of Liquid

Onion Extract Powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution was prepared by sterilization.

Preparation Example 5 Preparation of Health Food

Onion Extract Powder 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

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 mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

As described above, the onion extract of the present invention effectively prevents neuronal cell damage caused by cerebral ischemia, inhibits neuronal cell death caused by cerebral ischemia, and does not cause side effects in the human body. It can be usefully used as a food composition or a medical composition for prevention and improvement.

Claims (6)

Ischemic cerebrovascular disease prevention or improvement composition comprising onion (Allium cepa) extract as an active ingredient. The method of claim 1, Ischemic cerebrovascular disease prevention or improvement composition containing the onion extract at 0.1 to 99% by weight. The method of claim 1, The ischemic cerebrovascular disease is a disease selected from the group consisting of Alzheimer's, vascular dementia and Huntington's disease. The method of claim 2, The extract is a composition for preventing or improving ischemic cerebrovascular disease is an onion extract extracted with water or a low alcohol of 1 to 4 carbon atoms of 50 to 100% concentration. Food composition for preventing or improving ischemic cerebrovascular disease comprising 0.01 to 15% by weight of the composition according to any one of claims 1 to 4. The method of claim 5, The food composition is a food, food additives or beverages ischemic cerebrovascular disease food composition for preventing or improving.

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