KR100972023B1 - Composition for preventing or improving the brain ischemia disease containing extract of chrysanthemum indicum var. albescens - Google Patents

Abstract

The present invention relates to a composition for preventing or improving ischemic cerebrovascular disease comprising a white persimmon extract as an active ingredient, and more particularly, effectively prevents nerve damage in the CA1 region of the brain hippocampal tissue known to be sensitive to cerebral ischemia. It relates to a composition for preventing or improving ischemic cerebrovascular disease comprising a white persimmon extract harmless to.

White immunity, ischemic cerebrovascular disease, dementia, Alzheimer's disease

Description

Composition for preventing or improving cerebral ischemic disease comprising white extract of gamguk {COMPOSITION FOR PREVENTING OR IMPROVING THE BRAIN ISCHEMIA DISEASE CONTAINING EXTRACT OF CHRYSANTHEMUM INDICUM VAR. ALBESCENS}

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

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 span of Korean people is 77.5 years old, and the elderly population is rapidly increasing. Meanwhile, 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. 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, 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 no 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 the cells (Hara et al. al ., Brain Res. Bull., 29, pp 659-665 (1992); Nedergaard, Acta Neurol. Scand., 77, pp 81-101 (1988)) or 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), α-amino-3-hydroxy-5-methyl-4 -isoxazole-propionic acid (AMPA), and metabotropic glutamate receptor (MTA). Induces continuous activity of calcium, resulting in excessive cellular influx of calcium (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, 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.

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 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 preventing or improving ischemic cerebrovascular disease comprising a white gamguk extract as an active ingredient.

In addition, in order to achieve the above object, the present invention includes a composition for preventing or improving ischemic cerebrovascular disease as an active ingredient and provides a health functional food for preventing or improving ischemic cerebrovascular disease having a neuroprotective effect.

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

The inventors of the present invention, while studying a composition for preventing and improving cerebrovascular disease from a substance derived from natural plants that ensure human safety, preventing the damage of nerve cells in the CA1 region of the brain hippocampal tissue known to be sensitive to cerebral ischemia. It was confirmed that the effect is significantly increased compared to the conventional plant extracts, particularly yellow persimmon extract, which is related to the treatment of cerebrovascular disease, and completed the present invention based on this.

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

Yellow persimmon ( Chrysanthemum indicum ) is a perennial plant belonging to the family Asteraceae. The yellow persimmon is not only native to many domestic production, but is also grown.

In the present invention, white persimmon ( Chrysanthemum indicum var . albescens or Chrysanthemum indicum var . albescens Makino) is a variant of Gam-guk ( Deokanthema indicum f. albescens, Dendranthema indicum f. albescens (Makino) is also referred to as the scientific name such TBLee. Normally, the research on gam-guk ( Chrysanthemum) indicum ) has been performed only, and the research on the white persimmon is rarely carried out at home and abroad. The white persimmons to be extracted of the persimmon extract may be at least one selected from the group consisting of flowers, roots, stems, and leaves, and may be preferably flowers. White persimmon extract of the present invention, compared with the yellow persimmon extract, was found to have a remarkably superior neuronal protective effect of the brain hippocampal tissue CA1 region.

The extract of the present invention can be prepared by extracting with an extraction solvent or by adding a fractional solvent to the extract prepared by extracting with an extraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as alcohol having 1 to 5 carbon atoms such as methanol or ethanol, ethyl acetate or acetone, a nonpolar solvent of hexane or dichloromethane, or a mixed solvent thereof, and preferably 30 to 80% of ethanol. C1-C5 alcohol aqueous solution, More preferably, it may be 40-70% ethanol aqueous solution.

The fractional solvent may be water, butanol, ethyl acetate, chloroform, hexane or a mixture thereof.

White persimmon extract of the present invention may be prepared according to a conventional method for producing a plant extract. More specifically, the method may be performed by adding an extraction solvent to a white rice cake from which impurities are removed and performing an extraction process. The extraction process may be cold extraction, hot extraction, pressure extraction or ultrasonic grinding extraction. In addition, a fraction solvent is added to the extract prepared by the above extraction method, that is, crude extract, and then added in the order of hexane, chloroform, ethyl acetate, butanol, and water, and then separated into layers, followed by hexane fraction, chloroform fraction, ethyl acetate fraction, butanol. Processes for obtaining fractions and water fractions can be carried out.

In addition, the extract may be concentrated or removed by performing extraction or fractionation, followed by performing a filtration under reduced pressure or by further concentrating and / or lyophilizing. The obtained white persimmon extract can be stored in a deep freezer until use.

Ischemic cerebrovascular disease in the present invention refers to a term that collectively refers to a disease caused by the blood vessels of the brain blocked for some reason, the blood is not supplied to the brain, also referred to as cerebral ischemic 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 among brain cells due to decreased blood flow in the brain and normal supply of oxygen and glucose. In other words, examples of ischemic cerebrovascular disease may include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, white matter abnormality, and the like.

The white gakguk extract of the present invention anesthetizes gerbils to induce cerebral ischemia by ligation of the whole neck artery, and in the experiment of measuring the efficacy by staining neurons, the neuronal cell death suppression effect is significantly improved compared to the yellow gakguk extract. It was confirmed that there is, and has a remarkably excellent neuronal protective effect.

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

In addition to the white persimmon extract, 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 And a carbonation agent used for the carbonated beverage. The components may be added to the composition independently or in combination. The content of the additional ingredient is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the white persimmon extract.

The composition for preventing or improving ischemic cerebrovascular disease may be a therapeutic composition for treating or preventing ischemic cerebrovascular disease.

The pharmaceutical composition comprising the white persimmon extract as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation thereof.

Carriers, excipients and diluents that may be included in the composition comprising the white persimmon 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, It may be one or more selected from the group consisting of propylene glycol and liquid paraffin, but is not limited thereto. Any conventional carrier, excipient or diluent may be used. The ingredients may be added independently or in combination to the white gamgukg extract, which is the active ingredient.

Compositions comprising the white persimmon 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 therapeutic composition may comprise 0.001% by weight to 99.9% by weight, preferably 0.1% by weight to 99% by weight, more preferably 1% by weight to 50% by weight, based on the total weight of the composition. 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 white persimmon extract.

In addition, the therapeutic composition may further include conventional fillers, extenders, binders, disintegrating agents, surfactants, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers or preservatives, and when formulated, oral or parenteral You can use both phrases.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the antimicrobial composition. Prepared by mixing sucrose or 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, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.

In addition, the formulation of the therapeutic composition of the present invention may be in a preferred form depending on the method of use, and in particular employs methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be formulated. Examples of specific formulations may be granules, powders, syrups, solutions, suspensions, premises, acupuncture, tablets, suppositories, injections, spirits, capsules, pills, soft or hard gelatin capsules, and the like. Furthermore, the therapeutic compositions of the present invention are preferably using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). Can be formulated.

Preferred dosages of the therapeutic compositions of the present invention vary depending on condition and weight, extent of disease, drug form, route of administration, and duration, but may be appropriately selected by one of ordinary skill in the art to which the present invention belongs. have. Preferably, the therapeutic composition of the present invention is 0.0001 to 1000mg / kg per day based on the amount of white persimmon extract, it is preferable to administer at 0.01 to 100mg / kg to be more effective. 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.

In addition, to achieve the above object, the present invention includes a composition for preventing or improving ischemic cerebrovascular disease as an active ingredient and provides a food composition for preventing or improving ischemic cerebrovascular disease having a neuroprotective effect.

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 dietary supplements, beverages, food additives and beverage additives.

Examples of the food of the present invention include various foods, beverages, gums, teas, vitamin complexes, and health 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 ( For example, 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 ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup). The food, health functional food, beverages, food additives and beverage additives may be prepared by conventional manufacturing methods.

In the present invention, the health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition that 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 dietary supplement may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of dietary supplements.

In the present invention, the drink refers to a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional beverage. The beverage is not particularly limited to other ingredients other than including the white persimmon 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. have.

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 food composition of the present invention, in addition to the composition of the present invention for the production of natural fruit juices, fruit juice drinks, vegetable drinks It may further contain pulp for.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, 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 white persimmon extract of the present invention.

In the present invention, the health functional beverage is a biological defense rhythm control or disease prevention of a beverage group or beverage composition which has added value to the beverage by using physical, biochemical and biotechnological techniques to act and express the function of the beverage for a specific purpose. Means a beverage that is designed and processed to fully express the gymnastic function related to recovery and the like.

The health functional beverage is not particularly limited to other ingredients except the white persimmon 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 for the purpose of preventing or improving the ischemic cerebrovascular disease, the amount of white gamgukchi extract may comprise 0.01 to 15% by weight of the total food weight, the beverage composition based on 100 ml 0.02 to 5 g, preferably 0.3 to 1 g.

The composition and food comprising the white persimmon extract of the present invention as an active ingredient have an excellent neuronal cell protective effect of remarkably inhibiting neuronal cell death in the hippocampus region of male Mongolian gerbil (Harlan, USA) that induced ischemia. In addition, it was confirmed that the neuroprotective effect was significantly improved compared to the well-known yellow persimmon extract, which was well known in the animals administered the white persimmon extract.

As described above, the white gakguk extract of the present invention is more effective in preventing neuronal damage caused by cerebral ischemia than the yellow gakguk extract, and can inhibit neuronal cell death caused by cerebral ischemia, thereby preventing, treating or treating ischemic cerebrovascular disease. Not only can be usefully used for improvement and does not cause adverse effects on the human body, so can be widely used as a pharmaceutical composition or nutraceutical composition.

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

Example  1: yellow Country  And white Country  Preparation of Extract

To 200 g of each of the yellow and white persimmon buds purchased fresh, 1000 ml of 50% ethanol was added and extracted at 75 ° C. for 120 minutes. Collect the extract obtained by repeating the above procedure twice, concentrated under reduced pressure at 40 ℃ and freeze-dried the remaining water to obtain the final yellow dried dried extract (40.8g, 20.4%) and white dried dried extract (43.8g, 21.9%) Secured.

Example  2: yellow Country  And white Country  Of extract Cerebral ischemia  Measurement of neuronal neuroprotective effect

After anesthetizing the male Mongolian gerbil (Harlan, USA) of 65g to 75g in weight to expose the common carotid artery and ligation to induce cerebral ischemia, a method of staining and monitoring the hippocampal tissue of Mongolian gerbil The neuroprotective effect of yellow and white extract was confirmed.

2-1. Breeding of Experimental Animals

40 male Mongolian gerbil, Meriones unguiculatus (Halan, USA), weighing between 65 and 75 grams, with a constant light and dark cycle from 7 am to 7 pm, a temperature of 21 ° C. to 25 ° C. and 45% to 65 It was bred 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. yellow Immigration Division  White Country  Of extract Cerebral ischemia  Neuronal cell efficacy test

Dissolved 420 mg each of yellow and white persimmon extract of Example 1 in 50 ml of distilled water was orally administered to the experimental animals to 60 mg per 1kg body weight using the esophageal needle every day from a week ago. 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. 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 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. 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 settled in a 30% sucrose solution (in 0.1 M phosphate buffer) to settle down to the bottom, the brain tissue with a sliding microtome (Reichert-Jung, Germany) 30㎛ 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 extract was used. The normal group did not perform ischemia-reperfusion surgery but also administered any substance, including yellow or white gamma extract. Did not do it.

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. . FIG. 2 shows the results of photographing the experimental group administered with 60 mg / kg of the control group, the normal group, the white gamguk extract and the white gakguk extract.

2A and 2B show total hippocampal regions of a normal group and a control group, respectively, and E and F represent CA1 regions of hippocampus of a normal group and a control group, respectively. In addition, C and G of FIG. 2 show the entire hippocampal region and CA1 region of the hippocampus administered with 60 mg / kg of yellow persimmon extract, and D and H of the experimental group administered with 60 mg / kg of white persimmon extract The hippocampus and CA1 region of the hippocampus are shown. In order to verify the significance, Figure 2 was taken to pick the most general part of each group.

As can be seen in FIG. 2, the normal group was able to observe that the neurons are heavily stained throughout the hippocampus. However, it was observed that a large portion of neurons were not stained by neuronal cell death in the CA1 region due to cerebral ischemia in the control group not administered the extract.

On the other hand, in the experimental group administered with 60 mg / kg white persimmon extract, cells were stained in the entire hippocampus as in the normal group, but in the experimental group administered with 60 mg / kg yellow persimmon extract, cells were stained in a part of the inner part of the hippocampus. It was confirmed that it was not, and it was confirmed that some neuron deaths were advanced in the inner region of the hippocampus. From the above results, it was confirmed that the two extracts inhibit the neuronal cell death, but the white persimmon extract has a superior neuronal cell death inhibitory effect than the yellow persimmon extract.

In addition, a large number of neurons were observed in H photographing the CA1 region of the group administered with the white persimmon extract, compared to G photographing the CA1 region of the group administered with the yellow persimmon extract. Neuronal cell death inhibition was confirmed to be excellent.

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 measured tissue sections is shown in Figure 3 by the relative number.

As can be seen in Figure 3, the control group was observed about 12.3% of the number of CA1 domain cells of the hippocampus compared to the normal group, in the group administered with 60 mg / kg of yellow and white persimmon extract in a dose of compared to the control group Neuronal survival was confirmed to be excellent.

In particular, in the case of white persimmon extract, 78.9% of the neuronal cell survival rate was observed compared to the normal group, which is about 50% or more, that is, 1.5 times higher than the yellow persimmon extract which showed the neuronal cell survival rate of about 52.6% of the normal group. An improved superior neuronal protective effect was observed.

In addition, the neuronal survival rate of the experimental animals treated with white persimmon extract, which is recognized to have a superior neuronal cell protective effect about 1.5 times as compared to the yellow persimmon extract, is equivalent to that of the normal group, that is, about 80% level, such as cerebral ischemia. Even when this occurs, it is possible to suppress the death of neurons in a part or the whole of the brain, while in the case of yellow persimmon extract, only about 50% of the neurons were found to be protected.

When only about 50% of neurons are protected, when ischemic cerebrovascular disease is caused by cerebral ischemia, brain function related to exercise, language, vision or smell may be seriously deteriorated, but about 80% of neurons after cerebral ischemia If the degree is viable, even if cerebral ischemia occurs, the degradation of the functions is suppressed as much as possible, and when the ischemic cerebrovascular disease is induced by cerebral ischemia, the functional loss such as movement loss, numbness of the senses, speech impairment, visual and olfactory disorders is prevented. Because it can be effectively prevented or improved, white persimmon extract can be evaluated as having a very superior neuronal protective effect compared to yellow persimmon extract.

Example  3. yellow Country  And white Country  Changes of Glue Cell Activity Following Administration of Extracts

In animal experiments using gerbils, changes in glial activity of yellow and white persimmon extracts were confirmed by immunohistochemistry staining.

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

Tissue sections showing hippocampus complexes were selected and used in the experiments. The selected tissue sections were reacted with 0.3% H 2 O 2 (in PBS) for 30 minutes to remove endogenous peroxidase. 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 astrocytes, the tissue sections reacted with the 3% normal goat serum were reacted with rabbit anti-GFAP (Chemicon International, USA), which is a primary antibody diluted 1: 1000, and microprotrusions. In order to confirm the change of glial cells, the tissue sections were reacted with rabbit anti-Iba-1 (Wako, USA), which was 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 tissue sections that completed the reaction with GFAP and Iba-1 are shown in FIG. 4.

As shown in FIG. 4, in the experimental group to which the yellow persimmon extract and the white persimmon extract were administered, the expression of the glial cells was reduced compared to the control group to which the solvent was administered. In particular, the expression of GFAP, which is known as a marker of astrocytic cells, was observed. And it was confirmed that the expression of Iba-1 known as markers of microprotuberant glial cells decreased.

In particular, as shown in FIG. 4D confirming the expression of GFAP, in the case of white gamguk extract, the amount of GFAP expression was visually reduced compared to the yellow gamguk extract, and the expression level of Iba-1 was significantly reduced and fine. It was confirmed that the expression of dendritic glial cells can be significantly suppressed, white gamgi extract was found to have a very excellent effect on neuronal cell protection by inhibiting glial expression more effectively than the yellow gamgi extract.

Example  4. Using a rat Acute Toxicity  Test

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

4 mg, 28 mg, 70 mg, 140 mg and 280 mg of the white persimmon extract obtained in Example 1 were dissolved in 50 ml of distilled water in the experimental animal of Example 2, using a needle for esophagus daily for 0.5 days. After oral administration, the 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. Through oral administration to laboratory rats Acute Toxicity  Test

In order to confirm the acute toxicity of the white persimmon extract, the white persimmon extract obtained in Example 1 was dissolved in distilled water, and the specific pathogen-free (SPF) SD rats (Sprague-Dawley, Harlan, USA) of 6 weeks old Was administered by oral administration.

After the oral administration of 14 mg, 28 mg, 70 mg, 140 mg and 280 mg of white persimmon extract, respectively, in 1 ml of distilled water, the mortality, clinical symptoms and body weight of the animals were observed. A blood biochemical test and a blood biochemical test were carried out. The laboratory rats were examined by necropsy to observe the abdominal and thoracic organ abnormalities.

As a result, all experimental animals treated with white persimmon extract did not have any clinical signs or deaths that were remarkable, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

According to the above results, the white persimmon extract of the present invention did not show a toxicity change 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.

Production Example : White Soybean Extract  Used formulation

Production Example  One. Powder  Produce

White Persimmon 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.

Production Example  2. Preparation of Tablets

White Persimmon 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.

Production Example  3. Preparation of Capsule

White Persimmon 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.

Production Example  4. Liquid  Produce

White Persimmon Extract Powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water quantity

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution was prepared by sterilization.

Production Example  5. Preparation of dietary supplements

1000 mg white white extract powder

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 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.

1 is a schematic diagram showing a process of obtaining a yellow gazeuk extract and a white gakguk extract according to an embodiment of the present invention.

2 is a cresyl violet of hippocampal tissue of experimental animals 4 days after cerebral ischemia is induced in order to examine the protective effect of ischemic yellow and white extract according to one embodiment of the present invention. It is photographed by dyeing with).

Figure 3 is to determine the protective effect of ischemic cerebrovascular disease of the yellow and white extract according to one embodiment of the present invention, the survival rate of the neurons in the normal group to the number of cells surviving 4 days after induction of cerebral ischemia It is a graph of relative figures.

4 is a GFAP marker for glial cells of hippocampal tissues of experimental animals 4 days after cerebral ischemia induction, in order to investigate the protective effect of ischemic cerebrovascular extract of yellow and white extract according to one embodiment of the present invention. And Iba-1, which is a marker of microprogenitor cells, by immunohistochemistry.

Claims (4)

White Guam ( Chrysanthemum indicum var . albescens ) composition for preventing or improving ischemic cerebrovascular disease comprising the extract as an active ingredient. The method of claim 1, The ischemic cerebrovascular disease is a composition for preventing or improving ischemic cerebrovascular disease is any one selected from the group consisting of cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and leukemia. The method of claim 1, The white persimmon extract is a composition for preventing or improving ischemic cerebrovascular disease is an extract extracted with an aqueous alcohol solution of 1 to 5 carbon atoms of 30 to 80% concentration. Food composition for preventing or improving ischemic cerebrovascular disease comprising the composition according to any one of claims 1 to 3.

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