JP6329689B2 - A composition for preventing or treating degenerative brain disease, comprising Kanamura extract as an active ingredient - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain disease containing an extract of Humulus japonicus or a fraction thereof as an active ingredient, and more specifically, an inhibitory effect and oxidation of dopaminergic neuronal cell death. Pharmaceutical composition for preventing or treating degenerative brain disease, comprising Kanamura extract or a fraction thereof having an effect of protecting nerve cells against stress and improving cognitive ability and memory as an active ingredient, the pharmaceutical composition A method for treating degenerative brain disease comprising the steps of: administering Kanamgra extract or a fraction thereof for producing a pharmaceutical composition for preventing or treating said degenerative brain disease, and Kanamgra extract or The present invention relates to a health functional food for preventing or improving degenerative brain disease comprising a fraction as an active ingredient.

  For modern people, memory is becoming increasingly important in a rapidly changing life, and has become a major focus of interest from young people with high social learning to the elderly. Degenerative brain disease is known to result in memory loss, and oxidative stress is an important factor in degenerative brain diseases in the central nervous system such as Alzheimer's syndrome, Parkinson's syndrome, and Huntington's syndrome Became.

  In recent years, the number of patients with degenerative brain diseases such as dementia has increased rapidly due to an increase in the population of the elderly, and various treatment strategies have been established to improve and improve cognitive and learning functions that have declined due to dementia. Attempts have been made to develop effective and effective drugs. The memory improvement drugs that have been developed so far include acetylcholine precursors, receptor activators, acetylcholine esterase inhibitors, and the like. However, a therapeutic agent for treating the underlying cause of degenerative brain disease has not yet been developed, and as a general therapeutic agent, Aricept (Aricept), an acetylcholinesterase inhibitor, can be used. ), Exelon from Novartis, Reminyl from Janssen, and Ebixa from Lundbeck (N-methyl-D-aspartate receptor) antagonist mechanism recently approved by the US FDA ( Ebixa: Memantine). However, acetylcholinesterase inhibitors have the limitation that they cannot treat the underlying cause of Alzheimer's disease simply by improving the reduced cognitive ability. In addition, it is known that it is difficult to expect a fundamental therapeutic effect because only a temporary symptom relief effect is exhibited in some patients, and the medicinal effect does not last long. In addition to this, the long-term use of drugs is required due to the characteristics of degenerative brain disease. However, the pharmaceuticals have problems such as various side effects such as hepatotoxicity, vomiting and decreased appetite. Therefore, there is an urgent need to develop new therapeutic agents that can prevent the progression of degenerative brain disease. For this reason, many multinational pharmaceutical companies have invested enormously in research and development in this field, and in particular, β-comprising more than 40 amino acids that are estimated to be the cause of the underlying onset of Alzheimer's disease. The development of β or γ-secretase inhibitors that reduce the production of amyloid (β-amyloid) is the mainstream. In Korea, basic research on Alzheimer's disease has been carried out to some extent, but there is almost no treatment for dementia.

  Today, the Kampo medicine community is actively researching prescriptions of Kampo medicine as a treatment for Alzheimer's disease among degenerative brain diseases. Through such research, simple ingredients such as Onji, Sekishobu, Kajikazura, Koujin (Red Ginseng), Genjin (Ginseng), Jeonmyeontan, Tennohoshinjin It has been clarified that prescriptions such as (Tenno Prosthetic Dan) and Kihito (Kaisuito) have a certain effect on the treatment of Alzheimer's disease. However, it is not known about the prevention or treatment effect of degenerative brain disease of Kanamura.

  The present inventors can effectively suppress the onset and worsening of degenerative brain diseases such as Parkinson's disease and Alzheimer's disease, which have recently increased in incidence in modern people, and have side effects for treating this. As a result of diligent research efforts to develop a new preventive or therapeutic agent for degenerative brain disease that uses a substance that is not toxic to the human body even when ingested as a safe drug, Kanamgra extract is a dopaminergic neuron By suppressing death, having a neuronal protective effect against oxidative stress, and clarifying that it has an effect of improving cognitive ability and memory in animal models with degenerative brain diseases such as Parkinson's disease and Alzheimer's disease, It was confirmed that this can be used for the prevention or treatment of degenerative brain disease, and the present invention was completed.

  One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative brain disease comprising an extract of Humulus japonicus or a fraction thereof as an active ingredient.

  Another object of the present invention is to provide a method for treating degenerative brain disease comprising the step of administering the pharmaceutical composition.

  Another object of the present invention is to provide a health functional food for preventing or ameliorating degenerative brain disease comprising the extract or fraction as an active ingredient.

  Another object of the present invention is to provide a use of the extract of Kanamura or a fraction thereof for producing the pharmaceutical composition for preventing or treating the degenerative brain disease.

  In order to develop a substance that effectively suppresses the onset and exacerbation of degenerative brain disease, the present inventors conducted various researches on natural products that are highly safe to the human body. We focused on the extract of Humulus japonicus. It was confirmed that the canamgra extract suppresses dopaminergic neuron death and has a protective effect on neurons against oxidative stress. Moreover, the animal model which has degenerative brain diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, showed the improvement effect of cognitive ability and memory.

  Therefore, the Humulus japonicus extract is used to prevent or treat degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, Huntington's disease, etc. It was found that it can be used as an active ingredient of pharmaceutical compositions for medical use.

  In order to achieve the above-described object, the present invention provides, as one aspect, a pharmaceutical composition for preventing or treating degenerative brain disease, which comprises a humulus japonicus extract or a fraction thereof as an active ingredient. To do.

  The term “Humulus japonicus” in the present invention belongs to Cannabaceae, and is an annual plant of vine that is distributed mainly in Korea and East Asia. It is known that the fruit is used as a bitter stomachic agent and the whole plant with the fruit is used as a diuretic. However, the use for the treatment or prevention of diseases related to degenerative brain disease has not yet been disclosed, and the present inventors have not yet disclosed Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, mild cognitive impairment. It has become clear that it can be used in applications for the treatment or prevention of diseases associated with degenerative brain diseases including stroke and Huntington's disease. Further, in the present invention, the canamgra can be purchased commercially, collected from nature, or cultivated.

  The term “extract” of the present invention refers to a liquid component obtained by immersing a target substance in various solvents and then extracted at room temperature or in a heated state for a certain period of time. It means the resulting product such as solid content obtained by removing. Furthermore, in addition to the results, the results can be comprehensively interpreted as including all of the dilutions of the results, concentrated solutions thereof, crude purified products, purified products, and the like.

  In the present invention, the extract can be interpreted as a canamgra extract. The canamgra extract can be extracted from various organs of natural, hybrid, and variant plants, such as roots, aerial parts, stems, leaves, flowers, fruit trunks, fruit shells, as well as plant tissues. Extractable from the culture. The canamgra extract can be obtained by extraction with water or various organic solvents. At this time, the organic solvent used is not particularly limited as long as it can obtain an extract having a preventive or therapeutic effect on degenerative brain disease, but is preferably water, polar solvent or nonpolar It may be a solvent, more preferably water, a lower alcohol having 1 to 4 carbon atoms (such as methanol, ethanol, propanol or butanol), a mixed solvent thereof or the like, and most preferably methanol or its A mixed solvent may be used. Further, the method for obtaining the extract is not particularly limited as long as it can obtain an extract having a preventive or therapeutic effect on degenerative brain disease. Preferably, the root of the canamgrass, A cold extraction method in which stems, leaves, fruits, flowers, dried products and processed products thereof are immersed in the solvent and extracted at room temperature of 10 to 25 ° C, and a heat extraction method in which extraction is performed by heating to 40 to 100 ° C. Alternatively, an ultrasonic extraction method in which ultrasonic waves are added for extraction, a reflux extraction method using a reflux condenser, or the like may be used.

  The term “fraction” of the present invention means a product obtained by a fractionation method in which a specific component or a specific group is separated from a mixture containing various components.

  The fraction in the present invention can be interpreted as a fraction obtained by performing various fractionation methods on the canamgra extract. The fraction is obtained by performing various fractionation methods on the extract. The fractionation method is not particularly limited to this, but is a solvent fractionation method performed by treating with various solvents, an ultrafiltration fractionation performed by passing through an ultrafiltration membrane having a cut-off value of a certain molecular weight. And chromatographic fractionation methods that perform various chromatographies (made for separation by size, charge, hydrophobicity or affinity). In particular, the solvent used in the solvent fractionation method is not particularly limited, but a polar solvent or a nonpolar solvent can be used, and preferably a nonpolar solvent can be used. The solvent fractionation method can be performed by sequentially fractionating the extract using a nonpolar level solvent in the order from low to low solvent. For example, the extract can be obtained using nucleic acid or ethyl acetate. Sequential fractionation is possible.

  The term “degenerative brain disease” of the present invention means a disease that occurs in the brain among the degenerative diseases that occur with aging. It is known that the degenerative brain disease is caused by neurodegeneration associated with aging, protein aggregation due to genetic and environmental factors, and death of nerve cells, but the exact cause is still unclear. Not.

  In the present invention, the degenerative brain disease is not particularly limited, and examples thereof include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, mild cognitive impairment, stroke, Huntington's disease, and the like.

  The term “prevention” in the present invention means all actions to suppress or delay the onset of degenerative brain disease by administration of the pharmaceutical composition according to the present invention, and “treatment” means the above-mentioned pharmaceutical composition. By administration, it means all actions in which the symptoms of an individual suspected of degenerative brain disease and those who have developed improve or are advantageously altered.

  In the present invention, the prevention or treatment of the degenerative brain disease can be achieved by suppressing the killing of dopaminergic neurons by the extract and having a protective effect on neurons against oxidative stress, and behaviorally This can be achieved by showing an effect of improving cognitive ability and memory in an animal model having degenerative brain diseases such as Parkinson's disease and Alzheimer's disease.

  The term “dopamine” in the present invention is a neurotransmitter that transmits signals in the brain and is known to be related to movement and movement.

  The term “dopaminergic neuron death” in the present invention means the disappearance or degeneration of dopaminergic neurons that are densely located in the substantia nigra pars compacta of the midbrain. It is known that an animal model of induced Parkinson's disease can be produced by injecting 6-hydroxyldopamine (6-OHDA).

  The term “oxidative stress” in the present invention is an important factor causing degenerative brain disease. When the amount of active oxygen in the body increases, oxidative stress is induced. It is known that degenerative brain diseases such as Parkinson's disease and Alzheimer's disease can be induced.

  As described above, the extract of Kanamura or a fraction thereof provided in the present invention not only suppresses dopaminergic neuron death and has a protective effect on neurons against oxidative stress, but also administers the extract. Cognitive ability and memory can be improved in animal models with degenerative brain diseases such as Parkinson's disease and Alzheimer's disease.

  According to one embodiment of the present invention, as a result of obtaining a methanol extract from Canamgra and examining the efficacy of the obtained Canamgra extract, it is possible to suppress the generation of reactive oxygen species and to protect against the oxidative stress of neurons. (FIGS. 1a and 1b), showing the effect of suppressing inflammatory reaction (FIGS. 2a to 2g), showing the prevention and treatment effect of Parkinson's disease (FIGS. 3, 4, 5, 6a to 6d), Shows preventive and therapeutic effects (FIGS. 7, 8, 9a-9c, 10a to 10c, 11a-11c, 12a-12c and 13a-13c) and confirms that they show the preventive and therapeutic effects of Huntington's disease (FIG. 14) .

  Therefore, the canamgra extract or a fraction thereof has an effect of preventing or treating various degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, mild cognitive impairment, stroke and Huntington's disease. I found out.

  The pharmaceutical composition for preventing or treating degenerative brain disease of the present invention can further comprise a suitable carrier, excipient or diluent usually used in the manufacture of pharmaceutical compositions. Specifically, the pharmaceutical compositions are prepared by oral methods such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterilizations, respectively, by conventional methods. It can be used in the form of an injection solution. In the present invention, carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate Mention may be made of gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulating, it is prepared using a diluent or excipient such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant, and a surfactant that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients in the extract and fractions thereof. An agent such as starch, calcium carbonate, sucrose or lactose, gelatin or the like is mixed to prepare a preparation. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid preparations for oral use include suspensions, liquids for internal use, emulsions, syrups, etc., but various excipients other than water and liquid paraffin, which are commonly used simple diluents, Wetting agents, sweeteners, fragrances, preservatives and the like can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin and the like may be used.

  The content of the extract of Kanamura or a fraction thereof contained in the pharmaceutical composition of the present invention is not particularly limited, but is 0.0001 to 50% based on the total weight of the final composition. %, More preferably 0.01 to 20% by weight.

  The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, but the term “pharmaceutically effective amount” of the present invention is applicable to medical treatment or prevention. Meaning an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio, effective volume level refers to disease severity, drug activity, patient age, weight, health, gender, patient drug Sensitivity to, administration time, route of administration, and excretion rate of the composition of the present invention used, duration of treatment, presence or absence of drugs included in or simultaneously used in the composition of the present invention used, and others It can be determined according to factors well known in the medical field. The pharmaceutical compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Single or multiple doses can be administered. Considering all of the above factors, it is important to administer an amount that does not cause side effects and can achieve the maximum effect with a minimum amount.

  Regarding the dosage of the pharmaceutical composition of the present invention, for example, the pharmaceutical composition of the present invention can be administered to mammals including humans at 0.1 to 500 mg / kg body weight per day. In addition, the administration frequency of the composition of the present invention is not particularly limited, but can be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way.

  In another aspect, the present invention provides a method for treating degenerative brain disease comprising the step of administering the pharmaceutical composition to an individual who has developed degenerative brain disease in a pharmaceutically effective amount.

  As described above, the canamgra extract or fraction thereof provided in the present invention can be used as an active ingredient of a pharmaceutical composition for preventing or treating degenerative brain disease. It can be used to treat degenerative brain disease.

  The term “individual” of the present invention includes, without limitation, mammals including rats, domestic animals, humans and the like that may develop or develop degenerative brain disease.

  In the method for treating degenerative brain disease of the present invention, the administration route of the pharmaceutical composition may be any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration It can be administered through routes such as administration and rectal administration. However, when administered orally, gastric acid may denature the extract or fraction thereof, so the oral composition should be coated with an active agent or protected from gastrointestinal degradation. It must be shaped. The composition can also be administered by any device that allows the active agent to migrate to the target cells.

  Moreover, this invention provides the health functional food for prevention or improvement of a degenerative brain disease containing the extract of Kanamura or its fraction as another one aspect | mode.

  The extract of Kanamura or its fraction, which is an active ingredient of the pharmaceutical composition for preventing or treating degenerative brain disease, is derived from Kanamgra, which has been used as a traditional Chinese medicine and has proven its safety. Therefore, the extract of Kanamura or a fraction thereof can be eaten regularly, and can be produced and consumed in the form of a food that can prevent or ameliorate degenerative brain disease.

  The content of the extract of the canamgra or the fraction thereof contained in the food is not particularly limited, but may be contained in an amount of 0.001 to 50% by weight based on the total weight of the food composition, and more preferably. May be contained in an amount of 0.1 to 10% by weight. When a foodstuff is a drink, 1-10g may be contained on the basis of 100 ml, More preferably, it may be contained in the ratio of 2-7g. The composition can also include additional ingredients that are commonly used in food compositions and improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like can be included. Moreover, minerals, such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), can be included. It can also contain amino acids such as lysine, tryptophan, cysteine, valine and the like. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and advanced bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), dibutylhydroxy) Toluene (BHT, etc.), colorants (tar pigments, etc.), color formers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (eg, MSG sodium glutamate), sweeteners (durtine, cyclamate) , Saccharin, sodium, etc.), fragrances (vanillin, lactone, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glue), coating agents, gum bases, foam control Food additives such as additives, solvents and improvers can be added . The additive is selected according to the type of food and used in an appropriate amount.

  On the other hand, a health functional food for the prevention or amelioration of degenerative brain disease can be produced using the food composition for the prevention or amelioration of degenerative brain disease comprising the extract of Kanamura or a fraction thereof. .

  As a specific example, processed foods that prevent or ameliorate degenerative brain disease can be produced using the food composition. For example, confectionery, beverages, alcoholic beverages, fermented foods, canned foods, processed milk products, meat It can be produced as a health functional food in the form of processed food or processed noodle food. At this time, the confectionery includes biscuits, pie, cake, bread, candy, jelly, gum, cereal (including meal substitutes such as cereal flakes) and the like. Drinks include drinking water, carbonated drinks, functional ion drinks, juices (eg, apples, pears, grapes, aloe, citrus, peaches, carrots, tomato juices, etc.), amazake and the like. Sake includes sake, whiskey, shochu, beer, Western liquor, fruit liquor and the like. Fermented foods include soy sauce, miso, gochujang and the like. Canned products are canned fishery products (for example, canned tuna, mackerel, saury and turban shell), canned livestock products (canned beef, pork, chicken, turkey, etc.), canned agricultural products (canned corn, peach, pineapple, etc.) including. Milk processed foods include cheese, butter, yogurt and the like. Processed meat products include tonkatsu, beef cutlet, chicken cutlet, sausage, sweet and sour pork, nuggets, Nobianigui. Includes noodles such as hermetically sealed raw noodles. In addition, the composition can be used for retort foods, soups and the like.

  The term “functional food” in the present invention is the same term as food for special health use (FoSHU), and it effectively exhibits a bioregulatory function in addition to nutrition supply. In order to obtain a useful effect in the prevention or amelioration of degenerative brain disease, the food is a tablet, capsule, powder, granule, liquid, It can be manufactured in various forms such as a circle.

  In another aspect, the present invention provides the use of an extract of Kanamura or a fraction thereof for use in producing a pharmaceutical composition for preventing or treating the degenerative brain disease.

  The composition containing the extract of Humulus japonicus or a fraction thereof of the present invention as an active ingredient not only suppresses dopaminergic neuron death and has a protective effect on neurons against oxidative stress, but also Parkinson's disease and Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, and so on, in animal models with degenerative brain diseases such as Alzheimer's disease. It can be usefully used as a food or a medicine for the prevention or treatment of degenerative brain diseases including Huntington's disease.

It is a fluorescence micrograph which shows the effect of the canamgra extract on the production | generation of a reactive oxygen species. It is a graph which shows the result of having quantitatively analyzed the fluorescence level image | photographed with the said fluorescence microscope using the luminometer. It is a graph which shows the result of having compared the change of the intracellular TNF- (alpha) mRNA level by the treatment density | concentration of a canamgra extract in the microglia cell (microglia cell) strain | stump | stock with which the inflammatory reaction was induced | guided | derived by the lipopolysaccharide (LPS) process. It is a graph which shows the result of having compared the change of the intracellular IL-1 (beta) mRNA level by the treatment density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is a graph which shows the result of having compared the change of the intracellular IL-6 mRNA level by the processing density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is a graph which shows the result of having compared the change of the intracellular iNOS mRNA level by the processing density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is a graph which shows the result of having compared the change of the TNF- (alpha) protein level secreted extracellularly by the treatment density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is a graph which shows the result of having compared the change of IL-6 protein level secreted extracellularly by the treatment density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is a graph which shows the result of having compared the change of the NO protein level secreted extracellularly by the treatment density | concentration of the extract of Kanamura in the microglia cell line by which the inflammatory reaction was induced | guided | derived by LPS process. It is the graph which showed the result of the apomorphine induction rotation exercise | movement in the animal model of the Parkinson's disease mouse | mouth which induced the dopaminergic nerve cell specific cell death of the brain with 6-OHDA (6-hydroxyldopamine) by the treatment of the extract of Kanamura. It is a graph which shows the result of having measured the level of the expression level of tyrosine hydroxylase (THrosin hydroxylase, TH) which is dopaminergic neuron-specific protein by the treatment of the extract of Kanamura. It is a graph which shows the result of having compared the effect by the treatment density | concentration of a canamgra extract in the killing of the dopaminergic nerve induced | guided | derived by 6-OHDA treatment. Marker proteins involved in cell apoptosis in SH-SY5Y neurons treated with 6-OHDA and various concentrations (0, 50, 100, 200 μg / ml) of Canamgra extract, depending on the treatment concentration of Canamgra. It is a photograph which shows the Western blot analysis result which shows the change of an expression level. It is a graph which shows the change of the expression level of the cleaved caspase 9 (cleaved caspase 9) by the treatment density | concentration of canamgra obtained from the Western blot analysis result of the said FIG. 6a. It is a graph which shows the change of the expression level of the cleaved caspase 3 (cleaved caspase 3) by the processing density | concentration of canamgra obtained from the Western blot analysis result of the said FIG. 6a. It is a graph which shows the change of the expression level of the cut | disconnected PARP by the treatment density | concentration of canamgra obtained from the western blot analysis result of the said FIG. 6a. It is a graph which shows the improvement effect of the cognitive function and memory in the novel object recognition test (NORT) by Kanamura extract processing in the animal model of the Alzheimer disease onset mouse. It is a graph which shows the improvement effect of the spatial perception ability and the short-term memory decline in the Y-maze test of the canamgra extract in the animal model of the Alzheimer disease onset mouse | mouth. It is a photograph which shows the result of having performed the immuno-staining with respect to (beta) -amyloid in the brain of the animal model of the Alzheimer's disease onset mouse | mouth which is not administered Kanamura extract. It is a photograph which shows the result of having performed the immuno-staining with respect to (beta) -amyloid in the brain of the animal model of the Alzheimer disease onset mouse | mouth which administered Kanamura extract. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to (beta) -amyloid by administration of Kanamura extract in the brain of the animal model of an Alzheimer disease onset mouse | mouth. It is a photograph which shows the result of having performed the immuno-staining with respect to the phosphorylated tau protein in the brain of the animal model of the Alzheimer disease onset mouse | mouth which has not administered Kanamura extract. It is a photograph which shows the result of having performed the immuno-staining with respect to the phosphorylated tau protein in the brain of the animal model of the Alzheimer disease onset mouse | mouth which administered Kanamura extract. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to the phosphorylated tau protein by administration of Kanamura extract in the brain of the animal model of an Alzheimer disease onset mouse | mouth. It is a photograph which shows the result of having performed the immuno-staining with respect to the activated microglia cell (microglia cell) in the brain of the animal model of the Alzheimer disease onset mouse | mouth which has not administered Kanamura extract. It is a photograph which shows the result of having performed the immuno-staining with respect to the activated microglia cell in the brain of the animal model of the Alzheimer disease onset mouse | mouth which administered Kanamura extract. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to the activated microglia cell by administration of Kanamura extract in the brain of the animal model of the Alzheimer disease onset mouse | mouth. It is a photograph which shows the result of having performed the immuno-staining with respect to the activated astrocyte in the brain of the animal model of the Alzheimer disease onset mouse | mouth which has not administered Kanamura extract. It is a photograph which shows the result of having performed the immuno-staining with respect to the activated astrocyte glial in the brain of the animal model of the Alzheimer disease onset mouse | mouth which administered Kanamura extract. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to the activated astrocyte glial by administration of the canamgra extract in the brain of the animal model of an Alzheimer disease onset mouse | mouth. It is a graph which shows the result of having compared the mRNA level of TNF- (alpha) expressed in a brain by administering Kanamura extract. It is a graph which shows the result of having compared the mRNA level of IL-6 expressed in a brain by administering Kanamura extract. It is a graph which shows the result of having compared the mRNA level of IL-1 (beta) expressed in a brain by administering Kanamura extract. It is a graph which shows the result of having conducted the behavioral analysis by administration of the extract of Kanamura in the mouse | mouth with which Huntington's disease was induced.

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

Example 1: Manufacture of kanamgra extract The kanamgra extract was prepared by adding methanol to a ground or cut kanamgra dried sample, sonicating for 15 minutes, and then allowing it to stand at room temperature for 2 hours on one day. The extract obtained by repeating 10 times was filtered, concentrated, and then obtained by a method in which a sample frozen in a deep freezer was dried with a freeze dryer. The canamgra extracts obtained as described above were used in animal experiments and neuronal cell line experiments, respectively, with 0.5% carboxymethylcellulose (CMC) solution and dimethyl sulfoxide (DMSO). ) And dissolved.

Example 2: Effect of Kanamura extract on nerve cells In order to confirm the effect of Kanamura extract produced in Example 1 on nerve cells, reactive oxygen species known as the cause of the onset of various diseases were examined. The effects on production and induction of inflammatory response were evaluated.

Example 2-1: Effect on the generation of reactive oxygen species Neuro 2a cells, which are a neuronal cell line, were treated with Kanamura extract at a concentration of 400 μg / ml, cultured for 1 hour, and then t-butylhydro at a concentration of 300 μM. Peroxide (tert-butylhydroperoxide, t-BHP) was treated for 2 hours to induce oxidative stress in the neuronal cell line. Next, the medium of the neuronal cell line was replaced with a medium containing 2 ', 7'-dichlorodihydrofluorescein diacetate (DCF-DA) at a concentration of 10 μM, and further cultured for 1 hour. Then, the reactive oxygen species (ROS) level generated in the nerve cell line was measured using a fluorescence microscope and a luminometer (FIGS. 1a and 14b).

  FIG. 1a is a fluorescence micrograph showing the effect of the extract of Kanamura on the generation of reactive oxygen species, and FIG. 1b shows the result of quantitative analysis of the fluorescence level taken with the fluorescence microscope using a luminometer. It is a graph. As shown in FIGS. 1a and 14b, reactive oxygen species levels were significantly increased by t-BHP in the Neuro 2a neuronal cell line, but reactive oxygen species levels increased by t-BHP are evident by treatment with Kanamura extract It was confirmed that there was no effect on the reactive oxygen species level when treated with canamgra extract alone.

  Therefore, it was found that the extract of canamgra suppresses the generation of reactive oxygen species and exhibits a protective effect against oxidative stress of nerve cells.

Example 2-2: Effect on Inflammatory Response BV-2 microglia cells, a kind of microglia cell line, were treated with 1 μg / ml lipopolysaccaride (LPS). Induction of inflammatory response and treatment of 100 or 500 μg / ml of kanamgra extract, followed by intracellular proliferation of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and pro-inflammatory mediator protein (iNOS) The mRNA level and the protein level of the inflammatory cytokine secreted into the medium were measured and then compared (FIGS. 2a to 2g).

  FIG. 2a is a graph showing the results of comparison of changes in intracellular TNF-α mRNA levels depending on the treatment concentration of Kanamura extract in a microglial cell line in which an inflammatory response was induced by LPS treatment, and FIG. FIG. 2c is a graph showing the results of comparison of changes in intracellular IL-1β mRNA level depending on the treatment concentration of canamgra extract in a microglia cell line in which an inflammatory reaction was induced, and FIG. 2c is a microglia cell in which an inflammatory reaction was induced by LPS treatment Fig. 2d is a graph showing the results of comparison of changes in intracellular IL-6 mRNA level depending on the treatment concentration of the extract of Kanamura, and Fig. 2d shows the extract of Kanamura extract in a microglia cell line in which an inflammatory response was induced by LPS treatment. Of intracellular iNOS mRNA level by treatment concentration FIG. 2e is a graph showing the results of comparison, and FIG. 2e compares changes in the level of TNF-α protein secreted extracellularly depending on the treatment concentration of the extract of Kanamura in a microglial cell line in which an inflammatory response was induced by LPS treatment. FIG. 2f compares the changes in the level of IL-6 protein secreted extracellularly with the treatment concentration of the extract of Kanamura in microglia cell lines in which an inflammatory response was induced by LPS treatment. FIG. 2g is a graph showing the results, and FIG. 2g shows the results of comparing changes in the level of extracellularly secreted NO protein in the microglia cell line in which an inflammatory response was induced by LPS treatment, depending on the treatment concentration of the extract of Kanamura. It is a graph. As shown in FIGS. 2a-2g, inflammatory cytokines (TNF-α, IL-1β, and IL-6) expressed in microglial cell lines in which an inflammatory response was induced by LPS treatment, and pro-inflammatory mediator proteins ( It was confirmed that the intracellular mRNA level of iNOS) and the level of the protein secreted into the medium decreased as the treatment concentration of Kanamura extract increased.

  Therefore, it was found that the canamgra extract has an effect of suppressing the inflammatory reaction induced in the microglia cell line.

  Accordingly, it has been confirmed that the extract of Kanamura is effective in suppressing the generation of reactive oxygen species that cause abnormalities in nerve cells and the induction of inflammatory reactions. It was analyzed to be effective in treating the disease.

Example 3: Therapeutic effect of Kanamura extract on Parkinson's disease From the results of Example 2, it was analyzed that Kanamgra extract was effective in the treatment of various diseases derived from abnormalities in nerve cells. In order to confirm whether or not the drug has a therapeutic effect on degenerative brain disease, which is known as a disease caused by neuronal abnormalities, target animals with the onset of Parkinson's disease, a type of degenerative brain disease We tried to verify the therapeutic effect of Kanamura extract.

Example 3-1: Production of experimental group for induction of Parkinson's disease by administration of 6-hydroxyldopamine (6-OHDA) 9 weeks old C57BL / 6J male mice were used as experimental animals. The mice are bred while maintaining a 12-hour day / night cycle with free access to sterilized feed and water in a sterile (Specific pathogen free, SPF) environment maintained at a temperature of 22-24 ° C. did.

  The experimental animal group was divided into a control group to which 0.5% carboxymethyl cellulose (CMC) was administered and an experimental group to which 500 mg / kg Kanamgra extract was administered. At this time, the number of individuals in the control group and the experimental group was 5 and 8 respectively, and 0.5% CMC and 500 mg / kg Kanamura extract specifically killed dopaminergic neurons. Orally administered from 3 days before the administration of 6-OHDA.

  70-80% of the dopaminergic cells in the substantia nigra of the brain corresponding to the degree of progression of early Parkinson's disease are administered by administering an anesthetized drug administered with ketamine and rompun to the mouse. In order to eliminate this, 6-OHDA was directly injected into the brain. In order to prevent destruction of noradrenergic neurons, 25 mg / kg desipramine was administered intraperitoneally 30 minutes before 6-OHDA administration, and a total amount of 6 μg 6-OHDA was applied to the left brain striatum. (Brain fine injection coordinates: front and rear +1.3, left and right -1.8, depth -3.6). As described above, 6-OHDA was administered directly to the brain, the surgical site was sutured and disinfected, and then the body temperature of the mouse was maintained with a 37 ° C warmer.

  After inducing Parkinson's disease by the above-described surgical method, behavioral tests such as apomorphine-induced rotational test, and tyrosine hydroxylase (tyrosin hydrotylase), which is a dopaminergic neuron-specific protein, Until the degree of expression of (TH) was measured, the control group and the experimental group were fed with 0.5% CMC and 500 mg / kg Kanamgra extract, respectively. At this time, 0.5% CMC and 500 mg / kg canamgra extract were orally administered for two days after the operation when food intake was difficult.

Example 3-2: Behavioral evaluation using Parkinson's disease-inducing animal model The more the dopaminergic cell death by 6-OHDA, the more severe the lesion in one brain, and the behavioral rotation number in the experimental animal model It is known to increase. In contrast, 8 days after the administration of 6-OHDA, in order to evaluate the severity of abnormal motor regulation due to dopaminergic cell death, mice were injected with 1 mg / kg apomorphine into the abdominal cavity and then asymmetric rotational behavior Was observed.

  Specifically, after the administration of the apomorphine drug, the mouse was placed in a 20 cm diameter cylinder and the number of clockwise rotations for 1 hour was measured to evaluate the rotational behavior (FIG. 3).

  FIG. 3 is a graph showing the results of apomorphine-induced rotational movement in an animal model of Parkinson's disease mouse in which brain-specific dopaminergic neuron-specific cell death was induced with 6-OHDA by treatment with the extract of Canamula. As shown in FIG. 3, the rotational behavior of the experimental animals was statistically significantly reduced in the experimental group that ingested 500 mg / kg canamgra extract compared to the control group that ingested 0.5% CMC. Confirmed to do.

Example 3-3: Measurement of expression level of tyrosine hydroxylase (THrosin hydroxylase, TH) in Parkinson's disease-induced animal model By administering Kanamura extract, dopaminergic neuron death by 6-OHDA is suppressed in mice 10 days after 6-OHDA administration, 0.5% CMC administration group and 500 mg / kg Kanamgra extract administration experiment group mice in the striatal region of the mouse brain, dopaminergic properties The difference in the expression level of TH, which is a nerve cell specific protein, was analyzed by Western blotting (FIG. 4).

  FIG. 4 is a graph showing the results of measuring the level of expression of tyrosine hydroxylase (TH), which is a dopaminergic neuron-specific protein, by treatment with the extract of Canamula. As shown in FIG. 4, it was confirmed that the expression level of TH protein in the experimental group to which Kanamura extract was given was higher than that in the control group.

  Therefore, it was found that dopaminergic neuronal cell death was markedly suppressed by administration of Kanamura extract.

Example 3-4: Measurement of degree of dopaminergic neuronal cell death by treatment with 6-OHDA, which is a Parkinson's disease-inducing drug, 6 In order to confirm whether neuronal cell death by -OHDA is suppressed, DMEM / F-12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin is separated. Each well of the 96-well plate was inoculated with SH-SY5Y neuronal cell line to 1 × 10 ⁵ cells, and extracted from Canamgra at 6-OHDA 50 μM and various concentrations (0, 50, 100, 200 μg / ml). After treatment with the product, the cells were cultured for 24 hours, and the death level of the nerve cells was confirmed by MTT assay (FIG. 5). At this time, MTT assay was performed using 3- (4,5-Dimethylthiazol-2-yl) -2,5 diphenyltetrazole bromide (MTT) solution at a concentration of 0.5 mg / ml, and reacted at 37 ° C. for 4 hours. After discarding the solution and dissolving the formazan crystal with DMSO, the absorbance was measured with a 570 nm plate reader. At this time, cells cultured without any treatment were used as a control group.

  FIG. 5 is a graph showing the results of comparing the effects of treatment concentration of canamgra extract on the killing of dopaminergic neurons induced by 6-OHDA treatment. As shown in FIG. 5, neuronal cell death in the 6-OHDA 50 μM treatment group was significantly induced compared to the control group, but all experimental groups treated with Kanamura extract (50, 100, or 200 μg / It was confirmed that dopaminergic neuron death was suppressed in the ml treatment group).

Example 3-5: Measurement of protein change associated with dopaminergic neuronal cell death by treatment with 6-OHDA, a Parkinson's disease-inducing drug From each cell line obtained by the method of Example 3-4, cell apoptosis (apoptosis) Changes in the expression levels of marker proteins involved in (cleaved caspase 9, cleaved caspase 3, and cleaved PARP) were compared through Western blot analysis (FIGS. 6a-6d).

  FIG. 6a shows cell apoptosis in SH-SY5Y neurons treated with 6-OHDA and various concentrations (0, 50, 100, 200 μg / ml) of canamgra extract, depending on the treatment concentration of canamgra. FIG. 6b is a photograph showing the result of Western blot analysis showing the change in the expression level of the marker protein involved, and FIG. 6b is a cleaved caspase 9 (cleaved caspase 9) obtained by the treatment concentration of canamgra obtained from the result of Western blot analysis of FIG. 6a. Fig. 6c is a graph showing changes in the expression level of 9), and Fig. 6c shows changes in the expression level of cleaved caspase 3 according to the treatment concentration of canamgra obtained from the result of Western blot analysis in Fig. 6a. FIG. 6d is a graph showing the kana obtained from the Western blot analysis result of FIG. 6a. It is a graph which shows the change of the expression level of the cut | disconnected PARP (cleaved PARP) by the processing concentration of a mole.

  As shown in FIGS. 6a-6d, cleaved caspase 9, cleaved caspase 3 and cleaved PARP are cell self-destructive marker proteins expressed in dopaminergic neurons. It was confirmed that the expression level of (cleaved PARP) decreased in inverse proportion to the treatment concentration of canamgra extract.

  When the results of Examples 3-1 to 3-5 are combined, it can be seen that the extract of Kanamura shows the prevention and treatment effects of Parkinson's disease.

Example 4: Therapeutic effect of Kanamgra extract on Alzheimer's disease In order to verify the efficacy of Kanamgra extract for degenerative brain disease, Kanamgra on animals in which the onset of Alzheimer's disease, a type of degenerative brain disease, was induced. An attempt was made to verify the therapeutic effect of the extract.

Example 4-1 Production of an animal model experimental group of mice with Alzheimer's disease The animal model of mice with Alzheimer's disease (B6C3-) in which APPswe and PSEN1 genes, which are genes related to Alzheimer's disease, are overexpressed in the mouse brain Tg (APPswe / PSEN1dE9) 85DboJ, JAX, 004462) was examined for the therapeutic effect of Kanamura extract. The mouse animal model is characterized by markedly observed β-amyloid deposition in the brain from 6 months of age, and is characterized by cognitive impairment specific to Alzheimer's disease. Pathogen free (SPF) was bred while maintaining a 12 hour day-night cycle with free access to sterilized feed and water in a breeding facility.

  The experimental animal group is classified into a normal group (Non-Tg) mouse group (n = 20) that does not overexpress APP / PSEN1 and an Alzheimer disease-onset mouse group (n = 22) that overexpresses APP / PSEN1. Each of the groups was divided into a control group administered with 5% CMC and an experimental group administered with 500 mg / kg Kanamgra extract. The number of individuals in the control group and the experimental group of the Alzheimer's disease onset mouse group overexpressing APP / PSEN1 was 10 and 12, respectively. At this time, the mouse group used 5-month-old mice and orally administered 0.5% CMC and 500 mg / kg of kanamgra extract daily for 10 weeks.

Example 4-2: novel object recognition test (NORT)
A novel object recognition test (NORT) was performed to confirm the effects of Kanamgra extract on cognitive and memory enhancement of Alzheimer's cognitive impairment. Specifically, 5% CMC and 500 mg / kg of kanamgra extract were administered to each test group for 8 weeks as shown in Example 2-4, followed by NORT. On the first training day, the mouse was placed in a 41.5 cm × 20 cm × 21.5 cm white box and allowed to move freely for 10 minutes. After an adaptation period of 10 minutes as described above, return to the original cage and, on the second day, place two identical cylindrical wooden blocks on either side of the box so that the mouse can explore them. Exposed for 10 minutes. Twenty-four hours later, a new square block (new object) was placed in the box together with the cylindrical block (familiar object) that was familiar to the beginning, and then the movement of the mouse was observed. At this time, the time (sniffing time) which touched the block, made the nose squeak, and moved toward the block was measured. From the total number of times measured for the two blocks, the time for interest in the cylindrical block (familiar object) and the search time for interest in the square columnar block (new object) were measured (FIG. 7). At this time, the search time is (time for showing interest in the familiar object) / (time for showing interest in the familiar object + time for showing interest in the new object) × 100 (time for showing interest in the new object) / (familiar object) The time when the user is interested in the time + the time when the user is interested in the new object) × 100.

  FIG. 7 is a graph showing the effect of improving cognitive function and memory in a novel object recognition test (NORT) by treatment with the extract of Kanamura in an animal model of an Alzheimer disease-onset mouse. As shown in FIG. 7, in the case of the normal group (Non-Tg) mouse group that does not overexpress APP / PSEN1, it is a new object, a square columnar block, and a familiar object regardless of the administration of Kanamura extract. In the control group of Kanamgra extract untreated control group of Alzheimer's disease-onset mice overexpressing APP / PSEN1 while well recognizing both cylindrical blocks, Alzheimer's disease cognitive impairment was shown, and the search time for new objects was remarkable It was confirmed that the classification of the familiar object and the new object was not successful. On the other hand, in the experimental group treated with Kanamgra extract of the Alzheimer's disease-onset mouse group overexpressing APP / PSEN1, the novel object was compared with the control group not treated with Kanamgra extract of the Alzheimer's disease-onset mouse group overexpressing APP / PSEN1. It was confirmed that there was much higher interest in, and it was possible to reliably distinguish between familiar objects and new objects. It was also confirmed that the increase in exploratory behavior in such Alzheimer's disease animal mice was similar to that of normal group mice.

  Therefore, it can be confirmed that the extract of Kanamura is effective in the treatment of Alzheimer's disease. Furthermore, among the group of normal (Non-Tg) mice that do not overexpress APP / PSEN1, By confirming that the new object recognition time is increased as compared with the untreated control group, it was found that the extract of Kanamura also shows an effect of improving cognitive function and memory.

Example 4-3: Y-maze test
In order to confirm the effect of Kanamgra extract on the spatial perception and memory enhancement of spatial perception and memory loss induced by Alzheimer's disease, a Y-maze test (Y maze test) was performed.

  Specifically, the Y-maze test is an experiment for examining whether or not a laboratory animal's spatial perception ability and short-term memory recovery are useful. The maze is formed of a transparent acrylic plate (10 cm wide, 40 cm long, 25 cm high) and covered in four Y-shapes, and the mazes are arranged at a constant angle of 120 °. The test was carried out for 10 minutes, and after each maze was defined in areas A, B, and C, an experiment animal was placed in one area and the experiment was started to move freely through the maze. At this time, the number and order of entering each maze were measured, and the change behavior (spontaneous alteration,%) was evaluated (FIG. 8). At this time, when entering three different areas in order, one point (actual alteration: order of actual alteration, ABC, BCA, CAB, etc.) is recognized. The force was calculated by the formula of the total number of alteration actions (alteration) / total number of times of entry-2) × 100.

  FIG. 8 is a graph showing the effect of improving the spatial perception ability and short-term memory decline in the Y-maze test of Kanamura extract in an animal model of Alzheimer disease-onset mice. As shown in FIG. 8, in the case of an experimental group in which Alzheimer's disease-onset mice overexpressing APP / PSEN1 were administered with Kanamgra extract for 9 weeks, Alzheimer's disease-onset mice overexpressing APP / PSEN1 were treated with Kanamgra extract. It was confirmed that the change behavioral power increased statistically significantly compared to the control group that did not.

Example 4-4: Verification of the level of amyloid deposition in the brain In an animal model of an Alzheimer's disease-causing mouse, it will be confirmed whether or not β-amyloid deposition symptoms in the brain, which are characteristic of Alzheimer's disease, are changed by administration of Kanamura extract. I tried. Specifically, for 2.5 months, an experimental group in which the Kanamgra extract was administered to Alzheimer's disease mice at a concentration of 500 mg / kg / day and a control group that was not administered were prepared. The brain was removed from the specimen, fixed with 4% paraformform aldehyde, and then a 40 μm thick brain section was prepared. The brain sections prepared as described above were immunostained with Bam-10 antibody capable of detecting β-amyloid deposition, and immunostained with Bam-10 from the cerebral cortex of control group mice and experimental group mice. The ratio to the area was analyzed (FIGS. 9a-9c).

  FIG. 9a is a photograph showing the result of immunostaining for β-amyloid in the brain of an animal model of an Alzheimer disease-onset mouse to which Canamgra extract is not administered, and FIG. 9b is Alzheimer to which Canamgra extract is administered. FIG. 9c is a photograph showing the result of immunostaining for β-amyloid in the brain of an animal model of a disease-causing mouse, and FIG. 9c shows immunity against β-amyloid by administration of a canamgra extract in the brain of an animal model of an Alzheimer's disease-causing mouse. It is a graph which shows the result of having compared the change of a dyeing | staining level. As shown in FIGS. 9 a to 9 c, it was confirmed that the area of β-amyloid deposited area in the cerebral cortex was significantly reduced by administration of the extract of Kanamura.

Example 4-5: Verification of tau protein hyperphosphorylation in the brain In an animal model of Alzheimer's disease-onset mice, it was confirmed whether or not tau protein characteristic of Alzheimer's disease was changed by administration of hyperphosphorylated Kanamgra extract. I tried to do it. Roughly, the brain section prepared in Example 4-4 was subjected to immunofluorescence staining using AT8 antibody capable of detecting phosphorylated tau protein, and cerebral cortex of control group mice and experimental group mice. Were photographed with a fluorescence microscope, and the fluorescence values were quantitatively analyzed (FIGS. 10a to 10c).

  FIG. 10a is a photograph showing the result of immunostaining for phosphorylated tau protein in the brain of an animal model of an Alzheimer disease-onset mouse that has not been administered Kanamgra extract, and FIG. FIG. 10c is a photograph showing the result of immunostaining for phosphorylated tau protein in the brain of an animal model of Alzheimer's disease-onset mice administered, and FIG. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to the phosphorylated tau protein by administration of a thing. As shown in FIGS. 10a to 10c, it was confirmed that the hyperphosphorylation of tau protein in the cerebral cortex is significantly reduced by administration of the extract of Kanamura.

Example 4-6: Analysis of Specific Inflammatory Response in the Brain An attempt was made to confirm the effect of Kanamura extract on the inflammatory response shown in the cerebral cortex of an animal model of an Alzheimer disease-onset mouse.

Example 4-6-1: Effect on Levels of Activated Microglia Cells Iba-1 (ionized specifically binding to activated microglia cells was targeted on the brain section prepared in Example 4-4. Immunostaining was performed using calcium-binding adapter molecule 1) antibody, and the levels of microglia activated in the cerebral cortex of control group mice and experimental group mice were measured and then compared (FIGS. 11a to 11c). .

  FIG. 11a is a photograph showing the result of immunostaining for activated microglia cells in the brain of an animal model of an Alzheimer disease-onset mouse that has not been administered Kanamgra extract, and FIG. Fig. 11c is a photograph showing the result of immunostaining for activated microglia cells in the brain of an animal model of an Alzheimer's disease-onset mouse, and Fig. 11c shows the extraction of Canamula in the brain of an animal model of an Alzheimer's disease-onset mouse. It is a graph which shows the result of having compared the change of the immuno-staining level with respect to the activated microglia cell by administration of a thing. As shown in FIGS. 11 a to 11 c, it was confirmed that the level of microglia cells activated in the cerebral cortex was significantly reduced by administration of the extract of Kanamura.

Example 4-6-2: Effect on the level of activated astrocytes The target brain slice prepared in Example 4-4 specifically binds to activated astrocytes. Immunostaining was performed using Glial fibrillary acidic protein (GFAP) antibody, and the level of astroglial cells activated in the cerebral cortex of control and experimental mice was measured. Comparison was made (FIGS. 12a-12c).

  FIG. 12a is a photograph showing the results of immunostaining for activated astrocytes in the brain of an animal model of an Alzheimer disease-onset mouse that has not been administered Kanamgra extract, and FIG. 12c is a photograph showing the result of immunostaining for activated astrocytes in the brain of an animal model of an Alzheimer's disease-onset mouse to which a product is administered, and FIG. 12c is the brain of the animal model of an Alzheimer's-onset mouse FIG. 3 is a graph showing the results of comparing changes in the immunostaining level for activated astrocyte glia cells by administration of Kanamura extract. As shown in FIGS. 12a to 12c, it was confirmed that the level of astroglial cells activated in the cerebral cortex was remarkably reduced by administration of the extract of Kanamura.

Example 4-6-3: Effect on Inflammatory Cytokine Levels Inflammation known to be secreted by activated microglia cells and astrocyte cells from the brains of mice excised in Example 4-4 Sex cytokine (TNF-α, IL-6 and IL-1β) levels were measured by real-time PCR and compared (FIGS. 13a-13c). As a comparison group (Non-Tg), a brain extracted from a normal mouse to which nothing was administered was used, and as a control group, a brain extracted from an animal model of an Alzheimer disease-onset mouse to which nothing was administered. The brain extracted from an animal model of Alzheimer's disease-onset mice to which the extract of canamula was administered was used as an experimental group (canamgra).

  FIG. 13a is a graph showing the results of comparing the mRNA levels of TNF-α expressed in the brain by administration of the extract of Kanamura, and FIG. 13b shows IL- expressed in the brain by administration of the extract of Kanamura. Fig. 13c is a graph showing the result of comparing the mRNA levels of IL-1β expressed in the brain by administration of Kanamura extract. As shown in FIGS. 13a to 13c, the expression level of various inflammatory cytokines increased rapidly in the brain of the animal model of Alzheimer's disease-onset mice compared to the brain of normal mice. It was confirmed that the inflammatory cytokine level decreased.

  Therefore, it was found that Kanamura extract can suppress the inflammatory reaction induced at the time of Alzheimer's disease onset.

  Summarizing the results of Examples 4-1 to 4-6, Kanamura extract suppresses β-amyloid deposition in the cerebral cortex and hyperphosphorylation of tau protein that occurs at the onset of Alzheimer's disease, and suppresses inflammatory responses in the brain Therefore, it was confirmed that Kanamura extract has a therapeutic effect on Alzheimer's disease.

Example 5: Therapeutic effect of Kanamgra extract on Huntington's disease In order to verify the efficacy of Kanamgra extract on degenerative brain disease, Kanamgra was used in animals in which the onset of Huntington's disease, a type of degenerative brain disease, was induced. An attempt was made to verify the therapeutic effect of the extract.

  Toxic substances that damage brain cells in order to analyze behaviorally whether motor dysregulation induced by degenerative brain diseases such as Huntington's disease can be improved by administering Kanamgra extract 3-nitropropionic acid (3-NP), known as, was administered to experimental animals to induce Huntington's disease, and abnormal behavior was analyzed from a behavioral aspect.

  Specifically, C57BL / 6 male J mice aged 9 weeks were used as experimental animals, and the experimental animal group had a control group fed with a diet containing 0.5% CMC (carboxy methylcellulose) and a day. Tests were divided into experimental groups given 500 mg / kg of kanamgra extract. At this time, the number of individuals in the control group and the experimental group was 6 each, and 0.5% CMC and 500 mg / kg Kanamgra extract were extracted 5 days before administration of 3-NP that induces Huntington's disease. From the start of administration, the administration was continued until the time of drug administration. In both groups of experimental animals, 3-NP was administered twice at an interval of 12 hours at a concentration of 60 mg / kg, and again 12 hours later, it was again administered intraperitoneally at a concentration of 80 mg / kg. Evaluation was made through behavioral analysis (Figure 14). At this time, the behavioral evaluation was performed by observing behaviors such as contraction of the hind legs, kyphosis, abnormal tension of the hind legs, shaking the body on both sides, twisting the upper body in one direction, and the highest score for each action expression. Was evaluated by adding each score.

  FIG. 14 is a graph showing the results of behavioral analysis by administration of canamgra extract in mice in which Huntington's disease was induced. As shown in FIG. 14, it was confirmed that the mice administered with Kanamura extract had a statistically significant decrease in behavioral evaluation score.

As described above, it was confirmed that the canamgra extract has an improving effect on the behavioral abnormal symptoms shown at the onset of Huntington's disease, and thus it has been found that the canamgra extract has a therapeutic effect on Huntington's disease.

Claims (10)

A pharmaceutical composition for preventing or treating degenerative brain disease, comprising an extract of humulus japonicus or a fraction thereof as an active ingredient ,
The degenerative brain disease is a pharmaceutical composition, which is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, Huntington's disease and combinations thereof .   The composition according to claim 1, wherein the canamgra extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and combinations thereof.   The fraction is obtained by a method selected from the group consisting of the solvent fractionation method, the ultrafiltration fractionation method, the chromatography fractionation method, and a combination thereof with respect to the Kanamura extract. The composition of claim 1.   The composition according to claim 1, wherein the composition has an effect of suppressing death of dopaminergic neurons.   The composition according to claim 1, which has a nerve cell protective effect against oxidative stress.   The composition according to claim 1, wherein the composition has an effect of improving cognitive ability and memory ability.   2. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier. Prevention or treatment of degenerative brain diseases comprising administering the pharmaceutical composition of any one of claims 1-7 to an individual degenerative brain disease was developed except between humans in a pharmaceutically effective amount A method ,
The method wherein the degenerative brain disease is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, Huntington's disease and combinations thereof . A health functional food for preventing or ameliorating degenerative brain disease, comprising Kanamura extract or a fraction thereof as an active ingredient ,
The degenerative brain disease is a health functional food for improvement, which is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, Huntington's disease and combinations thereof . A feed composition for preventing or ameliorating degenerative brain disease comprising Kanamura extract or a fraction thereof as an active ingredient ,
The degenerative brain disease is a feed composition for improvement, which is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, Huntington's disease and combinations thereof .