KR20160008042A - Compostion for preventing or treating the neurodegenerative disease comprising Humulus japonicus extract as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain disease, containing a Humulus japonicus extract or a fraction thereof and, more specifically, to a pharmaceutical composition for preventing or treating degenerative brain disease or a health functional food for preventing or alleviating the degenerative brain disease, containing a Humulus japonicus extract or fraction thereof which is effective in inhibiting dopaminergic neuronal cell death, protecting neuron against oxidative stress, and improving cognitive abilities and memory. The composition containing a Humulus japonicus extract or a fraction thereof inhibits dopaminergic neuronal cell death, protects neuron against oxidative stress, and improves cognitive abilities and memory in an animal model having the degenerative brain disease such as Parkinson′s disease, Alzheimer′s disease, etc., thereby being able to be effectively used as a food or a drug for preventing or treating the degenerative brain disease including Alzheimer′s disease, Parkinson′s disease, Lou Gehrig′s disease, mild cognitive impairment, a stroke, Huntington′s disease, etc.

Description

[0001] The present invention relates to a composition for preventing or treating a degenerative brain disease comprising an extract of Huangshan japonica as an active ingredient,

The present invention relates to a method for the production of Humulus The present invention relates to a pharmaceutical composition for preventing or treating a degenerative brain disease comprising, as an active ingredient, an extract or a fraction thereof. More particularly, the present invention relates to a pharmaceutical composition for inhibiting dopaminergic neuronal cell death, The present invention also relates to a pharmaceutical composition for preventing or treating a degenerative brain disease comprising an extract of Hymenoptera or a fraction thereof having an effect of improving cognitive ability and memory, and a health functional food for prevention or improvement.

In modern life, memory is becoming increasingly important in a rapidly changing life, and it has become a major concern from young people with a lot of social skills to old age. Degenerative brain diseases are known to lead to memory decline and oxidative stress has been shown to be an important factor in degenerative brain diseases of the central nervous system such as Alzheimer's syndrome, Parkinson's syndrome, and Huntington's syndrome (Jin DQ et al, Biochemical and Biophysical Research Communications , 331: 1264-1269, 2005; Lim CS et al, Biological and Pharmaceutical Bulletin , 29: 1212-1216, 2006).

In recent years, the number of patients with degenerative brain disease such as dementia has increased due to an increase in the elderly population. Efforts have been made to develop various therapeutic strategies to improve and improve the cognitive function and learning function deteriorated due to dementia and to develop effective drugs have. There are acetylcholine precursors, receptor agonists, acetylcholine esterase inhibitors, and so on. However, there have not yet been developed therapeutic agents capable of treating the underlying cause of degenerative brain diseases. Common therapies that can be used as therapeutic agents include Aricept of Pythagoras, Exelon of Novartis, Reminyl and Ebixa (Memantine) from Lundbeck, a recent antagonist of the FDA-approved N-methyl-D-aspartate receptor. However, the acetylcholine degradation inhibitor improves the declining cognitive ability and limits the fundamental cause of Alzheimer's disease. In addition, it is known that in some patients only a temporary symptomatic relief effect is obtained, and its therapeutic effect is not long-lasting, so it is difficult to expect a fundamental therapeutic effect. In addition, due to the nature of the degenerative brain disease, long-term use of the drug is required, and the above-mentioned medicines have problems such as liver toxicity, vomiting, loss of appetite and various side effects. Therefore, it is urgent to develop a new therapeutic agent that can prevent the progress of degenerative brain diseases. To this end, many multinational pharmaceutical companies have invested heavily in research and development in this field. In particular, beta or amyloid, which reduces the amount of β-amyloid composed of 40 amino acids, which is presumed to be a fundamental cause of Alzheimer's disease, The development of gamma secretase inhibitors is predominant. In Korea, basic research on Alzheimer's disease has been done to some extent, but the development of dementia treatment itself is rarely done.

Recently, in Oriental medicine, studies on the prescription of herbal medicine as a treatment for Alzheimer's disease among degenerative brain diseases are actively conducted. Through these studies, it has been found that the prescription of Sangmyung, Gyungwangbomindan, Guvitang, etc. Has a certain effect on the treatment of Alzheimer 's disease. However, the effect of preventing or treating degenerative brain diseases of the hornblende is not known.

Under these circumstances, the present inventors have recently found that a safe drug which can effectively inhibit the onset and deterioration of degenerative brain diseases such as Parkinson's disease and Alzheimer's disease, which is increasingly prevalent in modern humans, and which has no side effects for treating such diseases, As a result of intensive researches to develop a preventive or therapeutic agent for a new degenerative brain disease using a substance which does not show toxicity to human body, the extract of Hwangsam Vine inhibits dopaminergic neuronal cell death and has a protective effect of neurons against oxidative stress , Parkinson's disease, and Alzheimer's disease, and thus it can be used for prevention or treatment of degenerative brain diseases. Thus, the present invention has been completed.

One object of the present invention is to provide a method of treating humulus The present invention also provides a pharmaceutical composition for preventing or treating degenerative brain diseases comprising, as an active ingredient, an extract or a fraction thereof.

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

The present inventors to develop a material that can effectively inhibit the onset and exacerbation of degenerative brain diseases, while trying to perform a variety of studies in a highly safe for the human body natural products, Humulus japonicus (Humulus japonicus ) extracts. It was confirmed that the extract of Hwangsam was inhibited dopaminergic neuronal cell death and had a protective effect on neuronal cells against oxidative stress. In addition, cognitive ability and memory improvement were shown in animal models with degenerative brain diseases such as Parkinson's disease and Alzheimer's disease.

Thus, the Humulus japonicus (Humulus japonicus extract can be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, Huntington's disease and the like.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease comprising an extract of Humulus japonicus or a fraction thereof as an active ingredient.

The term in the invention, "Humulus japonicus (Humulus japonicus "belongs to Cannabaceae and is an annual vine plant which is distributed throughout Korea and in East Asia. The stem of the stem is used as fiber, the fruit is used as an antiseptic, the fruit is used as a diuretic However, it has not been disclosed about the use of treatment or prevention of degenerative brain disease-related diseases and the like, and the inventors of the present invention firstly include Alzheimer's, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, Huntington's disease and the like The present invention provides a method of treating or preventing a degenerative brain disease-related disease. In the present invention, the horseradish peroxidase can be commercially purchased, or harvested or cultivated in nature.

The term "extract " in the present invention means a liquid component obtained by immersing a desired substance in various solvents and then extracting the mixture at room temperature or in a heated state for a certain period of time, a liquid component obtained by removing the solvent from the liquid component And the like. In addition, in addition to the above-mentioned results, the present invention can be broadly interpreted as including the diluted solution, the concentrate thereof, the controlled preparation thereof, and the purified product thereof.

In the present invention, the extract is a hull of Humulus japonicus ) extract. The hornblende extract can be extracted from various organs of natural, hybrid, and variant plants and can be extracted from plant tissue culture as well as root, top, stem, leaf, flower, fruit body, . The hornblende extract can be obtained by extraction with water or various organic solvents. In this case, the organic solvent to be used is not particularly limited as long as it can obtain an extract having an effect of preventing or treating degenerative brain diseases, but preferably it may be water, a polar solvent or a non-polar solvent, Water, a lower alcohol having 1 to 4 carbon atoms (such as methanol, ethanol, propanol or butanol), or a mixed solvent thereof, and most preferably methanol or a mixed solvent thereof. Also, the method for obtaining the above extract is not particularly limited as long as it is capable of obtaining an extract having an effect of preventing or treating degenerative brain diseases. Preferably, however, the roots, stems, leaves, fruits, flowers , A frozen extraction method in which a dried product or a processed product thereof is immersed in the solvent and extracted at a room temperature of 10 to 25 ° C, a heating extraction method in which heating is performed at 40 to 100 ° C, an ultrasonic extraction method in which ultrasonic waves are applied, And a reflux extraction method.

The term "fraction " in the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents.

In the present invention, the fractions can be interpreted as fractions obtained by applying the extract of Hornblende to various fractionation methods. The fraction of the extract can be obtained by applying the extract to various fractionation methods. The fractionation method is not particularly limited, but may be a solvent fractionation method which is carried out by treating various solvents, an ultrafiltration membrane having a constant molecular weight cut- , A chromatographic fractionation method in which various chromatographies (which are prepared for separation according to size, charge, hydrophobicity or affinity) are performed, and the like. 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 a nonpolar solvent can be preferably used. The solvent fractionation method may be carried out by sequentially fractionating the extract from a solvent having a high non-polarity level using a low solvent. For example, a method of sequentially fractionating the extract using nucleic acid or ethyl acetate may be used .

The term "degenerative brain disease" in the present invention is a disease that occurs in the brain of a degenerative disease caused by age, including but not limited to Alzheimer's, Parkinson's disease, Lou Gehrig's disease, Mild cognitive impairment, stroke, Huntington's disease, and combinations thereof.

Degenerative brain diseases are known to cause neuronal death due to protein aggregation due to neurodegeneration and genetic and environmental factors due to aging, but the exact cause is unknown yet.

The term "prevention" in the present invention means any action that inhibits or delays the onset of degenerative brain disease by administration of the pharmaceutical composition according to the present invention, and "treatment" Suspicion of brain disease, and any behavior that alleviates or alleviates symptoms of an onset individual.

In the present invention, the prevention or treatment of the degenerative brain disease can be achieved by inhibiting dopaminergic neuronal cell death by the extract, having a protective effect on neuronal cells against oxidative stress, and behaviorally treating Parkinson's disease and Alzheimer's disease May be achieved by exhibiting cognitive ability and memory improving effect in animal models with degenerative brain diseases such as diseases.

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 neuronal cell death" in the present invention refers to the disappearance or denaturation of dopaminergic neurons densely packed in the substantia nigra pars compacta of the midbrain, 6-OHDA (6-hydroxyldopamine) is known to be able to be manufactured by injecting.

The term "oxidative stress" in the present invention is an important factor that causes degenerative brain diseases. When the active oxygen is increased in the body, oxidative stress is induced, and the oxidative stress causes brain cell loss and Parkinson & It is known that degenerative brain diseases such as Alzheimer's disease can be induced.

As described above, Humulus japonicus (Humulus provided by the present invention japonicus extract or its fraction inhibits dopaminergic neuronal cell death and has protective effects on neuronal cells against oxidative stress as well as inhibition of neuronal cell death in animal models with degenerative brain diseases such as Parkinson's disease and Alzheimer's disease Ability and memory can be improved.

According to one embodiment of the present invention, apomorphine-induced rotational behavior in an animal model of Parkinson's disease caused by the dopamine neuronal cell death of brain with 6-OHDA following treatment with the extract of the present invention , And the rotational behavior was reduced by the extract (Fig. 1). In addition, it was confirmed that the expression amount of tyrosine hydroxylase (TH), a dopaminergic neuron specific protein, was significantly inhibited by treating the extract (FIG. 2). In addition, it was confirmed that the cognitive function improvement (FIG. 3) and the spatial perception ability and the short-term memory reduction in the Y-maze were improved in a mouse animal model with Alzheimer's disease (FIG.

On the other hand, it was confirmed that the treatment of the extract of the present invention inhibits the production of active oxygen species induced in nerve cells, thereby protecting the nerve cells against oxidative stress (FIG. 5).

Thus, the Humulus japonicus (Humulus japonicus extract or its fractions exhibit the effect of preventing or treating degenerative brain diseases including Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, Huntington's disease and the like.

The pharmaceutical compositions for the prevention or treatment of degenerative brain diseases of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension agents, solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Said Humulus japonicus (Humulus included in the pharmaceutical composition of the present invention The content of the extract or fraction thereof of japonicus is not particularly limited, but may be in the range of 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. And the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, The duration of the treatment, the factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition of the present invention is preferably, for example, 0.1 to 500 mg / kg of body weight per day for a day to a mammal including a human. The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

In another aspect of the present invention, the present invention provides a method for preventing or treating degenerative brain diseases, which comprises the step of administering the pharmaceutical composition to a subject having or likely to have a degenerative brain disease Or a method of treatment.

As described above, the Humulus japonicus (Humulus provided by the present invention japonicus or fractions thereof can be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of degenerative brain diseases, so that the composition can be used for preventing or treating degenerative brain diseases.

The term "individual" of the present invention includes, without limitation, mammals including rats, livestock, humans, and the like, who have or are at risk of developing degenerative brain diseases.

In the method of treating the degenerative brain disease of the present invention, the administration route of the pharmaceutical composition may be administered through any conventional route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered orally, intraperitoneally, intramuscularly, subcutaneously, intradermally, orally, intranasally, intracorporally, rectally, or the like depending on the purpose ≪ / RTI > However, the Humulus japonicus (Humulus by gastric acid when administered orally japonicus or fractions thereof may be denatured, the oral composition should be formulated so as to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

In another aspect of the present invention, there is provided a health functional food for preventing or ameliorating a degenerative brain disease comprising an extract of Humulus japonicus or a fraction thereof.

As described above, the hornblende can be added to health functional foods for the purpose of preventing or improving degenerative brain diseases.

When the composition of the present invention is used as a health functional food additive, the composition may be added as it is or may be used together with other health functional foods or health functional food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use. Generally, the composition of the present invention may be added in an amount of preferably not more than 15 parts by weight, more preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term intake intended for health control and hygiene, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, Alcoholic beverages, and vitamin complexes, and may include all the health functional foods in the conventional sense, and foods used as feeds for animals.

In addition, when the health functional food composition of the present invention is used in the form of a drink, it may contain various sweetening agents, flavoring agents, or natural carbohydrates as additional components such as ordinary beverages. The natural carbohydrates may be polysaccharides such as disaccharides such as monosaccharides such as glucose and fructose, maltose, sucrose, dextrin, cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be about 0.01 to 0.06 g, more preferably 0.03 to 0.05 g, per 100 ml of the composition of the present invention. The sweeteners may be natural sweeteners such as tau martin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

Humulus japonicus according to the present invention (Humulus japonicus extract or a fraction thereof as an active ingredient inhibits dopaminergic neuronal cell death and has a protective effect on nerve cells against oxidative stress as well as in an animal model with degenerative brain diseases such as Parkinson's disease and Alzheimer's disease Cognitive ability and memory, it can be effectively used as a food or medicine for the prevention or treatment of degenerative brain diseases including Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, and Huntington's disease.

FIG. 1 is a graph showing the results of apomorphine-induced rotational movement in an animal model of Parkinson's disease caused by dopaminergic neuron-specific cell death of brain with 6-OHDA according to the treatment of Rhodia japonica extract.
FIG. 2 is a graph showing the results of measurement of the expression level of tyrosine hydroxylase (TH), a dopaminergic neuron specific protein, according to the treatment of Rhodia japonica extract.
FIG. 3 is a graph showing the effect of improving the cognitive function and memory ability in a novel object recognition test (NORT) of a new object of the hornblende extract according to the present invention in a mouse animal model with onset of Alzheimer's disease.
FIG. 4 is a graph showing the spatial perception ability and the short-term memory depression improvement effect in the Y-maze test of the Horseradock extract according to the present invention in an animal model of mice with Alzheimer's disease.
FIG. 5 is a graph showing the results of behavioral analysis in an animal model of Huntington's disease caused by damaging brain cells with 3-NP according to the treatment of the Hymenoptera.
FIG. 6 is a photograph and a graph showing the effect of inhibiting the production of reactive oxygen species induced by neuronal cells, according to the treatment of Rhodia japonica extract.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Preparation of Hwangsam Vine Extract

The extracts obtained by pulverizing or cutting the dried hull gnaths were subjected to 10 days of incubation for 2 hours at room temperature by sonication for 15 minutes, filtered and concentrated. Later, the frozen sample was obtained by drying in a freeze dryer in a deep freezer. The above extracts of Solanum tubercanifolia were obtained by dissolving in 0.5% CMC (carboxy methylcellulose) solution and DMSO (dimethyl sulfoxide) for use in animal experiments and neural cell line experiments.

Example  2: Efficacy of Hansampigin extract for Degenerative Brain Disease

In order to examine the efficacy of Hansampigin extracts against degenerative brain diseases, the efficacy of Hanssemark extract was examined by using an animal model of Parkinson 's disease and mouse animal model of Alzheimer' s disease.

Example  2-1: 6- OHDA (6- hydroxyldopamine ) To induce Parkinson's disease induction group

Male C57BL / 6J mice at 9 weeks of age were used as experimental animals. The mice were allowed to freely consume sterilized feed and water in a specific pathogen free (SPF) environment maintained at a temperature of 22 to 24 ° C, and were maintained in a 12-hour day / night cycle.

Experimental animals were divided into two groups: a control group with 0.5% CMC (carboxy methylcellulose) and an experimental group with 500 mg / kg of Hansampigin extract. In this study, 5 and 8 animals were used in the control and experimental groups, respectively. The extracts of 0.5% CMC and 500 mg / kg of Hamsang Rhizoma extract were administered with 6-OHDA which can specifically uptake dopaminergic neurons They started to feed three days before.

The mouse was anesthetized with a ketamine and rumpun mixed drug. Experiments were performed to investigate the effect of 70 ~ 80% of the brain area on the progression of early Parkinson's disease, 6-OHDA was injected directly into the brain. To prevent noradrenergic neurons from being destroyed, 25 mg / kg of desipramine was given intraperitoneally 30 minutes before the administration of 6-OHDA, and a total of 6 ug of 6-OHDA was injected into the striatum of the left brain (Brain microinjection coordinates: forward + back +1.3, left-right -1.8, depth -3.6). As described above, 6-OHDA was directly administered to the brain, and the surgical site was closed and disinfected. Thereafter, the body temperature of the mice was maintained at 37 ° C in a warmer.

After the induction of Parkinson's disease by the above-described surgical method, a behavioral test such as an apomorphine-induced rotational test and the expression of tyrosine hydrotylase (TH), a dopaminergic neuron specific protein, 0.5% CMC and 500 mg / kg of hornblende extract were added to the control group and the experimental group, respectively, until the amount of the extract was measured. At this time, oral administration of 0.5% CMC and 500 mg / kg hornblende extract were administered for two days after the operation, which was difficult to take food.

Example  2-2: Behavioral assessment using Parkinson's disease-induced animal model

It is known that the more the dopaminergic cell death by 6-OHDA is, the more brain lesions become severe and the number of rotations is increased in the experimental animal model. After 8 days of 6-OHDA administration, 1 mg / kg of apomorphine was intraperitoneally injected into mice to evaluate the severity of exercise control ability by dopaminergic cell death, and asymmetric rotational behavior was observed .

Specifically, after administration of the above-mentioned apomorphine drug, mice were placed in a cylinder having a diameter of 20 cm, and the number of rotations in a clockwise direction for one hour was measured to evaluate the rotational behavior.

As a result, it was confirmed that the rotational behavior of the experimental animals was significantly reduced in the experimental group receiving the 500 mg / kg of the hanam extract (FIG. 1), as compared with the control group consuming the 0.5% CMC.

Example  2-3: Tyrosine hydroxylase in Parkinson's disease-induced animal models tyrosine  hydroxylase, TH ) ≪ / RTI >

In order to confirm the inhibition of dopaminergic neuronal cell death by 6-OHDA, 10% of 6-OHDA-treated mice were given 0.5% CMC control and 500 mg / In the striatum of the striatum, the difference in the expression level of the dopaminergic neuron - specific protein, TH, was analyzed by Western blot analysis.

As a result, it was confirmed that the dopamine neuronal cell death was significantly inhibited by the administration of the hornblende extract (FIG. 2) by confirming that the TH protein expression level of the test group fed with the hornblende extract was higher than that of the control group.

Therefore, based on these results, it was confirmed that the extract of Hwangsam Vinegalum has the preventive and therapeutic effect of Parkinson 's disease.

Example  2-4: Experimental group production of animal model mice with Alzheimer's disease

As an experimental animal, a mouse animal model in which the APPswe gene and the PSEN1 gene, which are genes related to Alzheimer's disease, were overexpressed in the brain of a mouse and the onset of Alzheimer's disease was purchased from Baxter Company of USA (B6C3-Tg (APPswe / PSEN1dE9) 85DboJ, JAX, 004462). The mouse has marked beta amyloid deposits in the brain at 6 months of age and is characterized by an Alzheimer-specific cognitive dysfunction.

Specifically, the mouse was obtained by crossing C57BL / 6J and C3H with F1 heterozygous mouse of APP / PSEN1 overexpressing mouse. After genotyping analysis, APP and PSEN1 gene (APP / PSEN1 overexpressing mouse) and normal mice were used for the experiment.

The mice were allowed to freely consume sterilized feed and water in a specific pathogen free (SPF) environment maintained at a temperature of 22 to 24 ° C, and were maintained in a 12-hour day / night cycle.

The experimental animal groups were tested by dividing into a group of non-Tg mice (n = 20) that do not overexpress APP / PSEN1 and a group of mice that developed AD / PSEN1 overexpressing Alzheimer's (n = 22) 5% CMC, and 500 mg / kg of Hamsangvin extract. The control group and the experimental group of the Alzheimer-caused mouse group overexpressing the APP / PSEN1 were carried out using 10 and 12 mice, respectively. At that time, the mouse group was used at 5 months of age, and oral administration of 0.5% CMC and 500 mg / kg of Hansampigin extract for 10 weeks.

Example  2-5: Cognitive testing of new things using an animal model of Alzheimer's disease novel object recognition test ; NORT )

A novel object recognition test (NORT) was performed to confirm the cognitive ability and memory enhancing effect of Horseshoe vinegar extract on Alzheimer 's cognitive impairment.

Specifically, 5% CMC and 500 mg / kg of Hamsanvolume extract were administered to each test group for 8 weeks as shown in Example 2-4, and then subjected to NORT. On the first training day, the mice were placed in a white box of 41.5 cm x 20 cm x 21.5 cm and allowed to move freely for 10 minutes. After the 10-minute adaptation period as described above, the mice were returned to the original cage. On the second day, two cylindrical blocks of the same cylindrical shape were placed on both sides of the box, and the mice were exposed for 10 minutes so that they could be detected. Twenty-four hours after this, a cylindrical block (a new object) with a square column was put together with the cylindrical block (the familiar object) which was originally seen, and the movement of the mouse was observed. At this time, the sniffing time was measured by touching the block, sniffing or moving towards the block. For both blocks, the time of interest in the cylindrical block (the familiar object) and the search time of interest in the square column block (the new object) were measured at the total measured number of times (FIG. 3).

Experimental results showed that, in the case of the non-Tg mouse group not overexpressing APP / PSEN1, regardless of the administration of the hornblende extract, it is possible to recognize both the square block as a new object and the cylindrical block as a familiar object On the other hand, in the untreated control group of Alzheimer's disease-overexpressing mice with overexpression of APP / PSEN1, the search time for a new object was remarkably reduced due to Alzheimer's cognitive impairment, and the distinction between a familiar object and a new object Respectively.

On the other hand, in the treatment group of Alzheimer's disease-overexpressing mouse with APP / PSEN1 overexpression, the interest in the new substance was much higher than that in the untreated control group of Alzheimer's disease-overexpressing mice with APP / PSEN1 overexpression , It was confirmed that the distinction between the familiar object and the new object was clearly good. In addition, it was confirmed that the increase in the search behavior in this animal of Alzheimer ' s disease was similar to that of normal group mice.

The search time is a time of interest in a familiar object / time of interest in a familiar object + time of interest in a new object × 100 (time of interest in a new object) / (interest in a familiar object) Time shown + time showing interest in new object) × 100.

From the above results, it was found that the horseradish peroxidase extract was effective in the treatment of Alzheimer's disease. In addition, the group administered with the Hanssemark extract of the non-Tg mouse group that did not overexpress APP / PSEN1, , It was found that the extract of Hwansamsun mulberry extract also improved the cognitive function and memory.

Example  2-6: Y-maze test using an animal model of Alzheimer's disease (Y maze test )

Y maze test was performed to confirm the spatial perception ability and memory enhancing effect of the hornblende extract on the loss of spatial perception ability and memory ability caused by Alzheimer's disease.

Specifically, the Y-maze test was conducted to investigate whether it could help the recovery of the spatial perception ability and short-term memory capacity of experimental animals. cm, length 40 cm, height 25 cm), and each maze is arranged at a constant angle of 120 ° with respect to each other. The test was conducted for 10 minutes. After setting each of the labyrinths as A, B, and C regions, the lab was placed in one area and the experiment was started to freely search the labyrinth. At this time, the number and order of entry into each maze were measured to evaluate the spontaneous alteration (%). One point (actual alteration, ABC, BCA, CAB, etc.) was recognized as a sequential entry into three different areas, but not consecutively.

The spontaneous alteration (%) was calculated by total alteration number / (total number of entry - 2) × 100.

As a result of the Y-maze test, it was found that in the case of the test group in which Alzheimer's overexpressing APP / PSEN1-overexpressing mice were treated with the horseradish peroxidase extract for 9 weeks, the APP / PSEN1-overexpressing Alzheimer- (Fig. 4). As shown in Fig.

From the above results, it was confirmed that the hornblende extract is effective for the treatment of Alzheimer's disease by confirming the improvement of the spatial perception ability and the short-term memory reduction by Alzheimer's disease.

Example  2-7: Huntington Disease  Production and behavioral analysis of animal models

In order to conduct a behavioral analysis to evaluate whether exercise control disorder caused by degenerative brain diseases such as Huntington's disease is improved by administering the extract of Hwangsan-vine, 3-nitropropionic acid (3 -nitropropionic acid (3-NP) was administered to experimental animals to induce Huntington's disease and behavioral aspects of abnormal behavior were analyzed.

Specifically, 9-week-old male C57BL / 6J mice were used as experimental animals. The experimental animals were divided into two groups: a control group fed with 0.5% CMC (carboxy methylcellulose) and a group fed with 500 mg / Respectively. At this time, the control group and the experimental group were carried out by using 6 rats, respectively. The 0.5% CMC and 500 mg / kg ratshunter extract were fed 5 days before 3-NP inducing Huntington's disease, Of the population. 3-NP was administered to the two groups of animals at a dose of 60 mg / kg twice at intervals of 12 hours, followed by intraperitoneal administration at a concentration of 80 mg / kg again after 12 hours. Were assessed through behavioral analysis.

Behavioral evaluation was performed by observing behaviors such as hind paw, vertebral hindlimb, hindlimb tension, wobbling of body on both sides, and swinging of upper body in one direction. The highest score of each behavior expression was 2 points, Respectively.

As a result of the behavioral analysis, it was confirmed that the behavioral evaluation score was statistically significantly reduced in the group administered with the hornblende extract (FIG. 5).

From the above results, it was found that the extract of Huangshan japonica has an effect of preventing and treating Huntington's disease.

Example  3: extract of hornblende Active oxygen species  Verification of production inhibition effect

In order to investigate the protective effect of Hwangsan-japonicus extract against neurons in the induction of neurotoxicity induced by active oxygen species, which is known to be a major risk factor of brain neurological diseases, t-BHP (tert-butylhydroperoxide) Causing oxidative neuronal damage.

Specifically, Neuro 2a cells were treated with 400 μg / ml of the extract of Neuros 2a and incubated for 1 hour. Then, 300 μM of t-BHP (tert-butylhydroperoxide) was treated to induce oxidative stress . Two hours after the induction of oxidative stress, in order to measure the reactive oxygen species (ROS) occurring in the cells, a medium containing 10 μM of DCF-DA (2 ', 7'-Dichlorofluorescein diacetate) Respectively. After the DCF-DA was treated and further cultured for 1 hour, the amount of active oxygen generated using a fluorescence microscope (FIG. 6A) and a luminometer (FIG. 6B) was analyzed.

As a result, it was confirmed that the production of reactive oxygen species was significantly increased by t-BHP in Neuro 2a neuronal cell line, and that the reactive oxygen species produced by t-BHP was significantly reduced by treatment with hornblende extract. In addition, it was confirmed that no active oxygen species were produced by the single treatment of the hornblende extract, and it was found that the hornblende extract itself did not induce oxidative stress and cytotoxicity (FIG. 6).

From the above results, it was confirmed that the production of reactive oxygen species by t-BHP in nerve cells was inhibited by the hornblende extract, and thus, the hornblende extract showed a protective effect against the oxidative stress of neurons.

Claims (9)

Humulus The present invention relates to a pharmaceutical composition for preventing or treating a degenerative brain disease comprising an extract or a fraction thereof as an active ingredient.
The method according to claim 1,
Wherein the extract is extracted using a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and combinations thereof.
The method according to claim 1,
Wherein said fraction is obtained by applying said Horseradish Vine Extract to a method selected from the group consisting of solvent fractionation, ultrafiltration fractionation, chromatographic fractionation and combinations thereof.
The method according to claim 1,
Wherein said degenerative brain disease is a disease selected from the group consisting of Alzheimer's, Parkinson's, Lou Gehrig's, mild cognitive impairment, stroke, Huntington's disease, and combinations thereof.
The method according to claim 1,
Wherein the composition has an inhibitory effect on dopaminergic neuronal cell death.
The method according to claim 1,
Wherein said composition has a neuroprotective effect on oxidative stress.
The method according to claim 1,
Wherein said composition has a cognitive ability and a memory improving effect.
The method according to claim 1,
Lt; RTI ID = 0.0 > pharmaceutically < / RTI > acceptable carrier.
Humulus japonicus extract or a fraction thereof as an active ingredient for preventing or ameliorating degenerative brain diseases.

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