KR20180130268A - Composition for preventing, alleviating and treating neurodegenerative diseases comprising Sargassum honeri extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases, and more specifically, relates to the pharmaceutical composition and a health functional food for preventing, ameliorating or treating the neurodegenerative diseases comprising sargassum horneri extract as an active ingredient. In order to achieve an object of the present invention, the present invention provides the pharmaceutical composition for preventing, ameliorating or treating neurodegenerative degeneracy comprising the sargassum horneri extract as the active ingredient.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating degenerative neurological diseases, which comprises extracts of horseshoe sauces as an active ingredient. (Composition for prevention, alleviating and treating neurodegenerative diseases comprising Sargassum honeri extracts)

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases, and more particularly, to a pharmaceutical composition and a health functional food for preventing, ameliorating or treating neurodegenerative diseases, .

Neurodegenerative Disease is a disease characterized by a decrease or loss of function of neurons, exercise control ability, cognitive function, sensory function, sensory function and autonomic dysfunction.

Recently, degenerative brain disease including senile dementia has become a social problem. Of these, cerebral vascular dementia due to Alzheimer's type dementia and extensive brain lesions caused by stroke or cerebral arteriosclerosis accounts for about 90% of all dementia diseases. Other cranial nerve diseases include Pick's disease, Croplets-Jacob disease, dementia caused by head injury, and Parkinson's disease.

One of the major mechanisms leading to degenerative brain disease is inflammatory reaction. The central nervous system of the brain is overlain by microglial cells, which form a line of defense against immune responses and bacterial invasion and wound. Microglia is a type of macrophage in the central nervous system that plays an important role in neuroinflammation and can be activated by various extrinsic and endogenous substances. Activated microglia are activated by the inflammatory cytokine TNF-a And IL-1 Nitric Oxide, prostaglandins, superoxide, and the like. The production of these substances induces an immune response in the short term, but excessive production or sustained production induces neuronal death of adjacent neurons, resulting in neural degeneration. In addition, these inflammatory mediators are regulated by mitogen-activated protein kinases (MAPKs). MAPKs are not only responsible for cellular responses to stimuli such as stress, mitosis-stimulating factors or proinflammatory cytokines, but also gene expression, differentiation and proliferation There is an association that regulates cellular functions including. In addition, MAPK has three major signaling stages, p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and p38 MAPK has been shown to produce high cytokines in the inflamed central nervous system ≪ / RTI >

In this way, microglial cells are present in the central nervous system (CNS) and brain and are involved in the protection and recovery of primary neurons. Activated microglia induce neuronal inflammatory responses, Many studies have been reported to lead to the cause of degenerative diseases. Microglial cells are activated by brain damage and various neurotoxins. Activated microglia are activated by nitric oxide (NO), reactive oxygen species (ROS), iNOS (inducible nitric oxide synthase) and COX IL-6, TNF-a, and IL-2 (cyclooxygenase-2) and proinflammatory cytokines (Graeber and Streit, 2010). In addition, neuronal degeneration becomes a vicious circle because the substances released by the dead neurons cause the microglial cells to re-activate. In fact, the activity of microglial cells has been reported to be related to various degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis (Wood PL, et al., 17 Neurol. 242, 1995).

Currently, benzimidazole, which inhibits the activation of microglial cells, is involved in neuroinflammatory patients (AIDS, dementia, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Down's syndrome, diffuse RI somatization, Huntington's disease, , Pick's disease, Alzheimer's disease, stroke, seizure, and tumors). Although the relationship between activation of microglial cells and degenerative neurological diseases has not yet been fully elucidated, it is generally believed that the activity of microglial cells is involved in the development and progression of degenerative neurological diseases. Therefore, inhibition of inflammatory response of microglial cells is suggested to be an effective treatment for relieving the progress of neurodegenerative diseases.

Therefore, it is expected that the control of neuroinflammation plays an important role in the prevention of degenerative neurodegenerative diseases, so that it is necessary to search for and study a substance having neuronal cell protective effect or neuroinflammation inhibitory effect.

Sargassum honeri is a seaweed having various activities in the body. According to the previously reported studies, it has been known that hoe saengmyunggi prevents osteoporosis, regulates blood pressure by releasing cholesterol, and prevents hyperlipemia. And functional foods have been reported to have skin aging, anti-acne, antiallergic and whitening effects. However, the antinociceptive activity and the mechanism of antinociceptive activity against Hornsby moths have not yet been clarified.

The present inventors have completed the present invention by discovering new uses of Sargassum honeri extract.

Accordingly, an object of the present invention is to provide a method for preventing, ameliorating or treating neurodegenerative diseases, which can prevent, ameliorate, or treat various neuroinflammatory diseases, particularly degenerative neurological diseases, A pharmaceutical composition and a health functional food.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing, ameliorating or treating a degenerative neurological disease comprising an extract of Horns mellifera as an active ingredient.

In a preferred embodiment, the horns extract inhibits expression of one or more selected from the group consisting of iNOS, COX-2, TNF-a and IL-6 genes involved in inflammatory responses in neuronal macrophages.

In a preferred embodiment, the degenerative neurological disease is selected from the group comprising multiple sclerosis, allergic myelitis, Parkinson's disease, Alzheimer's and Huntington's disease.

Also, the present invention provides a health functional food having an effect of preventing and improving degenerative neurological diseases, comprising an extract of Horns mellifera as an active ingredient.

In a preferred embodiment, it is a powder, a granule, a tablet, a capsule or a drink.

In a preferred embodiment, the degenerative neurological disease is selected from the group comprising multiple sclerosis, Parkinson's disease, Alzheimer's and Huntington's disease.

The present invention also provides a method for treating a neurodegenerative disease, comprising the step of administering any of the above-described pharmaceutical compositions to a mammal other than a human.

In a preferred embodiment, the degenerative neurological disease is selected from the group comprising multiple sclerosis, allergic myelitis, Parkinson's disease, Alzheimer's and Huntington's disease.

The horns of the present invention inhibit the expression of i-NOS and COX-2 mRNA, as well as the expression of i-NOS and COX-2, through inhibition of the p38 signal with the NF-κB signal in microglial cells And can be usefully used as a pharmaceutical composition and a functional food material for preventing, alleviating, or treating various neurodegenerative diseases using the same.

FIG. 1 is a graph showing the cytotoxicity test results of mouse BV-2 cells treated with extract of Horns mellifera.
FIG. 2 is a graph showing the inhibitory effect of LPS-induced NO production in mouse BV-2 cells treated with Horns mellifera extract.
FIG. 3 is a photograph showing the effect of suppressing the expression of i-NOS and COX-2 induced by LPS in mouse BV-2 cells treated with extracts of horseshoe crab.
FIG. 4 is a photograph showing the effect of suppressing the expression of proinflammatory gene mRNA induced by LPS in mouse BV-2 cells treated with extract of Horns mellifera.
FIG. 5 is a graph showing the inhibitory effect of LPS-induced cytokine production in mouse BV-2 cells treated with extracts of horseshoe crab.
FIG. 6A is a photograph showing the effect of inhibiting phosphorylation of ERK and P38 induced by LPS in mouse BV-2 cells treated with extract of Horns mellifera, and FIG. 6B is a photograph showing inhibition effect of phosphorylation of p65 induced by LPS And Fig. 6C is a photograph showing the effect of suppressing the expression of i-NOS and COX-2 protein induced by LPS.

Although the terms used in the present invention have been selected as general terms that are widely used at present, there are some terms selected arbitrarily by the applicant in a specific case. In this case, the meaning described or used in the detailed description part of the invention The meaning must be grasped.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

The technical feature of the present invention is that the extract of horseshoe monkeys inhibits the expression of i-NOS and the expression of COX-2, an enzyme which produces NO, which is active oxygen, in BV-2 microglia activated by LPS, And IL-6, and inhibited the activation of MAPK and NF-κB including ERK, p38, and p65, thereby reducing the expression of NO and proinflammatory cytokines. It is intended to provide a pharmaceutical composition and a health functional food which can prevent, ameliorate or treat neurodegenerative diseases, in particular degenerative neurological diseases, which can prevent, ameliorate or treat neurodegenerative diseases using novel uses of the extracts.

The microglial cell activation and degenerative neuropathy are not yet fully understood. However, microglial cell activity is thought to be related to the development and progression of degenerative neuropathy. Therefore, This is because it is known to be an effective treatment for relieving the progress of neurodegenerative diseases. Particularly, it has not yet been reported that the extract of Hornschus melliferae is useful for the improvement of neurodegenerative diseases. As described later in the present invention, the nerve inflammation caused by LPS-stimulated BV-2 microglia is inhibited .

Accordingly, the pharmaceutical composition for preventing, improving or treating neurodegenerative diseases of the present invention contains Sargassum honeri extract as an active ingredient.

As described above, the extract of horseshoe monkeys inhibited the expression of i-NOS and the expression of COX-2, which are enzymes that produce NO, which is an active oxygen in BV-2 microglia, and the expression of IL-6 and IL- And suppress the activation of MAPK and NF-κB including ERK, p38, and p65, thereby decreasing the expression of NO and proinflammatory cytokines. Therefore, the pharmaceutical composition of the present invention is orally or parenterally administered to mammals Can be used to prevent, ameliorate, or treat neurodegenerative diseases.

Herein, the extract of horseshoe crab can be separated using conventional extracting solvents according to a conventional method known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. As the extraction solvent, there can be used a solvent generally used in the extraction process, such as water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane and 1,3-butylene glycol And can be extracted using a solvent selected from the group consisting of. In one embodiment, the Horns Cyprinus mellifera extract may be a water-soluble extract of Horns mellifera, particularly an ethanol extract as described in the Examples.

The pharmaceutical composition of the present invention may contain the extract of Hornsby's moth alone, and may further include a pharmacologically acceptable carrier or excipient depending on the formulation, the method of use and the intended use. When provided as a mixture, the effective ingredient, horns mellifera extract, may be contained in the pharmaceutical composition in an amount of 0.01 to 99% by weight.

Examples of the carrier or excipient include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. These may be used alone or in combination, but not limited thereto, All excipients are available. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

When the pharmaceutical composition of the present invention is made into a pharmaceutical composition, it may further contain conventional fillers, extenders, binders, disintegrators, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives. The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by local application to the skin, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

In an individual dosage form, the content of the active ingredient Horns mellifera extract, which is an active ingredient thereof, may be an amount administered in a single dose, for example, a full day, a half, a third or a quarter of a usual daily dose. The dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the age, sex, condition of the patient, the degree of absorption of the active ingredient, the inactivity of the active ingredient, and the drug to be used together. For example, Gt; mg / ml, < / RTI > 1 to 5 times per day. On the other hand, the dosage of the pharmaceutical composition of the present invention on the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity. When an adult patient weighing 60 kg is used, It is generally 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once to several times a day at intervals of time according to the judgment of a doctor or pharmacist.

Next, the health functional food for the prevention and improvement of degenerative neurological diseases of the present invention has an inhibitory activity against neuronal inflammation caused by activated microglia as an active ingredient by containing the extract of Horns mellifera, which is contained in the above-mentioned pharmaceutical composition Can be used for the purpose of preventing and improving symptoms of various degenerative neurological diseases.

In the present invention, the term 'health functional food' means a natural product or a processed product containing one or more nutrients. Preferably, the function of the food is determined by physical, biochemical, biotechnological, Means a food group that has been imparted with added value to function and express, and a food which is designed and manufactured so that the body control function related to regulation of bio-defense rhythm of food composition, prevention and recovery of disease, etc. is sufficiently expressed in living body. The health functional food may include a pharmaceutically acceptable food supplementary additive and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of health functional foods.

Examples of the health functional food to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins and the like. In addition, it is also possible to use nutritional products such as special nutritious foods (eg crude oil, milk, baby food), health supplements, confectionery (eg snacks), dairy products (eg fermented milk, Beverages, two-oil, fermented beverages, etc.), but are not limited thereto. The food, beverage or food additives described above can be produced by a conventional production method.

The health functional food of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavor agent and a natural flavor agent, a colorant and an aging agent (cheese, chocolate etc.), a pectic acid and its salt, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, water, carbonating agents used in carbonated beverages and the like. These components can be used independently or in combination.

Thus, the health functional food of the present invention has various formulations as described above, and may have any one of powder, granule, tablet, capsule and beverage.

In one embodiment, when the health functional food of the present invention is implemented as a beverage, there are no particular restrictions on other components other than those containing the extract of the present invention as an essential ingredient. Various flavors or natural carbohydrates Or the like as an additional component. Examples of natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol, Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition, in the health functional food for the purpose of prevention and improvement of the degenerative neurological disease, the content of the extract of Horns mellifera, which is an effective ingredient, can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the extract in the health food may be 0.1 to 90 parts by weight of the whole food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

In addition, the method for treating degenerative neurological diseases of the present invention can treat degenerative degenerative diseases including oral or parenteral administration of the above-described pharmaceutical composition for treating degenerative neurological diseases of mammals other than humans. Herein, the degenerative neurological disease may be one or more selected from the group including multiple sclerosis, allergic myelitis, Parkinson's disease, Alzheimer's disease and Huntington's disease.

Example

The Sargassum honeri is washed three times with fresh water to remove seawater. The washed horns are dried in a drier at 90 ° C for 24 hours to remove moisture, and then blended using a blender. 100 g of Horn Seaweed powder is added to 900 ml of 70% ethanol and stirred. On the 2nd day, it is filtered on 3MM filter paper to collect Horn Seaweed extract. Repeat the above procedure 3 times to extract the horns of the horns for a total of 6 days. The obtained extracts were concentrated using a vacuum concentrator, and then all of the solvent was removed by using a freeze dryer to obtain powdered hoe saengmyungbom extract. The obtained composition was dissolved in 70% ethanol at a concentration of 300 / / ml to prepare a pharmaceutical composition.

Experimental Example 1

1. Cell culture

Mouse microglia, BV-2, was purchased from the American Type Culture Collection (Manassas, VA, USA). The medium used for cell culture is cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 200 IU / ml penicillin and 200 g / ml streptomycin under the conditions of 37 ° C. and 5% The extract of Horns mellifera was reduced to 100 mg / ml with 100% EtOH and dissolved in DMEM (final 100% EtOH concentration, 0.3%), Sargassum The control group without honeri treatment used 100% EtOH solvent.

2. Cytotoxic performance evaluation

Murine mcroglia cells BV-2 (2 × 10 ⁶ cells / well) were cultured in a 6-well plate for cell culture, Sargassum honeri (0 to 300 μg / ml) was incubated in the incubator for 24 hours after treatment. The cultured cells are centrifuged at 2000 rpm for 3 minutes at room temperature using a centrifuge, and washed 2 to 3 times with warm PBS. After that, staining was performed using Annexin V / PI Kit and measured using FC500 flow cytometry (Beckman coulter).

 Experimental results are expressed as mean (mean) S. SD (standard deviation) as a result of comparing the number of living cells to the control group (100% ETOH) as a percentage. The significance of the experimental results was compared at the p value <0.05.

As a result of measuring the cytotoxicity of the extract of Bombyx mori L. on BV-2 cells using Annexin V / PI, it was confirmed that they did not have toxicity to cells up to 300 μg / ml as shown in FIG. 1

Experimental Example 2

In order to confirm whether or not the extract of horseshoe crab moth is regulating the production of NO, an inflammatory mediator, the production of NO was measured in LPS-stimulated BV-2 microglia And the results are shown in Fig.

BV-2 microspheres (2 × 10 ⁴ cells / well) were cultured in 96-well plates and pretreated with various concentrations of Horns mellifera extract (0-300 μg / ml) for 2 hours. LPS Or untreated samples are incubated in a 37 ° C, 5% CO2 incubator for 22 h. After incubation, the medium and the same amount of Greiss reagent are mixed 1: 1 and incubated at room temperature for 15 minutes. After 15 minutes, the absorbance is measured at 540 nm using an ELISA microplate reader. (** p <0.01 vs LPS alone treatment group)

As shown in FIG. 2, the production of NO was increased in LPS compared to the control group, but the production of NO was inhibited in a concentration-dependent manner when the horseshoe crab extract (ESH) was treated at a concentration of 0 to 200 μg / ml p < 0.05). Thus, it can be seen that NO suppressed the production of NO from microglial cells stimulated by LPS at the non-toxic concentration (0-300 μg / ml) of the extract of hornbill.

Experimental Example 3

In order to confirm whether or not the extract of horseshoe crab mushroom suppresses the expression of i-NOS and the expression of COX-2, which are enzymes that produce NO, a reverse transcription polymerization reaction was performed as described below, and the results are shown in Fig. .

Murine microglia cells BV-2 (5 × 10 ⁵ cells / well) are cultured on a 12-well plate. The cultured cells are treated with concentration (0 to 300 μg / ml) for 2 hours, treated with LPS, and cultured for 6 hours. Using TRI reagent, isolate total RNA from cells according to the product test method. To synthesize cDNA, 0.1 μg total RNA, oligo dT, M-MLV RTase, dNTP and buffer solution for reaction are mixed and synthesized. To measure the mRNA levels of inflammatory cytokines including IL-1β, TNF-α and IL-6, primers for the target gene were prepared and used. Using the e-Taq DNA polymerase kit and primers, Gene Atlas G02 cDNA was amplified using gradient thermal cycler system (Astec Fukuoka, Japan). PCR reactions were identified by fluorescent stain and UV transilluminator.

The result of the expression of an inflammatory gene mRNA by reverse transcription polymerase chain reaction as shown in Figure 3, from the BV-2 cells stimulated by LPS i-NOS, mRNA expression of COX-2 is increased Hoe life is Sargassum extract (ESH ) Decreased mRNA expression of i-NOS and COX-2 in a dose-dependent manner. These results suggest that the extracts of horseshoe crab ( ESH) inhibit the expression of i-NOS and COX-2 mRNAs and inhibit NO production and have anti-neuronal activity.

Experimental Example 4

BV-2 cells were cultured on a 12-well plate and pretreated with Horseshoe salmon extract at a concentration of 0 to 300 μg / ml for 2 hours. BV-2 cells were stimulated with 200 ng / ml of LPS for 6 hours . The expression of inflammatory gene mRNA was observed by RT-PCR and the results are shown in Fig. As shown in FIG. 4, it can be seen that mRNA expression of IL-6, IL-1β and TNF-α, which are proinflammatory genes increased by LPS, is reduced.

Experimental Example 5

In order to confirm whether or not the extract of hornbill moth, Bombyx mori, reduces the production of inflammatory cytokines such as IL-6, TNF-α, and IL-1β known as cytokines secreted by microglial cells in the neuronal inflammatory reaction, The amount of cytokine produced in the BV-2 cell line stimulated by LPS after pretreatment of the extract of horseshoe monkeys was measured and the results are shown in FIG.

BV-2 cells were cultured in 96-well plates and pretreated with Houttuynia cordata (0-300 μg / ml) for 2 h. BV-2 cells were stimulated with 200 ng / ml LPS for 22 h Incubate in 37 ° C, 5% CO2 incubator. After this, the amount of cytokine production of TNF-α, IL-6 and IL-1β was recovered by measuring the absorbance at 450 nm with an ELISA microplate reader.

The IL-1β gene produced from BV-2 cells stimulated by LPS was not inhibited by the pretreatment of the extract of hornblende moth. On the other hand, as shown in FIG. 5, the gene expression of TNF-a and IL-6 was significantly inhibited by pretreatment of hornblende extract. Therefore, the extracts of hornbill moths were not effective in inhibiting the production of IL-1β, an inflammatory cytokine produced from BV-2 cells stimulated by LPS, whereas the production of TNF-α and IL-6 was inhibited by hornblende extract Inhibitory activity, thereby inducing antinociceptive activity.

Experimental Example 6

MAPKs such as p38, p65, and ERK play important roles in regulating cell growth, division, stress, and cytokine - mediated cellular responses, and are involved in signal transduction pathways that regulate inflammatory mediators through the activation of transcription factors. In addition, NF-κB is a transcription factor that regulates the intracellular synthesis of various molecules such as regulating the expression of inflammatory cytokines and i-NOS. In order to confirm the molecular mechanism of the inhibitory effect of the extract of horseshoe crab on the inflammation mediator in neurons, inhibition of LPS-induced MAPK phosphorylation and activation of NF-κB in BV-2 cells was analyzed by protein detection assay And the results are shown in Figs. 6A to 6C.

BV-2 microglial cells (2 × 10 ⁶ cells / dish) were cultured on a 100-mm culture dish and starvated with serum-free media (DMEM) for 4 hours. (SDS), 50 mM Tris (pH 8.0), and 0.1% sodium dodecyl sulfate (SDS). The cells were washed twice with cold PBS and washed twice with RIPA buffer ), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g / mL leupeptin, 1 g / mL pepstatin, 1 mM sodium orthovanadate, and 100 mM sodium fluoride) for 30 min at 4 ° C and the lysed cells were centrifuged The samples were centrifuged at 14,000 × g for 15 min and the proteins from the cell lysate were quantitated using BCA assay kit. Protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane at room temperature. % Skimmed milk solution and 2% BSA for 1 hour and 30 minutes. After the reaction, the primary antibody (Santa Cruz Biotechnology, CA, USA) was diluted 1: 2000 and reacted for one day. The rabbit, mouse antibody (Santa Cruze Biotechnology) secondary antibody conjugated with HRP was diluted 1: 5000 and reacted at room temperature for 2 hours. The band detection was performed by exposing the x-ray film using chemiluminescence (ECL) Respectively.

In order to examine NF-κB activity, LPS (200 ng / ml) was added to the cells for 15 minutes, 30 minutes, and 45 minutes. The same amount of cell protein was separated by SDS-PAGE, transferred to PVDF membrane, and analyzed by using ERK, p-ERK, p38, p-p38, p65, p-p65, i-NOS, COX- And then confirmed.

As shown in FIG. 6A, phosphorylation of p38, ERK was increased in BV-2 cells stimulated by LPS for 15 minutes, 30 minutes and 45 minutes, while the extract of horseshoe crab moth was increased by LPS-induced p38, ERK .

6 (b), the extracts of horseshoe crabs were pretreated for 2 hours at a concentration of 0 to 300 μg / ml, and the protein levels of i-NOS and COX-2 in BV-2 cells stimulated by LPS Expression was decreased in a concentration dependent manner.

On the other hand, as shown in FIG. 6C, phosphorylation of p65 was increased in the cytoplasm of BV-2 cells stimulated by LPS for 15 minutes, 30 minutes, and 45 minutes, Of phosphorylation was decreased. Indicating that it inhibits migration to the nucleus upon activation of NF-κB

These results demonstrate that the inhibitory effect of ESH on pro-inflammatory cytokine production in BV-2 cells induced by LPS is mediated via the p38, ERK and NF-κB signaling pathways.

From the above results, it was shown that the extract of hornbill moth, mycobacterium tuberculosis suppresses the expression of i-NOS and inhibits NO production. It is confirmed that the production of proinflammatory cytokines IL-6 and TNF-α is significantly inhibited Respectively.

MAPKs are composed of JNK, ERK, and p38, and play important roles in the regulation of various cellular processes such as cell differentiation, proliferation, and inflammation. Inactive MAPKs remain in the cytoplasm and are phosphorylated through LPS or other external stimuli Activation, however, is involved in the production of cytokines while moving to the nucleus. In the present invention, it was confirmed that the pretreatment of the extract with hoe saengmyung mungbean reduced the activity of ERK and p38.

In addition, NF-κB plays an important role in various immune responses, tumorigenesis and autoimmune diseases as well as inflammatory reactions. NF-κB is inactivated in association with Iκ-Bα, I? -B? Is phosphorylated to decompose NF-κB and the separated NF-κB is transferred into the nucleus and is involved in the production of NO and proinflammatory cytokines. However, in the present invention, NF- -B into the nucleus.

Thus, the present invention demonstrates that the extract of Hornschwiens japonica inhibits activation of MAPK and NF-κB including ERK and p38, thereby inhibiting NO and the proinflammatory cytokines IL-6 and TNF-α.

In the present study, it was reported that MAPK containing p38, JNK, and ERK plays an essential role in the production of IL-1β in LPS-stimulated microglial cells. In many studies, the activity of p38 .

Neuroinflammation in the central nervous system is mediated by the production of cytokines (IL-1β, IL-6, TNF-α), chemokines (CCL2, CCL5, CXCL1) ₂ ), and neurons and glial cells (astrocytes, microglia) activate NF-κB during brain injury. Activated NF-κB in neurons induces gene products or proteins of anti-apoptotic activity and is involved in regulating synaptic plasticity. NF-κB activity of neuroglial cells produces neurotoxins, reactive oxygen species that are toxic to neurons, and proinflammatory cytokines.

Thus, it was demonstrated that the LPS-induced phosphorylation of NF-κB was reduced by the extract of hornbill moth, and that the extract of hornbill moth moth was associated with the NF-κB signal and decreased the proinflammatory mediators by the p38 signal , It can be understood that the extract of Hornsby moth moth can be used as a functional food material as well as a new pharmaceutical composition for preventing and symptomatic treatment of neurodegenerative diseases by controlling neuroinflammation in degenerative neurological diseases.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.

Claims (8)

A pharmaceutical composition for preventing, ameliorating or treating a degenerative neurological disease comprising an extract of Hornsby moth, as an active ingredient.
The method according to claim 1,
Wherein the extract of Horns Cyprinus mellifera inhibits expression of at least one selected from the group consisting of iNOS, COX-2, TNF-alpha and IL-6 genes involved in inflammatory response in neuroblastoma cells. &Lt; / RTI &gt;
The method according to claim 1,
Wherein said degenerative neurological disease is selected from the group comprising multiple sclerosis, allergic myelitis, Parkinson's disease, Alzheimer's disease and Huntington's disease.
A health functional food having the effect of preventing and improving degenerative neurological diseases containing extract of hornbill moth as an active ingredient.
5. The method of claim 4,
Wherein the composition is a powder, a granule, a tablet, a capsule or a beverage.
5. The method of claim 4,
Wherein said degenerative neurological disease is selected from the group comprising multiple sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease.
A method for treating a neurodegenerative disease, comprising the step of administering the pharmaceutical composition of any one of claims 1 to 3 to a mammal other than a human.
8. The method of claim 7,
Wherein the degenerative neurological disease is selected from the group comprising multiple sclerosis, allergic myelitis, Parkinson's disease, Alzheimer's disease and Huntington's disease.

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