KR101915643B1 - Composition for preventing, alleviating and treating neurodegenerative diseases comprising pseudane-7 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases, and more particularly, to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases comprising pseudane-7 as an active ingredient and a health functional food.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating degenerative neurological diseases comprising pseudane-7 as an active ingredient. (Composition for prevention, alleviating and treating neurodegenerative diseases comprising pseudane-

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases, and more particularly, to a pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases comprising pseudane-7 as an active ingredient and a health functional food.

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.

On the other hand, pseudane-7 is a marine bacterium that is capable of fusogenic growth in seaweeds having antiviral activity, pseudoalteromonas sp. M2, and a previously reported study has not yet reported a mechanism other than that pseudane-7 has an activity of inhibiting melanin synthesis.

The present inventors have found that pseudoalteromonas sp. The present invention has been accomplished by finding a new use of pseudane-7, a secondary metabolite produced from M2.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing, ameliorating or treating a neurodegenerative disease which can prevent, ameliorate or treat various neuroinflammatory diseases, especially degenerative neurological diseases, comprising pseudane-7 or a pharmaceutically acceptable salt thereof as an active ingredient Compositions and health functional foods.

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 neurodegenerative diseases comprising pseudane-7 represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient do.

[Chemical Formula 1]

In a preferred embodiment, the pseudane-7 inhibits expression of one or more selected from the group consisting of iNOS, COX-2, IL-6 and IL-1 beta genes involved in inflammatory responses in neuritic germ cells.

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 present invention also provides a health functional food having the effect of preventing and / or improving degenerative neurological diseases comprising pseudane-7 represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In a preferred embodiment, the formulation of the health functional food 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 pseudane-7 or its pharmaceutically acceptable salt of the present invention inhibits i-NOS and COX-2 mRNA expression as well as i-NOS and COX-2 through inhibition of p38 signal with NF- And thus can be usefully used as a pharmaceutical composition and a functional food material for preventing, alleviating, or treating various neurodegenerative diseases using the same.

In FIG. 1, A is a structural formula of pseudane-7, and B is a graph of cell viability on MTT assay of mouse BV-2 cells treated with pseudane-7.
FIG. 2 is a graph showing the effect of inhibiting LPS-induced NO production in BV-2 cells treated with pseudane-7.
Fig. 3A is a photograph showing the effect of suppressing the expression of inflammatory gene mRNA induced by LPS in pseudane-7 treated mouse BV-2 cells. Fig. 3B is a photograph showing the expression of i-NOS and COX-2 of pseudane- The results are shown in Fig.
FIG. 4A is a photograph showing the effect of suppressing the expression of proinflammatory gene mRNA induced by LPS in pseudane-7 treated mouse BV-2 cells, and FIG. 4B is a graph showing the inhibitory effect of cytokine production.
FIG. 5A is a photograph showing the phosphorylation inhibition effect of ERK, JNK, and P38 induced by LPS in pseudane-7-treated mouse BV-2 cells, and FIG. 5B is a photograph of ERK 1/2 and p38 signal transduction inhibitor Which is a graph showing the NO production inhibition effect.
6 is a photograph showing the inhibitory effect of NF-κB activation in BV-2 cells treated with pseudane-7.

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.

Technical features of the present invention include pseudoalteromonas sp. M2 inhibited the expression of i-NOS and the expression of COX-2, an enzyme that produces NO, an active oxygen, in BV-2 microglia activated by LPS, and IL- 6 and IL-1β and inhibited the activation of MAPK and NF-κB including JNK, ERK, and p38, thereby reducing the expression of NO and proinflammatory cytokines. The pseudane- 7, which is capable of preventing, ameliorating or treating neurodegenerative diseases, particularly degenerative neurological diseases, which can prevent, ameliorate or treat neurodegenerative diseases.

In other words, inhibition of neuroinflammation by activated microglial cells is seen as mitigation of neurodegenerative diseases. As described below, pseudane-7 inhibits nerve inflammation in BV-2 microglia stimulated by LPS It was confirmed.

Accordingly, the pharmaceutical composition for preventing, ameliorating or treating neurodegenerative diseases of the present invention comprises pseudane-7 represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

As described above, pseudane-7 and / or a pharmaceutically acceptable salt thereof suppresses the expression of i-NOS and the expression of COX-2, which are enzymes that produce NO which is active oxygen in BV-2 microglia, -6 and IL-1β and inhibited the activation of MAPK and NF-κB including JNK, ERK, and p38, thereby reducing the expression of NO and proinflammatory cytokines. As a result, the pharmaceutical composition of the present invention can be orally or parenterally administered to prevent, ameliorate, or treat neurodegenerative diseases of mammals including humans.

The pharmaceutical composition of the present invention may contain pseudane-7 and / or a pharmaceutically acceptable salt thereof alone, and may further comprise a pharmacologically acceptable carrier or excipient depending on the formulation, the method of use, and the intended use have. When provided as a mixture, the active ingredient pseudane-7 and / or a pharmaceutically acceptable salt thereof 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.

The amount of pseudane-7 and / or a pharmaceutically acceptable salt thereof, which is an active ingredient thereof in the form of an individual dosage form, is in the amount administered in a single dose, for example, the total daily dose, 1/2, 1/3 or 1 / 4 times. 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 oral dosage amount of the pharmaceutical composition of the present invention may preferably be 0.00001-100 mg / kg (body weight) per day. The concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

Next, the health-functional food for the prevention and improvement of the neurodegenerative disease of the present invention comprises pseudane-7 and / or a pharmaceutically acceptable salt thereof contained in the above-mentioned pharmaceutical composition as an active ingredient, And thus can be used for the purpose of preventing and improving the symptoms of various neurodegenerative 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 pseudane-7 of the present invention and / or a pharmaceutically acceptable salt thereof can be added include various foods, beverages, gums, tea, and vitamin complexes. 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 pseudane-7 and / or a pharmaceutically acceptable salt thereof of the present invention as an essential ingredient, , As well as various flavoring agents or natural carbohydrates. 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, the health functional food for the purpose of prevention and improvement of the neurodegenerative diseases may contain the active ingredient pseudane-7 and / or its pharmaceutically acceptable salt at a concentration of 10 占 퐂 / ml or less. In some cases, it may be 0.00001-100 mg / kg (body weight) per day. The concentration of the active ingredient contained in the functional food of the present invention can be determined in consideration of the purpose of taking, the condition of the user, the period of time required, etc., and is not limited to a specific range of concentration.

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.

Data were analyzed by one-way ANOVA, followed by post-hoc test using LSD post-hoc test and analyzed by SPSS (IBM; Armonk, NY, USA). The result of the analysis is expressed as Mean (Mean) S. SD (Standard deviation). The significance of the experimental results was compared at the p value <0.05.

Example

Pseudoalteromonas sp. M2 strain was inoculated in 4 L of marine broth2216 (MB: Difco) medium and cultured in a shaking incubator at 22 degrees for 48 hours. The culture of the strain was repeated four times to cultivate a total of 16L. To obtain the secondary metabolite pseudane-7, the cell culture was centrifuged at 20000 g for 30 minutes to remove the precipitate. The supernatant was collected and treated with the same amount of ethyl acetate and stirred at 300 rpm for 15 minutes using a JEIO TECH RS-1 reciprocal shaker. The ethyl acetate layer (upper layer) was dried in vacuo using Speed-Vac and the extract (including pseudane-7) was diluted with 50% methanol. Each extract was purified by high-pressure liquid chromatography (HPLC) on a Water AutoPurification system (Xbridege prep C18 Columm (19 * 250 mm, 5 um) using QDa detector and gradient A (0.1% formic acid) and B (acetonitrile) min to obtain pseudane-7 having the structural formula shown in Fig. 1 (a). The initial gradient condition (90% A / 10% B) was used for 2.8 min, B was maintained at 65% for 5 min at 43 min, and A was reduced to 0% at 45 min.

Experimental Example 1

1. Cell culture

The mouse microsomal cell host BV-2 is cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 200 IU / ml penicillin, and 200 g / ml streptomycin at 37 ° C and 5% carbon dioxide. pseudane-7 was dissolved at a concentration of 20 mM in a DMSO: EtOH = 1: 9 ratio and dissolved in DMEM (final DMSO concentration 0.0025%, EtOH concentration 0.0225% v / v). Control groups without pseudane-7 treatment used DMSO and EtOH solvent.

2. Cytotoxic performance evaluation

Cytotoxicity was measured by MTT colorimetry. BV-2 microspheres (1 × 10 ⁴ cells / well) were cultured on a 96-well plate for cell culture and then treated with pseudane-7 (0 to 5 μM) did. After cultivation, pseudane-7 contained in the medium is removed, and 0.5 mg / ml MTT solution is added. After incubation at 37 ° C for 4 hours, MTT solution was removed, and formazan was confirmed to be formed. Then, formazan produced by mixing DMSO and ethanol 1: 1 was dissolved. The absorbance of the formazan solution is measured at 570 nm using an ELISA microplate reader. Experimental results are expressed as Mean (Mean) SD (Standard Deviation) as a result of comparing the number of living cells to the control group (0.0025% DMSO, 0.0225% 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 pseudane-7 to BV-2 cells using the MTT assay as described above, it was confirmed that they did not have toxicity to cells up to 5 μM as shown in FIG. 1 b.

Experimental Example 2

To determine whether pseudane-7 regulates the production of NO, an inflammatory mediator, NO production levels were measured in LPS-stimulated BV-2 microglial cells after treatment with pseudane-7 as follows The results are shown in Fig.

BV-2 microglial cells (2 × 10 ⁴ cells / well) were cultured in 96-well plates and pretreated with various concentrations of pseudane-7 (0 to 5 μM) for 2 hours. 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 <0.05 vs. control, * <0.05, ** p <0.001 vs LPS alone treatment group)

As shown in FIG. 2, the production of NO was increased in LPS compared to the control group, while the production of NO was inhibited at 2.5 and 5 μM when pseudane-7 was treated at concentrations of 0 to 5 μM (p < 0.05). Thus, it can be seen that NO produced from BV-2 cells stimulated by LPS is suppressed at a concentration (0.5 ~ 5 μM) of pseudane-7 without toxicity.

Experimental Example 3-1

In order to confirm whether pseudogene-7 inhibits 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. 3A .

BV-2 microglial cells (3 × 10 ⁵ cells / well) are cultured on a 6-well plate. The cultured cells are treated with concentration (0 to 5 μM) for 2 hours, treated with LPS, and cultured for 6 hours. Using Trizol 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.

As shown in FIG. 3A, the expression of the inflammatory gene mRNA by the reverse transcription polymerase chain reaction showed that pseudane-7 decreased the expression of mRNA of i-NOS and COX-2 increased by LPS. Expression of i-NOS and COX-2 was increased in BV-2 cells stimulated by LPS, and pseudane-7 was increased in i-NOS and COX-2 in a concentration-dependent manner.

Experimental Example 3-2

In order to confirm the effect of pseudane-7 on protein expression of i-NOS and COX-2, which are pseudogene-7 producing NO, western blots were performed as described below and the results are shown in FIG.

BV-2 microsphage cells (3 × 10 ⁵ cells / well) were cultured in a 60 mm culture dish and pseudane-7 was pretreated for 2 hours at a concentration of 0 to 5 μM. The cells are stimulated for 22 hours. The amount of equivalent cellular protein is separated by SDS-PAGE and transferred to a PVDF membrane. Attach i-NOS, COX-2 antibody to this membrane. β-actin was measured as a control.

As shown in FIG. 3B, protein expression of i-NOS and COX-2 was increased in BV-2 cells stimulated by LPS, pseudane-7 decreased in concentration-dependent expression of increased expression of i-NOS and COX-2 .

The results of Experimental Examples 3-1 and 3-2 show that pseudane-7 inhibits the expression of iNOS and COX-2 and thus has anti-neuronal inflammatory activity that can inhibit the production of NO.

Experimental Example 4-1

In order to confirm whether pseudane-7 reduces the expression of proinflammatory genes secreted by microglial cells in the neuronal inflammatory reaction, BV-2 cell line stimulated by LPS after pretreatment of pseudane-7 as described below The expression of inflammatory gene mRNA was measured by reverse transcription polymerase chain reaction and the results are shown in FIG. 4A.

BV-2 cells are cultured on a 6-well plate, and pseudane-7 is pretreated for 2 hours by concentration (0 to 5 μM), and BV-2 cells are stimulated with 200 ng / ml of LPS for 6 hours.

As shown in FIG. 4A, the expression of the inflammatory gene mRNA by the reverse transcription polymerase chain reaction showed that pseudane-7 decreased mRNA expression of IL-6 and IL-1β increased by LPS.

Experimental Example 4-2

In order to confirm whether pseudane-7 reduces the production of inflammatory cytokines such as IL-6, TNF-α, and IL-1β known as cytokines secreted by microglial cells in the neuroinflammation reaction, -7 was pretreated and the amount of cytokine production was measured in LPS-stimulated BV-2 cell line. The results are shown in FIG. 4B.

After BV-2 cells were cultured in 96-well plate, BV-2 cells were stimulated with 200 ng / ml of LPS for 22 hours after pseudane-7 was pretreated for 2 hours by concentration (0 to 5 μM) , 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.

As shown in FIG. 4B, the TNF-α and IL-6 genes produced from BV-2 cells stimulated by LPS were not inhibited by the pretreatment of pseudane-7. On the other hand, IL-1β gene expression was significantly inhibited by pretreatment of pseudane-7. Thus, pseudane-7 does not inhibit the production of inflammatory cytokines TNF-α and IL-6 produced from BV-2 cells stimulated by LPS, whereas IL-1β production is inhibited by pseudane-7 Inducing anti-neuronal inflammatory activity.

Experimental Example 5-1

MAPKs, such as p38, JNK, and ERK, play an important role in cell growth, division, stress, and regulation of cytokine-mediated cellular responses, and are involved in signal transduction pathways that regulate inflammatory mediators through the activation of transcription factors. In order to confirm the molecular mechanism of the inhibitory effect of pseudane-7 on the inflammatory mediator in neurons, LPS-induced MAPK phosphorylation level in BV-2 cells was confirmed by protein detection analysis as follows, Respectively.

BV-2 microglial cells (2 × 10 ⁶ cells / dish) were cultured in a 60 mm culture dish and starvated with serum-free media (DMEM) for 4 hours. Pseudane-7 (5 μM) After washing with cold PBS twice, the cells were washed twice with RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris After dissolving at 4 ° C for 30 minutes using 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g / mL leupeptin, 1 g / mL pepstatin, 1 mM sodium orthovanadate, and 100 mM sodium fluoride, the lysed cells were centrifuged at 4 ㅀ C , And centrifuged at 14,000xg for 15 minutes. Proteins from cell lysates were quantitated using the BCA assay kit. The same amount of protein samples were separated by 10% SDS-PAGE, transferred to PVDF membrane, and incubated in 5% skim milk solution and 2% BSA for 1 hour at room temperature . 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.

As shown in FIG. 5A, phosphorylation of JNK, p38 and ERK was increased in BV-2 cells stimulated by LPS for 5 minutes, 15 minutes and 30 minutes, while pseudane-7 was increased in LPS-induced JNK, p38, and phosphorylation of ERK.

Experimental Example 5-2

To confirm the effect of pharmaceutical inhibitors on BV-2 cells activated by the griess assay method to identify the major participating signaling mechanisms in regulating inflammatory mediators in neurons, the following experiment was conducted and the results are shown in Figure 5b Respectively.

BV-2 microglial cells (2 × 10 ⁴ cells / well) were cultured in 96-well plates and ERK 1/2 (U0126, final concentration 10 μM) was added to the cultured cells for 2 hours before pseudane- And p38 (SB203580, final concentration 10 μM) inhibitors were pretreated for 1 h. Cells were then stimulated with LPS (200 ng / ml) for 21 h. The supernatant was reacted with griess reagent 1: 1 at room temperature for 15 min and absorbance was measured at 450 nm with a microplate reader.

As shown in FIG. 5B, when SB203580 and pseudane-7 were treated together, the production of NO induced by LPS was effectively inhibited. On the other hand, when U0126 and pseudane-7 were treated together, LPS induced NO It did not reduce production. These results suggest that p38 MAPK signal plays an important role in the antinociceptive effect of pseudane-7.

Experimental Example 6

Since 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, it is necessary to determine whether the antineoplastic activity of pseudane-7 is due to the inhibition of NF-κB activation For confirmation, pseudane-7 was pretreated, and LPS-induced MAPK phosphorylation level in BV-2 cells for 30 minutes, 45 minutes and 60 minutes was confirmed by protein detection analysis, and the results are shown in FIG.

As shown in FIG. 6, phosphorylation of p65 was increased in BV-2 cells stimulated by LPS, whereas phosphorylation of p65 was decreased in the group treated with pseudane-7. Indicating that it inhibits the activity of NF-kB. These results demonstrate that the inhibitory effect of pseudane-7 on proinflammatory cytokine production in BV-2 cells induced by LPS is mediated through the NF-κB signaling pathway.

In the present invention, pseudane-7 reduced the expression of i-NOS and COX-2 as well as mRNA expression of i-NOS and COX-2 through the suppression of p38 signal together with NF-κB signal , and that NO synthesized by i-NOS was also reduced in a concentration-dependent manner and inhibited IL-1β and IL-6 proinflammatory genes and IL-1β production.

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 has been demonstrated through the present invention that phosphorylation of NF-κB induced by LPS is reduced by pseudane-7 and that pseudane-7 reduces the proinflammatory mediators via the p38 signal together with the NF-κB signal, -7 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)

The present invention includes pseudane-7 or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient,
[Chemical Formula 1]

Characterized in that said pseudane-7 inhibits expression of at least one selected from the group consisting of iNOS, COX-2, IL-6 and IL-1 beta genes involved in the inflammatory response in neuronal macrophage cells A pharmaceutical composition for preventing, ameliorating or treating neurological diseases.
delete 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.
The present invention includes pseudane-7 or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient,
[Chemical Formula 1]

Characterized in that said pseudane-7 inhibits expression of at least one selected from the group consisting of iNOS, COX-2, IL-6 and IL-1 beta genes involved in the inflammatory response in neuronal macrophage cells A health functional food having the effect of preventing and improving neurological diseases.
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 degenerative neurological disease comprising administering the pharmaceutical composition of claim 1 or 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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