WO2018008908A1 - Pharmaceutical composition for preventing or treating degenerative neuronal diseases, containing compound isolated from withania coagulans as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating degenerative neuronal diseases, containing Coagulansin A as an active ingredient. Coagulansin-A has effects of promoting the expression of HSP70 and inhibiting the expression of BACE1 so as to inhibit the formation and aggregation of Aβ, thereby being usable as an active ingredient of a pharmaceutical composition for preventing or treating degenerative neuronal diseases.

Description

Pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, containing as an active ingredient a compound isolated from vitania coagulans

The present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases containing a compound isolated from Vitania coagulans as an active ingredient.

Alzheimer's disease (hereinafter referred to as AD) is a degenerative neurological disease in which the brain selectively kills nerve cells as it ages. Histopathological features in the brain of AD patients include senile plaques that are deposited outside the cells in brain tissue and neurofibrillary tangles (NFTs) that accumulate like threads in nerve cells. The main components of the senile plaques are amyloid β petide (hereinafter referred to as Aβ), which is cleaved by proteolytic enzymes from amyloid precursor protein (hereinafter referred to as APP). Branch reports suggest that Αβ is the major cause of various pathological symptoms of AD.

Αβ is the protein C-terminal fragment remaining after the N-terminal fragment of APP is cut by the protease enzyme beta secretase (beta-secretase) is again broken down aspartic acid protein called gamma secretase (γ-secretase) The part that penetrates the membrane is cut by enzymes. It appears in the form of 40 to 42 or 43 amino acids, of which Aβ ₄₂ is known to play an important role in the deposition of age spots.

Heat-shock proteins (hereinafter referred to as HSPs) are chaperones that help the proteins have a three-dimensional structure, which prevents aggregation or misfolding of proteins in stress and abnormal environments. Do it. Among the chaperone molecules, the HSP70 family is known to play an important role in neuroprotection, but its expression is suppressed in the course of AD and other aging-related diseases, preventing them from functioning properly.

Withanolides are natural compounds of the steroidal lactones family found in plants belonging to the family Solanaceae . Vitalanides are extracted from the genus Withania , and the plants of this genus include Withania somnifera and Withania coagulans. coagulans ) species is famous. Vitania coagulans are native to Pakistan, Iran, Afghanistan, and India. The name coagulans is derived from the fruit's agglomerate properties, which are used to make cheese. Plants, roots, berries, seeds, leaves and stems have been used as traditional folk medicine, and the fruit is known to have a calming and diuretic effect and an effect on asthma.

The present inventors found that the coagulansin-A (Coagulansin-A; hereinafter referred to as Coa-A) compound extracted from Vit. Coagulus enhances the expression of HSP70 protein and inhibits the expression of BACE1 to inhibit the amyloid production pathway. Confirmed that the efficacy was completed the present invention.

One aspect of the present invention to provide a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect of the present invention to provide a health functional food for the prevention or improvement of degenerative neurological disease containing the compound represented by the formula (1) as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

As used herein, the term "degenerative neurological disease" refers to a disease causing degenerative changes in neurons of the central nervous system and causing various symptoms. Representative degenerative neurological disorders include Alzheimer's disease, Parkinson's disease, Progressive supranuclear palsy, Multiple system strophy, Olive nucleus-People-cerebellar atrophy , Shy-Drager syndrome, Striatonigral degeneration, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Essential tremor, Cortico-basal ganlionic degeneration, Diffuse Lewy body disease, Parkin-ALS-dementia complex of Guam, and Pick's disease. have.

Alzheimer's disease is the most common of the many conditions that cause dementia, with neuronal fibers twisted abnormally in elderly spots and nerve cells. In addition, there are many brain neurons that are important for maintaining memory and other intellectual abilities, and the amount of specific chemicals coming and going between these brain neurons is separated.

According to one embodiment, the neurodegenerative disease may be Alzheimer's disease or dementia induced by the accumulation of amyloid beta protein in brain cells, but the disease having the therapeutic and prophylactic effect of the compound of Formula 1 is not limited thereto. .

As used herein, the term "amyloid beta" is a protein produced by proteolytic enzyme action from an amyloid precursor protein, and is known to cause brain cell death while being deposited in brain tissue. When neurons in the cerebrum degenerate and die in large quantities, fibrous structures called 'senior spots' and 'nerve fibrous knots', in which Aβ is accumulated, are seen in the nerve cells, and nerve cells are killed by apoptosis. It is known.

According to one embodiment, the compound represented by Formula 1 may promote the expression of HSP70 in neurons, inhibit the expression of BACE1, and inhibit the inflammatory response of neurons.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, an 'effective amount' refers to an amount that exhibits a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state. Preferably the pharmaceutical composition of the present invention can vary the content of the active ingredient according to the degree of disease, according to one embodiment can be administered in an effective amount of 1 to 100 mg / kg / day in the form of parenteral administration have.

Another aspect of the present invention provides a health functional food for the prevention or improvement of degenerative neurological disease containing the compound represented by the formula (1) as an active ingredient.

[Formula 1]

The health food of the present invention may be added as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the kind of food. Examples of the food include drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins. Combinations and the like, and include all of the health foods in the conventional sense.

The compound of formula 1 according to the present invention may be added as it is to food or used together with other food or food ingredients, and may be suitably used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of the compound of formula 1 in the health food can be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is based on 100 ml in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g Can be added. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The health functional beverage composition of the present invention contains the compound of Chemical Formula 1 as an essential ingredient in the indicated ratio, and there are no particular limitations on other ingredients, and it may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. These components may be used independently or in combination, and the proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

According to one embodiment, the compound represented by Formula 1 may promote the expression of HSP70 in neurons and inhibit the expression of BACE1.

Since the compound of Formula 1 of the present invention promotes the expression of HSP70 and has the effect of inhibiting the production and aggregation of Aβ by inhibiting the expression of BACE1, it may be used as an active ingredient of a pharmaceutical composition for preventing or treating neurodegenerative diseases. .

1 is a diagram showing the survival rate of SH-SY5Y cells following coagulansin-A (Coa-A) and amyloid beta oligomer (AβO) treatment.

Figure 2 is a diagram confirming the change in the expression of proteins following Coa-A and AβO treatment.

Figure 3 is a diagram confirming the change in the expression of HSP70 protein by Coa-A and AβO treatment.

4 is a diagram confirming the expression change of HSP70 and BACE1 protein according to Coa-A administration in APP / PS1 mice.

Figure 5 is a diagram confirming the expression changes of HSP70 protein according to Coa-A administration in the cortex and hippocampus of APP / PS1 mice.

6 is a diagram confirming the action state of the amyloid production pathway according to Coa-A administration in APP / PS1 mice.

Figure 7 is a diagram confirming the action state of the non-amyloid production pathway following Coa-A administration in APP / PS1 mice.

8 is a check for the level of soluble Aβ, and Aβ _{1 -40 1 -42} according to the Coa-A therapy in APP / PS1 mice.

9 is a view showing the change in size and repeatability of Aβ spots according to Coa-A administration in the brain of APP / PS1 mice.

10 is a diagram confirming the change in the size and repeatability of Aβ spots according to Coa-A administration in the brain of APP / PS1 mice.

11 is a view showing the change in size and repeatability of Aβ spots according to Coa-A administration in the brain of APP / PS1 mice.

12 is a diagram confirming the changes in HSP70 and Aβ according to Coa-A administration in the brain of APP / PS1 mice.

Figure 13 is a diagram confirming the activity of microglia according to Coa-A administration in the brain of APP / PS1 mice.

14 is a diagram confirming the activity of astrocytes according to Coa-A administration in the brain of APP / PS1 mice.

Figure 15 is a diagram confirming the expression of inflammatory response related molecules according to Coa-A administration in the brain of APP / PS1 mice.

16 is a diagram confirming the expression of the exhibited synaptic protein and postsynaptic protein in the brain of APP / PS1 mice.

17 is a diagram confirming the expression of the exhibited synaptic protein and postsynaptic protein in the brain of APP / PS1 mice.

18 is a diagram confirming the escape time during the training period before the Coa-A administration in Morris water maze experiment.

19 is a diagram confirming the escape time during the training period before the Coa-A administration in Morris water maze experiment.

20 is a diagram confirming the probe test results after the completion of Morris underwater maze training.

21 is a view confirming the behavioral change rate according to the Coa -A administration in the Y-maze experiment.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

Experiment method

One. Coagulantine  A ( Coagulansin -A, Coa -A) and amyloid beta oligomers

Coa-A was provided by Professor Bushra Mirza of QUAD-I-Asam University (Islamabad, Pakistan) and the extraction method is disclosed in " J. Nat. Prod. 76, 22.28 (2013)".

Amyloid beta oligomers (denoted Aβ oligomers, hereinafter referred to as AβO) dissolve Aβ peptides (Sigma, USA) in hexafluoro propanol (HFIP), and disperse after sonication and filtering- Stored at 80.

2. Cell experiment

2-1. SH - SY5Y  Cell culture

SH-SY5Y cells, a human neuroblastoma, contain high concentrations of D-glucose, 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin). Dulbecco's modified Eagle medium) was incubated in humidified conditions containing 37 and 5% CO ₂ . AβO was treated at a concentration of 0.5 µm and Coa-A at 20 µg / ml.

2-2. MTT  assay

MTT experiments were performed using 3- [4, 5-dimethylthiazol-2-yl] -2 and 5-diphenyl tetrazolium bromide to measure cell viability.

Specifically, SH-SY5Y cells were cultured in a 96-well plate with 200 μl DMEM medium at 1 × 10 ⁵ / well concentration. After 48 hours, the cells were further incubated with AβO (0.5 μm) alone, Coa-A alone (10, 20 and 30 μg / ml), or medium containing Aβ _1-42 and Coa-A, respectively. After 24 hours, MTT solution dissolved in phosphate buffered saline (PBS) at a concentration of 5 ml / ml was added to each well, and further incubated at 37 to 4 hours. Thereafter, DMSO (dimethyl sulfoxide) was added to dissolve formazone and mixed for 10 to 20 minutes, and then absorbance (570 to 630 nm) was measured using ApoTox-Glo ^™ (Promega, USA).

3. Experimental animals  And drug treatment

APP / PS1 double transgenic mice are transgenic animals in which a human mutant APP gene and a mutant PS1 (preselin) gene are inserted, and exhibit an neuropathological characteristic of AD due to the significant development of geriatric spots and amyloid beta accumulation.

Eight-week-old male APP / PS1 double transgenic mice were purchased from Jackson Laboratory (Main, USA), and C57BL / 6J wild-type mice were purchased from Samtako Korea (South Korea) at 25 ° C. Breeding in a 12 hour light cycle and random feeding conditions. Every effort has been made to minimize the sacrifice and suffering of the animals, and the experimental procedure was carried out with the approval of the Animal Ethics Committee of Gyeongsang National University.

When the mice were 12 months old, they were randomly divided into four groups (n = 10) and used in the experiments: wild type mice with vehicle, wild type mice with Coa-A, APP / PS1 mice with vehicle, and APP / PS1 mice administered Coa-A. A vehicle (0.9% saline) or Coa-A (50 mg / kg) was administered intraperitoneally (inintraperiton, i.p.) every day for two weeks.

4. Protein Analysis

4-1. Weston Blotting

Protein concentration was measured using a BioRad protein assay kit (BioRad Laboratories, Calif., USA), and then 20 μg of protein was collected using 4-12% Bolt ^TM mini gel (Novex, Life Technologies, Kiryat Simona, Israel). Electrophoresis was transferred to the PVDF membrane. The membrane was then blocked with 5% (w / v) scheme milk to reduce nonspecific binding and reacted with the primary antibody at 4 overnight. The membrane was then washed, reacted with secondary antibodies and protein bands were identified according to the manufacturer's instructions using ECL detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden). All x-ray films were scanned and the intensity of the bands was analyzed using Sigma Gel program, version 1.0, SPSS, Illinois, USA.

Primary antibodies used were anti-HSP70, anti-p-IKK (Cell Signaling, Massachusetts, USA), anti-β-actin, anti-PSD95, anti-Iba-1, anti-GFAP, anti-amyloid C-terminus, Anti-BACE-1, anti-phospho-tau (Ser413), anti-Synaptophysin, anti-ADAM10, anti-p-NF-κB, anti-COX2, anti-iNOS, anti-IL-1β, anti-IDE (Santa Cruz Biotechnology, California, USA), anti-APP, anti-ADAM17 (Abcam technologies, UK) and anti-amyloid 6E10 (BioLegends, CA, USA).

4-2. Enzyme-linked immunosorbent assay

Mouse brain grinding soluble Aβ _{1 -42 1 -40,} and Aβ concentration in the solution (homogenate) using a Human Aβ _{1 -42,} and Aβ _{1 -40} EILSA kits (Invitrogen, CA, USA) was determined according to the manufacturer's instructions.

5. Brain Tissue Analysis

5-1. Brain Section Slide Production

For tissue analysis, ice-cold 4% paraformaldehyde was perfused to the mouse via perfusion, and the brain was post-fixed with 4% paraformaldehyde for 72 hours, followed by 72% in 20% sucrose. Stored for hours. Brains were frozen with O.C.T compound (A.O, USA) and coronal sections were cut to 14-μm thick using a CM 3050C cryostat (Leica, Germany). The cut coronal plane was attached by melting on a probe-on plus charged slide (Fisher, Illinois, USA).

5-2. Immunofluorescence  analysis

Fluorescent staining was performed to confirm protein expression in brain tissues, and staining was performed in parallel for dual staining.

Specifically, the slides were washed twice with 0.01 M PBS for 15 minutes each and reacted for 37 to 5 minutes by the addition of proteinase-k solution. After reacting with a blocking solution consisting of 5% normal goat serum, 0.3% Triton X-100 and PBS for 1 hour at room temperature, the primary antibody diluted 1: 100 was reacted overnight. The next day TRITC- or FICT-bound secondary antibody (Santa Cruz) was diluted 1:50 in PBS and reacted for 90 minutes, and then the slides were mounted with Prolong Antifade Reagent (Molecular Probe, Eugene, USA) for confocal laser- Observation was made with a scanning microscope (Flouview FV 1000, Olympus, Japan).

5-3. Thioflavin  S dyeing thioflavin  S staining)

Thioflavin S staining was performed to identify amyloid plaques in the brain.

Specifically, the slides were washed twice with 0.01 M PBS for 10 minutes, immersed in 1% thioflavin S solution and left at room temperature for 20 minutes. After immersing in 70% ethanol for 5 minutes, washed twice with water, counterstained with propidium iodide (PI) and then covered with a cover slip.

6. Morris Underwater Maze Experiment and Y-Maze Experiment

6-1. Morris Underwater Maze Experiment

In order to assess hippocampus-dependent learning and memory ability, a Morris underwater maze experiment was conducted.

Mice (n = 10 / group) were trained twice a day for three consecutive days, and the escape time for the mouse to find hidden escapes was calculated for more than 60 seconds in the Morris underwater maze. If the mouse did not find the shelters for 60 seconds, they were guided to the shelters to stay at the shelters for 30 seconds. After administering Coa-A or vehicle for 2 weeks, training was continued for 5 consecutive days and evacuation time was evaluated. Mice were allowed to rest for 3 days and then probe tests without hidden hides were recorded to record the time spent in the quadrant where the hides were.

6-2. Y-maze experiment

Y-maze experiments were conducted to assess hippocampal-dependent spatial memory capacity. After the training period, the mouse was placed in the center of the maze to allow free movement at random for 8 minutes, and the total number of branches and successful triples were recorded with the behavioral software system. The alteration behavior percentage (%) was calculated as [successful triple set (total number of branches-2) * 100. The percentage of alteration behavior rate is positively correlated with the spatial working memory capacity.

7. Statistical Analysis

The density of the protein bands is expressed as mean ± standard error mean (SEM) values in arbitrary units. Statistical analysis was performed by one-way analysis of variance (ANOVA) in all experiments, and further Student's t-test using Prism 5 (graph Pad Software, California, USA). The results of morphology analysis and amyloid spots were analyzed using Image J software (open source software provided by the National Institutes of Health, USA) and expressed as Integral Optical Densities (IODs). The results were considered significant when p <0.05, 0.01 and 0.001.

Experiment result

One. Coa Survival Analysis Results of Neurons by -A Treatment

MTT assay was performed to confirm cell viability change by Coa-A treatment. As a result, the experimental group treated with AβO (0.5 μm) significantly reduced the survival rate of SH-SY5Y cells compared to the control group, and the experimental group treated with Coa-A showed cells at all three concentrations (10, 20 and 30 μg / ml). It was confirmed that no toxicity appeared. On the other hand, the experimental group treated with AβO and Coa-A was confirmed that the cell survival rate was significantly increased compared to the experimental group treated with only AβO (FIG. 1).

2. Coa Analysis of Protein Expression Changes by -A Treatment

Expression changes of HSP70 and BACE1 proteins were confirmed by AβO (0.5 μm) treatment in SH-SY5Y cells.

As a result, in the experimental group treated with AβO, the expression of HSP70 protein was markedly decreased and the expression of BACE1 protein was increased at all 6, 12 and 24 hours (p <0.05, 0.01 and 0.001, respectively) time-dependently. (FIG. 2). On the other hand, the experimental group treated with Coa-A reversed the inhibition of expression of HSP70 protein by AβO treatment at all three concentrations (10, 20 and 30 µg / ml) and confirmed that the expression of HSP70 protein increased (at 24 hours). Most significant increase; p <0.001) (FIG. 3).

3. Coa Analysis of Protein Expression in Brain Tissue by A-A Administration

The effect of Coa-A on the expression of HSP70 in the Alzheimer's animal model APP / PS1 mice was confirmed.

Western blot results showed that the expression of HSP70 protein was reduced in APP / PS1 mice treated with vehicle compared to wild-type mice, whereas the expression of HSP70 protein was significantly increased in APP / PS1 mice treated with Coa-A. . In addition, it was confirmed that the expression of BACE1 protein was significantly inhibited in the brain of APP / PS1 mice when Coa-A was administered (FIG. 4).

In addition, the expression of HSP70 was increased in the hippocampus and cortex of APP / PS1 mice administered with Coa-A and decreased in APP / PS1 mice administered with vehicle through immunofluorescence staining (FIG. 5). ).

4. Coa In brain tissue by -A administration Of Aβ  Analysis of production and coagulation improvement effects

HSP70 protein is known to inhibit protein aggregation and protect neurons. Therefore, the effect of Coa-A on the production and aggregation of Aβ in APP / PS1 mice was investigated.

As a result of Western blot, it was confirmed that the activity of amyloidogenic pathway was significantly increased in APP / PS1 mice compared to wild type mice. Specifically, the amounts of amyloid precursor protein (APP), Aβ oligomer, fibrillar Aβ and phosphorylated tau protein were significantly increased. On the other hand, APP / PS1 mice administered with Coa-A for 2 weeks decreased the expression of the proteins and confirmed that the amyloidogenesis response was suppressed (FIG. 6).

In addition, Coa-A administration increased expression of α-secretase (s-APPα) genes such as A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and ADAM17, resulting in a non-amyloidogenic pathway. ) Was confirmed to act (Fig. 7).

In addition, a result of measuring the levels of soluble Aβ, and Aβ _{1 -40 1 -42} in the mouse brains grinding liquid, compared to the administration of vehicle APP / PS1 mouse soluble in an APP / PS1 mice administered the Coa-A Aβ _{1 - 40} and Aβ _{1 - 42} was found to have decreased (Fig. 8).

In addition, the comparison of thioflavin S and Aβ (C-terminal and 6E10) staining of brain tissues showed that the amount of Aβ spots and Aβ increased in the hippocampus and cortex of APP / PS1 mice compared to wild-type mice, and Coa- APP / PS1 mice administered A were found not only to reduce the amount of Aβ but also to reduce the size and repeatability of Aβ spots (FIGS. 9 to 11).

In addition, as a result of double staining of HSP70 and Aβ in brain tissues, it was confirmed that CoSP-A administration significantly increased HSP70 protein expression in the hippocampus and cortex of the brain and decreased aggregation of Aβ (FIG. 12).

5. Coa Inflammation in brain tissue by -A administration neuroinflammation ) Inhibitory effect analysis

Activated glial cells, including microglia and astrocytes, and associated neuroinflammation are one of the important features of the AD brain. Therefore, the effect of Coa-A administration on the activity and neuroinflammatory glial cells was confirmed.

Expression of microglia marker Iba1 (ionized calcium-binding adapter molecule 1) and astrocyte markers glial fibrillary acidic protein (GFAP) was confirmed by immunofluorescence staining. 'S brain has a significant number of activated glial cells. On the other hand, it was confirmed that glial cell activity was inhibited in the brain of APP / PS1 mice administered with Coa-A (FIGS. 13 and 14).

In addition, since NF-κB activation pathway is known to play an important role in the immune response, the effect of Coa-A administration was confirmed.

Western blot confirmed that phosphorylated NF-κB, phosphorylated IκB kinase (IKK), cyclooxygenase2 (COX2), and induced nitric oxide synthesis in vehicle-treated APP / PS1 mice compared to wild-type mice. The levels of enzymes (inducible nitric oxide synthetase) and interleukin-1 beta (IL-1β) were significantly increased (p <0.001). On the other hand, it was confirmed that the expression of the molecules was significantly reduced in APP / PS1 mice administered with Coa-A compared to vehicle-administered APP / PS1 mice (p <0.001) (FIG. 15).

6. Coa Pre- and by -A administration Postsynapse  Dysfunction) postsynaptic  dysfunction) improvement effect analysis

The effect of Coa-A on pre- and postsynaptic protein expression was confirmed.

Western blot analysis of brain pulmonary fluid showed that synaptophysin (SYP) and PSD95 (postsynaptic density protein 95) expression decreased in CoA-A in vehicle-treated APP / PS1 mice compared to wild-type mice. It was confirmed that expression was significantly increased in the APP / PS1 mice administered (Figure 16).

In addition, staining of SYP and PSD95 in the cerebral cortex and hippocampus of the teeth (dentate gyrus, DG) confirmed similar results to the above (FIG. 17).

7. Coa -A-Administration of Learning and Memory Improvement Effects

Maze experiments were conducted to evaluate learning and spatial memory.

In the training before vehicle or Coa-A administration, the escape time for wild-type mice and APP / PS1 mice to find hidden hides was compared to find that APP / PS1 mice were slightly higher. On day 2 and day 3, both mice showed reduced escape time, but significantly reduced escape time of wild-type mice compared to APP / PS1 mice (FIG. 18).

After estimating Coa-A for two weeks, the escape time was measured. On Day 1, the escape time of Coa-A-administered APP / PS1 mice was significantly reduced compared to the vehicle-administered APP / PS1 mice. It was confirmed that the learning ability was improved. In addition, as a result of the comparison from the second to the fifth day of training, the evacuation time of the APP / PS1 mice administered with Coa-A was significantly lower than that of the APP / PS1 mice administered with the vehicle (FIG. 19).

In order to confirm the spatial memory retention ability, a probe test was performed to remove the sheath and to measure the time to stay in the target quadrant where the sheath was. As a result, it was confirmed that the APP / PS1 mice administered Coa-A stayed longer in the target quadrant compared to the APP / PS1 mice administered vehicle (FIG. 20).

In addition, the Y-maze experiments were conducted to confirm the effect on short-term memory, and the altered behavior rate of APP / PS1 mice with vehicle was lower than that of wild-type mice. In comparison, it was confirmed that the altered behavior rate of APP / PS1 mice administered Coa-A was higher (FIG. 21).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (7)

1. A pharmaceutical composition for preventing or treating degenerative neurological diseases containing a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

   [Formula 1]

   
2. The pharmaceutical composition for preventing or treating degenerative neurological disease according to claim 1, wherein the compound represented by Chemical Formula 1 promotes expression of HSP70 and inhibits expression of BACE1.
3. The method of claim 1, wherein the neurodegenerative disease is stroke, stroke, memory loss, memory impairment, dementia, Parkinson's disease and Alzheimer's disease, characterized in that any one selected from the group consisting of prevention or treatment of neurodegenerative diseases Composition.
4. The method of claim 1, wherein the neurodegenerative disease is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that the disease induced by amyloid beta protein in brain cells.
5. According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that to inhibit the inflammatory response of nerve cells.
6. Health functional food for the prevention or improvement of degenerative neurological disease containing the compound represented by the formula (1) as an active ingredient:

   [Formula 1]

   
7. The health functional food for preventing or improving degenerative neurological disease according to claim 6, wherein the compound represented by Chemical Formula 1 promotes the expression of HSP70 and inhibits the expression of BACE1.

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