KR20200053370A - Composition for preventing or treating neurodegenerative disease comprising phlorotannin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating degenerative neurological diseases containing phlorotannin as an active component. Phlorotannin of the present invention has excellent activity of inhibiting Aβ-induced neurotoxicity, thereby being able to be usefully used in pharmaceutical medicines, functional foods, etc., for preventing or treating (alleviating) degenerative neurological diseases. Especially, phlorotannin of the present invention is a material derived from natural product, Ecklonia cava, thereby having stability without cytotoxicity. Therefore, the composition containing phlorotannin as the active component has an advantage of being safe even for a long-term use without side effects.

Description

Composition for preventing or treating neurodegenerative diseases comprising phlorotannin}

The present invention relates to a composition for preventing or treating degenerative neurological diseases, which includes polotanin as an active ingredient.

Neurons are constantly apoptosis in the process of regeneration and synapses, and apoptosis caused by stress and cytotoxic drugs is a major factor in degenerative brain disease. Among them, oxidative stress is known to have a lot to do with the causes of degenerative brain diseases such as Parkinson's disease, stress, aging, stroke, and Huntington's disease. According to a recent study, chronic stress and oxidative stress are hypothalamic. -Pituitary-adrenal cortex, hippocampus, striatum, black matter, and inducing oxidative stress in the whole cerebral cortex to increase apoptosis and reduce neurons and growth factors, leading to Parkinson's disease, stress, aging, stroke and Huntington's disease It is known to cause it.

In particular, free radicals released from oxygen are known to be the main causes of tissue damage, and the types of oxidative radicals related to neurotoxicity include hydrogen peroxide, hydrogen peroxide anion, and hydroxyl group, of which hydrogen peroxide is the most important precursor of highly reactive free radicals. It is known as a substance, and is considered to cause apoptosis of the central nervous system.

When oxidative stress is applied to cranial nerve cells, reactive oxygen species are triggered, which causes cytochrome C release and caspase-3 activation in mitochondria, leading to cell death. At the same time, ROS results in the activation of glutamate, particularly the NMDA receptor, resulting in an increase in Ca ²⁺ ions by the metabotrophical cascade, and an increase in intracellular Ca ²⁺ associated with ROS is also caspase-2. And activate DNA damage.

In addition, the world's aging population is increasing worldwide, and Korea has already entered an aging society in 2000, and in 2010, the proportion of the population aged 65 and over has risen to 11%. It is predicted that it will enter into an aging society in 2018 and an ultra-aging society in 2026, and the resolution of senile disease in the aging population is emerging as an urgent social problem.

In particular, Alzheimer's disease, which accounts for about half of senile dementia disease, currently has no effective treatment and prevention methods, and is one of the most urgently needed neurological diseases in an aging society. .

Korean Registered Patent No. 0838253

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a phlorotannin compound as an active ingredient.

In addition, another object of the present invention is to provide a health functional food for the prevention or improvement of degenerative neurological diseases comprising a phlorotannin compound as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a phlorotannin compound as an active ingredient.

 In one embodiment of the present invention, the compound may be eckol, dieckol or 8,8'-bieckol.

 In one embodiment of the present invention, the compound may be a compound isolated from persimmon.

In one embodiment of the present invention, the compound may be included in a concentration of 0.01 to 100 μM with respect to the composition.

In one embodiment of the present invention, the compound can inhibit the production of free radicals (ROS) or apoptosis by Aβ induction toxicity.

In one embodiment of the present invention, the compound can inhibit or inhibit the activity of TNF-α or IL-1β by Aβ induction toxicity.

In one embodiment of the present invention, the compound can inhibit or inhibit the activity of iNOS, COX-2 or NF-κB.

In one embodiment of the present invention, the degenerative neurological diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system brain dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, brain ischemia and dementia due to brain hemorrhage.

In addition, the present invention provides a health functional food for the prevention or improvement of degenerative neurological diseases comprising a phlorotannin compound as an active ingredient.

In one embodiment of the present invention, the compound may be eckol, dieckol or 8,8'-bieckol.

The fluorotannin of the present invention has excellent activity of inhibiting Aβ-induced neurotoxicity, and thus can be usefully used in pharmaceutical medicines, functional foods, etc. for the prevention or treatment (improvement) of degenerative neurological diseases including Alzheimer's. Particularly, the chlorotanin of the present invention is a substance derived from natural medicinal sensation, and since it has stability without cytotoxicity, the composition of the present invention including it as an active ingredient has a safe advantage for long-term use without side effects to the human body.

1 is a result of toxicity evaluation of eckol, dieckol and 8,8′-bieckol on PC12 cells.
Figure 2 is a result of confirming the cell viability for the beta-amyloid protein of eckol, dieckol and 8,8'-bieckol through MTT assay.
3 is a result confirming the effect of inhibiting Aβ _25-35 induced reactive oxygen species of eckol, dieckol and 8,8'-bieckol.
4 is a result confirming the effect on the cell cycle of eckol, dieckol and 8,8'-bieckol for PC12 cells treated with Aβ _25-35 .
5 is a diagram morphologically showing the effect of inhibiting Aβ _25-35 induced apoptosis of eckol, dieckol and 8,8′-bieckol through fluorescent staining.
6 is a diagram morphologically showing the effect of inhibiting Aβ _25-35 induced apoptosis of eckol, dieckol and 8,8'-bieckol through flow cytometry.
7 is a diagram showing mitochondrial membrane potential (MMP) for Aβ _25-35 of eckol, dieckol and 8,8′-bieckol.
8 is a view showing the effect of inhibiting the influx of Ca ^{2+ in} cells against Aβ _25-35 of eckol, dieckol and 8,8′-bieckol.
9 is a view showing the effect of reducing the Bax / Bcl-2 ratio of eckol, dieckol and 8,8'-bieckol on Aβ _25-35 .
10 is a diagram showing the inhibitory effects of eckol, dieckol and 8,8'-bieckol on caspase-8, -9, -3 and PARP-1 activity on Aβ _25-35 .
11 is a view showing the effect of inhibiting TNF-α activity on Aβ _25-35 of eckol, dieckol and 8,8'-bieckol.
12 is a view showing the effect of inhibiting IL-1β activity on Aβ _25-35 of eckol, dieckol and 8,8'-bieckol.
13 is a view showing the inhibitory effect of eckol, dieckol and 8,8'-bieckol on Aβ _25-35 induced NO production.
14 is a view showing the effect of inhibiting iNOS expression on Aβ _25-35 of eckol, dieckol and 8,8'-bieckol.
15 is a view showing the inhibitory effect of eckol, dieckol and 8,8'-bieckol on Aβ _25-35 induced PGE ₂ production.
16 is a diagram showing the effect of inhibiting COX2 expression on Aβ _25-35 of eckol, dieckol and 8,8'-bieckol.
17 is a result confirming the effect of eckol, dieckol and 8,8'-bieckol on Aβ _25-35- induced NF-kB and I-kB phosphorylation.
18 is a result confirming the inhibitory effect of eckol, dieckol and 8,8'-bieckol on the increased MAPK _S when Aβ _25-35 treatment.

The present invention relates to a novel use of polotanine, and has a feature of providing a composition for preventing or treating degenerative neurological diseases comprising polotanin as an active ingredient.

'Florotannin' of the present invention is a material isolated from brown algae among seaweeds and is known to have antioxidant, anti-inflammatory, UV protection, whitening and anti-diabetic effects, but can be used for degenerative brain diseases such as Alzheimer's dementia. No document has been reported so far.

Accordingly, the present inventors can effectively inhibit Aβ-induced neurotoxicity to chlorophyllin-derived chlorophyll during various studies for the treatment of Alzheimer's disease, and the difference between Aβ-induced neurotoxicity-inhibition differences according to the structural differences of polotanin components. The fact that it can be seen was first identified.

This fact can be confirmed through an embodiment of the present invention, in order to find out whether there is an inhibitory effect of free radicals of eckol, dieckol and 8,8′-bieckol, which are chlorotannin compounds on Aβ _25-35 induction toxicity. treated with Aβ _25-35 and then eckol, dieckol and 8,8'-bieckol subjected to a ROS assay targeted the control group not treated with the Aβ _25-35 and the group treated with different concentrations for each result, all three materials Aβ _{25 It} showed excellent inhibition against _-35 induced intracellular oxidative stress. In addition, dieckol and 8,8'-bieckol showed a lower ROS production amount than the positive control resveratrol in the 10 and 50 μ concentration sections, confirming that the oxidative stress inhibitory effect produced by Aβ _25-35 was excellent (Fig. 3).

In addition, if DNA damage occurs due to constant oxidative stress, cell cycle progress is temporarily stopped to repair the damaged DNA. During Aβ _25-35 treatment, the transition from G0 / G1 phase to S phase was suppressed, increasing G0 / G1 phase and reducing S phase, but eckol, dieckol and 8,8′-bieckol decreased G0 / G1 phase and arrested cell cycle Was restored (see Figure 4). In particular, it was found that dieckol most effectively restored the cell cycle.

In another embodiment of the present invention, as a result of confirming the protective effect of eckol, dieckol and 8,8′-bieckol against Aβ _25-35 induced cell death, when treated with Aβ _25-35 compared to the control group, fluorescence Strongly observed, apoptosis increased by more than 40% showed an excellent concentration-dependent effect upon pretreatment of polotannins, and at a concentration of dieckol 50μ, it was higher than that of the positive control resveratrol Aβ _{25 It} showed a great effect on _-35 induced apoptosis (see Fig. 5).

As a result of using a flow cytometer to specifically confirm the apoptosis inhibition pattern, it was found that late apoptosis was the most reduced by dieckol among the three chlorotanins, while initial apoptosis was most suppressed by 8,8′-bieckol. Could (see Figure 6).

In another embodiment of the present invention, as a result of confirming the protective effect of eckol, dieckol and 8,8′-bieckol on mitochondrial function induced by Aβ _25-35 through MMP (mitochondrial membrane potential), Aβ _25-35 treatment group Decreased the mitochondrial function compared to the control group, but dieckol and 8,8′-bieckol showed significant effect at 10μ. In particular, dieckol showed superior effects than terrestrial polyphenol resveratrol. By normalizing mitochondrial function, Ca ²⁺ levels in cells elevated due to Aβ _25-35 were decreased in a concentration-dependent manner upon treatment with polotannins. In the case of dieckol, it was found that it showed excellent effect in all concentration sections (see FIG. 7).

In another embodiment of the present invention, in order to confirm the mechanism of apoptosis according to the structure of polotanin, the ratio of Bax / Bcl-2 induced by Aβ _25-35 is markedly treated by eckol, dieckol and 8,8′-bieckol. It was confirmed that it was reduced. In particular, it was found that dieckol showed an excellent effect by reducing to a control level in all concentration sections.

Furthermore, in the present invention, it was confirmed that the inflammatory response increased by Aβ25-35 was effectively suppressed by fluorotannin treatment (see FIGS. 10 to 18).

Therefore, the present inventors have experimentally demonstrated that fluorotannin can effectively inhibit Aβ-induced oxidative stress and NF-κB signaling mechanism, and the neuroprotective effect of eckol, dieckol and 8,8′-bieckol is applied to each compound. It seems to be due to the present -OH group, and it was confirmed that the neuroprotective effect was superior in dieckol and 8,8'-bieckol, which are hexamers, than eckol, which is a trimer of phloroglucinol.

Therefore, the composition of the present invention containing polotanin as an active ingredient can effectively prevent or treat degenerative neurological diseases.

In the present invention, the polotanin compound may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid can be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The fluorotannin according to the present invention may be commercially available, or may be used that is isolated from nature or prepared by a chemical synthesis method known in the art.

The composition of the present invention is a pharmaceutical composition comprising the polotanin as an active ingredient and may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to these active ingredients, and as an adjuvant, disintegrant , Sweeteners, binders, coating agents, expanding agents, lubricants, lubricants or flavoring agents.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of tablets or capsules, the active ingredient can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and colorants can also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trachocanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by appropriate methods in the art.

In one embodiment of the present invention, the fluorotannin of the present invention may be included in the composition in a concentration of 0.01 to 100 μM.

Degenerative neurological diseases in which the pharmaceutical composition of the present invention may exhibit therapeutic effects include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, brain dysfunction of the immune system, progressive neurodegenerative disease , Metabolic brain disease, Niemann-Pick disease, dementia due to brain ischemia and brain hemorrhage, but is not limited thereto, and may be Alzheimer's disease in more detail.

In addition, the present invention provides the use of a composition comprising fluorotannin as an active ingredient for the manufacture of a medicament for the prevention or treatment of degenerative neurological diseases. The composition of the present invention containing the above-described polotanin as an active ingredient may be used for the manufacture of a medicament for the prevention or treatment of degenerative neurological diseases.

In addition, the present invention provides a method for preventing or treating degenerative neurological diseases, which includes administering fluorotannin to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

The term “therapeutically effective amount” as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, physician or other clinician, which It includes an amount that induces relief of symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective dose and the number of administrations for the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients in the composition, the type of formulation, and the patient's age, weight, general health status , Gender and diet, administration time, route of administration and composition, secretion rate, treatment duration, can be adjusted according to various factors, including drugs used simultaneously. In the treatment method of the present invention, in the case of adults, it is preferable to administer the polotanin of the present invention at a dose of 0.01 mg / kg to 250 mg / kg when administered once to several times a day.

In the treatment method of the present invention, the composition containing the polotanin of the present invention as an active ingredient is oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a conventional manner.

In addition, the present invention provides a health functional food for the prevention or improvement of degenerative neurological diseases containing polotanin as an active ingredient.

The health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and improving degenerative neurological diseases.

In the present invention, the term "health functional food" refers to food manufactured and processed using ingredients or ingredients having useful functionality for the human body according to Act 6727 on the Health Functional Food, and it is a nutrient for the structure and function of the human body. It means to ingest for the purpose of obtaining a useful effect for health purposes such as controlling or physiological action.

The health functional food of the present invention may include a normal food additive, and whether or not it is suitable as a food additive is related to the item according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. Judging by standards and standards.

Items listed in the "Food Additives Revolution" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamonic acid; Natural additives such as decolorant, licorice extract, crystalline cellulose, high pigment, and guar gum; And mixed preparations such as L-sodium glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar colorants.

For example, in the form of tablets, the health functional food is granulated by mixing the mixture of polotanin, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant, etc. Alternatively, the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules of capsules in the form of health functional foods can be prepared by filling a mixture of the active ingredient of the present invention with an additive such as an excipient, an excipient, and the like. The mixture mixed with the additive of can be prepared by filling a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring may be prepared by molding a mixture of a mixture of polotanin, an excipient, a binder, a disintegrant, etc., which is an active ingredient of the present invention, by a conventionally known method, and, if necessary, with a white sugar or other repellent It can be removed, or the surface can be coated with a material such as starch or talc.

The health functional food in the form of granules can be prepared in the form of a mixture of an active ingredient of the present invention, polotanin and excipients, a binder, a disintegrant, etc., in a conventionally known manner, and, if necessary, flavoring agents and mating agents And the like.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

<Example 1>

Materials and methods

<1-1> Cell culture

PC12 cells used in the experiment were pre-sold by ATCC, and the cells were added to RPM1 1640 medium with 10% fetal bovine serum (FBS), 5% horse serum, and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ conditions. Cultured in

<1-2> Cytotoxicity Assessment (MTT assay)

PC12 cells were put into a 96-well plate with 5 × 10 ⁴ cells / well using RPM1 1640 medium, cultured for 24 hours, and then added eckol, dieckol and 8,8′-bieckol at a concentration of 1,10,50,100µg / mL 1 After incubation for 48 hours, 50 μg of Aβ _25-35 aggregated for 48 hours was added, followed by incubation for 24 hours. Then, after reacting with the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazoliumbromide) solution for 3 hours and removing the supernatant, the resulting formazan was dissolved with DMSO to measure absorbance at 570 nm. Did.

<1-3> Measurement of inhibitory effect on reactive oxygen species (ROS assay)

In the concentration range of 1,10,50 μg / mL, which did not show cytotoxicity, it was confirmed that eckol, dieckol and 8,8′-bieckol inhibit ROS production. After culturing PC12 cells for 24 hours in a 96-well plate with blocked proximity light, fluorescent dye CH ₂ DCF-DA, which selectively stains ROS, was incubated for 30 minutes in a light-shielding state. Immediately after the reaction was completed, after washing with HBSS, fluorescence values were measured at a wavelength of 485 nm / 535 nm using a fluorescence measurement device, and the values were expressed relative to the Aβ _25-35 treatment group.

<1-4> Cell cycle measurement

After treatment of Eckol, dieckol and 8,8′-bieckol and Aβ _25-35 , cells attached to the wells were detached by adding trypsin-EDTA solution to make cell suspension with PBS. The cell suspension was fixed with 70% ethanol at -20 ° C for more than 3 hours, and then the Muse cell cycle solution was added and tested according to the manufacturer's instructions using a flow cytometer.

<1-5> Analysis of morphology and apoptosis inhibition of PC12 cells

The morphological changes of the cells were measured using Hoechst33342 dye. After dispensing the cells into 8-well plates, eckol, dieckol and 8,8′-bieckol were treated by concentration, Aβ _25-35 was treated after 1 hour, and cultured cells after 24 hours were fixed with 4% formaldehyde for 20 minutes. Washed with PBS. Hoechst33342 (1 μg / mL) was added and reacted at room temperature for 15 minutes, followed by observation using a fluorescence microscope (Olympus, Tokyo, Japan).

In addition, in order to specifically examine the pattern of apoptosis caused by Aβ _25-35 treatment, it was analyzed using an Annexin V & Dead Cell Kit (Merk Milipore, USA). The cultured sapo was collected with trypsin EDTA, collected, washed with PBS, and then annexin V & Dead Cell solution was added to react in the dark for 20 minutes, and then apoptosis type was analyzed using a flow cytometer (Milipore, USA).

<1-6> Evaluation of mitochondrial membrane potential (MMP)

PC12 cells were incubated for 24 hours in a 96-well plate blocked with proximity light, followed by adding eckol, dieckol and 8,8′-bieckol according to concentration, and adding Aβ _25-35 50μ aggregated for 48 hours after 1 hour incubation to add 24 Incubated for hours. Subsequently, the fluorescent dye rhodamine 123 was incubated for 30 minutes in a light-shielding state. Immediately after the reaction was completed, after washing with HBSS, fluorescence values were measured at a wavelength of 485 nm / 535 nm using a fluorescence measuring device.

<1-7> Measurement of intracellular calcium influx

eckol, dieckol and 8,8'-bieckol were confirmed to inhibit the influx of calcium into the cells. PC12 cells were incubated for 24 hours in a 96-well plate in which proximity light was blocked, followed by incubation of Fluo-3 / AM fluorescent dye containing 0.02% pluronic F-127 for 30 minutes in a light-shielding state. Immediately after the reaction was completed, after washing with HBSS, fluorescence values were measured at a wavelength of 485 nm / 535 nm using a fluorescence measuring device.

<1-8> NO production measurement

The produced nitrogen monoxide was measured by adding a Griess reagent in a cell culture medium to measure the amount of nitric oxide in the form of nitrite. 96-well by mixing 100 μl of cell culture solution with the same amount of Griess reagent [1% sulfanilamide, 0.1% N-1-napthylethylene diamine in 2.5% phosphoric acid] After reacting on the plate for 10 minutes, absorbance was measured at 570 nm. A standard curve was obtained using sodium nitrite, and compared with this, the amount of nitrogen monoxide produced was calculated.

<1-9> PGE ₂  Production measurement

Intracellular The concentration change of PGE ₂ was quantified using an assay kit (R & D System, Minneapolis, MN) using an enzyme-linked immunosorbent assay (ELISA) method. After adding PGE ₂ sample or sample to a 96-well plate to which Goat anti-mouse Ig was attached, 50 μg of each PGE ₂ -peroxidase conjugate was added and reacted at room temperature for 2 hours. After washing with PBS, the substrate solution was added and the reaction was stopped after 30 minutes in the dark. As a result, the absorbance was measured at 450 nm to calculate the PGE ₂ content contained in each cell culture solution using a standard calibration curve.

<1-10> Western blotting analysis

PC12 cells were placed in a 6-well plate at 2 × 10 ⁶ cells / well, and cultured for 24 hours, followed by eckol, dieckol, and 8,8′-bieckol, and after 1 hour of culture, Aβ _25-35 was added. Cells were washed twice with cold PBS, lysed for 1 hour using a lysis buffer containing protease inhibitor, and then centrifuged at 13,000 rpm for 10 minutes to separate only the protein supernatant. The protein concentration was quantified based on bovine serum albumin (BSA). The quantified protein was electrophoresed on a 10% polyacrylamide gel and transferred to a PVDF membrane at 15V and 300mA for 30 minutes. The protein-transferred membrane was placed in 1x PBST containing 5% skim milk powder, blocked for 1 hour, and then reacted with the primary antibody overnight. After the primary antibody reaction was completed, the membrane was washed 8 times with 1 × PBST, and then the secondary antibody was reacted at 4 ° C. for 2 hours and then washed with 1 × PBST. The protein was confirmed by Ez Capture ST (ATTO, Tokyo, Japan).

<Example 2>

Cell viability measurement result

As a result of measuring the toxicity of eckol, dieckol and 8,8′-bieckol in PC12 cells, it was found that eckol, dieckol and 8,8′-bieckol all had no effect on cell viability compared to the control group with no treatment up to 100 μ. It was understood (see Fig. 1).

As a result of investigating the cell protective effect of eckol, dieckol and 8,8′-bieckol by inducing Aβ _25-35 in PC12 cells, up to 66% of cells were killed when treated with Aβ _25-35 , but Aβ _25-35 in three active ingredients Was confirmed for the cell protective ability (see Figure 2).

<Example 3>

ROS inhibition effect measurement and cell cycle normalization evaluation result

In order to confirm the ROS inhibitory effect of eckol, dieckol and 8,8′-bieckol, Aβ _25-35 was treated with PC12 cells to induce the production of ROS, and then the inhibitory effect was confirmed. As a result, all three substances showed excellent inhibitory ability against Aβ _25-35 induced intracellular oxidative stress. In addition, dieckol and 8,8'-bieckol showed a lower ROS production amount than the positive control resveratrol in the 10 and 50 μ concentration sections, confirming that the oxidative stress inhibitory effect produced by Aβ _25-35 was excellent (Fig. 3).

When DNA damage occurs due to constant oxidative stress, cell cycle progress is temporarily stopped to repair the damaged DNA. During Aβ _25-35 treatment, the transition from G0 / G1 phase to S phase was suppressed, increasing G0 / G1 phase and reducing S phase, but eckol, dieckol and 8,8′-bieckol decreased G0 / G1 phase and arrested cell cycle Was restored (see Figure 4). In particular, it was found that dieckol most effectively restored the cell cycle.

<Example 4>

PC12 cell morphology and apoptosis inhibition pattern analysis results

The protective effect of eckol, dieckol and 8,8′-bieckol on Aβ _25-35 induced cell death was confirmed by Hoechst33342 staining of DNA fragments, one of the representative indicators of apoptosis (see FIG. 5).

When treated with Aβ _25-35 compared to the control group, it was observed that fluorescence was strongly observed, and apoptosis increased by 40% or more showed a concentration-dependent excellent effect upon pretreatment of polotannins, and at a concentration of dieckol 50 μ It showed a great effect on Aβ _25-35 induced apoptosis at a level superior to that of the positive control resveratrol.

The results of using a flow cytometer to specifically confirm the apoptosis inhibition pattern are shown in FIG. 6. Cells that have entered apoptosis are exposed to phospholipid phosphatidylserin (PS) on an extracellular surface and bind to annexin V, and when late apoptosis or death, the cell membrane is destroyed and PI enters the cell and is stained in the nucleus and can be observed. Aβ _25-35 significantly increased early and late apoptosis compared to the control group. Late apoptosis was the most reduced by dieckol among the three flolotanins, while early apoptosis was most inhibited by 8,8′-bieckol.

<Example 5>

Normalization of mitochondrial function and intracellular Ca ²⁺  Level check result

The protective effect of eckol, dieckol and 8,8'-bieckol on mitochondrial function induced by Aβ _25-35 was confirmed through MMP (mitochondrial membrane potential). As a result, the Aβ _25-35 treatment group decreased mitochondrial function compared to the control group, but dieckol and 8,8′-bieckol showed a significant effect at 10 μ. In particular, dieckol showed superior effects than terrestrial polyphenol resveratrol. By normalizing mitochondrial function, Ca ²⁺ levels in cells elevated due to Aβ _25-35 were decreased in a concentration-dependent manner upon treatment with polotannins. In the case of dieckol, it was found that it showed excellent effect in all concentration sections (see FIG. 7).

<Example 6>

Research on the mechanism of cell death

Apoptosis can be classified into exogenous and endogenous pathways. The exogenous pathway is induced by binding to the cell death receptor, where caspase-8 activated by the Fas associated death domain (FADD) causes apoptosis through caspase-3 at a lower level. Meanwhile, the endogenous pathway is activated by various cytotoxic stimuli, and the mitochondria play a major role. Mitochondrial dysfunction causes the release of cytochrome c from mitochondria to the cytoplasm, which in turn activates caspase-9 and caspase-3 cascades, leading to cleavage and apoptosis of poly ADP ribose polymerase (PARP).

Aβ _25-35 increased the Bax / Bcl-2 ratio at the protein level of PC12 cells (see Figure 9). All compounds decreased the Bax / Bcl-2 ratio increased by Aβ _25-35 , and in particular, dieckol decreased to the control level in all concentration sections and showed excellent effect.

dieckol and 8,8′-bieckol reduced the activity of the endogenous pathways caspase-9, -3, PARP-1 and the exogenous pathway caspase-8 against Aβ _25-35 induced apoptosis, while eckol reduced the endogenous pathway It was found that it suppressed apoptosis only (see FIG. 10).

<Example 7>

Inhibitory activity of inflammatory cytokines

TNF-α and IL-1β regulatory effects induced by Aβ _25-35 in PC12 cells are as follows.

The Aβ _25-35 treatment group increased TNF-α and IL-1β levels four times or more compared to the control group, and pretreatment with polotanins reduced inflammatory cytokine production in a concentration-dependent manner. eckol strongly inhibited TNF-α elevated by Aβ _25-35 at 50 μ. However, dieckol and 8,8'-bieckol showed strong inhibitory activity even at 1μ, and 10, 50μdieckol and 50μ8,8'-bieckol decreased TNF-α as a control level. Expression of IL-1β showed a pattern similar to TNF-α (see FIGS. 11 and 12).

<Example 8>

Nitrogen monoxide (NO) production and iNOS expression inhibitory effect

When the NO level increased by Aβ _25-35 treatment was pre-treated with 10, 50 μdieckol and 50 μ8,8′-bieckol, NO expression was significantly reduced to a level similar to that of the control group (see FIG. 13). However, eckol did not show a significant effect at 1 and 10 μ, and showed similar effects to dieckol and 8,8′-bieckol at 1 μ at 50 μ.

In the case of iNOS, which is a NO-producing enzyme, it was increased more than 3 times due to Aβ _25-35 (see FIG. 14). Pretreatment of eckol 50μdieckol and 8,8′-bieckol significantly confirmed the reduction of iNOS expression, but it was found that eckol of 1μ showed somewhat pharmaceutical effect.

<Example 9>

PGE ₂  Production and COX2 expression inhibitory effect

The effect of eckol, dieckol and 8,8′-bieckol on the production of PGE ₂ by Aβ _25-35 is shown in FIG. 15. Pretreatment of eckol, dieckol and 8,8'-bieckol decreased the concentration of PGE ₂ induced by Aβ _25-35 in a concentration-dependent manner. The decrease in PGE ₂ production was most pronounced in dieckol, showing a significant effect in all concentration sections, and at a concentration of 50 μ, similar to that of the positive control resveratrol.

As a result of investigating the effect of eckol, dieckol and 8,8′-bieckol on COX2 activity to investigate the mechanism of PGE ₂ inhibition, the increased expression of COX2 due to Aβ _25-35 has the strongest inhibitory effect on dieckol like PGE ₂ It was found that it was visible (see FIG. 16).

Through this, eckol, dieckol and 8,8'-bieckol inhibited COX2 and confirmed that PGE ₂ production was regulated.

<Example 10>

NF-kB and I-kB production inhibitory effect

As a result of treating Aβ _25-35 to PC12 cells, it was found that phosphorylation of p65 and I-kB, NF-kB subunits, was increased (see FIG. 17). dieckol significantly inhibited p65 protein levels compared to the Aβ _25-35 stimulation group. eckol and 8,8′-bieckol also attenuated p65 phosphorylation, but showed slightly lower effects than dieckol. Dieckol and 8,8′-bieckol decreased the level of pI kB at all concentrations, whereas eckol did not show a significant effect at 1 μ.

Through this, it was found that the polotanin component of persimmon inhibited p65 and I-kB phosphorylated with Aβ _25-35 to reduce excessive inflammatory response.

<Example 11>

MAPKs inhibitory effect

In the present invention, activation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPK known as the MAPKs family was measured. As a result, it was confirmed that the phosphorylation level of MAPKs was significantly increased when Aβ _25-35 was treated alone compared to the control group.

dieckol showed excellent phosphorylation inhibitory effect on all MAPKs, especially p-p38 recovered to the control level. 8,8′-bieckol significantly inhibited p-p38 and p-JNK protein expression, but did not show a significant effect in ERK. eckol showed p38 inhibitory effect at concentrations of 10 μ and 50 μ, but it was found to have no effect on ERK and JNK (see FIG. 18).

Through this, the inhibitory effect of the NF-kB pathway appears to be attributable to the inhibition of p38 phosphorylation.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (10)

A pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, which includes a phlorotannin compound as an active ingredient. According to claim 1,
The compound is characterized in that it is ekol (eckol), dieckol (dieckol) or 8,8'-biekkol (bieckol), a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases. According to claim 1,
The compound is characterized in that the compound isolated from persimmon, a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases. According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that contained in a concentration of 0.01 to 100μM relative to the composition. According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases characterized in that it inhibits the production of free radicals (ROS) or apoptosis by Aβ-induced toxicity. According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases characterized in that it inhibits or inhibits the activity of TNF-α or IL-1β by Aβ induction toxicity. According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases characterized in that it inhibits or inhibits the activity of iNOS, COX-2 or NF-κB. The method according to any one of claims 1 to 7,
The degenerative neurological diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, brain ischemia and brain hemorrhage. A pharmaceutical composition for preventing or treating degenerative neurological diseases, which is selected from the group consisting of dementia. A health functional food for the prevention or improvement of degenerative neurological diseases, which includes a phlorotannin compound as an active ingredient. The method of claim 9,
The compound is eckol, dieckol or 8,8′-bieckol, a health functional food for preventing or improving degenerative neurological diseases.

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