COMPOSITION FOR PREVENTING OR TREATING DEGENERATIVE NEURONAL DISEASE COMPRISING NOBILETIN AS ACTIVE INGREDIENT

The present invention refers to novel use of naringenin relates to active ingredient composition for preventing or treating neurodegenerative diseases including naringenin search number public box construction disclosed.

Of the present invention 'naringenin (nobiletin)' which is separated in citrus of obesity prevention of known as fat combustion cooled when it is judged that, when it is judged that the known but prevent cancer, neurodegenerative disease such as Alzheimer's dementia can be use in temporal document describes reported bar free.

The present invention the various studies for the treatment of Alzheimer's disease victims of the naringenin to automatically change the fact most at trillion number effectively billion A β neurotoxic induction was examined.

This allows the one in the embodiment of the present invention can be by selecting A β_25-35 Induced toxicity to naringenin active oxygen kind whether separate A β billion number effect_25-35 Then performs a processing according to a concentration and a group of A β with naringenin_25-35 The ROS assay tested embodiment a result in which the regulated, A β_25-35 In which A β relative to the controls_25-35 Oxidative stress was abruptly increases during processing. The number of generating ROS by the naringenin while cylindrical capable of effectively billion (reference 2 and 3 also).

In the embodiment of the present invention another process of naringenin in PC12 cells results identifying the type and volume of nuclear fragmentation, A β_25-35 PC12 cell toxicity (nuclear condensation) nuclear condensation, membrane bubble (membrance blebbing), cell shrinkage (cell shrinkage), such as morphological changes (nuclear fragmentation) nuclear parted naringenin while normal and similar form recovery processing has been his car (also 5 reference). In particular troop resveratrol (resveratrol) superior than positive contrast concentration 25 μm naringenin in cell death (apoptosis) was a 50 μm billion number. Further, flow cytometry (apoptosis) apoptosis through carefully the result as well as cell death (apoptosis) in a type naringenin even heat cell to selectively inhibit the intermediate schedulable his car (reference 6 also).

In the embodiment of the present invention another naringenin in the 25 aβ provided 35 with respect to the number increased by caspase-a 3 billion (7 also reference) activity depending on concentration.

In addition, to Go/G1 cell cycle progression is, for S, G2/M of periodically repeating order while proliferating cells to be coated. PC12 cells A β_25-35 In the Go/G1 progression into S confines the Go/G1 is increased S groups (arrest) is reduced. While, in processing of the present invention confines G0/G1 cell cycle for recovering and neutralize increased Go/G1 naringenin was again reduced his car (also 8 reference) is schedulable.

Further in the present invention increased by 25 aβ provided 35 inflammatory reaction by number has been confirmed that the naringenin processing is effectively billion (9 to 16 also reference).

The present invention is induced oxidative stress and the victims of the naringenin is A β signal transduction mechanisms may be effectively billion number NF provided κB to empirically demonstrated.

The naringenin effective in preventing or treating neurodegenerative diseases including of the present invention composition active ingredient can be.

Naringenin in the present invention said be a compound represented by formula 1.

<Formula 1>

The present invention according to formula 1 compound salt, preferably can be used in form of a pharmaceutically acceptable salt thereof. Said salts include pharmaceutically acceptable acid addition salt formed by free acid (free acid) preferably, said free acid include organic acid and an inorganic acid can be used. the organic acid has the number one but not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, tree flow five high-performance, benzoic acid, a central, meta sulfonic acid, glycolic acid, succinic acid, 4 - toluene sulfonic acid, glutamic acid and aspartic comprises. In addition said inorganic acid is the number one is but not limited to, hydrochloric acid, sulfonic acid, comprising sulfuric acid and phosphoric acid.

The naringenin represented by condensed water is formed using the present invention according to formula 1 may be, or natural or synthetic MethodsIn separated from publicly known chemical contrast can be prepared by the number employed.

Compositions of the present invention composition including about number such as active ingredient in addition to active ingredient naringenin said therapeutically suitable physiologically acceptable auxiliary number about number using number can be tank, said auxiliary number number include excipients, disintegrating number, sweetener number, number coupled, coating number, expansion number, number lubrication, bow [thayk[thayk] number or flavor number can be use.

Said therapeutic composition to said substrate in addition to the active number about number administration with about dermatologically acceptable carrier comprising at least one additional therapeutic composition can be preferably about number 1 number number under anger.

Number number number number of form about said granular compositions, acid number, positive number, coated tablets, capsule number, number left, number solution, syrup, juice, suspension number, oil number, number or injectable liquid droplets can be like number. For example, the form number number according to the number of positive number or capsules, effective component is ethanol, glycerol, water such as oral, about number can be combined with a non-toxic dermatologically acceptable inert carrier. In addition, desired or if necessary, a suitable binding number, lubricating number, number of the leaving group can be in admixture number in addition disintegrating. A suitable binding number is the number the isn't the thing one, starch, gelatin, glucose or beta - natural sugar such as lactose, corn sweetener number, acacia, such as sodium or [thu[thu] lacquer can [su[su] the [ley which comes the [thu and natural and synthetic gum, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride etc.. The disintegrating number is the number one but not limited to, starch, methyl cellulose, agar, it cuts the toe knit, etc. and xanthan gum. Liquid solution there is to a water and it stands about number number number of acceptable therapeutic composition which carrier include, sterilization and biocompatible provided, saline, sterile water, ringer's solution, buffer saline, albumin injection solution, dextran lactose solution, malto dextrin solution, glycerol, ethanol and of these components can be mixed at least 1 component, antioxidant number needed, buffer, addition of other usual number such as bacteriostatic number can be added. In addition dilution number, dispersion number, number surfactants, number and lubrication number added coupled added to aqueous solution, suspension, emulsion for injection such as number type, pill, capsule, granule or positive number to number number can be under anger. Further a detail suitable method to Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA method according to the disclosure in response to the disease or components can be preferably according to number number.

In in one embodiment of the present invention, the composition of the present invention 0 naringenin. 01 to a concentration of 100 μm can be included.

The pharmaceutical composition of the present invention effect of neurodegenerative diseases about number, Alzheimer's disease, Parkinson's disease, diseases rugate spinning, hunting tone diseases, amyotrophic lateral side three stiffening symptoms, multiple sclerosis, immune system or more brain function failure, progressive neurodegenerative diseases, metabolic brain, only - fick bottle sludge, such as dementia due to brain ischemia and cerebral hemorrhage is cited, be a limited to but not limited to more particularly Alzheimer's disease.

In addition, the present invention refers to number of naringenin for numerical control machine preventive or therapeutic drug for neurodegenerative diseases including use of such active ingredient number under public affairs substrate. the active ingredient of a medicament for the prevention or treatment of neurodegenerative diseases including of the present invention composition naringenin number can be uses for numerical control machine.

In addition, the present invention refers to the prevention or treatment of neurodegenerative diseases including administering to a mammal a naringenin method number under public affairs substrate.

Where the methods comprise terms used in "mammal", observing or experiments it is the context of the mammal, preferably human said substrate.

"Therapeutically effective amount" terms used in here forward user, veterinarian, physician or other clinical considered by the organized orgin, biological or medical active ingredient or drug number in animals or humans to a DNA compositions means that the amount of water, for the relief of symptoms of the disease which becomes to be therapeutically or disorders comprising inducing amount. Active ingredient a therapeutically effective dose and the desired effect of the present invention treatment administration will be varied in accordance with the skilled nontrivial disclosed. The optimal dosage is administered can be easily determined by one skilled, type of disease, disease severity, and at least a composition containing other components content, number of types, and patient age, weight, general health, sex and dietary, administration time, routes and compositions secretion rate, a treatment period, including simultaneous use of various factors can be adjustable. In the treatment method of the present invention, in the case of adult, 1 1 of the present invention many times to times when administered naringenin a, 0. 01 mg/kg - 250 mg/kg administered in an amount preferably.

The treatment method of the present invention as the active ingredient of the present invention composition including naringenin in oral, rectal, intravenous, in artery, intraperitoneally, intramuscular, in the sternum, transdermal, topical, intraocular or syringes can be administered over a conventional manner.

In addition, the present invention refers to health food for preventing or ameliorating neurodegenerative diseases including naringenin active ingredient number under public affairs substrate.

Health food product may be a neurodegenerative and prevention of hearing loss and improvement of the present invention, positive number, capsules, powder, granules, liquid, like in the form of ring can be number bath and treating.

In the present invention referred to as "health food" it health food law number 6727 cottonseed functionality using raw materials or components having call according to number bath and treating food it is, such as a nutrient for the structural and functional unit to adjust the human physiological action is taken which is useful for application health letter pattern means that the substrate.

Health food of the present invention comprises additives can be conventional food, food additive as other suitable regulation whether a free, Korea Food and Drug Administration acknowledged food additive of lost motion such as corresponding items according to a general regulation and general test and reference standard determined by each other.

Said valve to a "food additive lost motion" items include e.g., ketones, glycine, citric acid calcium, nicotinic acid, acid chemical composition; small dark blue, extract, crystalline cellulose, quantity dye, guar gum natural additive; sodium number number L - glutamic acid, alkali number added noodles, the preservation number number, mixing such as tar dye number number number number or the like is cited.

For example, health food of the present invention in the form of a positive number less than 75 number naringenin active ingredient, coupled number, number and other disintegrating addition number granulation into conventional method and then mixed with mixture, like bow [thayk[thayk] number filled or compression molding, can be compression-molding said mixture directly. In addition said health food product may be in the form of a positive number and the like needed number bee disapproval.

In the form of number by a cylindrical hard capsules of the present invention to production cost also health food ingredient such as filling the high pressure liquid coolant mixture mixed with addition of naringenin less than 75 number number number can be, naringenin is also mixed with 100 weight number number number less than 75 such as gelatin mixture to high pressure liquid coolant also can be number instead of the number. Said plasticizer such as glycerin or sorbitol is also number number according to need, colored number, number can be preserved and the like.

Health food product may be in the form of active ingredient of the present invention ring number naringenin with excipients, binding number, a publicly known method such as an orally disintegrating number obtained by mixing a mixture can be formed into tank new number, pin number or other number number needed to avoid the problems of white sugar, or starch, for coating the surface of a substance such as the [khu which burns and disapproval.

Granules of the present invention number health food ingredient naringenin with excipients, binding number, publicly known method such as a high pressure liquid coolant sprayed mixture obtained by mixing a disintegrating number to number can be in particulate, if necessary flavour number, number can be bee and the like.

Said beverage current health food, meat, chocolate, food, confectionery, pizza, if, other noodles, gum type, candy current, ice cream flow, alcohol beverage flow, vitamin composite number and health support food etc. disclosed.

Hereinafter, the present invention broadcast receiver through more detailed in the embodiment. The present invention is more specifically account for these in the embodiment is, in the embodiment of the present invention range and not the limited to these.

<In the embodiment 1>

Experiment material and experiment method

<1-1> Cell culture

PC12 cells (ATCC CRL 1721) at each of the present invention is using the cost of chromium that 95% air and 5% CO₂ 37 °C rat adrenal of affinity of such transgenic animals induced in 5% horse serum conditions, including RPMI1640/10% FBS and 1% penicillin-resistant Streptococcus pneumoniae (Hyclone Laboratories, INc) Streptococcus 7.5 by him as a medium.

<1-2> Cell survival rate (MTTAssay) Method

PC12 cells to 1 × 10 RPMI1640 optimum medium 96 well plate⁵ Culturing cells/well into 24 time after time and concentration of 0, 1, 10, 25 μm and incorporated into culture nor post-A β 1 naringenin_25-35 Then adding a 50 μm so that the 24 time him as. Then, MTT (3 - [4, 5 a-dimethylthiazol - 2 a-yl] - 2, 5 - diphenyl tetrazoliumbromide) reacting a supernatant solution 10 μl into and out of the stand-alone generated number 3 time after dissolving in DMSO formazan (formazan) 570 nm were measured absorbance.

<1-3> Active oxygen kind Billion number effect measuring (ROSAssay) Method

(ROS) at doses of 0, 1, 10, 25 μm cytotoxic evaluation of the same number has been confirmed that the activity of naringenin billion generating active oxygen kind. For the measurement of fluorescent dye CH ROS₂ Using DCF provided DA has been confirmed. PC12 cells 24 to 96 well plate (well plate) in the vicinity off light CH culture nor post-incubation time₂ In 30 minutes for shielding DCF provided DA him as. Reaction end immediately after, washed with HBSS 485 nm/535 nm wavelength fluorescence measuring device after using accumulated on the ROS A β by measuring the coupled fluorescent DCF provided DA_25-35 Processing digitized from the relative qualifications with respect to shown.

<1-4> PC12 cells and cell death inhibitors in the form of analysis method aspect

Cell morphological changes were measured using Hoechst 33342 dye. 8 well plate (well plate) cells to naringenin which is included in the processing according to a concentration, 1 A β has been completed_25-35 4% formaldehyde for processing after some time to 20 minutes to an culture cells 24 was washed with PBS. Hoechst 33342 (1 μg/mL) is added 15 minutes (Olympus, Tokyo, Japan) at room temperature value of 28 to observed using fluorescence microscopy.

In addition, A β_25-35 Processing aspect caused cell death (Merk Milipore, USA) using specifically separate Annexin V & Dead Cell Kit was analyzed. Culture cells washed with PBS by recovering trypsin EDTA into and out of the annexin V & Dead Cell after 20 minutes in the dark place reacting out behind, analyzer (Milipore, USA) was analyzes a type apoptosis using flow cytometry.

<1-5> Caspase-3 activity measuring method

6 - well plate (well plate) PC12 cells to 1 × 10⁶ After some time after the naringenin is provided which reduces to a location A β cells/well_25-35 The meeting. 24 hours after the cells washed with PBS and yet trypsin non-EDTA, 10 minutes on ice with respect to the added lysine cis buffer (lysis buffer) reaction. After crushing the cell to obtain a cyclone reactor 13,000 rpm, 96 well plate in DTT solution and DEVD -pNA which fit value of 28 to 405 nm which has been toasted in time using ELX808 pOf extinction price NA were measured.

<1-6> Cell cycle measuring method

A β naringenin and_25-35 After processing of a cell suspension to PBS solution to the well cells attached to trypsin-a EDTA to have been produced. The cell suspension in 70% ethanol from 3 hours to an -20 °C, Muse cell cycle solution utilizing the number according to the instruction of the radiation was filled flow cytometry experiments.

<1-7> NO Amount measuring method

The Griess reagents required to nitrite nitric oxide generated cells were measured amounts in the form of method (nitrite) nitric oxide quality. 100 μl of culture on Griess reagent comparable [1% sulfanyl neel amide (sulfanilamide), 0. 1% N-a 1 - me [phu ethylene diamine (N-a 1 provided napthylethylene diamine) in 2. 5% phosphoric acid (phosphoric acid)] by reacting 10 minutes in 96 well plate by mixing in absorbance 570 nm were measured. Sodium nitrite (Sodium nitrite) is determined using a standard curve is generated by a nitric oxide donor was compared with the quantity.

<1-8> PGE2 Amount measuring method

The changes in concentration of intracellular PGE2 ELISA (enzyme a-linked immunosorvent assay) assay kit (R&D Systems, Minneapolis, MN) using his method using a sample reservoir. With 96 well plate Goat anti-a mouse Ig PGE₂ After introducing or sample, each 50 μl at room temperature into the lung rock it is sour, oh PGE2 - 2 with respect to the temporal number conjugates. Then PBS via washing, substrate solution in 30 minutes into the reaction reaction place a piezoelectric element. 450 nm absorbance measuring results in each cell culture PGE2 content standard calibration curves contained in the bill.

<1-9> The [wey the [su it shook off blot Analysis method

PC12 cells to 3 × 10 6 well plate (well plate)⁶ And incorporated into the cells/well 24 hours, after which the naringenin frame and beany 1 A β_25-35 Was added. Cells to 2 times (protease inhibitor) number number number including cold PBS washed and proteasome using 1 billion (lysis buffer) lysine cis buffer time cells in 10 minutes after dissolving 13,000 rpm centrifuge separating supernatant protein was only. BSA (bovine serum albumin) protein concentration was a standard measurement. 10% polyacrylamide gel electrophoretic and PVDF film (poly a-vinylidene difluoride membrane) quantifying a protein of 15V to, 300 mA to transition with respect to the 30 minutes. Protein before film including removal of fat gushing oil 5% after 1 time blocking (blocking) is placed 1X PBST, 1 difference stored at reaction with respect to the antibodies. 1 to 8 times difference antibody substances after washing the films 1X PBST, 2 2 4 °C 1X PBST was then washed in the temporal difference antibodies. (ATTO, Tokyo, Japan) results Ez Capture ST protein therefrom.

<In the embodiment 2>

Experiment result

<2-1> Naringenin The result of measurement of survival rates of apoptosis caused by processing

PC12 cell survival in a cell of the present invention on the payout rate for extracting naringenin is from 1 μm 200 μm after processing result to the cytotoxic verifying, PC12 cells to cytotoxic concentration of naringenin is 25 μm can be ascertained not been (also reference 2). The, influence cell survival rate will not affect the concentration with respect to a method is A β is 25 μm is fed to the naringenin_25-35 Cellular response to protective decline-induced toxicity in the line therefrom.

First, MTT assay results through heat, A β_25-35 By stimulated PC12 cells live show rapid rate but, with respect to the recovery rate from naringenin is 1 μm cell survival. Flow cytometry on a metal thin film excellent in charge to achieve an enhanced effect can be ascertained even result all concentration section been (reference 3 also).

<2-2> Naringenin Treatment Active oxygen kind Identifying billion number effect

A β of naringenin_25-35 By active oxygen kind billion (reactive oxigen species; ROS) number effect a separate cell elements accumulated in a ROS CH₂ The dyed DCF provided DA were measured.

As a result, 4 also compared to controls such as A β_25-35 Strong fluorescence was observed intracellular ROS accumulation processing group represented, this prior to naringenin to 1, 10, 25 μm 88 by clicking. 86 ± 3. 70, 76. 34 ± 4. 62, 70. 69 ± 1. To know that were reduced to 86% (reference 4 also).

<2-3> Naringenin The type and volume of PC12 cells inhibiting cell death process of identifying aspect

During cell death (apoptosis) appearing at the morphological change is processed hoechst 33342 fluorescent dye to specifically bind to DNA is observed under fluorescence microscopy. As a result, controls 7. 50 ± 1. A β compared to 2%_25-35 The 38. 3 ± 2. 6% cell death (apoptosis) but to the naringenin concentrations causing reduced significantly and the number of. In particular, the troop resveratrol (resveratrol) superior than positive contrast concentration 25 μm naringenin in cell death (apoptosis) is changed to a 50 μm billion number his car (also 5 reference).

See specifically inhibiting apoptosis aspect of naringenin on initial cell death marker annexin V flow cytometry cell to cell death marker 7 provided AAD controller and by selecting a result, A β_25-35 Cell death (apoptosis) EDM electrode both initial and late, the initial as well as cell death (apoptosis) naringenin intermediate cell to selectively inhibit the schedulable his car (reference 6 also).

<2-4> Naringenin Treatment Caspase-3 active confirmation

A β_25-35 In the form of cell death (apoptosis) due to cell death for identifying modulators for modulating index is a representative one of the caspase-a 3 were measured. As a result, A β_25-35 Processing only when confirmed is activated caspase-a 3 cream, this naringenin concentration depending on his car that reduced caspase-a 3 (reference 7 also).

<2-5> Naringenin Identifying normalizing process of PC12 cells of cell cycle

For Go/G1 cell cycle progression is, for S, G2/M of periodically repeating order while proliferating cells to be coated. PC12 cells A β_25-35 In the Go/G1 progression into S confines the Go/G1 is increased S groups (arrest) is reduced. While, in processing of the present invention confines G0/G1 cell cycle for recovering and neutralize increased Go/G1 naringenin was again reduced his car (also 8 reference) is schedulable.

<2-6> Naringenin Treatment of inflammatory cytokine production billion number identifying processing

A β_25-35 In response to antigenic stimulation produced by the inflammation mediated cytokine TNF a-α on analyzing result IL-a beta 1, generation of A β TNF a-α_25-35 10 times compared to controls when it has processed a plasmid but, when processing a number concentration of at least 10 μm naringenin were to know that the effective billion (also reference 9).

In addition, A β_25-35 Raised 10 μm or more due to generation of IL-a beta 1 making sure that the capable of naringenin processing also reduced (10 also reference).

<2-7> Naringenin Treatment NO Generating and INOS Expression identifying number billion

A top NO cells by inducing inflammation in excess of cell damage and death mediates Alzheimer's important could be bonded each other. The PC12 cells for measuring NO A β_25-35 The stimulating then, be used as the generation of the naringenin concentration NO, A β_25-35 10 times during processing alone increased NO compared to 1 μm and 10 μm or more even low concentrations of generating positive contrast in about half naringenin reduces transient similar level is inhibin number 50 μm resveratrol troops billion (p<0. 001) (reference 11 also).

The result of the expression of iNOS NO generation mechanisms in order to identify the guide 12 billion number also such as disclosed. PC12 cells A β 1 naringenin a processing time_25-35 Then irradiated with the stimulating expression of iNOS result, A β_25-35 For derived iNOS to naringenin ratio different mistletoe. The, inhibiting iNOS A β to the naringenin_25-35 A large number of generated excessively by NO billion play a role to determine the other.

<2-8> Naringenin Treatment PGE2 Generating and COX-2 billion number confirming expression

PC12 cells A β_25-35 PGE2 has been inducing generation of processing, be used as the number employed for generating naringenin of PGE2 billion, billion when the number concentration depending on PGE2 production, in particular, been observed potent PGE2 billion number generating active at high concentrations (13 also reference).

PGE2 identifying result of expression of COX-a 2 generating aspect, as in Figure 14 the A β_25-35 Processing regulated expression in the group consisting only german silver compared to 286. 7 ± 25. Been increased 9%. However naringenin according to a concentration (1, 10, 25 μm) when it has processed, even lightly 134. 5 ± 25. To reduce COX-a 2 1% expression, 10 μm in 111. 3 ± 24. 3% reduces the powerful COX-a 2 inhibiting capacity inhibin receptor.

<2-9> Naringenin Treatment NF-ΚB And I -ΚB Confirming generation of

Inflammatory cytokines and move into and when activated NF provided κB iNOS, COX-a 2 induces the production of enzymes such as inflammatory response is induced by ultimately are (Noh et al, 2011).

If A β in experiments_25-35 Processing when the number incremented by irradiating NF provided κB billion activity, in the presence of 10 μm or more in an effective ingredient NF provided κB subunit p65 naringenin result in his car (also 15 reference). In an activation process should be phosphate residues serine (serine) NF provided κB IκB is off from the substrate. A β_25-35 When the IκB phosphorylation promoting processing, naringenin was number is a billion. The cell in which the expression of the naringenin result NF provided κB, from affecting IκB degradation means other.

<2-10> Naringenin Treatment MAPKs Identifying inhibitors

An upper mechanism for identifying ERK1/2 mediated activation NF provided κB, JNK, phosphorylation of shape p38 MAPKs were measured.

As a result, A β_25-35 Upon activation of the MAPKs due to increased expression of the naringenin on p-a JNK p provided p38 billion number but, in the significant difference could not be p a-ERK(16 also reference). The naringenin viewed till these results by number billion JNK MAPKs NF provided κB during expression, suggesting a potential for a higher signal transduction pathway p38 MAPKs is to serve as a substrate.

The number into the tank by the present invention to the preferred embodiment the transformed for flaws. The present invention is in the field of the present invention is provided essentially from deviating from a person with skill in the art of the present invention is embodied in the form of modified inputted properties may be understand it will rain. The definitive aspect as well as the descriptive disclosure in the embodiment are contemplated aspect should. The aforementioned range of the present invention description and claim rather than as shown, and the present invention is in a range equal to all differences may be carried on an will be interpreted.