KR101717352B1 - Composition for preventing and treating neurodegenerative diseases comprising gold nanoparticles and anthocyanins conjugates - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating degenerative brain diseases comprising a combination of anthocyanin and gold nanoparticle as an active ingredient, and a health functional food.
The anthocyanin and gold nanoparticle binding agent (anthocyanin-AuNP) of the present invention reduces the level of amyloid beta in the brain and increases the number of neurons in comparison with administration of anthocyanin alone in a relatively small amount, The composition comprising the compound has an excellent effect for the prevention, improvement and treatment of degenerative brain diseases.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating degenerative brain diseases, which comprises a complex of anthocyanin and gold nanoparticles, and more particularly, to a composition for preventing and treating neurodegenerative diseases,

The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating degenerative brain diseases comprising a combination of anthocyanin and gold nanoparticle as an active ingredient, and a health functional food.

Alzheimer's disease (AD) is a disease that causes gradual memory impairment and cognitive impairment. It is known to progress to an onset of illness, to have no fundamental treatment, and to shorten the average life expectancy compared with normal age groups. In this disease, amyloid beta (A [beta]) peptides are deposited throughout the brain, resulting in specific pathologies such as neurodegeneration, synaptic disorder, inflammation and production of reactive oxygen species.

Meanwhile, the blood-brain barrier (BBB) is a capillary wall in the brain. It serves as a physical and functional barrier to isolate the brain parenchyma from systemic circulation, thereby effectively protecting the brain from circulating toxins . However, charged molecules and molecules with a size greater than 700 daltons can not pass through the cerebral vascular barrier, which is a barrier to pharmacological treatment of brain diseases such as Alzheimer's disease. To address this problem, various drug delivery strategies have been attempted to control and target drug delivery to the brain and to provide effective treatment continuously, but no clear solution has been made to date.

Anthocyanin is a flavonoid polyphenol found in fruits, flowers, and leaves of plants. It is known to have effects on memory and cognitive ability (B. Shukitt-Hale etc., Nutr. Neurosci 12, 135-140 (2009)), and its effect has been confirmed in an animal model of Alzheimer's disease (PH Shih, J. Nutr. Biochem., 21, 598-605 (2010)).

Accordingly, the present inventors have made intensive efforts to develop a therapeutic agent for degenerative brain diseases such as Alzheimer's that can pass through the blood-brain barrier. As a result, they have found that gold nanoparticles (AuNP) and anthocyanins (anthocyanin) to produce an anthocyanin-AuNP conjugate. The present inventors completed the present invention by confirming that the conjugate is effective in the treatment of degenerative brain diseases caused by amyloid beta.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, which comprises a combination of anthocyanin and gold nanoparticle as an active ingredient.

It is another object of the present invention to provide a health functional food for preventing or ameliorating degenerative brain diseases, which comprises a combination of anthocyanin and gold nanoparticle as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, which comprises, as an active ingredient, a combination of anthocyanin and gold nanoparticle as an active ingredient.

In the present invention, the term "anthocyanin" is a water-soluble coloring matter glucoside present in a plant, and is a purple color, red color, or blue color depending on the acidic concentration of the cell liquid, the chemical structure of the dye compound, And the like. In recent years, it has been known that anthocyanins have various physiological activities. For example, an aging-inhibiting action, an antibacterial action, a mutagenic action, a cholesterol-lowering action, a vision improvement effect, a vascular protection function and an anti-

The anthocyanin may be extracted from plants or chemically synthesized. The plants include all plants that produce anthocyanins, such as black rice, black beans, black currant, chokeberry, black chokeberry, Cranberries, audi, cherries, raspberries, blueberries, blackberries, eggplants, acai, corn or grapes.

According to one embodiment of the present invention, the black bean skin is extracted under the condition of 1% HCl-MeOH solvent, and the extract is subjected to cationic compound containing an anthocyanin component from a nonionic compound using a cation exchange resin such as Ambrite XAD 7 or the like . Then, ionic substances including anthocyanin components were separated by elution at 1% HCl-MeOH condition to remove nonionic water-soluble and non-polar substances. Next, the above compound was subjected to size exclusion chromatography using Sephadex LH20 as a filler in a mixed solvent of 1% HCl-MeOH / water, and then an anthocyanin compound was purified using a reversed-phase adsorbent C18 Sep Pak.

The anthocyanin compounds include, for example, delphinidine 3-O-glucoside, cyanidine 3-O-glucoside and petunidin 3-O-glucoside ), But the present invention is not limited thereto. The respective formulas of the delphinidine 3-O-glucoside, cyanidine 3-O-glucoside and petunidin 3-O-glucoside are as follows: As shown below.

[Chemical Formula 1]

In the present invention, the term "gold nanoparticle" is a particle made of gold atom, also referred to herein as AuNP. The gold nanoparticles may be particles having a diameter of 0.1-100 nm, specifically 1-50 nm, but are not limited thereto. The size of the gold nanoparticles can be varied according to various conditions and can be appropriately selected by those skilled in the art depending on the purpose.

The gold nanoparticles are not particularly limited, but may be PEGylated to enhance residence time in a living body to which the conjugate is administered. Pegylated gold nanoparticles are referred to herein as PEG-AuNP.

The term "Pegylation " of the present invention means a processing method for improving the retention time of blood of the binding body by introducing polyethylene glycol into the gold nanoparticle. Specifically, by pegylating gold nanoparticles with polyethylene glycol, the hydrophilicity of the surface of the nanoparticles is increased, and immune function including macrophages in vivo which predates and extinguishes pathogens, waste materials, The rapid disintegration in the body through the so-called stealth effect, which prevents recognition from the body, can be prevented. Therefore, the blood retention time of the conjugate can be improved by the pegylation.

In one specific embodiment of the present invention, in order to confirm whether gold nanoparticles have safety suitable for use as a composition for preventing or treating degenerative brain diseases, PEG-AuNP was treated with cells and MTT assay and ApoTox-Glo ^™ Triplex As a result of the analysis, it was confirmed that the cells were not cytotoxic at all concentrations (0.5% to 10%) treated (FIGS. 1 and 2).

In the present invention, the term "combination of anthocyanin and gold nanoparticles" means a combination of anthocyanin and gold nanoparticles. The binding includes both physical and chemical bonding between gold nanoparticles and anthocyanin, It is a concept that includes the encapsulation of anthocyanin inside nanoparticles. At this time, as described above, the gold nanoparticles may be pegylated.

In an embodiment of the present invention, anthocyanin and pegylated gold nanoparticles (hereinafter referred to as "anthocyanin-PEG-AuNP") are bound to anthocyanin by binding pegylated gold nanoparticles (PEG- . Specifically, anthocyanin was dissolved in ethanol, and the solution was mixed with PEG-AuNP and centrifuged several times, followed by drying to prepare an anthocyanin-PEG-AuNP conjugate (FIG. 3).

The term "degenerative brain disease" in the present invention means a generic term for various diseases related to the brain. Specifically, in the present invention, the degenerative brain disease may be a disease in which brain cell damage is caused by amyloid beta peptide-induced oxidative stress. More specifically, the degenerative brain disease may be a dementia, Alzheimer's disease, stroke, paralysis, Parkinson's disease, Huntington's disease, Or Creutzfeldt-Jakob disease, and more specifically, but not exclusively, Alzheimer's disease.

In the present invention, the term "Alzheimer" is one of the major causes of dementia mainly caused by the degenerative brain disease in the elderly, and is a disease in which mental function declines gradually as brain tissue loses function in the process of aging. The illness causes serious disturbances in memory and emotion. Alzheimer's and dementia are sometimes identified, but dementia is caused not only by Alzheimer's disease but also by adult diseases such as hypertension, diabetes and heart disease. In neuronal mitochondria of the Alzheimer model, accumulation of amyloid beta has been found to accumulate, resulting in increased production of reactive oxygen species in mitochondria, leading to oxidative damage, and this oxidative damage is known to be associated with the onset of Alzheimer's disease.

As used herein, the term "prevention" refers to the administration of a pharmaceutical composition containing the combination of anthocyanin and gold nanoparticles according to the present invention as an active ingredient to a subject suspected of having degenerative brain disease to inhibit or delay the onset of degenerative brain diseases It can mean all the acts that you do.

The term "treatment" as used in the present invention means all the actions of causing the composition of the present invention to be administered to a subject suffering from a degenerative brain disease so that the symptom of the disease is improved or improved.

As used herein, the term "individual" may refer to all animals, including humans, who have developed or are at risk of developing degenerative brain diseases. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The pharmaceutical composition of the present invention can be used as a single preparation and can be used as a combination preparation containing a drug known to have an effect of treating a known degenerative brain disease and can be formulated using a pharmaceutically acceptable carrier or excipient , Or may be manufactured by inserting it into a multi-dose container.

As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound being injected, without stimulating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The carrier may comprise a non-naturally occuring carrier. In addition, if necessary, other conventional additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. A diluent, a dispersant, a surfactant, a binder, a lubricant, Pills, capsules, granules or tablets, and the like.

In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of anthocyanin and gold nanoparticles. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and is generally in the range of 0.001 to 1000 mg / kg, 200 mg / kg, more preferably 0.1 to 100 mg / kg, may be administered once to several times per day. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the specific composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer an amount that can achieve the maximum effect in a minimal amount without causing side effects, and can be readily determined by those skilled in the art.

The term "administering" as used herein refers to the introduction of a pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be oral or parenteral May be administered via various routes.

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be conventionally used in the art. As a non-limiting example of such a mode of administration, the compositions may be administered orally or parenterally. The pharmaceutical composition according to the present invention can be manufactured into various formulations according to the intended administration mode.

The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.

The anthocyanin-AuNP conjugate of the present invention decreases amyloid beta (A [beta]) levels or increases the number of neurons in the brain when administered to a patient suffering from or susceptible to degenerative brain disease Thereby preventing or treating degenerative brain diseases.

In a specific example of the present invention, it was confirmed that an anthocyanin-PEG-AuNP conjugate was treated with an Alzheimer's disease model mouse treated with A? _{1 -42} to be effective in the prevention and treatment of degenerative brain diseases. More specifically, when the handle-AuNP -PEG conjugate anthocyanins in the mouth, through the Y- maze test spatial working memory capacity is improved (FIG. 4), Aβ _{1 -42} increased by treatment with Aβ, BACE-1 And the level of phosphorylated tau protein was markedly reduced by Western blot analysis (Fig. 5).

Particularly, the decrease effect of Aβ levels was more remarkable in the case of the combination treatment than in the case of treatment with anthocyanin alone, and this aspect was confirmed by immunofluorescence analysis of Aβ in CA1, CA3, DG region and cortex of hippocampus (Figs. 6 and 7).

Furthermore, the anthocyanin-PEG-AuNP conjugate restores synaptic dysfunction induced by A? _{1 -42} treatment (FIGS. 8 and 9) and restores the abnormal PI-3K / Akt / p-GSK- (Fig. 10), confirming the effect of preventing neurodegeneration (Figs. 11 and 12).

Taken together, the anthocyanin-PEG-AuNP conjugate showed superior therapeutic efficacy in Alzheimer's model mice induced by A? _{1 -42} treatment, which is less than anthocyanin treated alone, It was confirmed that this effect was more remarkable than anthocyanin alone treatment. Thus, the composition comprising the combination of anthocyanin and gold nanoparticles of the present invention as an active ingredient can be used as a composition for preventing and treating degenerative brain diseases.

Another aspect of the present invention provides a health functional food for preventing or ameliorating degenerative brain diseases, which comprises a combination of anthocyanin and gold nanoparticle as an active ingredient.

The combination of anthocyanin and gold nanoparticles, and degenerative brain diseases have been described above.

The anthocyanin may be selected from the group consisting of black rice, black beans, black currant, chokeberry, black chokeberry, cranberry, audi, cherry, raspberry, blueberry , Blackberries, eggplant, acai, grapevines, or grapes, and has proved its stability for a long time. In addition, it was confirmed that the gold nanoparticles were free from cytotoxicity through a specific example of the present invention (FIGS. 1 and 2). Accordingly, the combined anthocyanin and gold nanoparticles of the present invention can be prepared in the form of foods that are common but can prevent or ameliorate degenerative brain diseases.

Functional food is the same term as food for special health use (FoSHU). It refers to foods that have been processed so that the biocontrol functions are efficiently displayed in addition to nutritional supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and circles to obtain a useful effect for preventing or improving degenerative brain diseases.

At this time, the content of the combined anthocyanin and gold nanoparticles contained in the health functional food is not particularly limited, but is preferably 0.01 to 100% by weight, more preferably 1 to 80% by weight based on the total weight of the health functional food .

Similar to the pharmaceutical composition of the present invention, the health functional food reduces the level of amyloid beta (A [beta]) in the brain when ingested by patients suffering from degenerative brain disease or who are likely to develop degenerative brain diseases Or increasing the number of neurons to prevent or improve degenerative brain disease.

The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. Also, unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is used for a long time by using food as a raw material, and it is excellent in portability.

The anthocyanin and gold nanoparticle binding agent (anthocyanin-AuNP) of the present invention reduces the level of amyloid beta in the brain and increases the number of neurons in comparison with administration of anthocyanin alone in a relatively small amount, The composition comprising the compound has an excellent effect for the prevention, improvement and treatment of degenerative brain diseases.

FIG. 1 shows MTT analysis of PEG-AuNP treated at different concentrations (0.5%, 1%, 5%, and 10%).
Figure 2 is a graphical representation of Apo-Tox Glo ^TM Triplex analysis is performed. Cell viability, cytotoxicity and cell schedules of HT22 were determined by treating PEG-AuNP with concentrations (0.5%, 1%, 5%, and 10%). n = 3.
FIG. 3 is a view showing an anthocyanin-PEG-AuNP conjugate. FIG. (A and B) The morphology of the nanoparticles was observed using a transmission electron microscope (TEM, Tecnai-12, 120 kV). (C) TEM analysis revealed anthocyanin-PEG-AuNP complex in brain tissue.
Figure 4 is a check of the spontaneous alternation level (spontaneous alteration) by administering the -PEG-AuNP conjugate anthocyanins in mice treated with Aβ _{1 -42} and perform the Y- maze test. A [beta] _{i- 42} was maintained in the mice for 7 days with or without treatment. After 7 days of treatment with A? _{1 -42} , Anthocyanin and anthocyanin-PEG-AuNP conjugates were administered to mice for 14 days. The Y-maze test was used to measure the percentage of spontaneous alternation behavior for each group for 8 minutes. n = 15. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
5 is Aβ _{1 -} is a ₄₂ treated mice confirm the effects of the anthocyanin-AuNP -PEG conjugate for Aβ, BACE-1 and tau protein hyperphosphorylation the screen level. Western blot results of A [beta], BACE-1 and p-Tau (Ser413) in mouse hippocampus are shown. Bands were quantitated using Sigma Gel software and plotted. β-actin was used as a loading control. Density values were expressed as mean ± SEM relative to the respective expressed proteins, based on the expression level of the control group (n = 10 mice / group). * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
6 is Aβ _{1 - 42} is treated mice with Aβ _{1 -42} and anthocyanins even making sure of the Aβ immunofluorescence to see the immunoreactivity in the hippocampus of -PEG-AuNP conjugate treated mice. 10 x magnification. Scale bar = 100 占 퐉. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
7 is Aβ _{1 - 42} is treated mice with Aβ _{1 -42} and anthocyanins even making sure of the Aβ immunofluorescence to see the immunoreactivity in the cortex of the -PEG-AuNP conjugate treated mice. 10 x magnification. Scale bar = 100 占 퐉. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
8 shows the expression levels of synaptophysin, SNAP23, PSD95, p-GluR1 (Ser845), and p-CREB (Ser133) according to treatment with anthocyanin-PEG-AuNP conjugate in mouse hippocampus. Bands were quantified using Sigma Gel software and plotted as a graph. β-actin was used as a loading control. Density values were expressed as mean ± SEM relative to the respective expressed proteins, based on the expression level of the control group (n = 10 mice / group). * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
FIG. 9 shows immunofluorescence reactivity of PSD95 and SNAP23 in CA1, CA3 and DG regions of hippocampus. 40 x magnification. Scale bar = 100 占 퐉. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
Fig. 10 shows the expression levels of p-PI3K, p-Akt (Ser473) and p-GSK3? (Ser9) in the mouse hippocampus. Bands were quantified using Sigma Gel software and plotted as a graph. β-actin was used as a loading control. Density values were expressed as mean ± SEM relative to the respective expressed proteins, based on the expression level of the control group (n = 10 mice / group). * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
Fig. 11 shows the expression levels of caspase-9, caspase-3 and PARP-1 in mouse hippocampus. Bands were quantified using Sigma Gel software and plotted as a graph. β-Actin was used as a loading control. Density values were expressed as mean ± SEM relative to the hippocampal proteins indicated, respectively (n = 10 mice / group), based on the expression level of the control. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
Figure 12 is a photograph of Nissl staining in the hippocampus DG, CA3, CA1 region of Aβ _{1 -42} treatment group. Scale bar = 200 占 퐉. * Significant differences from control; # Significant difference from A? _{1 -42} treated mice, significance = P <0.05.
13 is a mouse model for Aβ _{1 -42} Fig. 5 schematically shows the effect of anthocyanin-PEG-AuNP conjugate.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  1. Reagents

β- amyloid _1-42 and Aβ _{42 -1} peptide was purchased from Sigma-Aldrich (Saint Louis, MO , USA). An aqueous suspension of 5 nm pegylated gold nanoparticles (PEG-AuNP) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA). The AuNP was stored at 4 [deg.] C to prevent aggregation.

Example  2. Extraction of anthocyanin

After extracting the black bean husk with 1% HCl-MeOH solvent, the extract was treated with cation exchange resin such as Ambrite XAD 7 or the like to obtain a cationic compound containing the anthocyanin component of the composition of the present invention from a non- To remove water-soluble and non-irritating substances, ionic substances including anthocyanin components were separated by elution under 1% HCl-MeOH condition. In the second step, the compound was subjected to size exclusion chromatography using Sephadex LH20 as a filler in a mixed solvent of 1% HCl-MeOH / water and then subjected to size exclusion chromatography using C18 Sep Pak, a reversed-phase adsorbent, with delphinidin-3-glucoside, cyanidin- -Glucoside and the lung-tunin-3-glucoside.

Example  3. MTT  analysis

To evaluate the cytotoxicity of PEG-AuNP, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) analysis was performed on various concentrations of PEG-AuNP. In a 96-well plate, 1 x 10 &lt; ⁵ &gt; HT22 hippocampal neuronal cells were inoculated in 200 [mu] l DMEM (Dulbecco's modified Eagle's medium) per well. After 48 hours of culture, the cells were treated with 0.1%, 0.5%, 5%, and 10% PEG-AuNP, respectively, and cultured for 12 hours. Then, MTT dissolved in PBS (phosphate buffered saline) at a concentration of 5 mg / ml was added to each well and cultured at 37 ° C for 4 hours. Then formazan dissolved in dimethyl sulfoxide (DMSO) was added to each well, and the plate was stirred in the shaker for 10 minutes to 20 minutes. The absorbance was then measured at 550 to 570 nm (L1) and 620 to 650 nm (L2) using a scanning microplate reader. The L2 absorbance is used to measure defects in cell debris and wells. The absorbance of each well was corrected (A = L1 - L2) and the cell viability (%) was calculated as (corrected absorbance of treatment group well / corrected absorbance of control well) X100.

Example  4. ApoTox - Glo ^TM Triplex analysis

Cells was carried out toxicity, viability, and ^{caspase-3/7-Glo TM} ApoTox Triplex analyzed to determine the activity of (caspase ^3/7).

1% penicillin / streptomycin and 10% FBS (fetal bovine serum) to mouse 200 ㎕ DMEM containing hippocampal neuronal (HT22) cells (2 x 10 ⁴ pieces) and the inoculated in a 96-well plate, 5% CO _2, And cultured in a 37 ° C incubator. After 48 hours of incubation, the cells were treated with PEG-AuNP at various concentrations (0.5%, 1%, 5%, and 10%).

For analysis, 20 [mu] l of viable / cytotoxic reagent containing GF-AFC and bis-Aaf-R110 substrate was added to the wells and mixed for 30 seconds at 500 rpm using orbital shaking, Lt; 0 &gt; C for 1 hour. Fluorescence was measured at a wavelength of 400 nm / 505 nm and a wavelength of 485 nm / 520 nm to measure cytotoxicity for viability measurement. GF-AFC (glycyl-phenylalanyl-aminofluorocoumarin) substrates enter living cells and release AFCs during degradation by proteases, so the activity of living cells was measured. bis-alanylalanyl-phenylalanyl-rhodamine 110) was degraded by the protease of dead cells and released R110 rather than living cells, and the protease released from dead cells was measured using this.

Then, we use the tetra peptide (tetrapeptide) luminometer transgenic (luminogenic) Case Paget ^3/7 substrate consisting of SEQ ID NO DEVD to measure Paget Case ^3/7 activity. 100 [mu] l of caspase-Glo reagent was added to all wells and mixed using orbital shaking at 500 rpm for 30 seconds. Then, after incubation at room temperature for 30 minutes, luminescence was measured to detect caspase ^3/7 activity.

Example  5. Anthocyanin-PEG- AuNP  Preparation and Characterization of Conjugates

A 5 nm gold nanoparticle aqueous solution was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The gold nanoparticles were coated with polyethylene glycol (PEG). And stored at 4 ° C to prevent aggregation of AuNP.

A specific method for preparing PEG-AuNP and anthocyanin conjugate is as follows. After 13 mg of anthocyanin was dissolved in 15 ml of ethanol, the solution was mixed with 40 ml of PEG-AuNP conjugate and stirred at 60 for 3 hours. The solution was then allowed to cool at room temperature and centrifuged at 4000 rpm to remove unattached anthocyanins. The procedure was repeated three times. The solution was then evaporated using a rotary evaporator under vacuum. The morphology of the nanoparticles was observed using a transmission electron microscope (TEM, Tecnai-12, 120 kV) and the encapsulated anthocyanin was confirmed in the nanoparticles.

Example  6. Experimental animal and drug treatment

Male C57BL / 6N mice were purchased from Samtako Bio (Korea) at 8-10 weeks of age (average weight 25 g) and were raised in an environment with a nominal / cancer cycle of 23 hours, 10% humidity and 12 hours / 12 hours. The mice were allowed to use water and feed freely.

The mice were divided into five groups as follows (n = 15 per group). (1) treatment with 0.9% saline (control) (C), (2) beta-amyloid, A? Treatment, (3) 4) treatment with beta-amyloid and anthocyanin (Aβ + Anth 12 mg / kg / day), (5) treatment with Aβ + AuNP (100 μl) + anthocyanin (6 mg / kg / day).

The drug treatment process is as follows. A stock solution of Aβ _{1 -42} peptide (1 mg / ml) was prepared in sterilized saline. Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; to make peptide aggregates. The agglomerated _{1 -42} Aβ peptides or control (0.9% NaCl, 3 ㎕ / 5 minute / mouse) using a Hamilton micro-syringe (microsyringe) was administered to the brain blood vessels (intracerebroventricular, icv). Anesthesia was anesthetized with a combination of 0.05 ml rumpun per 100 g body weight and 0.1 ml zoliti per 100 g body weight and was measured in steriotaxic coordinates for bregma (0.2 mm anterior, AP, (mediolateral, ML) = 1 mm, dorsoventral (DV) = 2.4 mm) was injected into the ventricle (icv) with a Hamilton microsyringe. During this process, the body temperature of the animals was adjusted because anesthesia caused hypothermia, which could cause tau hyperphosphorylation.

AuNP suspensions Aβ _{1 -42} was intravenously via the tail vein 7 days after 0.90 mg / kg body weight density after injection. Mice were inhaled and anesthetized with isoflurane to relax the muscle. Thereafter, AuNP (100 [mu] l) suspension was slowly injected using a 1 ml insulin syringe. In addition, Aβ _{1 -42} scan was injected 7 days after, anthocyanins 12mg / kg / day anthocyanins -PEG-AuNP 6mg / kg / day respectively for 14 days. Animals were sacrificed for biochemical and immunohistochemical analysis when behavioral analysis was complete.

Example  7. Behavioral analysis (voluntary behavior change in Y-maze test)

A behavioral test of the mice was performed using the Y-maze device (n = 15 / group). The maze was made of wood painted in black paint. Each arm of the maze is 50 cm long, 20 cm high, and 10 cm wide. Each mouse was placed in the middle of the Y-maze instrument and allowed to move the maze freely three times for an 8-minute period. The entry into the arm of the Y-maze device was visually observed, and the spontaneous alteration was defined as the consecutive entry of the mouse into the three arms in the triplicate set. The change behavior (%) was evaluated as [Successful triplet set (successive entry with three different arms) / (total arm entry -2)] x100.

Example  8. Western blot  analysis

Protein expression levels were measured using Western blot analysis. After behavioral analysis the mice were sacrificed. The brain was quickly removed, the hippocampus was incised, and the brain was cooled with dry ice. The hippocampus was crushed in 0.2 M PBS supplemented with protease inhibitor. Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA) was used to measure the protein concentration of the milled liquor.

4-12% Bolt ^TM under the same experimental conditions the same amount of protein (15-30 ㎍) Mini gel and MES SDS running buffer 1 x (Novex, Life Technologies). Prestained protein ladders (GangNam stain ^TM ) capable of handling a wide range of molecular weights (10-245 kDa) interon Biotechnology) as a control for molecular weight. Membranes were blocked and 5% skim milk was used to reduce nonspecific binding. The membrane was then incubated overnight at 4 째 C with primary antibody (1: 1000 dilution). To detect the protein, secondary radiolabeled horse radish peroxidase conjugated antibody was used and the protein was detected using ECL detection reagent. The X-ray film was scanned and the optical density of the developed band on the film was analyzed by densitometry using a computer-based sigma gel program version 1.0 (SPSS, Chicago, IL, USA). Antibodies used were as follows: anti-BACE-1, anti-Ap, anti-p-Tau (Ser413), anti-PARP-1, anti- SNAP23, anti-PSD95, anti- -GSK3? (Ser9), anti-p-GluR1 (Ser845), anti-beta-Actin (Santa Cruz Biotechnology), anti-p-CREB (Ser133), anti-synaptophysin, , Anti-pAkt (Ser473), and caspase-9 (Cell Signaling).

Example  9. Tissue recovery  And sample preparation

After behavioral analysis, mouse brain slices were analyzed in the control, Aβ treated, Aβ and anthocyanin treated, and Aβ and anthocyanin-PEG-AuNP conjugate treated groups. After transcardially 1 X phosphate buffered saline (PBS) was perfused, 4% cold paraformaldehyde was perfused. The brains were then fixed overnight in 4% paraformaldehyde and transferred to a 20% sucrose solution for 72 hours. The brain was transferred to O.C.T. (A.O., USA) solution to freeze the brain, and then cut into a 14-μm-thick coronal plane using a CM 3050C cryostate (Leica, Wetzlar, Germany). The sections were thaw-fixed on a positive charge slide (Thermo Fisher Scientific Inc., Waltham, Mass., USA).

Example  10. Immunofluorescence analysis

The tissue-lined slides were washed with 0.01 M PBS twice for 10 min each. The slides are then immersed for 90 minutes in a blocking solution of standard chlorine / bovine serum and 0.3% Triton X-100 dissolved in PBS. Primary antibodies (mouse monoclonal antibody A [beta] and SNAP23 from Santa Cruz Biotechnology, rabbit polyclonal antibody PSD95 from cell signaling) were diluted 1: 100 in PBS and incubated overnight at 4 [deg.] C. The slides were then placed in a secondary antibody (Santa Cruz Biotechnology) labeled with FITC and TRITC diluted 1:50 in PBS for 2 hours at room temperature and the slides were washed twice with PBS for 5 minutes each. For double immunofluorescence, the slides were incubated overnight with another primary antibody and the secondary antibody incubation and washing steps were repeated as in the previous procedure. Slides were mounted with anti-fade reagent (Molecular Probe, Eugene, OR, USA). The staining patterns of dual immunofluorescence and single immunofluorescence were observed using a confocal laser microscope (Flouview FV 1000; Olympus, Tokyo, Japan).

Example  11. Nissl  dyeing

Nissl staining was performed to measure histological examination and loss of neurons. The 14-μm slice slides in 0.01 M PBS were washed twice for 15 minutes each and stained with 0.5% cresyl violet solution (including a few drops of glacial acetic acid) for 10-15 minutes. The slice was then washed with distilled water and dehydrated with increasing ethanol concentrations to 70%, 95%, and 100%, placed in xylene, and covered with cover slip using mounting solution. The number of cells in the cortex, CA1 and CA3 regions of the hippocampus was counted using computer image analysis.

Experimental Example  1. Anthocyanin-PEG- AuNP  In vitro binding of the conjugate ( in vitro ) Cytotoxicity

To evaluate the cytotoxicity of gold nanoparticles, MTT and ApoTox-Glo ^TM in the immortalized hippocampal neuron HT22 cell line Triplex Assay (Promega) was performed. Cytotoxicity of PEG-AuNP at 0.5%, 1%, 5%, and 10% concentrations was evaluated to determine cell viability. As a result of MTT analysis, there was no significant difference in cell survival rate at all concentrations, and it was found that PEG-AuNP was not toxic (Fig. 1).

Next, in order to analyze cell survival rate, cytotoxicity, and apoptosis (using apoptosis marker ^3/7 ) in neuron HT22 cells, ApoTox-Glo ^TM Triplex assays were performed. Similar to the MTT assay results, ApoTox-Glo ^TM Triplex assays also showed no significant differences in cell viability, cytotoxicity, and apoptosis at all concentrations (0.5%, 1%, 5%, and 10%) of PEG-AuNP (FIG. 2). The above results confirm that PEG-AuNP is not toxic to cells.

Experimental Example  2. Anthocyanin-PEG- AuNP  Characterization of the combination

To prepare the anthocyanin-PEG-AuNP conjugate, spherical gold nanoparticles were selected to distribute uniformly throughout the cerebrovascular barrier and coated with PEG to prevent aggregation.

As a result of observation of the anthocyanin-PEG-AuNP conjugate by TEM, it was confirmed that the hydrodynamic diameter of the PEG-AuNP containing anthocyanin was about 20 nm and had a size distribution similar to a regular shape (see FIGS. 3A and 3B ). Furthermore, anthocyanin-PEG-AuNP conjugates were observed in hippocampal tissues of mice (FIG. 3C).

Experimental Example  3. Aβ _{One -42} - Anthocyanin-PEG- AuNP  Effect of combination

In order to evaluate the effect of anthocyanin-PEG-AuNP conjugates on memory-impaired mouse models induced by A [beta] _{i- 42} injection, the number of consecutive entries into three arms was observed using the Y- (N = 15 / group) were calculated.

Spontaneous behavior change rate (%), which indicates spatial working memory function, is a form of spatial work short-term memory. Aβ after administration of the _1-42, The percentage of spontaneous behavioral changes was significantly reduced in Aβ _{1 -42} treated mice compared to controls, indicating that memory impairment was induced by Aβ _1-42 . Anthocyanin treatment of (12 mg / kg / day) and anthocyanins -PEG-AuNP (6 mg / kg / day) was treated only Aβ _{1 -42} Mice treated with anthocyanin-PEG-AuNP conjugate showed the largest increase in spontaneous behavioral change, even at lower doses than with anthocyanin alone (Fig. 4).

Experimental Example  4. Anthocyanin-PEG- AuNP  Cohesive Aβ , BACE -1 and phosphorylated Tau  Confirming protein level reduction effect

Aggregated A [beta] peptides are one of the key and important features of Alzheimer's disease. After injection of A? _{1 -42} into the brain, Aβ expression levels were analyzed in the brain using Western blot analysis.

As a result, Aβ _{1 -42} and treated mice showed an increase in Aβ levels than the control group, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) treated mice only Aβ _{1 -42} Compared with the control group. In particular, when the anthocyanin-PEG-AuNP conjugate was treated, it was confirmed that the level of Aβ was most remarkably reduced even at a lower dose than when anthocyanin alone was treated (FIG. 5).

In addition, immunofluorescence analysis of Aβ _{1 -42} compared to the control group by-product was confirmed to be the Aβ immunofluorescence reactivity in hippocampal CA1, CA3, DG area of the treated mice improved. Anthocyanin treatment of (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) was treated only Aβ _{1 -42} Aβ immunoreactivity was significantly decreased in the hippocampal CA1, CA3, and DG regions and cortex compared to mice. Especially, in the cortex, anthocyanin-PEG-AuNP conjugate was treated with anthocyanin alone (Fig. 6 and Fig. 7), despite a dose lower than that of Aβ.

On the other hand, _{A? 1-42} The expression level of BACE-1 is known to be increased, and the expression of BACE-1 in Aβ _{1 -42} -treated mice was also increased in the Western blot compared to the control. However, the process of the anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) reduced the expression levels of BACE-1 than in mice treated only Aβ _{1 -42} significantly (Fig. 5).

Further, the increase of Aβ is known to be activating the screen hyperphosphorylation of tau protein, Aβ _{1 -42} in order to evaluate them - Western blot of the tau protein is phosphorylated at the Ser413 treated mice was carried out. As a result, Aβ _{1 -42} injection increased the phosphorylation of tau protein Ser413 compared to the control group. However, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) treated mice is that the level of Ser413 phosphorylation of tau protein significantly decreased compared to mice treated only Aβ _{1 -42} (Fig. 5).

Taken together, the above results, anthocyanins or anthocyanin-AuNP -PEG conjugate sikimyeo significantly reducing the level of Aβ _{1 -42} by treatment Aβ, BACE-1, and phosphorylation of tau protein (Ser413), in particular anthocyanins -PEG-AuNP conjugate Significantly reduced levels of A [beta] despite lower doses than anthocyanin alone.

Experimental Example  5. Anthocyanin-PEG- AuNP  Cohesive Aβ _{One -42} -Induced synaptic dysfunction reduction

To determine the effect of anthocyanin (12 mg / kg / day) and anthocyanin-PEG-AuNP (6 mg / kg / day) on Aβ-induced synaptic deficits, Aβ induced synaptic dysfunction, Synaptic synaptophysin, post-synaptic synaptosomal associated protein (SNAP-23), postsynaptic density protein (PSD95), and AMPA (α- amino-3-hydroxy-5-methylisoxazol-4-propionic acid) receptor (AMPAR) protein.

Western blot analysis showed that synaptophysin expression level was decreased in Aβ _{1 -42} -treated mice compared to the control group. However, the process of the anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) has to overcome the effects of Aβ _{1 -42,} Significantly increased the level of synaptophysin expression (Figure 8).

Similarly, Aβ _{1 -42} - but in the treated mice the level of expression of PSD95 and SNAP23 reduced compared to the control group, and anthocyanins Anthocyanins -PEG-AuNP conjugate increased the level of expression of PSD95 and SNAP23 significantly (Figure 8 ).

Furthermore, the expression of SNAP-23 and PSD95 in hippocampal CA1, CA3, and DG regions was examined using immunofluorescence analysis. Treatment of Aβ _{1 -42} is sikyeoteuna reduced both the expression of the two proteins in the CA1, CA3, DG part of the hippocampus, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) is SNAP-23 And PSD95 expression levels (Fig. 9).

On the other hand, synaptic damage induced by A? _{1 -42} decreases AMPAR and phosphorylation at the Ser845 residue of AMPA receptor (GluR1) plays an important role in regulating the post-synaptic glutamate receptor trafficking system , And expression level of Ser845-phosphorylated GluR1 was analyzed. As a result, compared with the control group Aβ _{1 -42} - a Although Ser845 is phosphorylated GluR1 is reduced in treated mice, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) is Ser845 phosphorylation GluR1 (Fig. 8). &Lt; / RTI &gt;

In addition, p-CREB (phosphorylated cAMP response element binding protein) was an important transcription factor related to memory function and synapse formation, and decreased p-CREB (Ser 133) in Aβ _{1 -42} -treated mice compared to the control group. Treatment with anthocyanin-PEG-AuNP (6 mg / kg / day) significantly improved the expression level of p-CREB (Ser133) The expression level of pCREB (Ser133) was further increased compared with the treatment with anthocyanin alone (FIG. 8).

Experimental Example  6. Aβ _{One -42} Induced by Abnormal PI-3K / Akt / p- GSK - 3β  The anthocyanin-PEG- AuNP  Confirm the effect of the combination

Unusual PI-3K / Akt / GSK3β I signaling pathway is known to affect the hyperphosphorylation of Tau protein Chemistry and neurodegeneration, the present inventors have found that Aβ _{1 -42} - in relation to the tau phosphorylation in the treated mice the PI-3K / Akt / GSK3? Signaling pathway was investigated. The effect of anthocyanin-PEG-AuNP conjugates on GSK3?

As a result, phosphorylated phosphatidylinositol 3-kinase (p-PI3K) was significantly decreased in Aβ _{1 -42} -treated mice compared to the control group, but anthocyanin (12 mg / kg / PEG-AuNP (6 mg / kg / day) significantly increased the level of p-PI3K (Figure 10).

Similarly, Aβ _{1 -42} compared to the control - but the phosphorylation of Ser473 of (as protein kinase B also named) treated mice significantly reduced in Akt, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) overcome the A [beta] effect and significantly increased the phosphorylation of p-Akt-Ser473 (Fig. 10).

As described above, it is known that activation of Akt by phosphorylation of the Ser473 residue of Akt phosphorylates Ser9 of GSK3? To decrease the activity of GSK3 ?. Similar to the p-Akt result, phosphorylation of GSK3β Ser9 is compared with the control group Aβ _{1 -42} - decreased in treated mice, treatment of anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / day) It was confirmed that the phosphorylation of GSK3? Ser9 was significantly increased (Fig. 10).

Experimental Example  7. Aβ _{One -42} &Lt; RTI ID = 0.0 &gt; anthocyanin-PEG- AuNP  Effect of combination

Aβ _{1 -42-} mediated apoptosis plays an important role in neurodegeneration. It is known that activation of the PI-3K / Akt pathway prevents apoptosis induced by Aβ by blocking apoptotic markers. Western blot analysis for caspase-9 and caspase-3, which play an important role in apoptosis, was performed to examine the expression of apoptotic cascade proteins.

As a result, expression of both caspase-9 and caspase-3 was increased in Aβ _{1 -42} -treated mice compared with the control group, but anthocyanin (12 mg / kg / day) and anthocyanin-PEG- ) both Aβ _{1 -42} - has reduced the level of Case and Case Paget Paget -9 -3 significantly in treated mice (Fig. 11).

On the other hand, it is known that Aβ peptide induces hyperactivity of PARP-1, which causes DNA damage, resulting in neural degeneration of hippocampus, and the expression of PARP-1 was examined by Western blotting. As a result, it was confirmed that PARP-1 partitioned in hippocampus of Aβ _{1 -42-} treated mice was increased, but treatment of anthocyanin (12 mg / kg / day) and anthocyanin-PEG-AuNP (6 mg / kg / Significantly reduced PARP-1 levels in the hippocampus compared to the group treated with A? _{1 -42} alone (FIG. 11). These results indicate that anthocyanin and anthocyanin-PEG-AuNP conjugates have the effect of restoring apoptosis and neurodegeneration induced by A? _{1 -42} .

Furthermore, Aβ _{1 -42} - nissl the staining was performed to evaluate the protective effect of anthocyanins and anthocyanin -PEG-AuNP combination in the hippocampus of a mouse treated.

As a result, but in a process only Aβ _{1 -42} as compared to control mice DG, CA3, the number of neurons in the CA1 region significantly decreased, anthocyanin (12mg / kg / day) and anthocyanins -PEG-AuNP (6mg / kg / Day) significantly increased the number of neurons, especially the anthocyanin-PEG-AuNP conjugate, which increased the number of neurons to a level similar to that of the control group, resulting in a significantly more pronounced anthocyanin- (Fig. 12).

In summary, the anthocyanin-PEG-AuNP conjugates of the present invention are not cytotoxic and inhibit neurodegeneration by regulating Aβ accumulation and related signal transduction pathways in Aβ-induced mouse Alzheimer's disease model, (Fig. 13). These results are remarkable in spite of lower doses than when anthocyanin alone was treated, suggesting that the anthocyanin-PEG-AuNP conjugate of the present invention has excellent effects on neurodegenerative diseases such as Alzheimer's disease.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (7)

A pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising, as an active ingredient, a conjugate of anthocyanin and pegylated gold nanoparticles isolated from a black bean skin extract,
The anthocyanins isolated from the black bean husk extract are delphinidin 3-O-glucoside, cyanidine 3-O-glucoside and petunidin 3- 0-glucoside). &Lt; / RTI &gt;
delete delete The composition according to claim 1, wherein the degenerative brain disease is any one selected from the group consisting of dementia, Alzheimer's, stroke, stroke, Parkinson's disease, Huntington's disease, peak disease and Creutzfeldt-Jakob disease.
A health functional food for preventing or ameliorating a degenerative brain disease comprising, as an active ingredient, a combination of anthocyanin and pegylated gold nanoparticles isolated from a black bean skin extract,
The anthocyanins isolated from the black bean husk extract are delphinidin 3-O-glucoside, cyanidine 3-O-glucoside and petunidin 3- 0-glucoside). &Lt; / RTI &gt;
delete 6. The health functional food according to claim 5, wherein the degenerative brain disease is any one selected from the group consisting of dementia, Alzheimer's disease, stroke, paralysis, Parkinson's disease, Huntington's disease, peak disease and Creutzfeldt-Jakob disease.

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