KR20150007860A - Therapeutic agent for treating neurological disorders comprising anthocyanin and GABAB receptor agonist - Google Patents

Abstract

Provided is a composition including anthocyanin and a GABAB1R agent as active ingredients to prevent and treat nervous diseases caused by neuronal cell death. Also, provided is a method for preventing and treating nervous diseases using the same.

Description

[0002] Therapeutic agents for treating neurological disorders comprising anthocyanin and GABAB receptor agonist include anthocyanin and GABAB receptor agonists as active ingredients.

The present invention relates to a neuroprotective composition, and more particularly, to a therapeutic agent for a neurological disease comprising an anthocyanin and a GABAB receptor agonist as an active ingredient.

Gamma-aminobutyric acid (GABA) receptors are widely distributed in the central nervous system and regulate nerve activity, particularly in degenerative brain diseases and pathophysiologic disorders. For example, overexpression of the GABA B receptor has been associated with Down's syndrome (Tyler K. et al., J. Neurophysiol. , 97: 892-900, 2007), atypical seizures (Stewart LS, et al., Epilepsy Behav ., 14 (4): 577-81, 2009), Alzheimer's (Palop JJ, et al., Neuron , 55: 697-711, 2007; Williams C., et al., PLoS One . 2009), depression (Slattery, et al., J. Pharmacol. Exp. Ther. , 312 (1): 290-6, 2004).

Neuroprotective effects of anthocyanins have been reported in various literature (Tarozzi et al ., Neurosci Lett . 424 (1): 36-40, 2007; Chen et al ., Neurotox Res . 17 (1): 91 -101, 2010), and its specific protective mechanism has not been elucidated yet.

The neurotoxic effects of conventional anthocyanins have been demonstrated in various literature, but have not yet been elucidated in connection with its specific signaling mechanism. Further, the neuroprotective effect and the relationship with GABAB receptors have not been elucidated.

It is an object of the present invention to provide an excellent neurological disease treating agent, a health functional composition containing the same, and a neuroprotective method by remarkably elevating the neuroprotective effect of anthocyanin by identifying a specific signal mechanism. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a composition for preventing and treating neurological diseases, comprising an anthocyanin and a GABA (gamma-aminobutyric acid) B receptor agonist as an active ingredient.

In this composition, the GABA B receptor agonist is selected from the group consisting of baclofen, 1,4-butanediol, GBL (gamma-butyrolactone), gamma -hydroxybutyric acid (GHB) gamma -hydroxyvaleric acid, GVL (gamma -valerolactone), lesogaberan, phenibut, and the like.

The composition of claim 1 wherein the disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia of HIV, epilepsy, schizophrenia, depression, bipolar disorder, neurogenic disorders, autism, stroke, Lou Gehrig's disease, Huntington's disease and multiple sclerosis Or two or more.

In the composition, the anthocyanin may be isolated from plants or chemically synthesized, and the plant may be any plant that produces anthocyanins, such as black beans, black currants, black currant, chokeberry, black chokeberry, cranberry, audi, cherry, raspberry, blueberry, blackberry, eggplant, acai, mulberry or grape.

At this time, the composition according to an embodiment of the present invention may preferably contain anthocyanin and gamma-aminobutyric acid (GABA) B receptor agonist in an amount of 0.1 to 50% by weight based on the total weight of the composition.

In addition, it may further contain at least one active ingredient which exhibits the same or similar function as the GABA B receptor agonist, but preferably does not exceed the content of the active ingredient.

The composition may be administered orally or parenterally at the time of clinical administration and may be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, May be administered by injection, and may be used in the form of a conventional pharmaceutical preparation.

Each oral composition according to one embodiment of the present invention further comprises an inert ingredient, including a pharmaceutically acceptable carrier.

In practical use, the composition according to one embodiment of the present invention may be combined with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending upon the preparation desired, for example, for oral or parenteral administration (including intravenous administration).

In addition, the composition according to one embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

According to one embodiment of the present invention as described above, a nerve protection composition can be realized. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a graph showing that an anthocyanin protective effect of anthocyanin is mediated by GABA1R. FIG. 1 a shows the results of Western blot analysis of the expression level of apoptotic protein FIG. 1 (b) is a graph showing the quantitative expression of Bax protein in comparison with Bcl-2 expression level in the results of FIG. 1a. FIG. 1 (c) , caspase-3 and PARP-1.
FIG. 2 shows the results of the FACS analysis of the anthocyanin-protecting effect of anthocyanin according to an embodiment of the present invention. FIG. 2B shows the result of the FACS analysis quantitatively Graph.
FIG. 3 is a photograph showing the protective effect of anthocyanin using a cytomorphological analysis method.
FIG. 4 is a graph showing the results of analysis of GABAB1R and its lower signal transduction mechanism during ethanol-induced cell death. FIG. 4a shows the decrease in GABA1R expression level by GABAB1R siRNA treatment according to one embodiment of the present invention, FIG. 4B shows the result of Western blot analysis for the change of GABAB1R expression level by the combination of agonist or antagonist of GABAB1R together with GABAB1R siRNA.
FIG. 5A is a result of analysis of GABAB1R lower signal changes in anthocyanin neuronal cell protection mechanism, FIG. 5A is a result of confirming the expression level of a lower signal substance using Western blot analysis, FIG. 5B is a result of FIG. As shown in Fig.
FIG. 6 shows the result of analysis of an anthocyanin protective mechanism against ethanol induced Ca ²⁺ homeostasis.
FIG. 7 shows the result of analysis of an anthocyanin protective mechanism against the mitochondrial membrane potential (?? M) of ethanol induced. FIG. 7A is a result of analysis using a flow cytometer, FIG. 7B is a graph to be.

The terms used in this document are defined as follows.

As used herein, "pharmaceutically acceptable carrier" is a term referring to a composition, specifically a component other than the active ingredient of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.

Example 1: Animal breeding

Sparague-Dawley rats (250 g, Gyeongsang National University, Department of Neurobiology) are female and female (n = 10) Sprague-Dawley rats are housed under controlled temperature, light cycle (8: 00-20: 00) Respectively. The modified day was calculated as 0.5 day of pregnancy and pentobarbital sodium was intravenously administered at a rate of 3 mg / 100 g body weight at 17.5 days (GD 17.5 days) after pregnancy, and then the head was decapitated ). All experimental procedures were approved by the animal ethics committee of the Department of Biology, Gyeongsang National University (IACUC).

Example 2: Primary cell culture of primary hippocampus

The hippocampal tissue of the GD 17.5-day-old embryo obtained from the pregnant female Sprague Dawaur rats sacrificed in Example 1 above was isolated. The separated hippocampal tissues were treated with 0.25% trypsin EDTA for 20 minutes, physically separated in a Hank's buffer (pH 7.4) containing no calcium and magnesium, and centrifuged Respectively. Then, 10% heat inactivated fetal bovine serum, 1 mM pyruvate, 4.2 mM sodium bicarbonate, 20 mM HEPES, 0.3 g / L bovine serum albumin, 50 U / ml cell culture plate resuspended in DMEM (Dulbecco's modified Eagle medium) medium containing 50 mg / L penicillin and 50 mg / L streptomycin and coated with 0.02 g / L poly-lysine or Lt; ⁶ > cells / ml in a chamber slide. The cells were incubated at 37 ° C in a humidified condition with 5% CO ₂ . To prevent neuroglial cells from being cultured, the cells were cultured in a medium supplemented with 100 [mu] M cytosine [beta] -D-arabinofuranoside (Sigma, USA) for 12 hours. After 12 hours, the medium was replaced with the above-mentioned medium.

Example 3: Synthesis of GABAB1R siRNA

The GABAB1R cDNA plasmid (Novartis Pharma, Basel, Switzerland) was digested with XbaI and EcoRI and then the insert was isolated from the pCI vector. The PCR primers used for GABAB1R cDNA amplification were as follows, and the T7 promoter sequence was placed at the 5 'end:

SEQ ID NO: 1: 5'-CGGTAATACGACTCACTATAGGGAGACGCTACCATCCAACAGACCA-3 '; And

SEQ ID NO: 2: 5'-GCGTAATACGACTCACTATAGGGAGATCCTGTGAGCTCATGTTGGAA-3 '.

The PCR reaction amplified the 420 bp fragment with the highest silencing activity from GABAB1R cDNA ranging from 1,096 bp to 1,516 bp. The PCR product was used as a template for dsDNS synthesis and was synthesized using MEGA script ^® RNAi kit (Ambion, Austin, TX, USA). Short-length fragments for transduction were then treated with ShortCut RNAi kit (New England Biolabs, Buckinghamshire, UK).

The siRNA produced via the process of liposomes (Liposome) solution (DMEM containing Lipofectamine2000 ^TM, Invitrogen, Carlsbad, CA, USA) and equivalent amount of dsRNAs was allowed to stand for a (21 bp) for 5 minutes at room temperature, respectively, and mixed 20 minutes Lt; / RTI > The mixture was added to cells (1 x ¹⁰⁶ cells / ml), where the cells were starved in DMEM medium without antibiotics and serum for 24 hours, and finally with 40 nM siRNA concentration in DMEM The medium was added. The siRNA used as a negative control was purchased from Qiagen. Negative control siRNA was also purchased from Qiagen.

Example 4: Extraction of anthocyanin

The anthocyanin used in one embodiment of the present invention was extracted from Korean black beans provided by the Institute of Industrial Science and Technology, Gyeongsang National University.

To extract anthocyanin, 1,500 g of black soybean was firstly extracted by repeating the procedure of stirring in 1,500 ml of 95% methanol / 1% HCl for 72 hours under dark condition at room temperature and for three times. The methanol extract was concentrated to 150 mL using a rotary evaporator, and loaded onto an XAD-7 column. Thereafter, the eluate was washed with distilled water until it became a pale red color. Thereafter, the column was washed with ethyl acetate until the lower part of the column except the 1 cm layer became purple. Then, 95% methanol / 1% HCL was added to the column to elute anthocyanin until the color of the whole column became red. The eluate containing anthocyanin was made into a volume of 100 ml using a rotary evaporator, and then a fine material was removed using a 0.45 μm filter. The anthocyanin concentrate was loaded on a Sephadex column and eluted with a 50% methanol / 50% distilled water / 1% HCl solution until obtaining 800-1,000 ml of reddish eluate. The red anthocyanin eluate was evaporated to dryness using a rotary evaporator, and the resulting anthocyanin powder was stored at -20 ° C until immediately before use in the following experiment.

Experimental Example 1: Analysis of an anthocyanin protective mechanism against ethanol-induced apoptosis

1-1: Analysis of cell death kinetics induced by ethanol

In order to analyze the inhibitory effect of anthocyanin on cell death induced by ethanol according to an embodiment of the present invention, after the treatment according to the experimental group of the primary hippocampal cell of Example 2, the level of apoptotic marker protein was Western blotted (E), 100 mM ethanol (E), 100 mM ethanol + 0.1 mg / mL anthocyanin (E + An), 100 mM ethanol + 40 nM GABAB1R siRNA (E + siRNA) and 100 mM ethanol + 0.1 mg / mL anthocyanin + 40 nM GABAB1R siRNA (E + siRNA + An). In this experimental group, the treatment sequence was such that siRNAs were transfected into cells and after 48 hours, they were replaced with normal medium and ethanol and / or anthocyanins were reacted for 20 minutes each in the order described above.

Thereafter, the cells were lysed using a cell lysis buffer (Cell Signaling No. 9803, Danvers, MA, USA), and the cell lysate was centrifuged at 12,000 rpm for 10 minutes at 4 ° C for 2 minutes, The liquid was separated. Protein concentrations were measured using a Bio-Rad protein assay kit. 30 μg of the whole protein was loaded on a 12% SDS-polyacrylamide gel, transferred to a PVDF (polyvinylidene fluoride) membrane, and then reacted using a primary antibody (Ullah N, et al ., Neuropharmacology 61: 1248-1255, 2011). The primary antibodies used were as follows: guinea-pig anti-rat GABAB1R (1: 500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti- rat PKA-α (1: 500 dilution, Santa Cruz Biotechnology ), Rabbit anti-rat CaMKII (1: 500 dilution, Cell Signaling). Rabbit anti-rat p-CREB (1: 500 dilution, Cell Signaling). Bax and Bcl-2 rabbit anti-rat (1: 500 dilution, Santa Cruz Biotechnology), PARP-1 (1: 500 dilution, Santa Cruz Biotechnology), rabbit anti- , Cell signaling) or anti-actin (1: 1000 dilution, Sigma-Aldrich, Jerusalem, Israel; used as loading control). The primary antibody was reacted at 4 ° C for 24 hours and then the blot was washed and incubated with a goat anti-mouse, mouse anti-goat or goat anti-rabbit IgG HRP as a secondary antibody for 2 hours at room temperature And then the antigen was detected using ECL (enhanced chemiluminescence) (Western blotting detection reagents, Amersham Pharmacia Biotech, Piscataway, NJ, USA).

When the same membrane was to be reused, the blots were stripped and reacted with a new primary antibody. To this end, the membranes were washed with TBST (Tris-buffered saline) solution containing 0.1% (v / v) Tween-20, and the membranes were washed with ReBlot Plus Strong Antibody stripping solution (Millipore, Temecula , CA, USA). Thereafter, the cells were washed 4 times for 5 minutes using TBST and reacted using a new antibody. Western blot analysis was performed using a computer-based Sigma Gel system (SPSS Inc., Chicago, IL, USA). Evolution of the band was expressed as mean ± SEM.

As a result, the level of intracellular apoptotic protein Bax was remarkably increased in 20 minutes after exposure to ethanol, while the level of anti-apoptotic protein Bcl-2 protein was remarkably decreased. Significantly increased Bax / Bcl-2 levels (* p < 0.05) and marked differences (see FIG. 1).

It is known that PARP-1 is isolated by activated caspase 3, resulting in apoptosis and gangrene cell death (Sairanen T, et al ., Acta Neuropathol . 118: 541-552, 2009). Thus, we measured the levels of activated caspase-3 and isolated PARP-1 using Western blot.

As a result, activated caspase-3 levels and isolated PARP-1 levels were observed in ethanol treated cells (see FIG. 1). The above results indicate that ethanol increases the level of the cell death markers, that is, cell death.

On the other hand, after ethanol treatment, when anthocyanin was treated for 20 minutes, increased Bax levels and reduced Bcl-2 levels were recovered by ethanol treatment, indicating a protective effect. In addition, it decreased Bax / Bcl-2 levels, which is due to the reduction of activated caspase-3 and isolated PARP-1 levels (see FIG. 1). These results demonstrate that anthocyanin has a protective effect against ethanol-induced apoptosis.

1-2: Analysis of protective effect of anthocyanin using apoptosis assay

Next, the present inventors confirmed whether or not anthocyanin shows a protective effect against ethanol-induced apoptosis by using apoptosis assay.

The primary hippocampal cells of Example 2 were treated with drugs according to the following experimental groups and then subjected to apoptosis analysis (control (C): 100 mM ethanol (E); 50 μM baclofen (Ba); 0.1 100 mM ethanol + 0.1 mg / mL anthocyanin (An +); 100 mM ethanol + 40 nM GABAB1R siRNA (E + siRNA); and 100 mM ethanol + 0.1 mg / 40 nM GABAB1R siRNA (E + siRNA + An)). Among the above experimental groups, the C, E, Ba, An, and E + An groups that were not transfected with siRNA treated the vehicle used to deliver the siRNA. Treatment according to the experimental group was performed after 48 hours of siRNA transfection. For each experimental group, 20 minutes after ethanol treatment, anthocyanin was treated at 37 ° C for 20 minutes. The bar graph shows the percentage of cells killed in each experimental group. Data were presented as the average of three repeated experiments performed on each of the three plates. M1 refers to the cell cycle arrest phase during mitosis. * Means p < 0.05 compared to the control group, and # means p < 0.05 compared to the ethanol treated group.

The cells were resuspended in 100 μl PBS, fixed in 1 ml 70% ethanol (?? 20 ° C) and washed with PBS. Each pellet was resuspended in 250 μl PBS containing 1 mg / mL RNase and reacted on ice for 30 minutes. After that, the cells were allowed to react with 250 μl of PI (Propidium iodide) solution at room temperature for 30 minutes, and the cells were used for FACS analysis.

As a result, the proportion of dead cells in the ethanol-treated group was significantly higher in the non-transfected group than in the control and type texture-conditioned group (see FIG. 2). When the ethanol treatment was followed by anthocyanin treatment for 20 minutes, the proportion of apoptotic cells in the non-transfected group was significantly reduced. However, this effect was not observed in the group transfected with GABAB1R siRNA. These results imply that GABAB1R is essential for the cytoprotective effect of anthocyanins.

1-3: Protection effect of anthocyanin using cytomorphological analysis

To determine the level of apoptosis, morphological analysis was performed. The present inventors analyzed cells using FJB (Fluoro-Jade-B) and PI (Propidium iodide). FJB (Flouro-Jade B) staining was performed as described in Ullah I, et al., BMC Neuroscience 13:11, 2012). The hippocampal neurons of Example 1 in which GABAB1R siRNA was transduced or not introduced were reacted in a chamber coated with poly-D-lysine for 48 hours. Then, the cultured cells were treated with 100 mM ethanol (E), 100 mM ethanol, 100 mM ethanol, 0.1 mg / mL anthocyanin (E + An), 100 mM ethanol + 40 nM GABAB1R siRNA (E + siRNA); 100 mM ethanol + 0.1 mg / mL anthocyanin + 40 nM GABAB1R siRNA (E + siRNA + An) and 100 mM ethanol + 0.1 mg / mL anthocyanin + 50 μM baclofen , Ba) (E + An + Ba)) and allowed to react at 37 ° C for 12 hours. Among the above experimental groups, the C, E, and E + AN groups that did not transduce the siRNA treated the vehicle used to deliver the siRNA. In the E + An + Ba group, ethanol was treated for 20 minutes, then anthocyanin and baclofen were treated together for 20 minutes.

The cells were then fixed in 4% paraformaldehyde for 5 minutes and stored at -70 ° C. until used in the experiment. The slides were allowed to dry for 3 hours in air and then reacted for 10 minutes in 0.06% potassium permanganate solution, washed with distilled water, 0.0004% FJB (Calbiochem, San Diego, California, USA) After 20 minutes of reaction in the solution, it was washed three times with distilled water. The slides were dried at 55 占 폚 for 10 minutes, and then observed using a confocal microscope FITC filter (Olympus Fluoview FV1000, Japan). For PI (propidium iodide) staining, the slides were slowly immersed in PI solution (1 μg / mL in PBS), allowed to react at room temperature for 20 minutes, and washed twice with PBS for 10 minutes each. A glass cover slip was placed on the slide and a mounting medium was placed just before observing with a confocal microscope.

As a result, as shown in FIG. 3, among the cells of Example 2, the number of cells killed by ethanol treatment in the non-transfected cells was remarkably increased compared to the control. On the other hand, in the case of the cells treated with anthocyanin for 20 minutes after the ethanol treatment, the cell death was remarkably reduced compared to the cells treated with ethanol alone, and the cytoprotective effect of the anthocyanin was observed in combination with baclofen + An + Ba). This reaffirms the protective effect of ethanol induced induction of anthocyanin on apoptosis and demonstrates that the combined treatment of GABAB agonist and anthocyanin can significantly increase the cytoprotective effect. On the other hand, no cell death was observed in the cells treated with anthocyanin and GABAB1R siRNA after ethanol treatment. The white arrow on the drawing indicates the nucleus of the neuron. Means a magnification of 40 times, and a scale bar means 20 μm.

To summarize the above results, the degree of apoptosis was increased by ethanol treatment, which was confirmed by the expression level and morphological analysis of the apoptotic protein, and anthocyanin showed a cytoprotective effect, GABAB1R &lt; / RTI &gt; can be carried out by treating GABAB1R siRNA or GABAB1R agonist with anthocyanin. That is, GABAB1R is indispensably required for the cytoprotection of anthocyanin, and it is the first result that proves that the combination treatment of anthocyanin and GABAB1R agonist can significantly increase the protective effect.

Experimental Example 2: Analysis of GABAB1R and its sub-signaling mechanism in ethanol-induced apoptosis

2-1: Analysis of GABAB1R expression levels

In order to elucidate the cell death mechanism of ethanol induction, the inventors analyzed the expression level of GABAB1R protein and expression of its lower signaling molecules.

First, the primary hippocampal cell of Example 2 was treated with GABAB1R siRNA and then the level of expression of GABAB1R protein was confirmed. As a control group, control siRNAs were treated. As a result, GABAB1R siRNA was transfected into the primary hippocampal cell of Example 2, as shown in FIG. 2, and the level of GABAB1R was decreased by Western blotting. No change in the level of GABAB1R was observed in cells transfected with the control siRNA (see Fig. 4A).

Next, we transfected GABAB1R siRNA into hippocampal cells and after 48 hours, the GABAB1R agonist or antagonist was treated and the expression level of GABAB1R was observed. The expression level of GABAB1R was confirmed by Western blotting after 50 μM of baclofen (Ba) agonist GABAB1R and 100 μM of inhibitor phaclofen at 37 ° C. for 20 minutes.

As a result, as shown in FIG. 4B, the level of GABAB1R reduced by GABAB1R siRNA treatment was slightly increased by baclofen, and the level of GABAB1R expression was decreased by treatment with paracopene (See FIG. 4B).

2-2: Analysis of GABAB1R downstream signaling

The effect of the anthocyanin on the GABAB1R and GABAB1R sub-signal levels by ethanol treatment was confirmed by using proteins extracted from the primary hippocampal cells of Example 2 above. 100 mM ethanol + 0.1 mg / mL baclofen (Ba), 0.1 mg / mL anthocyanins (An), 100 mM ethanol (E) Anthocyanin (E + An); 100 mM ethanol + 40 nM GABAB1R siRNA (E + siRNA); and 100 mM ethanol + 0.1 mg / mL anthocyanin + 40 nM GABAB1R siRNA (E + siRNA + An).

As a result, as shown in FIG. 5, in the primary hippocampal cell of Example 2, the ethanol treatment increased the level of GABAB1R and markedly increased the expression level of its lower signal transduction molecules, CaMKII and p-CREB, PKA-a increased its expression. In the ethanol + anthocyanin-treated group, GABAB1R, CaMKII and p-CREB levels were lower than in the ethanol-treated group, but PKA-α levels were higher. These results indicate that anthocyanins interfere with the process of signaling ethanol induced. At this time, baclofen as an agonist for GABAB1R was treated for comparison (see FIG. 5).

Experimental Example 3: Analysis of an anthocyanin protecting mechanism against ethanol induced Ca2 + homeostatic change

Ca ²⁺ homeostasis plays an important role in the release of neurotransmitters and development of neurons, and it is known that when homeostasis is not maintained, neuronal death is induced. It is known that ethanol-induced neurotoxicity is associated with abnormalities in intracellular Ca ²⁺ concentration regulation (Naseer MI, et al., Synapse, 64: 181-190 , 2010).

The intracellular free Ca ²⁺ concentration was measured using Fura-2AM (fura-2 acetoxymethyl ester), a fluorescent Ca2 + detector, under drug treatment or untreated conditions. Specifically, 1 × 10 ⁶ cells were treated with drugs and siRNAs according to each experimental group and repeated 3 times (control (C): 100 mM ethanol (E); 50 μM baclofen (Ba) 100 mM ethanol + 0.1 mg / mL anthocyanin (An +); 100 mM ethanol + 40 nM GABAB1R siRNA (E + siRNA); and 100 mM ethanol + 0.1 mg / 40 nM GABAB1R siRNA (E + siRNA + An)). Among the above experimental groups, the C, E, Ba, An, and E + An groups that were not transfected with siRNA treated the vehicle used to deliver the siRNA. In the experimental group, the treatment was carried out by transfecting the cells with siRNAs, and after 48 hours, they were exchanged into the normal medium and reacted for 20 minutes each in the order of ethanol and / or anthocyanin. After treatment according to the experimental group, the cells were washed twice with Krebs buffer and cultured in DMEM medium containing 5 mM Fura-2 AM in a humidified incubator containing 5% carbon dioxide at 37 DEG C for 60 Min. &Lt; / RTI &gt; Then, the cells were washed twice with a Locke's solution (pH 7.8), and the Ca2 + Fura-2AM fluorescence signal was measured with an emission spectrophotometer (LS50B, Perkin Elmer, Boston, Mass.). The above 340 nm / 380 nm fluorescence ratio was averaged based on a 2-second time interval and observed. The fluorescence signals obtained were analyzed using a computer with general imaging software or a MicroVAX II computer with origin 7 software. Intracellular calcium concentration was calculated using the Grynkiewicz equation of the following equation (Grynkyewicz G, et al., J. Biol. Chem., 260: 3440-3450, 1985).

In the above formula (1), Kd is the dissociation constant of Fura-2AM Ca ²⁺ bond, which is calculated as 225 nM in the intracellular environment, R means fluorescence ratio of 340 nm / 380 nm; Rmin means the R value when the intracellular concentration of Ca ²⁺ is 0, Rmax means the R value when the Ca ²⁺ is saturated (using calcium chloride), Sf ^{2 means} the intracellular concentration of Ca ²⁺ Quot; means the wavelength at 380 nm when &lt; RTI ID = 0.0 &gt; 0 &lt; / RTI &gt; Sb2 means the wavelength at 380 nm for saturated Ca &lt; ^{2 + &} gt ;.

As a result, as shown in Fig. 6, the concentration of Ca ²⁺ in the non-transfected group was remarkably increased by ethanol treatment for 20 minutes as compared with the control group. In the non-transfected group, the increase in Ca ²⁺ concentration by ethanol was not observed at all when the anthocyanin was treated for 20 minutes after the ethanol treatment (see FIG. 6). The protective effect of these anthocyanins on ethanol induced Ca ²⁺ uptake was not present in the GABAB1R siRNA treated group, which implies that GABAB1R is essential for the protective effect of anthocyanin on neurons.

In addition, baclofen, a GABAB1R agonist, reduced the intracellular Ca ²⁺ concentration. These results demonstrate that ethanol increases intracellular Ca ²⁺ concentration, whereas anthocyanin inhibits this increase, and that this is manifested through GABAB1R. In summary, anthocyanin has protective effects against ethanol induced cell death It is possible.

Experimental Example 4: Analysis of an anthocyanin protective mechanism against ethanol induced mitoccal membrane potential (?? M)

The abnormal increase of intracellular Ca ²⁺ levels by ethanol impairs the mitochondrial membrane potential (ΔΨM), which causes cytochrome c release, resulting in apoptosis signaling (Simasko MS, et al., Brain Res., 824: 89-96, 1999).

The primary hippocampal cells of Example 2 were treated with drugs according to the following experimental groups and then subjected to apoptosis analysis (control (C): 100 mM ethanol (E); 0.1 mg / ml anthocyanins GABAB1R siRNA (E + siRNA) and 100 mM ethanol + 0.1 mg / mL anthocyanin + 40 nM GABAB1R siRNA (E + siRNA + An) ). Among the above experimental groups, the C, E, An, and E + An groups that did not transduce siRNA treated the vehicle used to deliver the siRNA. Treatment according to the experimental group was performed after 48 hours of siRNA transfection. For each experimental group, 20 minutes after ethanol treatment, anthocyanin was treated at 37 ° C for 20 minutes. Data were presented as the average of three repeated experiments performed on each of the three plates. M1 refers to the cell cycle arrest phase during mitosis. * Means p < 0.05 compared to the control group, and # means p < 0.05 compared to the ethanol treated group.

The mitochondrial membrane potential (ΔΨm) was analyzed using a JC-1 mitochondrial membrane potential detection kit (Biotium Inc., Hayward, CA, USA). After transfection or non-introduction of siGABAB1R RNA into GD 17.5 hippocampal cell of Example 1, the drug was treated according to the experimental group. The cells were then harvested and stained for 15 min at 37 ° C with JC-1 drug, washed twice with 1 × assay buffer, and analyzed by FACSCalibur Flow Cytometer (Becton Dickinson, San Jose , CA, USA). The assay was repeated three times and it is known that JC-1 aggregates in the normal polarized mitochondria and emits red fluorescence at a wavelength of 590 nm. The JC-1 monolith from the depolarized mitochondria emits green fluorescence at 530 nm. The red and green fluorescence was measured on FL-1 (green) and FL-2 (red) channels, respectively, on a flow cytometer. The cells were then observed using a "dual band pass" filter in a fluorescence microscope designed to simultaneously detect fluorescence and rhodamine dyes.

As a result, as shown in Fig. 7, the proportion of cells with low mitochondrial membrane potential (24.04%) was higher in the non-transfected group treated with ethanol than the control group (11.56%). In the group treated with anthocyanin for 20 minutes, the ratio of cells with low mitochondrial membrane potential was very low (15.86%) as compared with the group treated with ethanol alone. These results indicate that anthocyanins inhibit the toxicity of ethanol. On the other hand, in the cells transfected with GABAB1R siRNA, anthocyanin did not show the effect of restoring the mitochondrial membrane potential to normal (see Fig. 4). In other words, the mitochondrial membrane potential restoration effect of anthocyanin appears through GABAB1R.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Therapeutic agents for treating neuropsychiatric disorders          comprising anthocyanin and GABAB receptor agonist <130> PD13-0777 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 1 cggtaatacg actcactata gggagacgct accatccaac agacca 46 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 2 gcgtaatacg actcactata gggagatcct gtgagctcat gttggaa 47

Claims (5)

A composition for preventing and treating neurological diseases, comprising an anthocyanin and a gamma-aminobutyric acid (GABA) B receptor agonist as an active ingredient. The method according to claim 1,
The GABA B receptor agonists include baclofen, 1,4-butanediol, GBL (gamma -butyrolactone), gamma -hydroxybutyric acid (GHB), gamma -hydroxyvaleric acid (GHV) Wherein the composition is at least one selected from the group consisting of GVL (gamma -valerolactone), lesogaberan, and phenibut. The method according to claim 1,
Wherein the neurological disorder is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia of HIV, epilepsy, schizophrenia, depression, manic depression, neurogenic disorders, autism, stroke, Lou Gehrig's disease, Huntington's disease, Composition. The method according to claim 1,
Wherein the anthocyanin is derived from a plant. The method according to claim 1,
The plant may be selected from the group consisting of black beans, black currant, chokeberry, black chokeberry, cranberry, audi, cherry, raspberry, blueberry, blackberry, Wherein the composition is acai, corn or grape.

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