KR102206745B1 - Therapeutic agent for treating neurological disorders comprising anthocyanin and GABAB receptor agonist - Google Patents

Abstract

The present invention provides a composition comprising an anthocyanin and a GABAB1R agonist as active ingredients and a method for preventing and treating neurological diseases using the same in order to provide a preventive and therapeutic effect of neurological diseases caused by neuronal cell death. .

Description

Therapeutic agent for treating neurological disorders comprising anthocyanin and GABAB receptor agonist as active ingredients

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

GABA (gamma-aminobutyric acid) B receptor is widely distributed in the central nervous system and regulates nerve activity, and is particularly closely related to degenerative brain diseases and pathophysiological disorders. For example, overexpression of the GABA B receptor is associated with Down syndrome (Tyler K. et al., J. Neurophysiol. , 97:892-900, 2007), atypical absence 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 ., 4(3):e4936, 2009), depression (Slattery DA., et al., J. Pharmacol. Exp. Ther. , 312(1):290-6, 2004).

The neuroprotective effect of anthocyanin has been published in various literatures (Tarozzi et al ., Neurosci Lett . 424(1):36-40, 2007; Chen et al ., Neurotox Res . 17(1):91) -101, 2010), and its specific protection mechanism has not been identified.

Conventional neuroprotective effects of anthocyanins have been proven through various literatures, but their specific signaling mechanisms have not been identified, and furthermore, the relationship between these neuroprotective effects and GABAB receptors has not been identified.

An object of the present invention is to provide an excellent therapeutic agent for neurological diseases, a health functional composition comprising the same, and a neuroprotective method by remarkably increasing the neuroprotective effect of anthocyanin by identifying a specific signaling mechanism. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

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

In the composition, the GABA B receptor agonist is baclofen, 1,4-butanediol (1,4-Butanediol), GBL (γ-Butyrolactone), GHB (γ-Hydroxybutyric acid), GHV ( γ-Hydroxyvaleric acid), GVL (γ-Valerolactone), lesogaberan (lesogaberan), phenibut (phenibut) may be one or more selected from the group consisting of.

In the composition, the neurological disease is one selected from the group consisting of Alzheimer's disease, Parkinson's disease, HIV dementia, epilepsy, schizophrenia, depression, manic depression, neurogenic disorder, autism, stroke, Lou Gehrig, Huntington's disease, and multiple sclerosis. Or it may be two or more.

In the composition, the anthocyanin may be isolated from a plant or chemically synthesized, and the plant may be any plant that produces anthocyanin, for example, black bean, black currant ( black currant), chokeberry, black chokeberry, cranberry, mulberry, cherry, raspberry, blueberry, blackberry, eggplant, acai, grapefruit or grape.

At this time, the composition according to an embodiment of the present invention may preferably include anthocyanin and GABA (gamma-aminobutyric acid) 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 one or more active ingredients exhibiting the same or similar functions as GABA B receptor agonists, but it is preferable not to exceed the content of the active ingredients.

The composition can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular irradiation or intrathoracic It can be administered by injection, and can be used in the form of a general pharmaceutical formulation.

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

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

In addition, the composition according to an 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. Meanwhile, the dosage may be appropriately adjusted according to the patient's age, sex, and condition.

According to an embodiment of the present invention made as described above, a composition for neuroprotection can be implemented. Of course, the scope of the present invention is not limited by these effects.

1 is a result of demonstrating that the neuronal protective effect of anthocyanin according to an embodiment of the present invention occurs through GABA1R, and FIG. 1A is a result of analyzing the expression level of apoptotic protein using Western blot analysis. , FIG. 1B is a graph quantitatively showing the expression of Bax protein compared to the Bcl-2 expression level among the results of FIG. 1A, and FIG. 1C is a graph showing the expression level of beta-actin in the result of FIG. 1A, Bax, Bcl-2, It is a graph that quantitatively shows the expression levels of caspase-3 and PARP-1.
Figure 2 is a result of demonstrating the neuronal protective effect of anthocyanin according to an embodiment of the present invention using the apoptosis assay method, Figure 2a shows the result of FACS analysis, Figure 2b shows the result of Figure 2a quantitatively It is a graph.
3 is a photograph of an analysis of the protective effect of anthocyanins using a cell morphological analysis method.
FIG. 4 is a result of analyzing GABAB1R and its sub-signaling mechanisms during ethanol-induced apoptosis, and FIG. 4A is an analysis of the reduction of GABA1R expression level by GABAB1R siRNA treatment according to an embodiment of the present invention using Western blot Figure 4b is a result of analyzing the change in the expression level of GABAB1R by the combination treatment of GABAB1R agonist or inhibitor (antagonist) with GABAB1R siRNA using a Western blot analysis method.
Figure 5 is a result of analyzing the GABAB1R sub-signal change in the neuronal protective mechanism of anthocyanin, Fig. 5a is a result of confirming the expression level of the sub-signal substance using western blot analysis, and Fig. 5b is the result of Fig. 5a It is a graph showing quantitatively.
6 is a result of analysis of the protective mechanism of anthocyanins against changes in ethanol-induced Ca ²⁺ homeostasis.
7 is a result of an anthocyanin protection mechanism analysis for ethanol-induced mitochondrial membrane potential (ΔΨM) abnormalities, FIG. 7a is a result of analysis using a flow cytometer, and FIG. 7b is a graph quantifying the result of FIG. 7a.

The terms used in this document are as follows.

The term "pharmaceutically acceptable carrier" as used herein refers to a composition, specifically, a component of a pharmaceutical composition other than an active substance. 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 through examples and experimental examples. However, the present invention is not limited to the Examples and Experimental Examples disclosed below, but may be implemented in various different forms. The Examples and Experimental Examples below make the disclosure of the present invention complete, and common knowledge It is provided to fully inform the possessor of the scope of the invention.

Example 1: Animal breeding

Female (n=10) Sparague-Dawley rats (250 g, Gyeongsang National University, Neurobiology Laboratory) are temperature-controlled, and reared in light cycle (8:00-20:00), free diet conditions. I did. The day of fertilization was calculated as 0.5 day of pregnancy, and pentobarbital sodium was administered intravenously at the rate of 3 mg/100 g body weight on the 17.5 day after pregnancy (GD 17.5 day), and then the head was decapitation. ) And sacrificed. All experimental procedures were approved by the animal ethics committee (IACUC) of the Department of Applied Biological Sciences in the Department of Biology at Gyeongsang National University.

Example 2: Culture of primary hippocampal cells (primary cell culture)

The hippocampal tissue of a GD 17.5 day old embryo obtained from the pregnant female Sprague Dawry rat sacrificed in Example 1 was isolated. The separated hippocampal tissue was treated with 0.25% trypsin EDTA for 20 minutes, and after physically separating the tissue into cells in a Hank's buffer of pH 7.4 that did not contain cold calcium and magnesium, centrifugation was performed. I did. 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 penicillin, and resuspended in DMEM (Dulbecco's modified Eagle medium) medium containing 50 mg/L streptomycin, and a cell culture plate coated with 0.02 g/L poly-lysine, or It was planted in a chamber slide at a rate of 1×10 ⁶ cells/ml. Cells were cultured at 37°C with 5% CO ₂ conditions and humidity. To prevent cultivation of neuroglia, 100 μM cytosine β-D-arabino furanoside (cytosine β-D-Arabino Furanoside, Sigma, USA) was added thereto for 12 hours. After 12 hours elapsed, it was replaced with the above-described medium.

Example 3: GABAB1R siRNA synthesis

GABAB1R cDNA plasmid (Novartis Pharma, Basel, Switzerland) was digested using XbaI and EcoRI, and then an insert was isolated from the pCI vector. The PCR primers used for GABAB1R cDNA amplification are as follows, and the T7 promoter sequence was constructed to be located at the 5'end:

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

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

The PCR reaction amplified a fragment of 420 bp having the highest silencing activity from GABAB1R cDNA having a size of 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). Thereafter, short-length fragments for transduction were processed using the ShortCut RNAi kit (New England Biolabs, Buckinghamshire, UK).

The siRNA prepared through the above process is a liposome solution (DMEM containing Lipofectamine2000 ^TM , Invitrogen, Carlsbad, CA, USA) and the same amount of dsRNAs (21 bp), respectively, left at room temperature for 5 minutes, and then mixed for 20 minutes. Reacted for a while. The mixture was added to cells (1×10 ⁶ cells/ml), at which time the cells were starved in DMEM medium containing no antibiotics and serum for 24 hours, and at this time, DMEM to finally reach 40 nM siRNA concentration. Medium was added. SiRNA used as a negative control for this was purchased from Qiagen. In addition, a negative control siRNA was purchased from Qiagen.

Example 4: Extraction of anthocyanins

Anthocyanin used in an embodiment of the present invention was extracted from Korean black beans provided by the Labor Institute of Gyeongsang National University.

In order to extract anthocyanin, first, 1,500 g of black beans were extracted by repeating the process of stirring for 72 hours at room temperature under dark conditions in 1,500 ml of 95% methanol/1% HCl. The methanol extract was concentrated using a rotary evaporator to make 150 mL, and then loaded onto the 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 portion excluding the lowermost 1 cm layer turned purple. Thereafter, until the color of the entire column turned red, 95% methanol/1% HCL was added to the column to elute anthocyanin. After making 100 ml of the eluate containing anthocyanin using a rotary evaporator, fine substances were removed using a 0.45 μm filter. The anthocyanin concentrate was loaded on a Sephadex column, and eluted using a 50% methanol/50% distilled water/1% HCl solution until 800-1,000 ml of a red color eluate was obtained. After the red anthocyanin eluate was evaporated and dried using a rotary evaporator, the obtained anthocyanin powder was stored at -20°C until immediately before use in the following experiment.

Experimental Example 1: Analysis of protective mechanisms of anthocyanins against ethanol-induced apoptosis

1-1: Ethanol-induced apoptosis level analysis

In order to analyze the apoptosis inhibitory effect of anthocyanin according to an embodiment of the present invention induced by ethanol, after treatment according to the experimental group on the primary hippocampal cells of Example 2, the level of apoptosis marker protein was determined using Western blot. (Control (C); 100 mM ethanol (E); 0.1 mg/mL anthocyanin (An); 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 the experimental group, the treatment sequence was siRNA transduced into cells, 48 hours later, exchanged for normal medium, and reacted with ethanol and/or anthocyanin for 20 minutes in the order described above.

Thereafter, the cells were lysed using a cell lysis buffer (Cell Signaling no. 9803, Danvers, MA, USA), and then the cell lysate was centrifuged twice for 10 minutes at 4°C and 12,000 rpm, and the upper layer The liquid was separated. Protein concentration was measured using a Bio-Rad protein assay kit. 30 μg of the total protein was loaded onto a 12% SDS-polyaclyamide gel and separated, transferred to a polyvinylidene fluoride (PVDF) 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-rat caspase-3 (1:500 dilution) , 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, after which the blot was washed, and goat anti-mouse, mouse anti-goat or goat anti-rabbit IgG HRP was used as a secondary antibody for 2 hours at room temperature. After reaction, the antigen was detected using ECL (enhanced chemiluminescence) (Western blotting detection reagents, Amersham Pharmacia Biotech, Piscataway, NJ, USA).

In addition, when the same membrane was reused, the blot was stripped and then reacted using a new primary antibody. To this end, specifically, after washing the membrane using TBST, a Tris-buffered saline (TBS) solution containing 0.1% (v/v) Tween-20, the membrane was washed with ReBlot Plus Strong Antibody stripping solution (Millipore, Temecula). , CA, USA) was used to stripping (stripping). Thereafter, it was 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) to analyze the thickness of the band. The intensity of the band was expressed as mean±SEM.

As a result, the level of Bax, an intracellular apoptotic protein, significantly increased after 20 minutes of exposure to ethanol, whereas the level of Bcl-2 protein, an anti-apoptotic protein, was remarkably decreased. The Bax/Bcl-2 level was statistically significantly increased (*p<0.05), and a significant difference was shown (see FIG. 1).

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

As a result, activated casphase-3 levels and isolated PARP-1 levels were observed in ethanol-treated cells (see FIG. 1). The above result means that the level of apoptosis marker is increased by ethanol, that is, apoptosis is increased.

On the other hand, after the ethanol treatment, when anthocyanin was treated for 20 minutes, the increased Bax level and the decreased Bcl-2 level were recovered by the ethanol treatment, showing a protective effect. It also reduced Bax/Bcl-2 levels, which is due to a decrease in activated casphase-3 and isolated PARP-1 levels (see Figure 1). These results prove that anthocyanin has a protective effect against apoptosis by ethanol.

1-2: Analysis of the protective effect of anthocyanins using apoptosis assay method

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

After the primary hippocampal cells of Example 2 were treated with drugs according to the following experimental groups, apoptosis analysis was performed (control group (C); 100 mM ethanol (E); 50 μM baclofen, Ba); 0.1 mg/mL anthocyanins (An); 100 mM ethanol + 0.1 mg/mL anthocyanins (E+An); 100 mM ethanol + 40 nM GABAB1R siRNA (E+siRNA); and 100 mM ethanol + 0.1 mg/mL anthocyanins + 40 nM GABAB1R siRNA (E+siRNA+An)). Among the experimental groups, C, E, Ba, An, and E+An groups that did not transduce siRNA were treated with a vehicle used to deliver siRNA. Treatment according to the experimental group was performed 48 hours after siRNA transfection. In each experimental group, the treatment sequence was 20 minutes after ethanol treatment, followed by treatment with anthocyanins at 37°C for 20 minutes. The bar graph represents the percentage of dead cells in each experimental group. Data are presented as the average of the results of 3 repeated experiments performed on each of the 3 plates. M1 refers to the cell cycle arrest phase in the mitosis process. * Means p<0.05 compared to the control group, and # means p<0.05 compared to the ethanol-treated group.

After obtaining the cells, 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. Then, after reacting with 250 μl PI (Propidium iodide) solution at room temperature for 30 minutes, the cells were used for FACS analysis.

As a result, the proportion of killed cells in the ethanol-treated group was significantly higher in the non-transfected group compared to the control group and the left group (see Fig. 2). When anthocyanin was treated for 20 minutes following ethanol treatment, the proportion of apoptotic cells in the non-transfected group was significantly reduced. However, this effect was not observed in the group transduced with GABAB1R siRNA. These results mean that GABAB1R is essentially required for the cytoprotective effect of anthocyanins.

1-3: Analysis of the protective effect of anthocyanins using cell morphological analysis

In order to measure the level of apoptosis, morphological analysis was performed. The present inventor analyzed cells using FJB (Fluoro-Jade-B) and PI (Propidium iodide). Flouro-Jade B (FJB) staining was performed as described in the following literature (Ullah, I. et al., BMC Neuroscience 13:11, 2012). The hippocampal nerve cells of Example 1, transduced or not transduced with GABAB1R siRNA, were reacted for 48 hours in a chamber coated with poly-D-lysine. Thereafter, the cultured cells were transferred to each experimental group (control group (C); 100 mM ethanol (E); 0.1 mg/mL anthocyanin (An); 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)), the drug was treated and reacted at 37° C. for 12 hours. Among the experimental groups, the C, E, and E+An groups not transduced with siRNA were treated with vehicles used to deliver siRNA. In the E+An+Ba group, ethanol was treated for 20 minutes, then anthocyanin and baclofen were treated together, followed by treatment for 20 minutes.

Thereafter, the cells were fixed in 4% paraformaldehyde for 5 minutes, and then stored at -70°C until used in experiments. The slides were dried in air for 3 hours, then reacted for 10 minutes in a 0.06% potassium permanganate solution, washed with distilled water, 0.1% acetic acid containing 0.0004% FJB (Calbiochem, San Diego, California, USA). After reacting in the solution for 20 minutes, it was washed three times with distilled water. The slide was dried at 55° C. for 10 minutes, and then observed using a confocal microscope FITC filter (Olympus Fluoview FV1000, Japan). For PI (propidium iodide) staining, slides were slowly immersed in a PI solution (1 μg/mL in PBS), reacted for 20 minutes at room temperature, and washed twice in PBS for 10 minutes. A glass cover slip was placed on the slide, and a mounting medium was placed immediately before observation with a confocal microscope.

As a result, as shown in FIG. 3, among the cells of Example 2, it was observed that the number of cells killed by ethanol treatment in the non-transfected cells was significantly increased compared to the control group. On the other hand, in the case of cells treated with anthocyanin for 20 minutes after ethanol treatment, apoptosis was significantly reduced compared to the cell group treated with ethanol alone, and the cytoprotective effect of this anthocyanin was observed in combination with baclofen (E +An+Ba) remarkably appeared. This proves that the protective effect of ethanol-induced anthocyanin on apoptosis against apoptosis is re-proven, and at the same time, the combination treatment of GABAB agonist and anthocyanin can significantly increase the protective effect of cells. On the other hand, after ethanol treatment, no apoptosis was observed in the cells transduced with anthocyanin treatment and GABAB1R siRNA. The white arrow on the drawing indicates the nucleus of the nerve cell. It means a magnification of 40 times, and the scale bar means 20 μm.

Summarizing the above results, the level of apoptosis was increased by ethanol treatment, which could be confirmed through the expression level and morphological analysis of the apoptotic protein. In response, anthocyanin exhibits a cytoprotective effect, and this protective mechanism is It was demonstrated by treatment with GABAB1R siRNA or GABAB1R agonist with anthocyanins that this can be done through GABAB1R. That is, GABAB1R is essential for the cellular protective mechanism of anthocyanin, and it is the first result of demonstrating 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 mechanisms upon ethanol-induced apoptosis

2-1: Analysis of GABAB1R expression level

In order to elucidate the mechanism of ethanol-induced apoptosis, the present inventors analyzed the expression level of the GABAB1R protein and its sub-signaling molecule.

First, the primary hippocampal cells of Example 2 were treated with GABAB1R siRNA, and then the expression level of the GABAB1R protein was confirmed. Control siRNA was treated as a control. As a result, as shown in FIG. 2, after transduction of GABAB1R siRNA into the primary hippocampal cells of Example 2, it was confirmed that the level of GABAB1R decreased through Western blot. In the cells to which the control siRNA was introduced, no change in the level of GABAB1R was observed (see FIG. 4A).

Subsequently, the present inventors transduced GABAB1R siRNA into hippocampal cells, and 48 hours later, treated with a GABAB1R agonist or inhibitor, and observed changes in the expression level of GABAB1R. After treatment with 50 μM of a GABAB1R agonist baclofen (Ba) and 100 μM of an inhibitor of paclofen at 37° C. for 20 minutes, the expression level of GABAB1R was confirmed through Western blot.

As a result, as shown in Figure 4b, the GABAB1R level decreased by the GABAB1R siRNA treatment slightly increased the expression level of GABAB1R by baclofen, and the expression level of GABAB1R decreased by the paclofen treatment. It could be confirmed (see Fig. 4b).

2-2: GABAB1R sub-signaling analysis

It was confirmed using the protein extracted from the primary hippocampal cells of Example 2 that the effect of anthocyanin on the sub-signaling steps of GABAB1R and GABAB1R by ethanol treatment was offset. At this time, the experimental group is as follows (control group (C); 100 mM ethanol (E); 50 μM baclofen, Ba); 0.1 mg/mL anthocyanins (An); 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)).

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

Experimental Example 3: Analysis of the protective mechanism of anthocyanins against changes in ethanol-induced Ca2+ homeostasis

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

The concentration of free Ca ^{2+ in} cells was measured using a fluorescent Ca2+ detector, Fura-2AM (fura-2 acetoxymethyl ester) under drug-treated or untreated conditions. Specifically, 1×10 ⁶ cells were treated with drugs and siRNA according to each experimental group, and the experiment was repeated three times (control group (C); 100 mM ethanol (E); 50 μM baclofen, Ba); 0.1 mg/mL anthocyanins (An); 100 mM ethanol + 0.1 mg/mL anthocyanins (E+An); 100 mM ethanol + 40 nM GABAB1R siRNA (E+siRNA); and 100 mM ethanol + 0.1 mg/mL anthocyanins + 40 nM GABAB1R siRNA (E+siRNA+An)). Among the experimental groups, C, E, Ba, An, and E+An groups that did not transduce siRNA were treated with a vehicle used to deliver siRNA. In the experimental group, the treatment sequence was siRNA transduced into cells, 48 hours later, exchanged for normal medium, and reacted in ethanol and/or anthocyanin sequence for 20 minutes, respectively. After treatment according to the experimental group, the cells were washed twice using Krebs buffer, and in DMEM medium containing 5 μM Fura-2AM in a humid, 37° C., incubator containing 5% carbon dioxide for 60 minutes During incubation. Thereafter, the cells were washed twice with Locke's solution (pH 7.8), and the Fura-2AM fluorescence signal of Ca ²⁺ was subjected to a luminescence photometer (LS50B, Perkin) having an excitation wavelength of 340 nm to 380 nm and an emission wavelength of 510 nm. Elmer, Boston, MA). The 340 nm/380 nm fluorescence ratio was averaged and observed based on a 2 second time interval. The obtained fluorescence signal was analyzed using a computer equipped with general imaging software or a MicroVAX II computer equipped with origin 7 software. The intracellular calcium concentration was calculated using the Grynkiewicz equation of Equation 1 below (Grynkyewicz G, et al., J. Biol. Chem., 260:3440-3450, 1985).

In Equation 1, Kd is the dissociation constant of the Fura-2AM Ca ²⁺ bond, calculated as 225 nM in the intracytoplasmic environment, and R means 340 nm/380 nm fluorescence ratio; 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), and Sf2 is the intracellular concentration of Ca ²⁺ When is 0, it means the wavelength at 380 nm; Sb2 means the wavelength at 380 nm for saturated Ca ²⁺ .

As a result, as shown in Fig. 6, the concentration of Ca ²⁺ was significantly increased compared to the control group by ethanol treatment for 20 minutes in the non-transfected group. In the non-transfected group, an increase in the Ca ²⁺ concentration due to 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 the anthocyanin on the increase in Ca ²⁺ of ethanol-induced ethanol was not found in the GABAB1R siRNA-treated group, and these results imply that GABAB1R is essentially required for the neuroprotective effect of anthocyanin.

In addition, baclofen, a GABAB1R agonist, decreased the concentration of Ca ²⁺ in cells. These results demonstrate that ethanol increases the intracellular Ca ²⁺ concentration, whereas anthocyanin inhibits this increasing effect, which is shown through GABAB1R. Taken together, anthocyanins have a protective effect against ethanol-induced apoptosis. It suggests that it can provide.

Experimental Example 4: Ethanol-induced mitochondrial membrane potential ( Δ Analysis of protective mechanisms of anthocyanins against ΨM) abnormalities

An abnormal increase in intracellular Ca ²⁺ levels caused by ethanol damaged the mitochondrial membrane potential (ΔΨM), and this damage releases cytochrome-c, resulting in apoptosis signaling mechanism. It is known to activate (Simasko MS, et al., Brain Res., 824:89-96, 1999).

After the primary hippocampal cells of Example 2 were treated with drugs according to the following experimental groups, apoptosis analysis was performed (control group (C); 100 mM ethanol (E); 0.1 mg/mL anthocyanins, An); 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) ). Among the experimental groups, C, E, An, and E+An groups that were not transduced with siRNA were treated with vehicles used to deliver siRNA. Treatment according to the experimental group was performed 48 hours after siRNA transfection. In each experimental group, the treatment sequence was 20 minutes after ethanol treatment, followed by treatment with anthocyanins at 37°C for 20 minutes. Data are presented as the average of the results of 3 repeated experiments performed on each of the 3 plates. M1 refers to the cell cycle arrest phase in the mitosis process. * Means p <0.05 compared to the control group, and # means p <0.05 compared to the ethanol treatment group.

Mitochondrial membrane potential (ΔΨm) was analyzed using a JC-1 mitochondrial membrane potential detection kit (Biotium Inc., Hayward, CA, USA). After transduction or non-transduction of siGABAB1R RNA into GD 17.5 hippocampal cells of Example 1, drugs were treated according to the experimental group. Thereafter, the cells were obtained and stained for 15 minutes at 37° C. using JC-1 drug, washed twice using 1× assay buffer, and FACS analysis (FACSCalibur Flow Cytometer; Becton Dickinson, San Jose , CA, USA) in 0.5 ml. The analysis was repeated three times, and it is known that JC-1 aggregates in normal polarized mitochondria and emits red fluorescence at a wavelength of 590 nm. The JC-1 monologue that escapes from the depolarized mitochondria emits green fluorescence at 530 nm. The red and green fluorescence were measured in the FL-1 (green) and FL-2 (red) channels, respectively, in a flow cytometer. Thereafter, the cells were observed using a "double band pass" filter in a fluorescence microscope designed to detect fluorescence and rhodamine dyes simultaneously.

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

The present invention has been described with reference to the embodiments shown in the drawings, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Therapeutic agent 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)

Anthocyanin extracted from black beans; And the group consisting of Alzheimer's disease, Parkinson's disease, HIV dementia, epilepsy, schizophrenia, depression, manic depression, neurogenic disorder, autism, stroke, Lou Gehrig, Huntington's disease, and multiple sclerosis, including baclofen as an active ingredient. In the composition for the prevention and treatment of neurological diseases selected from,
Step 1 of extracting the anthocyanin extracted from the black beans by repeating the process of stirring the black beans in 95% methanol/1% HCl for 72 hours at room temperature under dark conditions 3 times;
A second step of concentrating the black bean extract using a rotary evaporator and loading it onto a column;
A third step of washing with distilled water until the eluate of the second step turns red, and then washing with ethyl acetate until a portion except for the lowermost 1cm layer of the column turns purple;
Then, a fourth step of eluting anthocyanins by putting 95% methanol/1% HCL into the column until the color of the entire column becomes red;
A fifth step of removing fine substances from the eluate containing anthocyanin using a rotary evaporator and a 0.45 μm filter;
6 step of loading the anthocyanin concentrate onto a column and eluting with a 50% methanol/50% distilled water/1% HCl solution until 800 to 1,000 ml of a red color eluate is obtained;
And anthocyanin obtained through 7 steps of evaporating the red anthocyanin eluate using a rotary evaporator to obtain a dried anthocyanin powder,
The composition is characterized in that to protect the nerve cell damage caused by ethanol, a composition for preventing and treating neurological diseases. delete delete delete delete

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