KR101968019B1 - Composition comprising glyceollins with cognition-improving effects and the use thereof - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating cognitive impairments containing bean-derived glyceollin and a composition for improving memory or learning ability. More particularly, the present invention relates to the composition for preventing, alleviating or treating cognitive impairments and the composition for improving memory or learning ability, which have an effect of improving cognitive function such as memory degradation due to a powerful antioxidant effect of glyceollin, which is a bean-derived isoflavone component, and an acetylcholinesterase inhibiting activity.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for improving cognitive function comprising glycerol derived from soybean as an active ingredient,

The present invention relates to a composition for improving cognitive function comprising soybean-derived glyceollin, and more particularly, to a composition for improving cognitive function comprising glycerol, which is a transcription factor of Nrf2 The present invention relates to a composition for improving the cognitive function characterized by inhibiting the activity of acetylcholine esterase and consequently having an effect of improving cognitive function such as memory loss.

In general, phytoalexin is a low-molecular-weight antimicrobial substance that is produced by plants to fight fungi when they are infested with various insects or fungi. These substances are not found in plants before infection, but within a few hours after infection It is synthesized at a very high rate and acts on the infected area and has toxic properties against various bacteria and fungi. These phytoalexins are self-produced by protecting plants. However, recently, the functions such as antimicrobial activity of these substances have been discovered, and a lot of studies have been actively carried out to use them for stable and inexpensive medicines, dyes, flavors and directions .

In particular, there are various phytoalexins in soybeans, and many studies have been conducted on them. It has been known that when the edible fungus is treated in the germination of soybean, special functional substances that do not exist in the soybean itself are produced. Among them, studies on glyceolins, a kind of phytoalexin produced in soybean, are actively conducted.

Glyceollins are one of phytoalexins, such as isoflavones, which are low molecular weight substances produced when plants are stressed by microbial infections or by substances such as inorganic salts. Phytoalexin (JA Agric Food Chem. Jun., 48, 6, 2167-72, 2000). Recently, anticancer and anti-inflammatory effects have been known, and new functionalities For example, in case of resveratrol of grape, phytoalexin is one of the most important phytoalexins. In addition to antioxidant and anticancer effects, it is also known as a protein deacetylating enzyme, a protein deacetylating enzyme produced in some tissues of the body such as the brain, It has been known that activating the pathway of sirtuin lowers caloric intake and prolongs the life span. However, there are few studies on other functions of phytoalexin.

In addition, glyceollins are substances derived from soybeans by fungal infections and do not exist in ordinary soybeans, and soybean is used as an edible sauce such as Asp. Sojae, Asp. Oryzae, , Or R. oligosporus, are known to be capable of inducing the production of phytoalexins, such as glycerol and oxooctadecadienoic acids (Boue et al., J Oct. 17, 55 (21), 8589-95, 2007). Recently, it has been reported that these glycerol It has been reported that it has the effect of preventing the progression of cancer such as breast cancer and ovarian cancer. However, other functions of glycerol other than the above functions have not been disclosed at all

Therefore, the inventors of the present invention conducted an experiment to determine whether glycerol, an active antioxidant enzyme inducer, could inhibit oxidative stress-induced neuronal damage, Acetylcholinesterase inhibition. Furthermore, the present inventors completed the present invention upon confirming that the increase in the memory decline is significantly reduced when glycerol is treated in an animal model in which memory deterioration has been induced.

Korean Patent Publication No. 10-2010-0115840 Korean Patent Publication No. 10-2017-0030806

It is an object of the present invention to provide a composition for preventing, improving or treating cognitive dysfunction comprising glycerol.

Another object of the present invention is to provide a drug for preventing, ameliorating or treating cognitive dysfunction including glycerol.

It is another object of the present invention to provide a food for preventing or improving cognitive dysfunction which comprises glycerol.

Another object of the present invention is to provide a composition for improving memory or learning ability comprising glycerol.

In order to solve the above problems, the present invention provides a composition for preventing, ameliorating or treating cognitive dysfunction including glycerol.

In one embodiment of the present invention, the glycerol is derived from a plant and is preferably, but not limited to, derived from soy.

In one embodiment of the present invention, the glycerol is obtained by inoculating mycelium with fungi and isolating the glycerol from the soybean having an increased content of glycerol.

In one embodiment of the present invention, the fungus is Aspergillus oryzae, Aspergillus sojae or Rhizopus oligosporus, preferably an aspergillus scent But is not limited thereto.

In one embodiment of the present invention, the glycerol is glycerol Ⅰ, glycerol Ⅱ, glycerol Ⅲ, IV or a mixture thereof, preferably glycerol Ⅰ, but is not limited thereto.

In one embodiment of the present invention, the composition inhibits acetylcholinesterase activity and has a strong antioxidant function.

In another aspect, the present invention is directed to a pharmaceutical composition for preventing, ameliorating or treating cognitive dysfunction comprising glycerol.

In another aspect, the present invention relates to a composition for preventing or ameliorating cognitive dysfunction comprising glycerol.

In another aspect, the present invention relates to a composition for enhancing memory or learning ability comprising glycerol.

The composition for preventing, improving or treating cognitive dysfunction and the composition for improving memory or learning ability comprising glycerol of the present invention has an acetylcholinesterase inhibitory effect and a strong antioxidant ability and thus can prevent, And further has an effect of remarkably improving the memory capacity.

Figure 1 shows the results of confirming whether cytotoxicity induced by glutamate is improved by glycerol. Unlike the wild type, the Nrf2 knockout model showed that the cytotoxic mitigation effect of glycerol was inhibited.
Figure 2 is the result of confirming the effect of inhibitors that inhibit the cytotoxicity induced by glutamate by glycerol.
FIG. 3 shows the results of confirming the effect of inhibitors which inhibit glutamate-induced intracellular substrate-induced production of active oxygen by glycerol.
FIG. 4 shows the results of confirming the effect of the inhibitor inhibiting the intracranial transcription signal of Nrf2 delivered by glycerol.
FIG. 5 shows the results of confirming the action of inhibitors (MAPK, PI3K, JNK) inhibiting the transcription factor activity of glycerol.
FIG. 6 is a diagram illustrating an entire process of an experiment in which the cognitive function-improving effect of glycerol using a mouse model causing cognitive dysfunction was confirmed.
Figure 7 shows the results of passive avoidance experiments using a mouse model that induced cognitive dysfunction.
FIG. 8 shows the result of a spatial perception test (Y-Maze) using a mouse model that caused cognitive dysfunction.
FIG. 9 shows results of a Morris underwater maze model experiment using a mouse model that caused cognitive dysfunction.
Fig. 10 shows the results of experiments on the action of glycerol, which inhibits acetylcholinesterase, from cells isolated from cerebral or hippocampal tissues.

The terms used in the present invention are described as follows.

The term "cognitive impairment " as used in the present invention means a symptom or a disease that does not normally perform cognitive functions such as memory processing, perception or problem solving, and specifically refers to working memory, attention and vigilance, Memory, visual learning and memory, reasoning and problem solving, such as executive functions, processing speed or societal perception.

The terms " pharmaceutically acceptable "and" pharmaceutically acceptable "in the present invention are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term "prophylactic " means any act that inhibits the symptoms of a particular disease or delays its progress by administration of the composition of the present invention.

The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease upon administration of the composition of the present invention.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The present invention relates to a composition for improving cognitive function or memory ability comprising glycerol. Glycerol is a kind of phytoalexin derived from soybean. It is known as a substance having antioxidant, antibacterial and other effects. In the present invention, glycerol has a strong antioxidative activity and has a protective effect on cranial nerve cells, It is intended to be used as a composition having the above-mentioned use.

In the present invention, fungal mycelia were used to separate glycerol from soybeans. Glycerol is a phytoalexin substance that is not present in natural soybeans but is derived from soybeans by fungal infections. The inventors of the present invention have filed a patent application for a method of mass-producing glycerol from soybeans in the previous study (Patent Application No. 10-2015-0128174), and the glycerol of the present invention But are not limited thereto.

The glycerol of the present invention is obtained by inoculating fungal mycelium into soybeans, cultivating the soybean fungus, increasing the content thereof, and separating it. The fungus may be Aspergillus oryzae, Aspergillus sojae or Rhizopus oligosporus, preferably aspergillus, but is not limited thereto .

Glycerol I is the most important form of glycerol separated by this method, and glycerolines II, III and IV, which are three isomers thereof, exist in trace amounts (Clinical Cancer Research 2006; 12 (23) December 1 , 2006, 7159-7164, Antiestrogenic Glyceollins Suppress Human Breast and Ovarian Carcinoma Tumorigenesis). The active ingredient "glycerol" in the present invention is meant to include all of glycerolin I, glycerolin II, glycerolin III, glycerolin IV or mixtures thereof, more preferably glycerolin I.

The glycerol of the present invention is specifically characterized by inhibiting acetylcholinesterase. In normal people who do not have cognitive problems in general, neurotransmitters such as acetylcholine are secreted from the brain neurons and memory is maintained and learning becomes possible. However, when there is a problem in cognitive ability such as dementia, the cognitive function is deteriorated due to destruction of the nerve cell that secretes acetylcholine. When the acetylcholinesterase inhibitor is administered to these patients, the acetylcholine concentration in the synaptic gap is increased To improve cognitive function.

In the present invention, the composition may be formulated into a pharmaceutical composition, a food additive, a food composition (in particular, a functional food composition), a feed additive or the like depending on the intended use or aspect. Also, the content of glycerol as an active ingredient may be adjusted in various ranges depending on the specific form of the composition, the purpose of use, and the manner of use.

The pharmaceutical compositions of the present invention may include carriers, diluents, excipients, or a combination of two or more thereof, which are commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, and can be used by mixing, for example, saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as antioxidants, buffers, and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. In addition, the composition of the present invention may further contain one or more active ingredients showing the same or similar functions. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Further, it can be advantageously formulated according to each disease or ingredient, using appropriate methods in the art, or using methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA

The composition of the present invention may be administered orally or non-orally (for example, topical application, intravenous, subcutaneous, intramuscular, intraperitoneal or topical application) or by oral administration according to the intended method. The appropriate dosage of the pharmaceutical composition of the present invention may be determined depending on the patient's body weight, age, sex, health condition, diet, method of administration, administration time, administration method, excretion rate and severity of disease. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies.

The food composition of the present invention may be in the form of a pill, a powder, a granule, an infusion, a tablet, a capsule, or a liquid. Examples of the food include meat, sausage, bread, chocolate, candy, snack, Noodles, other noodles, gums, dairy products including ice cream, soups, beverages, tea, functional water, drinks, alcoholic beverages and vitamin complexes. In addition to the active ingredient, the food composition of the present invention may contain a pharmaceutically acceptable carrier, various flavors or natural carbohydrates as an additional ingredient. In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame may be used as the flavor.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

1. Materials and Preparation

≪ 1-1 > Reagents and compounds

All cell culture media were obtained from Welgene (Daegu, Korea). Neurobasal medium, fetal bovine serum (FBS), N2 supplements and B-27 supplements were purchased from Gibco (GIBCO BRD, Gaithersburg, Md.). Most of the chemicals used in this study were obtained from Sigma (St Louis, MO, USA). Antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, Calif.).

<1-2> Isolation of glycerol

Glyceollins were isolated and purified by Professor Lim Soon Sung of the Department of Food and Nutrition, Hallym University. Glyceolin composed of glyceollin I, II, III and IV was prepared according to the method described in the previously disclosed patent (KR application 10-2015-0128174). Soybean extract containing glycerol was inoculated with Aspegillus sojae in soybean seeds and cultured in a dark room at 26 ° C for 3 days. After homogenization in 80% aqueous ethanol, the filtered extract was lyophilized. The ethyl acetate soluble fraction of the crude extract was isolated via silica gel column chromatography. One fraction was analyzed by TLC and HPLC to confirm the presence of glycerol, and the fractions were evaporated under reduced pressure.

<1-3> Cell culture

The glutamate-sensitive mouse hippocampal neuronal cell line HT22 and human neuroblastoma cell line SHSY5Y were cultured in a 5% CO _2, 37 ° C temperature condition FBS 10%, penicillin 100 units / mL, and streptomycin 100 μg / mL. The cultured cells were subcultured at a 1: 8 ratio every two days.

<1-4> Cerebral Cortex Neuron Isolation

Cerebral cortical neurons (nerve cells) were prepared as follows. C57BL / 6 wild-type and Nrf2 knockout mice A 15-week-old fetal cortex of pregnancy was dissected and stored in cold phosphate buffered saline. Cortical tissues were dissociated into single cells through a 70-μm nylon cell filter (SPL Life Sciences, Pocheon, Korea). The cell suspension was centrifuged at 1,100 x g for 3 minutes, and the resulting pellet was re-inoculated into the plate medium. (DMEM / F12, N2 supplement, non-essential amino acid (5 mL / L), HEPES (3.9 g / L) sodium bicarbonate (3.7 g / L), sodium pyruvate (55 mg / L) and pH 7.2-7.4). The cells were inoculated in a suitable culture plate coated with poly-L-lysine at a density of 3 × 10 ⁵ cells / well and cultured in a CO ₂ incubator (5% [v / v], 37 ° C.). After neurons were attached to the culture plates, they were replaced with neurobasal medium containing B-27 supplement to inhibit the growth of contaminated non-neuronal cells and supplemented with 0.5 mM glutamate. The cultured cells were replaced with new neurobasal medium every other day. All analyzes were performed one week after incubation so that neurons could be performed after maturation.

2. Identification of antioxidant activity of glycerol on neurons

<2-1> Oxytocis (excitotoxicity) analysis

The C57BL / 6 wild-type and Nrf2 knockout cerebral cortical neurons isolated from mice in Experimental Example 1 were confirmed by glutamate-induced cytotoxicity by several methods as follows.

HT22 cells and primary cultured cerebral cortical neurons were inoculated into a 48-well plate with DMEM medium containing 10% FBS at an initial density of 2 x 10 ³ cells / well and incubated at 37 ° C with 5% CO ₂ Lt; / RTI &gt; for 24 hours to 1 week. The medium was then removed and replaced with DMEM medium containing glycerol at a concentration of 1 to 10 [mu] g / mL. Then, 5 mM glutamate was added to each plate.

After 24 hours, the medium was removed from the wells and 3- [4,5-dimethylthiazol-2-yl] - 2,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide 2,5-diphenyltetrazolium bromide, MTT) solution (containing 0.25 mg / mL in the medium) was added to terminate the cell culture. After 2 hours, the solution was removed and 200 mu l of DMSO was added. After 10 minutes, the absorbance was measured at 570 nm by a microplate reader (Tecan, Australia). The cell proliferation of each group was calculated by comparing the absorbance of the control group and the treatment group.

<2-2> Effects of MAPK, PI3K, Nrf2 and HO-1 on glutamate-induced oxytocin attenuation by glycerol

MAPK, PI3K and HO-1 inhibitors were treated at different concentrations in the culture medium of HT22 cells, and the cells were treated with glutamate and glycerol as described above. MAPK, PI3K and HO-1 inhibitors are known to inhibit the action of glycerol, which inhibits glutamate-induced apoptosis in cells, in a concentration-dependent manner. After this material treatment, intracellular excitotoxicity was measured.

 <2-3> Construction of antioxidant response factor (ARE) by transfection of cell lines

HT22 or SHSY5Y cells were transfected into a 6-well plate using 100 ng of pGL4.37 [luc2P / ARE / Hyg] vector (Promega Corp., Madison, Wis., USA) using lipofectamine 2000 (Invitrogen, Carlsbad, CA) Transfected. The cells transfected with the gene were selected by treating with 0.2 mM hygromycin. To establish a stable cell line, both HT22-ARE and SHSY5Y-ARE cell lines were subcultured 5 times by treatment with 0.2 mM hygromycin at a ratio of 1: 5 every 2 days.

<2-4> Antioxidant response factor (ARE) - Reporter gene analysis

Cultured for 12 hours in HT22-ARE or ARE-SHSY5Y cells inoculated in 12-well plate with DMEM medium containing 10% FBS to an initial density of 1x10 ⁴ cell / well and the atmosphere consisting of 37 ℃, 5% CO ₂ Respectively. The medium was then removed and the MAPK inhibitor regulated and replaced with serum-free DMEM medium containing various concentrations of glycerol. After incubation for 16 hours, cells were harvested and luciferase activity was measured according to the protocol provided by the manufacturer (Promega Corp., Madison, Wis.).

<2-5> Measurement of ROS (reactive oxygen species) level of intracellular matrix

Oxidative stress was measured quantitatively in cells by a modified 2,7-dichlorofluorescein diacetate (DCFDA) assay according to Wang and Joseph (Wang and Joseph, 1999). DCFDA is a probe applied to the detection of ROS and electron transfer processes and is a non-fluorescent cell permeable compound used in flow cytometry. Cells were inoculated into 96-well plates (Nunc, Rochester, NY, USA) with black bottom and cultured for 24 hours with DMEM containing 10% FBS. Cells were further incubated for 12 additional hours in the absence and presence of glycerol. Then, dichlorofluorescein (DCFDA) dissolved in dimethylsulfoxide (DMSO, final concentration 10 μM) was added and the cells were cultured for 30 minutes. Fluorescence was measured at 485 and 535 nm using a fluorescent microplate reader (Infinite 200; Tecan, Grodig, Austria). Most of the steps including incubation, washing and fluorescence measurements of the reaction mixture containing the dye and the oxidizing agent were carried out in the dark. The fluorescence intensities of the samples were expressed as relative percentages relative to negative control to which no glycerol and glutamate were added.

<2-6> Western blot analysis

The cells were washed with 12% SDS-polyacrylamide gel (1.5M Trizam base, 10% sodium dodecyl sulfate, 30% acrylicamide, 10% ammonium sulfate, TEMED) to confirm expression of HO-1 and accumulation of Nrf2 in the nucleus And transferred to a PVDF membrane (Millipore, Bedford, MA, USA) at 100 V for 70 minutes. Membranes were blocked with 1% BSA-TBS / T for a minimum of 2 hours. (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). The membranes were then incubated with anti-HO-1, anti-Nrf2, anti-lamin B or anti-beta-actin antibodies diluted 1: 1,000 at 4 ° C and washed three times at 10 intervals in TBS / T And then incubated for 4 hours with anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG conjugated with horseradish peroxidase. Protein bands were then detected using Super Signal® West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) after washing three times at 10 min intervals in TBS / T. Densitometry analysis was performed using MultiGauge 3.0 software (Fujifilm, Tokyo, Japan).

<2-7> Immunocytochemistry

HT22 cells were seeded onto a 24-well plate containing sterile coverslips at a density of 3x10 ⁴ cells / well. After 24 hours of incubation, the cells were exposed to glycerol for 6 hours. Cells were washed with TBS, fixed with 3.7% paraformaldehyde and blocked with 1% bovine serum albumin dissolved in TBS. Cells were incubated with anti-rabbit IgG, anti-Nrf2 and anti-Keap1 (100 ng / ml) combined with fluorescein isothiocyanate (FITC), anti-goat IgG- conjugated Texas red and 4 ', 6- diamidino- Lt; / RTI &gt; After washing twice with TBS, cell slides were treated with fluorescence mounting media (Dako A / S, Glostrup, Denmark) and observed under fluorescence microscopy (Eclipse 80i, Nikon, Tokyo, Japan)

<2-8> Statistical analysis

Data were tested by Duncan's multi-range testing followed by analysis of variance using SPSS Statistics 20 software (SPSS Inc., Chicago, IL, USA). The significance level was set at p <0.05.

3. Experimental Results

<3-1> Effect of glycerol on cytotoxicity induced by glucosamine

To investigate the effect of glycerol on cytotoxicity induced by glutamate, mouse primed cortical neurons isolated from Experimental Example 1 were used. As shown in Fig. 1, when the glutamate was added, the cell viability of both the wild type and Nrf2 knockout cells was found to be lowered to 60% or less (white bars). Glycerol was concentration-dependent in the range of 1-10 μg / mL Inhibited glutamate-induced excitotoxicity in cortical neurons isolated from fetal brain of C57BL / 6 wild-type mice. In contrast, the protective effect of glycerol was not observed in cortical neurons isolated from the fetal brain of C57BL / 6 Nrf2 - / - mice. (Figure 1 black bar)

<3-2> Effects of MAPK, PI3K, Nrf2 and HO-1 on glycerol action

Proliferation inhibition by glutamate was restored to the control level without glutamate treatment when glycerol was treated at a concentration of 10 μg / mL in HT22 cells. However, the effect of glycerol is counteracted when the HO-1 inhibitor (40 μM SnPP) or an ERK inhibitor (10-30 μM SP600125) or a JNK inhibitor (10-30 μM PD98059) (Fig. However, p38 inhibitors (10-30 μM SB203580) and PI3K inhibitors (10-30 μM LY294002) did not affect inhibition of cell growth in the presence of glycerol and glutamate (Figure 2A).

When HT22 cells were treated with glutamate at a concentration of 25 mM for 24 hours, cell death, shrinkage, DNA condensation, and cell volume changes resulted in altered cell volume. However, when treated with glycerol, the cell loss due to glutamate as described above was reduced. That is, the effect of glycerol to reduce apoptosis was not significant when co-treated with ERK inhibitor (10-30 μM SP600125), JNK inhibitor (10-30 μM PD98059) or HO-1 (40 μM SnPP) inhibitor . (Figure 2B).

<3-3> Inhibition of intracellular ROS levels by glycerol

As shown in FIG. 3, it was confirmed that the level of intracellular ROS was inhibited by glycerol. Through the above experiment, it was confirmed that when HT22 cells were exposed to glutamate (5 mM) for 6 hours, ROS production was increased by about 50% as compared with the control group. (Figure 3A), whereas the addition of ERK, JNK, and HO-1 inhibitors offset the inhibitory effect of glycerol on glutamate-mediated ROS production. (Fig. 3B).

<3-4> Effects of ERK and JNK Inhibitors on Nrf2 Transcription by Glycerol

Accumulation of Nrf2 in the nucleus was improved by treating glycerol for 6 h (Fig. 4A). In other words, glycerol increased the rate of Nrf2 transfer to the nucleus, which was confirmed by immunocytochemistry (Fig. 4B). On the other hand, when ERK or JNK inhibitor were treated simultaneously, Nrf2 metastasis decreased. In addition, the expression of Keap1 in the cytoplasm decreased when treated with 10 μg / mL of glycerol, and Keap1 was restored to the control level by incubation with ERK or JNK inhibitor (Fig. 4).

<3-5> Effects of MAPK and PI3K Inhibitors on ARE-Mediated Transcriptional Activity Induced by Glycerol

In order to examine whether glycerol interacts with ARE to induce HO-1, SHSY5Y-ARE and HT22-ARE cells containing the cis-element, reporter ARE-luciferase gene in the promoter region of antioxidant enzymes, And stimulated with glycerol for 16 hours. The ARE-luciferase activity induced by glycerol increased depending on the treatment concentration of glycerol, but the activity was decreased in the HT22-ARE cell line, especially when treated with ERK or JNK inhibitor (Fig. 5) .

<3-6> Induction of HO-1 by glycerol

To determine whether glycerol modulates the expression of HO-1, SHSY5Y cells were transfected with HO-1 siRNA for a transient period of 48 hours. After transfection, cells were treated with glycerol for 24 hours. As a result, the expression of HO-1 was induced by glycerol in a concentration-dependent manner in SHSY5Y cells. However, in the HO-1 siRNA knockout cells, the expression of HO-1 was not increased, despite treatment with glycerol.

4. Identification of the improvement of cognitive ability of glycerol by animal model

<4-1> Passive avoidance experiment

As shown in Fig. 6, a mouse model for cognitive ability improvement was designed. A 7-week-old mouse was injected intraperitoneally (1 time) with scopolamine at a dose of 1 mg / kg body weight to produce a memory deficient animal model.

Passive avoidance experiments were performed using the mouse model. The passive avoidance test was conducted to assess the learning and memory of glycerol. This is a way to test the memory by measuring the amount of time you spend in the bright room, then letting the mouse flow into the dark compartment, letting the mouse emerge into a bright space where no current flows. As a result, as shown in Fig. 7, when the scopolamine was administered to mice, the time spent in the dark compartment of the device became shorter, whereas the glycerol (once dose, 10 mg / kg body weight) When injected with scopolamine after 2 doses, the time spent in the bright space was significantly longer. That is, it was confirmed that the reduction of memory by scopolamine was suppressed by the pre-administration of glycerol.

<4-2> Y-maze test

The Y-maze test is a method of testing the will to search for a new environment of the mouse. It is a method of measuring the relative frequency of an animal to identify surrounding cues and sequentially enter the maze. Mice have a tendency to instinctively explore new environments, but the ability to detect new environments (spontaneous alteration,%) is significantly reduced for memory-impaired mice treated with scopolamine. On the other hand, it was confirmed that the memory instability of oral administration of glycerol was restored to the search instinct. (Fig. 8)

<4-3> Experiment of Underwater Maze Model

Morris underwater maze model was used to test cognitive ability and short term memory. That is, water (22 ± 1 ° C) is filled up to a height of 90 cm in diameter and 45 cm in height and 45 cm in height. After training the mouse to find the target, the time ) And latency time near the target were measured. As a result, as shown in Fig. 9, it was observed that when scopolamine was treated, the time for finding the target was significantly longer and the time for staying in the vicinity of the target was remarkably decreased. On the other hand, after administration of glycerol twice a day for 2 days before the experiment, the time to reach the target was shortened and the stay time was longer when the scopolamine was administered. This demonstrates that glycerol has a short-term memory improvement effect.

&Lt; 4-4 > Acetylcolyesterase (AChE) Inhibitory Activity

AChE analysis was carried out with minor modifications according to Ellman's method. The cortex and hippocampal cells were isolated from the fetal brain at 15 days of gestation with normal mouse (type C57BL / 6), and the tissues were suspended in 10 mL of phosphate buffer (containing 12.5 mM phosphate buffer, pH 7.0, 400 mM NaCl) Homogenized and then centrifuged at 1000 x g for 10 minutes at &lt; RTI ID = 0.0 &gt; 4 C. &lt; / RTI &gt; The supernatant was used as an enzyme source for analysis. Add 25 μL of Ellman's reagent (10 mM 5,5'-dithio-bis [2-nitrobenzoic acid] and 15 mM sodium bicarbonate), and mix at room temperature for 30 minutes at 25 ° C. Add 100 μL of the compound, 100 μL of 100 mM sodium phosphate buffer, Lt; / RTI &gt; In addition, the mixture was reacted with 10 [mu] l of 75 mM acetyl thiocholine iodide for 10 minutes. Absorbance was measured at 415 nm using a microplate reader (Sunrise TECAN, Salzburg, Austria).

As a result, as shown in FIG. 10, it was confirmed that glycerol inhibited enzyme activity in a concentration-dependent manner, and this AChE inhibitory activity is a major mechanism for improving cognitive function.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

A composition for preventing, ameliorating or treating cognitive dysfunction, comprising glyceollin which is a mixture of glycerol Ⅰ, glycerol Ⅱ, glycerol Ⅲ, glycerol IV. The method according to claim 1,
Wherein the glycerol is derived from a soybean. The method according to claim 1,
Wherein said glycerol is isolated from soybean cultured by inoculating mycelium with mycelium of soybean, said composition being for preventing, improving or treating cognitive dysfunction The method of claim 3,
The fungal mycelium is selected from the group consisting of Aspergillus oryzae, Aspergillus sojae, and Rhizopus oligosporus. Therapeutic composition delete The method according to claim 1,
A composition for preventing, improving or treating cognitive dysfunction, characterized in that the composition inhibits acetylcholinesterase A medicament for preventing, ameliorating or treating a cognitive disorder comprising the composition of any one of claims 1 to 6. A food for preventing or improving cognitive dysfunction comprising the composition of any one of claims 1 to 6. A composition for enhancing memory or learning ability comprising glyceollin, which is a mixture of glycerol Ⅰ, glycerol Ⅱ, glycerol Ⅲ, glycerol IV. 10. The method of claim 9,
Wherein said glycerol is isolated from soybeans cultured by inoculating mycelium with mycelium of soybean. 9. A memory or learning ability enhancing food comprising the composition of claim 9.

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