KR20170086012A - A composition for improving learning ability comprising osmotin - Google Patents

Abstract

The present invention relates to a composition for improving memory performance or learning ability, and more particularly, to a composition for health functional foods and a pharmaceutical composition for enhancing memory ability or learning ability including osmotin. When the composition containing osmotin according to the present invention is used, the activity of AMPK and SIRT1 can be increased to restore memory or learning ability defects, and thus it can be usefully used as a composition for improving memory or improving learning ability.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for enhancing memory ability or learning ability including osmotin,

The present invention relates to a composition for improving memory performance or learning ability, and more particularly, to a composition for health functional foods and a pharmaceutical composition for enhancing memory ability or learning ability including osmotin.

Human cells recapitulate differentiation and destruction, and cell growth can be promoted through the natural environment, food, exercise and training processes, and there are a myriad of substances around our lives that delay cell destruction. When selected and consumed, they can maintain healthy brain function with longevity, but they can cause disease when they are not. It is no exaggeration to say that the brain function is the most central to man. When the activity of the brain cell is active, the mental power is enhanced, the will and determination are strengthened, and the face and the whole body are full of energy. For example, in children, when the activity of brain cells is vigorous, not only the differentiation of brain cells, but also growth and judgment are enhanced, memory is improved, and learning ability is improved. On the other hand, as the person gets older, the brain function is lowered, the concentration is weakened, and memory and learning ability are lowered by external factors such as stress.

Human cells recapitulate differentiation and destruction, and cell growth can be promoted through the natural environment, food, exercise and training processes, and there are a myriad of substances around our lives that delay cell destruction. When selected and consumed, they can maintain healthy brain function with longevity, but they can cause disease when they are not. It is no exaggeration to say that the brain function is the most central to man. When the activity of the brain cell is active, the mental power is enhanced, the will and determination are strengthened, and the face and the whole body are full of energy. For example, in children, when the activity of brain cells is vigorous, not only the differentiation of brain cells, but also growth and judgment are enhanced, memory is improved, and learning ability is improved. On the other hand, as the person gets older, the brain function is lowered, the concentration is weakened, and memory and learning ability are lowered by external factors such as stress.

On the other hand, vertebrate hormone adiponectin plays a role in regulating energy metabolism through the AMPK / SIRT1 pathway in skeletal muscle and liver, and neuroprotective ability has been reported for several neurotoxins (X. Shen, et al J. Am. J. Physiol. Gastrointest. Liver Physiol . 298, G364-G374 (2010)). Osmotin is a structurally and functionally similar tobacco protein with vertebrate adiponectin, and the fundamental role of osmotin in plants appears to be a defense against pathogens. Osmotin orthologs are widely found in edible fruits and vegetables and are classified by their serological relevance, such as (PR) -5 family or thaumatin-like proteins associated with structural similarity and onset. In this regard, the present inventors have recently reported that osmotin can protect against neurodegeneration induced by glutamate and ethanol during development of rodent brain. (Korean Patent Publication No. 10-2011-0085260). However, the neurodegeneration is a limited one induced by glutamate and ethanol, and it is possible that such neurodegeneration may lead to memory or learning deficits, but that osmotins have a direct effect on memory and learning ability It was not. The aim of this study was to investigate whether osmotin, a structurally and functionally similar protein with adiponectin, could improve memory or improve learning ability, in view of the low serum adiponectin levels observed in cognitive impairment patients with memory and learning impairment .

Accordingly, the present inventors have found that osmotin treatment inhibits the progression of memory and learning deficits in a dual transgenic mouse line expressing APPsw and PS1 (presenilin-1) showing symptoms of memory and learning ability deterioration, . These results suggest that osmotin may be used as a composition for improving memory or learning ability through the AMPK / SIRT1 pathway.

It is an object of the present invention to provide a composition for health functional foods for improving memory performance or improving learning ability including osmotin.

Another object of the present invention is to provide a pharmaceutical composition for enhancing memory ability or learning ability including osmotin.

In one aspect, the present invention provides a composition for health functional foods for improving memory performance or improving learning ability including osmotin.

The term "osmotin" in the present invention is a protein contained in a large amount in aged fruit such as grape, and was first found about 30 years ago. It has been reported that osmotin functions in a similar manner to a protein that inhibits obesity and diabetes in the human body. It has been known that it can be used as a therapeutic composition for some cancers. However, Is not known at all.

The inventors of the present invention for the first time confirmed that the osmotin has a defensive function against memory and learning ability defects and can improve the defects. Specifically, osmotin protects against memory and learning abilities in APP / PS1 mice, an animal model with memory and learning deficit symptoms, and affects energy metabolism in memory and learning abilities in the Invivo and Invitro Induced the activity of AMPK and SIRT1 protein. In addition, it has been proved through concrete experiments that the above-described mouse model has excellent effects on memory enhancement and learning ability such as not only inhibiting cholesterol biosynthesis but also increasing the number of functional synapses.

Osmotin is a protein consisting of about 150 to 250 amino acids depending on the individual, and may be composed of amino acids lower or higher depending on the kind of plant. These osmotins are known to be contained in aged fruit of plants, such as Nicotiana tabacum, Citrus sinensis, Rosa roxburghii, Solanum tuberosum, Piper colubrinum, Solanum tuberosum, Ricinus communis and Arabidopsis thaliana, The above examples do not limit the kind of osmotin used in the present invention. The osmotin of the present invention is not limited in its origin, and osmotin derived from various plants as described above may be used, and may be artificially synthesized and used according to a known genetic engineering method.

Preferably, the osmotin of the present invention can be administered in an amount of 0.001 to 1000 μg / g, more preferably 0.1 to 500 μg / g, more preferably 1 to 100 μg / Lt; / RTI > g / g, and most preferably 10 to 20 g / g. In addition, when administered at a dose of 0.001 μg / g or less, the effect on the enhancement of cognitive function is insignificant, and even when administered at 1000 μg / g or more, the effect of the cognitive enhancement is not exhibited. However, the dose of osmorein can be determined by a person skilled in the art according to various factors such as the kind or volume, characteristics, weight and the like of the cell or individual to be administered.

In the present invention, the term "memory enhancement" or "improvement of learning ability" means all actions in which memory or learning ability is advantageously changed by administration of the composition for health functional food according to the present invention.

In a specific embodiment of the present invention, it was confirmed that the deficiency was recovered when osmotin was administered to APP / PS1 mouse which is an animal model of memory and learning ability deficiency (Fig. 1).

On the other hand, when the composition for health functional foods of the present invention is used as a food additive, the composition may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, it may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, natural sweeteners such as disaccharides such as maltose and sucrose and dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the 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 ratios of these additives can also be appropriately selected by those skilled in the art.

In one embodiment of the present invention, the APP / PS1 mouse, which is a cognitive-impaired mouse model, is administered with a MWM (Morris Water Maze) test and a Y-maze spontaneous alternation test, And a defense function against learning ability reduction (FIGS. 1 to 3).

On the other hand, AMP-activated protein kinase (AMPK) and SIRT1 (the NAD ⁽⁺⁾ -dependent protein deacetylases sirtuin 1) are energy sensing enzymes of cells. Their activity leads to the activity of the metabolic pathway leading to energy production. The energy metabolism of the brain is closely related to memory and learning ability. The composition of the present invention can enhance memory and learning ability by inducing the activity of AMPK and SIRT1. In a specific example of the present invention, it was confirmed that the reduced AMPK and SIRT1 activity was restored upon treatment with osmotin (Figs. 4 to 7).

In another embodiment of the present invention, hippocampal cells cultured in DIV21 are treated with osmotin and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated mEPSC (miniature excitatory postsynaptic currents ) Showed that osmoreine had an effect of increasing the number of functional synapses without affecting the synaptic strength in hippocampal neurons (Fig. 11).

Therefore, the osmotin-containing composition of the present invention can be utilized as a composition for health functional foods for improving memory or improving learning ability.

In another aspect, the present invention provides a pharmaceutical composition for enhancing memory or learning ability comprising osmotin.

Improvement of learning ability or learning ability of osmoreine has been described above.

In the present invention, the term "pharmaceutical composition" means a preparation for the purpose of preventing or treating disease, and can be formulated into various forms according to ordinary methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated in the form of external preparations, suppositories, and sterilized injection solutions.

In addition, depending on the respective formulation, a carrier known in the art such as a pharmaceutically acceptable carrier such as a buffering agent, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, a natural or non- As shown in FIG.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level will depend on the health condition, severity, activity of the drug, The method of administration, the time of administration, the route of administration and the rate of release, the duration of the treatment, the factors including the combined or concurrent drugs, and other factors well known in the medical arts.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

When the composition containing osmotin according to the present invention is used, the activity of AMPK and SIRT1 can be increased to restore memory and learning ability defects, and thus can be usefully used as a composition for improving memory or learning ability.

Figure 1 shows the escape time from the training period (submerged platform) of the MWM test. Arrows indicate processing time. Data represent mean ± SEM values (n = 10 / group).
Figure 2 shows the mouse resting and MWM probe test for two days after training. It shows the time consumption, expressed as a percentage, in the quadrant where the platform was during the MWM probe test (platform removed). Data represent mean ± SEM values (n = 10 / group). valence; *** p < 0.001.
Figure 3 shows the results of a Y-maze spontaneous shift test performed to evaluate the effect of osmotin treatment on short-term memory in dual transgenic APP / PS1 AD-model mice. Data represent mean ± SEM values (n = 10 / group). valence; *** p < 0.001.
Figure 4 shows Western blot analysis of p-AMPK and SIRT1 levels in hippocampus and cortical extracts of 9- and 12-month old untreated WT and vehicle (Veh) -or osmotin (Os) -treated APP / PS1 and APPsw mice Lt; / RTI &gt; Osmotin (15 μg / g of bw) was administered twice per day for two days. beta -actin signal as a loading control. The graph shows the band density relative to the band of WT in the western blot. The bars are mean ± SEM values (n = 5 / group). valence; *** p < 0.001.
Figure 5 shows measurements of AMPK activity in brain homogenates of WT and APP / PSl mice. The experiment was performed three times and shows representative data of one of the three repeated experiments. valence; *** p < 0.001.
Figure 6 shows measurements of SIRT activity in brain homogenates of WT and APP / PSl mice. The experiment was performed three times and shows representative data of one of the three repeated experiments. valence; *** p < 0.001.
Figure 7 shows Western blot analysis of p-AMPKa (Thr ¹⁷² ) and AMPKa and beta -actin (loading control) in hippocampus extract of APP / PS1 mice treated with 9 months old WT and vehicle (Veh) or osmotin (Os) Lt; / RTI &gt;
Figure 8 shows the effect of NMDAR1, NMDAR2, SNAP25, which is a presynaptic marker, and synaptopahedin, and synaptic flank markers, on APP / PS1 mouse hippocampal extracts treated with 9 month old untreated WT and vehicle (Veh) or osmotin (Os) &Lt; / RTI &gt; SNAP23 and PSD95 levels. A representative photograph and a graph showing the band density relative to the WT band are shown. The bars are mean ± SEM values (n = 5 / group). Significance: ** P <0.01, *** P <0.001.
Figure 9 shows SNAP23 (green, FITC-labeled), synaptophysin (red, TRITC-1) in DG and CA1 regions of APP / PS1 mouse brain hippocampus treated with 8 month old untreated WT and vehicle (Veh) Label) and DAPI (blue). The graph represents a quantitative analysis of the corresponding fluorescence signal. valence; ** P &lt; 0.01, *** P < 0.001.
Figure 10 shows SNAP23 (green, FITC-labeled), synaptophysin (red, TRITC-labeled) and DAPI (green) in the APP / PS1 mouse brain cortex treated with 8 months old untreated WT and vehicle (Veh) Blue). &Lt; / RTI &gt; The graph represents a quantitative analysis of the corresponding fluorescence signal. valence; ** P &lt; 0.01, *** P &lt; 0.001.
Figure 11 is a record of hippocampal neurons cultured in a group treated with vehicle and osmotin (8, 16, 32 nM). (a) is a representative record of spontaneous unitary EPSC. (b) shows the cumulative amplitude distribution of mEPSC (b, left) and the simplified mEPSC amplitude (b, right) from all recorded neurons. (c) shows the cumulative frequency distribution (c, left) of mEPSC and the simplified mEPSC frequency (c, right) from all recorded neurons. P> 0.05 between the mean mEPSC amplitude of the osmotin treated group and the control group. valence; **, P &lt; 0.01.

Hereinafter, the present invention will be described in more detail through Experimental Examples and Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Osmotin

The osmotin used in the present invention was purified to apparent homogeneity (= 99%) from culturing Nicotiana tabacum cv. Wisconsin 38 cells by adapting to NaCl and then further purified by repeated recrystallization. The endotoxin content of osmotin was <0.03 EU / mg protein as measured by the LAL assay kit (Genescript).

Example 2: Mouse strain and drug treatment

A double transgenic B6.Cg-Tg (APPswe, PSENdE9) 85Dbo / Mmjax memory and learning disabled mice with male C57BL / 6J mice (WT) and genes were purchased from Jackson Laboratory. The double transgenic mouse is a Swedish mutant (human) containing a chimeric mouse-human amyloid precursor protein as well as a human Presenilin 1 protein (PS1-dE9) mutation in CNS neurons Mo / HuAPP695swe). A transgenic C57BL / 6J-Tg (NSE-APPsw) KLAR mouse overexpressing a Swedish (K670N / M671L) mutant containing a mutant form of the human amyloid precursor protein in a neuron-specific enolase promoter It was purchased from the Korea Food & Drug Administration. Both of these transgenic mice exhibited symptoms of memory and learning disability, and thus were used as models of the present invention. The mice were randomly fed with water and 25 ℃ for 12 hours / 12 hours. The experiment was approved by the Animal Ethics Committee of Gyeongsang National University, Department of Applied Life Sciences.

Mice were divided into groups as follows before treatment: WT mice were randomly divided into two groups - saline treated group and osmotin treated group. Transgenic mice (single or double) were randomly divided into three subgroups: saline treated group, 2 osmotin treated group (two consecutive days) and 4 osmotin treated group (treated every other day). Osmotin (15 mg / g body weight) was dissolved in saline and administered intraperitoneally. The mice were sacrificed at the age of 8, 9, 11 and 12 months.

Example 3: Behavioral Experiment

Behavioral experiments were performed with 9-month old mice. Morris water maze (MWM) experiments were performed to evaluate hippocampal-dependent long-term spatial learning that can identify memory and learning abilities. The MWM device consists of a circular water tank (100 cm diameter X 40 cm high) filled with water (22 ± 1 ° C, depth 26 cm) made opaque with the addition of non-toxic water-soluble white ink. Experimental equipment was placed in a behavioral lab to prevent hazards during training and experimentation. Each mouse (n = 10 / group) had two training sessions per day for six days starting from two different quadrants per training session. The escape time from the water by finding a submerged escape platform was calculated for 60 seconds. If the mouse does not find the platform immersed in 60 seconds, put the mouse on the platform and stay on the platform for 10 seconds. On the third day of training, mice were intraperitoneally injected with vehicle or osmotin (15 mg / g b. W.) And the training was continued until day 6. The mice were then allowed to rest for two days, and on day 9, a probe experiment without a water immersed platform was performed. Time consumption was recorded in the quadrant where the platform existed.

To evaluate memory and learning abilities, a Y-maze experiment was performed on hippocampal-dependent spatial memory with or without osmotin treatment three days after treatment. The goal of this experiment is to move the mouse over a white Y-maze with three arms (50 cm long x 10 cm wide x 20 cm high) at a 120 ° angle. Place each mouse in the Y-maze and adapt to the new environment for at least 10 minutes before beginning the experiment. After the adaptation period, place the mouse at the center of the Y-maze, looking at the intersection of two randomly selected arms, and move around the maze for 8 minutes. The total number of times the arm entered the arm and the number of times it entered all three arms were recorded in the behavioral software system. The percentage of shifts was calculated as [the number of times all the arms went into the arm / the total number of times the arms went into the arm - 2] x 100. The alternation percentage has a positive correlation with the spatial working memory function.

Example 4: Western blot

A specific method of Western blotting for measuring the degree of protein expression in the present invention is as follows. The protein concentrations of the cell lysates were measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA). The same amount (20 mg) of the protein was subjected to SDS-PAGE with a 4-12% Bolt ^™ mini gel (Novex, Life technologies) and transferred to the PVDF membrane. To reduce nonspecific binding, the membrane was blocked with 5% defatted milk and reacted with primary antibody overnight at 4 ° C. The primary antibodies used are as follows: wherein -β- actin, wherein -PSD95, wherein - Ke SPA kinase-3, wherein -p-Akt (Ser ^473), wherein -AMPK, wherein -p-AMPK (Thr ¹⁷²⁾ Anti-BSA-1, anti-SREBP1 & 2, anti-NMDAR1 & 2, anti-p-tau (Ser ⁴¹³ ), anti- -PARP-1, anti-sinaep who topa, wherein -SNAP-25, wherein -SIRT1, wherein -ADAM10, wherein -p-GSK3β (Ser ^9), and wherein -SNAP23 (Santa Cruz Biotechnology, Santa Cruz, CA, USA ), Anti-APP, anti-sAPP-a and anti-ADAM17 (Abcam technologies). After washing and reacting with secondary antibody conjugated with horseradish peroxidase, protein bands were detected using ECL reagent according to the manufacturer's instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). The X-ray film was developed and the intensity of the band was analyzed by density measurement using Sigma Gel software (SPSS, Chicago, IL, USA).

Example 5: Immunofluorescence and histochemical analysis

Immunofluorescence or histochemical analysis was performed to confirm protein expression in tissues. To obtain mouse brain tissue sections, mouse brains were first fixed by transcardial perfusion with ice-cold 4% paraformaldehyde. Brains were fixed with 4% paraformaldehyde for 72 hours and then transferred to 20% sucrose at 4 ° C for 72 hours. The brain was fixed with OCT (Optimal Cutting Temperature) compound (AO, USA), frozen with liquid nitrogen and cut into coronal planes with a CM3050C cryostat (Leica, Germany) (14 mm) . The tissue sections were placed on a ProbeOn Plus charge slide (Fisher, USA).

Specific experimental methods of immunofluorescence analysis are as follows. Slides containing tissue sections were washed twice in 0.01 M PBS for 15 minutes. Protease K was poured on the slides and the slides were incubated at 37 ° C for 5 minutes. The tissue sections were then incubated in blocking solution (5% standard goat serum, 0.3% Triton X-100, PBS) for 1 hour. The slide was incubated overnight with the primary antibody (as described in the 'Western Blot') and incubated with secondary TRITC- or FITC-conjugated antibodies (1:50 dilution in PBS; Santa Cruz) for 90 minutes at room temperature Lt; / RTI &gt; For double staining, incubation was performed at the same time. The slides were fixed with Prolong Antifade reagent (Molecular Probe, Eugene, OR, USA). All stained slides were confirmed using a confocal laser scanning microscope (Flouview FV 1000).

Amyloid plaques were visualized in the brain slices after thiopurin S staining. The brain slice is washed twice with 0.01M PBS for 10 minutes, and then dipped in fresh 1% thiopurin S solution at room temperature for 10 minutes. The sections were then soaked in 70% ethanol for 5 minutes, washed twice with water, contrasted with PI (propidium iodide) and covered with cover slip.

In morphological analysis, the signal was analyzed with Image J software (open source software provided by the National Institutes of Health) and expressed as IOD (Integral Optical Density). The percentage of amyloid plaque area within a given area was also quantified using image J software.

Example 6: Electrophysiology

Electrophysiological analyzes were performed to identify functional synapses in neurons. First, invasive hippocampal neurons isolated from E19 Sprague-Dawley rats were cultured at a density of 150 cells / mm ² . The mEPSC (AMPA) -mediated miniature excitatory postsynaptic currents were recorded using conventional whole-cell techniques. When the internal solution was filled, the electrode resistance was set to 3 to 5 M OMEGA. Measurements were performed using an Axis patch 200A patch-clamp amplifier (Axopatch 200A patch-clamp amplifier, Molecular Devices, Sunnyvale, Calif.). Digitization of voltage, command, membrane voltage and current was controlled using the Digidata 1322A interface with Clampex 0.2 (Molecular Devices, Sunnyvale, Calif.) Of the pClamp software package on IBM-compatible computers. Data were analyzed using Clampfit (Molecular Devices, Sunnyvale, CA) and Prism 4.0 (GraphPad, San Diego, Calif.). The current was filtered by a low-pass filter at 2 kHz using a four-pole Bessel filter in the amplifier. AMPAR-mEPSC was electrophysiologically separated by adding tetrodoxotin to 1 mM tetraethoxysilane and maintaining a Cl ^- equilibrium potential of -70 mV in the internal and external solution compositions. Intracellular recording solutions (patch electrodes) include (mM): 125 Cs methanesulfonate, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP and 5 QX- with CsOH, 285 mosmol- ¹ ). The extracellular recording solution contains the following (mM): 134 NaCl, 5.4 KCl, 2.5 CaCl 2, 1.2 MgCl 2, 10 D- glucose, and 10 (pH 7.4 with NaOH) HEPES . For each cell, the data was analyzed using a template-based miniature synaptic current detection algorithm that was filtered at 2KH and run in Clampfit 9.0 software (Molecular Devices, Union City, CA). Each estimated mEPSC detected by the software was visually determined according to whether its approximate shape was expected to occur in a synapse. 300 consecutive mEPSCs satisfying the rise time criteria were analyzed from each cell. The AMPAR-mediated EPSC amplitude was measured at -70 mV current peak. Data are presented as mean ± SEM values with the number of experiments given in parentheses. The cumulative probability curves of mEPSC were calculated using Clampfit 9.0 software and Prism 4.0 (GraphPad, San Diego, Calif.). Student t - test was used to compare the two groups. Statistical significance was p <0.05.

Example 7: Enzyme analysis

CycLex AMPK kinase assay kit was used to measure the level of AMPK activated in transgenic mouse brain homogenate and SHSY5Y cell lysates according to the manufacturer's instructions. The AMPK activity of brain homogenate and cell lysate was recorded at an absorbance of 450 nm.

SIRT activity was measured in mouse brain homogenate and SHSY5Y cell lysates using the HDAC Fluorimetric assay kit (Enzo Lifesciences) according to the manufacturer's instructions. Enzyme activity was measured at fluorescence emission of 440/460 nm and excitation wavelength of 350/380 nm.

Experimental Example 1: Confirmation of defense function against osmotin memory and cognitive decline in APP / PS1 mouse

The present inventors first examined the effects of hippocampal-dependent memory and learning abilities on dual genetically modified APP / PS1 memory and learning deficit model mice using the MWM (Morris Water Maze) test and the Y-maze spontaneous alternation test (Fig. 1 to Fig. 3). The time to escape from the water to the platform was slightly different on the first day of training compared to that of wild type (WT) mice without genetically modified strains, but the difference was remarkably increased from the second day. These results indicate that the spatial memory and learning ability defects occur in the APP / PS1 mouse compared to the wild type. At this time, osmotin was administered to APP / PS1 mice from the third day of training to confirm the effect of osmotin. As a result, osmotin-treated APP / PS1 (Os) mice showed a sharp decrease in the escape time on the fourth day, and subsequently (4 and 5 days) And there was a difference in escape time between the untreated group (FIG. 1). It was confirmed that osmotin treatment can reduce spatial memory and learning ability defects.

Further, in order to perform a probe test in which the platform was removed after the above test, the tested mice were given a rest period of two days after the training period. In the probe test, the time spent in the quadrant where the platform was present was 35.80%, 17.60% and 24.58% for APP / PS1 mice treated with WT, untreated osmotin and osmotin (FIG. 2) Lt; RTI ID = 0.0 &gt; APP / PS1 &lt; / RTI &gt;

Next, a Y-maze test was performed to confirm the effect of osmotin on short-term spatial memory loss in APP / PS1 mice. Voluntary changes were calculated by counting the number of times that all three arms had entered and the total number of times each arm had entered. After 3 days of treatment, the percentage of change in APP / PS1 mice treated with WT, osmotin-treated and osmotin was shown in the graph of Fig. 3, respectively. Spontaneous changes in osmotin-treated mice were reduced compared to WT, but the spontaneous rate of change was recovered in mice treated with osmotin (FIG. 3). Indicating that the osmotin treatment reduced short-term spatial memory deficits in APP / PS1 mice.

In conclusion, osmotin treatment restored the long-term spatial memory, short-term spatial memory, and learning ability of APP / PS1 mice, and thus it was confirmed that the osmotin treatment is excellent as a composition for health functional foods for improving memory or learning ability.

Experimental Example 2: Confirmation of the induction effect of osmotin on AMPK and SIRT1 activity

AMPK-activated protein kinase (SAMP) and SIRT1 (the NAD ⁽⁺⁾ -dependent protein deacetylases sirtuin 1) are energy sensing enzymes in cells. Their activity leads to the activation of metabolic pathways involved in energy production. Western blot analysis of the activity of AMPK and SIRT1 was performed to determine how osmotin treatment affects energy metabolism in the brain. Analysis showed that levels of activated AMPK (p-AMPKa (Thr ¹⁷² )) and SIRT1 in hippocampus and cortex were lower in APP / PS1 and APPsw mice than in WT (Fig. 4). Likewise, the activity of AMPK and SIRT1 in the brain homogenate of mice was examined using an enzyme assay kit. As a result, the activity of each enzyme in the brain tissue of APP / PS1 mouse was found to be lower than that of WT (FIGS. 5 and 6 6). These results imply that the energy metabolism of the brain in the memory and learning deficit models APP / PS1 and APPsw mice is defective.

However, when osmotin was treated, the level of p-AMPKa was increased in the hippocampus of 9-month old WT mice (Fig. 7). Osmotin treatment also led to an increase in the levels of p-AMPKa and SIRT1 in the cortex and hippocampus of 9- and 12-month old APP / PS1 and APPsw mice (Fig. 4) compared to the untreated OSMOTINE group.

Likewise, we observed increased activity of AMPK and SIRTl in brain homogenates of 9- and 12-month old APP / PS1 mice treated with osmotin as compared to the non-osmotinized group (Figures 5 and 6 ).

Taken together, these results suggest that osmotin prevents the reduction of AMPK and SIRT1 activity, suggesting that osmorein can promote energy metabolism in neurons or defend the reduction of energy metabolism. And promoting energy metabolism in neurons, it is confirmed that the composition containing osmotin of the present invention has an excellent effect on memory enhancement and learning ability improvement.

Experimental Example 3: Confirmation of effect of osmotin on loss of NMDA receptor and loss of NMDAR activity in APP / PS1 mouse

Expression levels of N-methyl-D-aspartate receptors NMDAR (NR1) and NMDAR2 (NR2), which regulate synaptic plasticity, synaptic plasticity and memory function in relation to memory and learning ability, The levels were elevated in osmotin treated APP / PS1 _(OS) mice, whereas low levels were observed in the brain tissues of osmotin-untreated APP / PS1 mice. SNAP-23 (synaptosomal-associated protein of 23 kDa) and SNAP-25 play an important role in the trafficking of NMDAR on the cell surface, suggesting that osmotin-treated APP / PS1 mouse brain hippocampus and cortical tissue extract The levels of SNAP23, SNAP25, synaptophysin and PSD95 were lower in WT than in Western blot analysis (FIG. 8) and immunofluorescence analysis of tissue sections (FIGS. 9 and 10) / PS1 mice. &Lt; / RTI &gt; This tendency was observed in mice between 8 and 9 months. In the hippocampus in particular, this tendency towards synaptophysin and SNAP23 was observed in the DG and CA1 regions (Fig. 9). As a result, it was confirmed that osmotin inhibits loss of NMDA receptors and loss of NMDAR activity in APP / PS1 mice, indicating that osmotin enhances memory and enhances learning ability.

Experimental Example 4: Determination of the effect of osmotin on the number of functional synapses without affecting synaptic strength in hippocampal neurons

To investigate whether os- motin could have an effect on the plasticity of functional synapses, since functional synapses affect memory and learning abilities, we treat osmotin (8 to 32 nM) for 24 hours, or untreated osmotin, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were measured in hippocampal cells cultured in DIV 21. Osmotin (16 and 32 nM) increased the mean frequency of mEPSC via AMPAR compared to the osmotin untreated control; (Osmotin untreated, 15.6 ± 3.2 Hz; osmotin 8 nM, 17.1 ± 1.0 Hz; osmotin 16 nM, 25.0 ± 2.1 Hz; osmotin 32 nM, 34.0 ± 2.0 Hz; n = 7 for all groups). However, there was little effect on the mean amplitude of AMPAR-mediated mEPSC in all osmotin treated groups, indicating that osmotin treatment did not induce a change in postsynaptic strength (osmotin treatment , 9.7 ± 0.4 pA; osmotin 8 nM, 10.3 ± 1.5 pA; osmotin 16 nM, 10.8 ± 1.5 pA; osmotin 32 nM, 10.5 ± 0.4 Hz; n = 7 for all groups (FIG. In conclusion, treatment with osmotin can enhance cognitive abilities by increasing the number of functional synapses without inducing significant changes in synaptic connectivity.

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

Claims (6)

Compositions for health functional foods for improving memory or learning ability, including osmotin.
The composition of claim 1, wherein the osmotin increases the activity of AMPK or SIRT1.
3. The composition of claim 1, wherein the composition enhances or enhances reduced memory or learning abilities by reducing activity of SIRT1, loss of NMDA receptor or activity, or reduction of functional synapses.
4. The composition of claim 3, wherein the decrease in activity of SIRT1, loss of NMDA receptor or activity, or decrease in functional synapse is a non-alcoholic factor.
2. The health functional food composition according to claim 1, wherein the osmotin is administered in an amount of 10 to 20 占 퐂 / ml.
A pharmaceutical composition for improving memory or learning ability comprising osmotin.

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