JP7383412B2 - New memory applications for AGIQ-containing compositions - Google Patents

Description

The present invention relates to new uses of α-glycosylisoquercitrin.

In living organisms, molecules called active oxygen are produced during the metabolic process. Active oxygen species damage proteins and DNA in the cytoplasm either directly or through multiple routes, so living bodies are equipped with antioxidant defense functions against them. However, when the balance between production and defense is disrupted, cell and tissue damage progresses, leading to a state called oxidative stress, which contributes to various diseases such as lifestyle-related diseases, cancer, and stroke. This is becoming clear.

In the brain, there is an important region involved in learning and memory called the hippocampus in the temporal lobe. Since new neurons are produced in the hippocampus after birth, there is a high oxygen demand, and active oxygen is generated locally in the hippocampus. Therefore, it can be said that the hippocampal region is constantly exposed to oxidative stress. Therefore, by suppressing the oxidative stress state in the hippocampal region using, for example, antioxidants, it is expected to prevent or ameliorate diseases related to brain function, including memory and learning, or improve brain function. From this perspective, for example, Patent Document 1 states that Polygonami minas extract (plant extract) containing quercetin-3-glucuronide, quercitrin, etc. as active ingredients promotes cognitive function due to its high active oxygen absorption ability, It has been described to improve memory and learning abilities. Further, Patent Document 2 describes that a pinus bark extract containing flavonoid compounds such as catechin and quercetin is effective in improving or preventing a decline in human cognitive ability (memory, learning ability). Furthermore, ginkgo biloba, which is known to improve cognitive function, is known to contain quercitrin and kaempferol, which are known to have antioxidant and radical (nitric oxide, etc.) scavenging effects ( Non-patent document 1). It has also been reported that the action of antioxidants produces neuroprotective effects in the hippocampus and enhances synaptic plasticity, thereby improving memory and learning ability caused by nerve injury (Non-patent Document 2). ).

Special Publication No. 2015-526511 Special Publication No. 2008-542263

Fujio Shimura, “Herbal nutritional supplements aimed at improving brain function,” Journal of Nutrition, Vol. 58, No. 4, 151-160 (2000) Ma et al., “Melatonin Alleviates the Epilepsy-Associated Impairments in Hippocampal LTP and Spatial Learning Through Rescue of Surface GluR2 Expression at Hippocampal CA1 Synapses”, Neurochem. Res. 42(5):1438-1448 (2017)

As described above, antioxidants are known to have many pharmacological uses. Among antioxidants, the present invention provides new information regarding α-glycosyl isoquercitrin (hereinafter simply referred to as "AGIQ"), which has been eaten as a food material and is highly absorbable into the body. The challenge is to provide applications. In particular, an object of the present invention is to provide a new use for AGIQ related to higher brain functions centered on the hippocampus.

The present inventors have conducted intensive studies to solve the above problems, and found that by continuously ingesting AGIQ in rats, markers of synaptic plasticity were reduced in the hippocampus, as shown in the experimental example described below. A significant increase in the number of c-FOS positive cells was observed, and an increase in mRNA expression of a glutamatergic neuron receptor gene called Grin2d was also observed in the amygdala. c-FOS is an indicator of neuronal activation, and its expression is known to be important for memory formation in various types of learning. GRIN2D is also known to function in enhancing synaptic plasticity associated with long-term memory. Furthermore, it was confirmed that the ability of rats to continuously ingest AGIQ to learn to erase fear memories was significantly enhanced in a contextual fear conditioning test. Furthermore, these series of phenomena were unique to rats that were continuously ingested with AGIQ, and were not observed in rats that were continuously ingested with α-lipoic acid, which has an antioxidant effect like AGIQ. From these results, although it is not necessarily clear how the increase in the number of c-FOS-positive cells and the enhancement of Grin2d expression are related to the promotion of extinction learning of fear memory, AGIQ ingestion may significantly improve extinction learning of fear memory. It was suggested that the facilitative effect may be related to improved synaptic plasticity in the hippocampus and amygdala. Moreover, this is not observed among antioxidants in general, but is thought to be due to the action mechanism unique to AGIQ. From these series of findings, the present inventors have discovered that AGIQ may effectively prevent or improve the onset of post-traumatic stress disorder (PTSD), which is a psychological aftereffect caused by fearful memories.

The present invention was completed through further studies based on the above series of findings, and includes the following embodiments.
Item 1. A composition for enhancing c-FOS expression in the hippocampus, which contains AGIQ as an active ingredient.
Item 2. A composition for enhancing Grin2d expression in the amygdala, which contains AGIQ as an active ingredient.
Item 3. A composition for promoting learning to erase fear memories, containing AGIQ as an active ingredient.
Item 4. A composition for preventing or improving the onset of post-traumatic stress disorder, which contains AGIQ as an active ingredient. Hereinafter, this may be simply referred to as "composition for anti-post-traumatic stress disorder" or "composition for anti-PDST."
Item 5. Item 5. The composition according to any one of Items 1 to 4, which is a pharmaceutical, quasi-drug, or food or drink that is orally ingested or administered.

An experimental scheme in an experimental example is shown. Specifically, it is a diagram showing a continuous administration schedule of mixed diet administration of α-glycosylisoquercitrin (AGIQ) or α-lipoic acid (ALA) to rats (mother rat, baby rat). The results of a contextual fear conditioning test conducted in an experimental example are shown. In the figure, "Acquisition" is the time of acquisition of fear memory, "Extinction 1", "Extinction 2", and "Extinction 3" are the time points when only the conditioned stimulus was given 4 days, 6 days, and 8 days after fear conditioning, respectively. (when the fear memory is erased). The vertical axis indicates the ratio (%) of freezing action time to trial time. The trial time refers to the time the animal was placed in the experimental box, which in this case was 5 minutes. An overview of hippocampal formation in male rats stained with hematoxin and eosin is shown. Magnification ×40, bar length 200 μm. The magnification of the inserted part in the figure is ×400, and the bar length is 50 μm. In the figure, "Cornu ammonis" (abbreviation CA) is Ammon's horn, "Granule cell layer" is the granule cell layer, "Subgranular zone" is the subgranular cell layer, "Molecular cell layer" is the molecular cell layer, and "Hilus" is Each means a gate.

The composition for increasing c-FOS expression, the composition for increasing Grin2d expression, the composition for promoting extinction learning of fear memory, and the composition for anti-post-traumatic stress disorder, all of which are targeted by the present invention, are effective against AGIQ. It is characterized by being an ingredient. The active ingredient AGIQ and its uses will be explained in order below. In the case of the animal species rat, the gene name is usually written in italics with only the first letter capitalized, and when the molecule name is written as an abbreviation that follows the gene name, it is written in all capital letters and non-italics. In this book, gene names are underlined instead of italicized.

(1) α-glycosylisoquercitrin (AGIQ)
AGIQ is a flavonoid glycoside having the structure shown by the following formula.
In the formula, Glc represents a glucose residue, and n represents an integer of 1 or more. Specifically, as shown in the above formula, AGIQ is a glucose residue of isoquercitrin (hereinafter also simply referred to as "IQC") in which one glucose (Glc) is beta-bonded to the 3rd position of querserin, and further It is a flavonoid glycoside with a structure in which one or more glucose residues are attached via an α-1,4 bond. Although the number n is not limited, it is preferably 20 or less, more preferably 10 or less, particularly preferably 7 or less. Note that AGIQ may be a single compound represented by the above chemical formula, or may be a mixture of compounds where n is 1 or more. Preferably, it is a mixture of compounds in which n is 1 to 10, more preferably n is 1 to 7. Further, the mixture AGIQ may include IQC in which n is 0 in the above formula.

AGIQ is not limited in any way by its form or manufacturing method. For example, the production method is not limited, but can be exemplified by a method in which rutin extracted from the buds and flowers of a deciduous tree called Japanese apricot is used as a raw material and rutin is treated with an enzyme. Although not limited, specifically, as described in Japanese Patent Publication No. 54-32073 or WO2005/030975 pamphlet, a reaction substrate such as isoquercitrin and starch or a glucose donor containing the same can be used. A method is exemplified in which a glycosyltransferase such as α-glucosidase or cyclomaltodextrin glucanotransferase is applied to a solution containing α-glucosidase to transfer equimolar or more glucose residues to isoquercitrin, and then amylase is applied. Ru. AGIQ produced by this method is also called enzyme-modified isoquercitrin (EMIQ). Note that AGIQ and EMIQ have the same meaning, and in the present invention, AGIQ is used to include EMIQ. Note that AGIQ produced by the glycosyltransferase and subsequent amylase treatment can be purified, if necessary, by further subjecting it to an optional purification treatment before contacting it with a porous synthetic adsorbent or the like. The form of AGIQ prepared in this manner is not particularly limited, and it can be prepared in the form of a solution, a concentrate, a dried product, or a dry powder.

Such AGIQ can be easily obtained commercially, such as Sunmelin (registered trademark) AO-3000, Sunmelin (registered trademark) Powder C-10, Sunemic (registered trademark) P15, and Sunmelin (registered trademark) P15. An example is the product name Sunemic (registered trademark) R-20 (all of the above are manufactured by San-Ei Gen FFI Co., Ltd.).

(2) Composition for enhancing c-FOS expression AGIQ is useful as an active ingredient of a composition for enhancing c-FOS expression in the hippocampus. In other words, a composition having AGIQ can be effectively used as a composition for enhancing c-FOS expression in the hippocampus. As shown in the experimental examples described later, AGIQ administration increases the expression of Fos mRNA (expression at the gene level) in the hippocampus, and accordingly increases the expression of c-FOS (expression at the protein level). -The number of FOS positive cells increases. Therefore, in the present invention, "enhanced c-FOS expression" means expression at the protein level, but also includes enhanced expression at the gene level.

The hippocampus is an important brain region located in the temporal lobe and is involved in cognition, learning, and memory formation. In the hippocampus, new neurons are produced after birth in the subgranular zone (SGZ) within a small region called the dentate gyrus (DG). This is called biological neurogenesis. Neurogenesis involves several processes, including self-renewal of stem cells, continuous division of progenitor cells to produce new granule cells, subsequent cell differentiation, and migration into the granule cell layer (GCL). It consists of multiple processes including developmental stages. In the hippocampal dentate gyrus, some GABAergic interneurons stimulate cell populations of the granule cell lineage and control neurogenesis in the SGZ. One type of GABAergic interneuron produces the extracellular glycoprotein Reelin. Reelin is essential for the migration and correct positioning of neurons, including the granule cell lineage. In addition to inputs from GABAergic interneurons, various types of nerves from outside the SGZ make neural connections to neurons within the hippocampal dentate gyrus. For example, glutamatergic neural input to the SGZ is important for maintaining proper division and differentiation of the granule cell lineage. Neonatal hippocampal neurons function as granule cells, but their function is different from that of surrounding mature granule cells, and is more similar to that of young neurons that are often in the developmental stage, and is rich in synaptic plasticity. Newly generated neurons are rich in synaptic plasticity mediated by NMDA-type glutamate receptors, and are responsible for pattern separation in spatial memory. Furthermore, new neurons have the function of updating memories and alleviating the stress response that organizes past memories.

c-FOS is one of the marker genes for synaptic plasticity. In addition, c-FOS is expressed at the mRNA level after about 30 minutes and at the protein level after 1 to 2 hours in neurons that responded to the stimulus during behavioral experiments related to memory and anxiety. It is used as an indicator of neuronal activity (see special feature "Information Transmission and Diseases in the Brain and Nervous System", Experimental Medicine Special Issue Vol. 28, No. 5 (2010)). As shown in the experimental examples below, it was confirmed that the number of c-FOS-positive cells was significantly increased in the hippocampus, which is an important area of the brain involved in cognition, learning, and memory formation, in the AGIQ administration group. Ta. From this, AGIQ (administration/intake) is considered to be useful for improving brain neuron activity and/or synaptic plasticity. In contrast, no increase in c-FOS-positive cells was observed in the group treated with ALA, which has an antioxidant effect like AGIQ. From this, the effect of enhancing c-FOS expression at the protein level in the hippocampus and the effects derived from this (e.g., improving brain neuron activity and/or synaptic plasticity) are among the antioxidants. However, it is recognized that this is an action and effect unique to AGIQ.

Incidentally, increased expression of c-FOS in the hippocampal dentate gyrus is known to be important for context learning. Specifically, in a novel object/novel context search test using rats, Arias N. et al. found that c-FOS positivity in the hippocampal dentate gyrus was found to be higher when the object and context were changed compared to when only the object was changed. It has been reported that the number of cells increases significantly (Arias N et al., Behavioral Brain Research 2015, 292, 44-49). Furthermore, in the literature by Richard L. Dees et al., it has been reported that severe damage to the hippocampal dentate gyrus causes impairment in the exploration of novel objects containing contextual information (Richard L. Dees et al., Neurobiology of Learning and Memory 2013, 106, 112-117). Furthermore, increased expression of c-FOS in the hippocampal dentate gyrus is said to be related to fear learning (recall of fear memory). Specifically, Xu Liu et al. found that by optogenetically labeling neurons that expressed c-FOS during fear conditioning and forcibly activating the same neurons by laser irradiation to the hippocampal dentate gyrus, It has been reported that fear responses can be elicited even in contexts that do not normally elicit fear responses (Xu Liu et al., Nature 2012, 484, 381-385).

As described above, the composition for enhancing c-FOS expression of the present invention can enhance c-FOS expression in the hippocampus and exhibit various effects derived therefrom. As mentioned above, c-FOS is an indicator of neuronal activation and is thought to be involved in memory formation during learning. Enhancement of c-FOS expression in the hippocampus is not observed with ALA administration, but is uniquely observed with AGIQ administration, so the effects derived from it are unique to AGIQ and not observed with other antioxidants. It is considered to be included. Although it is known that c-FOS expression in the hippocampal dentate gyrus increases after fear conditioning or extinction learning, it is not known that increased c-FOS expression is related to extinction learning of fear memory. However, since c-FOS is involved in memory formation, it is inferred that it is also involved in the promotion of fear learning, especially extinction learning, which will be described later.

(3) Composition for enhancing Grin2d expression AGIQ is useful as an active ingredient of a composition for enhancing Grin2d expression in the amygdala. In other words, a composition having AGIQ can be effectively used as a composition for enhancing Grin2d expression in the amygdala. In addition, Grin2d expression here means the expression of Grin2d mRNA (expression at the gene level).

GRIN2D is one of the NMDA-type receptors on glutamatergic neurons, and is known to function in enhancing synaptic plasticity associated with long-term memory. As shown in the experimental examples described below, in the amygdala, increased expression of Grid2d was observed only in the AGIQ administration group, but not in the ALA administration group. Therefore, regarding long-term memory, it is thought that there is a mechanism of action unique to AGIQ that cannot be explained solely by its antioxidant effect.

Thus, according to the composition for enhancing Grin2d expression of the present invention, it is possible to enhance Grin2d expression in the amygdala and to exhibit various effects derived therefrom. The mechanism of fear memory storage in the amygdala is thought to be long-term potentiation (LTP) via glutamatergic neural input. Since NMDA-type glutamate neural input is essential for the generation of LTP, GRIN2D may be involved in strengthening extinction memory via LTP. In fact, it is known that fear conditioning and LTP generation are inhibited in Grid2b knockout mice, which is one of the NMDA-type glutamate receptor subtypes, and conversely, overexpression of GRIN2B promotes fear learning. Are known. Furthermore, it is generally believed that NMDA-type glutamate receptors in the amygdala are involved in the acquisition or extinction of fear.

From the above, the composition for enhancing Grin2d expression of the present invention not only enhances Grin2d expression in the amygdala, but also has various effects derived therefrom, such as long-term memory enhancement and fear memory erasure as observed in the experimental examples described below. It is thought that it acts to strengthen learning. In addition, the enhancement of Grin2d expression in the amygdala is not observed with ALA administration, but is uniquely observed with AGIQ administration, so the effects derived from it are unique to AGIQ, which are not observed with other antioxidants. It is considered to be included.

(4) A composition for promoting learning to erase fear memories AGIQ is useful as an active ingredient of a composition for promoting learning to erase fear memories. In other words, a composition having AGIQ can be effectively used as a composition for promoting extinction learning of fear memory.

The fear conditioning test consists of two stages: learning to acquire a fear memory using electric shock, and then learning to erase that fear memory. Normally, when given an electric shock, animals become immobile due to fear and exhibit freezing behavior. This is the acquisition of fear memory. Even after that, they usually exhibit freezing behavior due to fear when placed in the same environment even if no electric shock is given. However, if you continue for several days without applying electric shocks, they will learn that no electric shocks will come and their freezing behavior will decrease. As shown in the experimental examples described below, the three-way study included an untreated control group, an AGIQ administration group, and a group in which α-lipoic acid, which has an antioxidant effect similar to AGIQ, was administered instead of AGIQ administration (ALA administration group). No difference was observed in the acquisition of fear memory. On the other hand, regarding extinction learning of fear memories, a significant decrease in freezing behavior was observed in the AGIQ administration group. On the other hand, since no decrease in freezing behavior was observed in the ALA-administered group, it is considered that the promotion of extinction learning of fear memories is due to AGIQ administration, that is, a result based on the mechanism of action unique to AGIQ.

However, the facilitation of extinction learning of fear memories observed in the AGIQ administration group may be due to the aforementioned improvement in synaptic plasticity in the hippocampus and/or amygdala. There have been many reports that enhancement of synaptic plasticity in the hippocampus by antioxidants improves memory and learning abilities that have deteriorated due to nerve damage caused by toxic substances. In contrast, the experiments conducted in the present invention were conducted on normal animals (normal mammals) without administering toxic substances, so the results are not limited to the animals concerned. , it can also be applied to humans.

(5) Composition for anti-post-traumatic stress disorder AGIQ is useful as an active ingredient of a composition for anti-post-traumatic stress disorder. In other words, a composition having AGIQ can be effectively used as a composition for treating post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a human mental illness caused by fearful memories. Specifically, it is the mental aftereffects caused by traumatic events that cause strong stress, such as earthquakes, accidents, violence, and abuse. For example, an experience of receiving a strong shock (stress) (a fear experience) is remembered over and over again, and the person continues to feel fear. Although abnormalities in fear memory control are thought to be related to the onset of PTSD, the details are still unclear, and elucidation of the mechanism of PTSD onset is expected from the perspective of fear memory control.

As shown in the experimental examples described later and as described above, AGIQ has the effect of promoting extinction learning of fear memories. Although PTSD symptoms generally improve as time passes after a traumatic event, it is known that in one-third of cases, symptoms persist even 6 years or more after onset. By applying the composition of the present invention to a person with PTSD symptoms, it is possible to hasten the relief of PTSD symptoms based on the effect of the active ingredient AGIQ that promotes learning to erase fear memories. become. In other words, the composition of the present invention can help alleviate PTSD symptoms at an early stage. Furthermore, the present invention can be applied to people who have received strong shock (stress) due to traumatic events, etc. (those with fear memories) even if they have not yet developed PTSD, thereby promoting the erasure of fear memories. However, it is also possible to prevent the onset of PTSD.

Treatment for PTSD has not yet been established, and antidepressants called selective serotonin reuptake inhibitors and serotonin-norepinephrine reuptake inhibitors are currently used. The anti-PTSD composition of the present invention may contain only AGIQ as an active ingredient, but since the mechanism of action is different from the above-mentioned antidepressants, one or both of the anti-PTSD It is thought that by using it in combination, high effects can be obtained with a small amount.

(6) Oral compositions (food and beverages, quasi-drugs, pharmaceuticals)
The composition for enhancing c-FOS expression, the composition for enhancing Grin2d expression, the composition for promoting extinction learning of fear memory, and the composition for anti-post-traumatic stress disorder described above are all prepared in the form of an oral composition. and can be administered orally to a subject. Oral compositions include foods and drinks, quasi-drugs, and pharmaceuticals. Preferably it is food and drink. In addition, in this specification, the term "administration" includes both the meanings of ingestion and administration. The subject can include mammals including humans. Preferably it is a human. In addition, depending on the intended use (for increasing c-FOS expression, for increasing Grin2d expression, for promoting extinction learning of fear memory, and for anti-post-traumatic stress disorder), the subjects to be administered are selected and set. You can also.

The content of AGIQ in the oral composition of the present invention is at most 100% by mass, and is not particularly limited as long as it achieves the above-mentioned effects. Preferably, it is 1% by mass or more in solid terms. More preferably, the content is 1 to 80% by mass in terms of solids, still more preferably 1 to 50% by mass, particularly preferably 1 to 30% by mass.

When the oral composition of the present invention is administered to humans (adults) in particular, the total amount of AGIQ to be incorporated is not limited, but is preferably in the range of 1 mg to 30 g per day for an adult weighing 50 kg. Selective settings can be made. In order to obtain effective effects, it is preferable to continue administering the drug over a long period of time.

When the oral composition of the present invention is manufactured and provided in the form of food or drink, the form may be tablets, capsules, powders, granules, drinks (including solutions and suspensions), etc. It can also be provided in the form of formulations like so-called supplements, as well as beverages such as soft drinks, tea drinks, coffee drinks, non-alcoholic drinks, beer-taste drinks, dairy products such as yogurt and lactic acid bacteria drinks, seasonings, processed foods, It can be manufactured and provided in the form of general food and drink such as desserts and confectionery (eg, gum, candy, jelly), but is not limited thereto. Note that the food and drink products targeted by the present invention include health foods. Health foods are generally considered to be ``general foods that are widely sold and used as foods that contribute to maintaining and promoting health.'' The oral composition of the present invention is useful as a health food because it can promote human health through the physiological action of AGIQ contained as an active ingredient. There is an ``insurance functional food system'' that allows foods that meet government-defined standards and standards to display health functions. Includes food. Health foods also include ``nutritional supplements,'' ``health supplements,'' ``functional foods,'' ``foods for special purposes,'' and ``health-oriented foods,'' and supplements are also included in health foods. Note that the health foods targeted by the present invention may have the form of general food and drink products, or may have the same formulation form as pharmaceuticals such as tablets, capsules, powders, and granules. You can.

In addition to AGIQ, the food and drink products targeted by the present invention may include edible acids, fatty acids, saccharides, sugar alcohols, alcohols, and antioxidants, as necessary, as long as they do not interfere with the effects of each use. One or more of the following may be blended: agents, high-intensity sweeteners, proteins, peptides, amino acids, vitamins, minerals, nutritional components, thickening stabilizers, and surfactants. In addition, excipients, binders, emulsifiers, tonicity agents, buffers, solubilizing agents, preservatives, stabilizers, antioxidants, coloring agents, and coagulants that are included in formulations. , a coating agent, etc. may be added as appropriate.

In addition, when the oral composition of the present invention is manufactured and provided in the form of a drug or quasi-drug, the form may be tablets, capsules, powders, granules, drinks (solutions or suspensions). It can be provided in a standard formulation such as (including). In this case, the pharmaceuticals and quasi-drugs targeted by the present invention may contain any medicinal ingredients other than AGIQ, as necessary, as long as they do not interfere with the effects of each use. In addition, excipients, binders, emulsifiers, tonicity agents, buffers, solubilizers, preservatives, stabilizers, antioxidants, colorants, and coagulants used in formulation. , a coating agent, etc. may be added as appropriate.

Hereinafter, the present invention will be specifically explained using examples. However, the present invention is not limited to these Examples in any way. In the following experiments, "%" means "% by mass" and "parts" means "parts by mass" unless otherwise specified.

The following experiment showed that alpha-glucosylisoquercitrin (AGIQ), an antioxidant, was continuously administered to rats from the developmental period to adulthood to show its beneficial effects on brain function. This was done to find out whether or not it would bring about this. Specifically, neurobehavioral tests related to spatial learning and long-term associative learning; immunostaining targeting the prefrontal cortex, hippocampal dentate gyrus, and amygdala; glutamatergic signals and cholinergic signals related to synaptic plasticity using real-time RT-PCR. , the expression of nerve growth factors and immediate early genes was investigated. As a control for comparing the neuroprotective effect of AGIQ, α-lipoic acid (DL form) (hereinafter referred to as "ALA"), which is known as an antioxidant like AGIQ, was used. Since ALA is a highly lipid-soluble antioxidant and is known to easily pass through the blood-brain barrier, it is considered to be an effective comparative substance for evaluating neural effects on the brain.

All test procedures were conducted in accordance with the "Guidelines for the Proper Conduct of Animal Experiments" (Science Council of Japan, June 1, 2006) and the protocol approved by the Tokyo University of Agriculture and Technology Animal Health Use Review Committee. did.

Experimental example (1) Test method (1-1) Administration of AGIQ Female Sprague Dawley (SD) rats 1 day after mating were purchased from Japan SLC (Hamamatsu) and randomly divided into 3 groups (untreated control group [ [n = 13], AGIQ administration group [n = 19], and ALA administration group [n = 19]). In the following experiment, the AGIQ administration group was fed a mixed feed containing AGIQ at a concentration of 0.5%, and the ALA administration group was fed a mixed feed containing ALA at a concentration of 0.2%. In addition, an untreated control group was fed normal food containing neither AGIQ nor ALA. The doses of AGIQ and ALA mentioned above were determined based on the dietary dose that suppressed the carcinogenic promotion effect in a two-stage liver carcinogenicity test using rats (Kimura, M, et al., Exp. Toxicol. Pathol. 65, 979-988, 2013;Fujii, Y. et al.,Chem.Biol.interact. 205, 108-118, 2013;Fujii, Y. et al., Toxicology 305, 30-40, 2013).

Administration of the mixed diet was started on the 6th day of pregnancy (GD6), and AGIQ or ALA was administered to the rat (mother) on the 6th day of pregnancy, and the mixed diet administration was continued until the 21st day after birth (PND21), which is the time of weaning. (period of administration to mothers: from pregnancy to weaning). The rat pups (male offspring) in each group (AGIQ administration group, ALA administration group) were divided into two groups: a group to which AGIQ or ALA was administered (administration group) and a group not administered (non-administration group). AGIQ or ALA was continuously administered in the diet to the group (male rats) from the 21st day (PND21) to the 77th day (PND77) after birth (see FIG. 1).

(1-2) Behavioral experiment (Y-maze and contextual fear conditioning test)
A Y-maze and a contextual fear conditioning test were conducted as behavioral experiments on male rat rats (7 rats/group) from postnatal day 60 (PND60) to postnatal day 70 (PND70). The Y-maze test was conducted to evaluate the spatial learning ability of rats, and the contextual fear conditioning test was conducted to evaluate memory and learning ability.

In the contextual fear conditioning test, on the first day, a situation (context) including a place was given as a conditioned stimulus and an electric shock was given as an unconditioned stimulus, so that the subjects learned the association between the conditioned stimulus and the unconditioned stimulus. One day after fear conditioning, only the conditioned stimulus was given to remind the subjects of the unconditioned stimulus, induce a state of not moving as a fear response (freezing), and measure the duration (acquisition of fear memory). Furthermore, after 4 days (Extinction 1), 6 days (Extinction 2), and 8 days (Extinction 3), only the conditioned stimulus is given, allowing the child to learn that there is no need to respond to the conditioned stimulus and reinforcing the fear response. We investigated extinction learning (elimination of fear memory) in which fear gradually diminishes. All mothers were euthanized on postnatal day 21 (PND21) and their pups were euthanized on postnatal day 77 (PND77).

(1-3) Genetic analysis The fear memory circuit consists of connections between three areas: the hippocampus, the amygdala, and the prefrontal cortex. Therefore, we removed the brains from 77-day-old male rat rats (PND77) from each group (untreated control group, AGIQ administration group, ALA administration group) and conducted genetic analysis using the hippocampus, amygdala, and prefrontal cortex. did. Specifically, to evaluate the effects of AGIQ or ALA on synaptic plasticity, mRNA expression analysis in the hippocampal dentate gyrus, amygdala, and prefrontal cortex was performed using real-time RT-PCR (6 animals/group). ). The target genes are as follows.
Acetylcholine receptors: Chrm1 , Chrm2 , Chrna7
Glutamate receptors: Gria1 , Gria2 , Gria2a , Gria2d ,
Glutamate transporters: Slc17a6 , Slc17a7 ,
Immediate early genes related to neuroplasticity: Fos , Arc
Neurotrophic factor relationship: Bdnf , Ntrk2

Table 1 shows the base sequences of the primer sets used for mRNA expression analysis (real-time RT-PCR) of each gene and the official name of each gene.

(1-4) Immunohistological staining and apoptosis analysis For the hippocampal dentate gyrus, immunohistochemical staining and apoptosis analysis were performed. For immunohistochemical staining, brain sections (3 μm thick) that were processed according to the standard method and embedded in paraffin were stained with Vectastain (registered trademark) Elite using 3,3'-diaminobenzidine (DAB)/H ₂ O ₂ as a coloring dye. Immunohistochemical detection was performed using an ABC kit, followed by hematoxylin counterstaining and observation under a microscope. Apoptosis analysis in the SGZ of the dentate gyrus was performed by TUNEL (TdT-mediated dUTP nick end labeling) assay. The TUNEL assay was performed using ApopTag In Situ Apoptois Detection Kit (manufactured by Millipore) using DAB/H ₂ O ₂ as a coloring dye. Immunohistological staining and apoptosis analysis were performed to determine granule cell lineage indicators (GFAP, SOX2, DCX, NeuN) in the subgranular cell zone (SGZ) or granule cell layer (GCL), and cell proliferation and apoptosis indicators (PCNA) in the SGZ. , TUNEL-assay), GABA interneuron indicators (Reelin, Parvaibumin) in the hilus of the dentate gyrus, distribution of cells expressing the earliest gene products (ARC, c-FOS, COX-2) that function in synaptic plasticity in the GCL (10 animals/group).

Table 2 shows the antibodies used for immunohistological staining.

The results obtained in the above experiments were compared between the untreated control group and each administration group (AGIQ administration group, ALA administration group), and statistical analysis was performed. For statistical analysis, first examine homoscedasticity using the Barrett test, then perform the Dunnett test if the variances are equal, and the Steel test if the variances are ^unequal . Or less than 0.01 ( ^** ), it was determined to be statistically significant.

(2) Test results (2-1) Behavioral experiment (Y-maze and contextual fear conditioning test)
Among the behavioral experiments, no difference was observed between the untreated control group and the treated groups (AGIQ-administered group and ALA-administered group) in the Y-maze test that evaluates the spatial learning ability of rats. This suggests that antioxidants such as AGIQ do not affect spatial learning ability.

On the other hand, as a result of a contextual fear conditioning test that evaluates memory and learning ability, no major difference was observed between the untreated control group and the treated groups (AGIQ administration group and ALA administration group) regarding the acquisition of fear memory. Regarding fear memory extinction learning ability, a decrease in freezing behavior was observed in the AGIQ administration group. In particular, a significant decrease in freezing behavior was observed 8 days after fear conditioning (46.97% in untreated control group vs. 17.80% in AGIQ vs. 48.89% in ALA) (p value: less than 0.05). The results are shown in Figure 2. As shown in Figure 2, no decrease in freezing behavior was observed in the ALA administration group, which has an antioxidant effect like AGIQ, so the enhancement (promotion) of fear memory extinction learning ability is due to the antioxidant effect. Rather, it is thought that this is due to the mechanism of action unique to AGIQ.

(2-2) Gene analysis Table 3 shows changes in transcriptional expression of each gene in the hippocampal dentate gyrus. The meanings (official names) of the abbreviations of each gene are the same as in Table 1, except for " Gapdh " and " Hprt1 ." In the table, the " Gapdh " column shows the values normalized to the endogenous control (housekeeping gene) Gapdh , and the "Hprt" column shows the values normalized to the endogenous control (housekeeping gene) Hprt1 . are shown respectively.

As can be seen from these results, it was confirmed that the expression of the following genes was significantly increased in the hippocampal dentate gyrus in the AGIQ administration group and the ALA administration group.
AGIQ administration group: Fos (1.75-fold increase ^** ), Slc17a6 (3.84-fold increase ^** ), Chrm2 (2.00-fold increase ^** ), Ntrk2 (1.31-fold increase ^* )
ALA administration group: Fos (1.64-fold increase ^* ), Slc17a6 (3.90-fold increase ^** ), Chrm2 (2.19-fold increase ^** ), Ntrk2 (1.44-fold increase ^** ).

Table 4 shows changes in transcriptional expression of each gene in the amygdala. The meaning (official name) of each gene abbreviation is the same as in Tables 1 and 3.

As can be seen from this result, it was confirmed that the expression of Grin2d was significantly increased in the amygdala in the AGIQ administration group (1.5-fold increase ^* ). However, in the AGIQ administration group, no significant difference was observed in relation to the untreated control group regarding the searched genes. Furthermore, in the prefrontal cortex, no significant difference was observed in the searched genes in both the AGIQ administration group and the ALA administration group in relation to the untreated control group.

(2-3) Immunohistological staining and apoptosis analysis Figure 3 shows an outline of the hippocampus of a male rat in a hematoxin-eosin stained section. The number of cells in the hilus of the hippocampal dentate gyrus (encircled by the dotted line in Figure 3) that showed immunoreactivity for Reelin or Parvaibumin was measured and normalized to the number per unit area of the hilus of the dentate gyrus. (No./mm ² hilar region). Only small cells showing positive immunoreactivity for Reelin or Parvaibumin were enumerated (RELN ⁺ cells, PVALB ⁺ cells), and larger cornu ammonis (CA)3 neurons were excluded. The inserted part in FIG. 3 is an enlarged image of the granule cell layer (GCL) and the subgranular zone (SGZ). Count the number of cells immunohistochemically positive for GFAP, SOX2, DCX, NeuN, ARC, FOS or COX2, as well as proliferating or TUNEL ⁺ apoptotic cells in the GCL and SGZ and normalize to SGZ length. (No.mm SGZ length).

Table 5 shows the measurement results for the untreated control group, the AGIQ administration group, and the ALA administration group.

As can be seen from this result, it was confirmed that c-FOS positive cells were significantly increased in the AGIQ administration group (1.79-fold increase ^** ). However, regarding cells that are positive for other GFAP, SOX2, DCX, NeuN, PCNA, TUNEL, Reelin, Parvaibumin, ARC, or COX-2, the number of positive cells was lower than that of the untreated control group. There was no significant difference. Furthermore, in the ALA-administered group, there was no significant difference in the number of positive cells for all molecules including c-FOS compared to the untreated control group.

(3) Discussion From the above, although no difference was observed in fear memory acquisition in the AGIQ administration group and the untreated control group, promotion of extinction learning was observed 8 days after conditioning. On the other hand, in the ALA-administered group, no difference was observed in both fear memory acquisition and extinction learning compared to the untreated control group. From this, it is considered that the enhancement (promotion) of the fear memory erasure learning ability is an action specific to AGIQ that is not limited to just an antioxidant action.

In gene expression analysis, in the hippocampal dentate gyrus, expression of the same genes such as the earliest gene Fos , glutamate transporter, acetylcholine receptor, and BDNF receptor was increased in both the AGIQ-administered group and the ALA-administered group. However, no increase or decrease in positive cells was observed in any immunohistochemical staining in the ALA-administered group. On the other hand, in the AGIQ administration group, c-FOS-expressing granule cells among the earliest gene products related to synaptic plasticity increased, which was consistent with the results of genetic analysis (increase in the expression level of Fos mRNA). Furthermore, in the AGIQ administration group, a significant increase in the expression level of the glutamate receptor Grin2d in the amygdala was observed. GRIN2D is one of the NMDA-type glutamate receptors and is known to function in enhancing synaptic plasticity associated with long-term memory. As shown in the above results, in the AGIQ administration group, promotion of extinction learning of fear memory was observed, and enhancement of c-FOS-related synaptic plasticity was observed in the absence of electrical stimulation.

Fear conditioning tests are discussed in the context of post-traumatic stress disorder (PTSD), a mental illness caused by fearful memories. In other words, abnormalities in fear memory control are thought to be related to the onset of PTSD, and elucidation of the mechanism of PTSD onset is expected from the perspective of fear memory control (Johansen et al., 2011; Johansen et al., 2012). Additionally, cognitive behavioral therapy ``sustained exposure therapy'' is currently known as an effective treatment for PTSD. It is expected that this drug will promote the effects of sustained exposure therapy and exhibit efficacy as a therapeutic agent for PTSD. The only treatments currently used for PTSD are antidepressants called SSRIs (selective serotonin reuptake inhibitors) and SNRIs (serotonin-norepinephrine reuptake inhibitors). The c-FOS-related synaptic plasticity-enhancing effect in granule cells of AGIQ of the present invention is thought to be effective against PTSD through a mechanism different from that of these PTSD therapeutics, and AGIQ alone or in combination with the above-mentioned PTSD therapeutics is thought to be effective against PTSD. By doing so, it is thought that a higher therapeutic effect on PTSD can be exhibited.

SEQ ID NOS: 1 to 30 show the base sequences of primers used in real-time RT-PCR to analyze the mRNA expression of each gene in the hippocampal dentate gyrus, amygdala, and prefrontal cortex. Specifically, SEQ ID NO: 1 and 2 are forward and reverse primers for Arc ; SEQ ID NO: 3 and 4 are forward and reverse primers for Bdnf ; SEQ ID NO: 5 and 6 are forward and reverse primers for Chrm1 ; SEQ ID NO: 7 and 8 are forward and reverse primers for Chrm2 ; SEQ ID NOS: 9 and 10 are forward and reverse primers for Chrna7 ; SEQ ID NOS: 11 and 12 are forward and reverse primers for Fos ; SEQ ID NOS: 13 and 14 are forward and reverse primers for Gapdh . Reverse primer; SEQ ID NOs: 15 and 16 are forward and reverse primers for Gria1 ; SEQ ID NOs: 17 and 18 are forward and reverse primers for Gria2 ; SEQ ID NOs: 19 and 20 are forward and reverse primers for Grin2a ; SEQ ID NOs: 21 and 22 are forward and reverse primers for Grin2d ; SEQ ID NOs: 23 and 24 are forward and reverse primers for Hprt1 ; SEQ ID NOs: 25 and 26 are forward and reverse primers for Ntrk2 ; SEQ ID NOs: 27 and 28 are forward and reverse primers for Slc17a7 . ; and SEQ ID NOs: 29 and 30 indicate forward and reverse primers for Slc17a6 (see Table 1).

Claims (5)

A composition for enhancing c-FOS expression in the hippocampus, which contains α-glycosylisoquercitrin as an active ingredient. A composition for enhancing Grin2d expression in the amygdala, which contains α-glycosylisoquercitrin as an active ingredient. A composition for promoting learning to erase fear memories, which contains α-glycosylisoquercitrin as an active ingredient. A composition containing α-glycosylisoquercitrin as an active ingredient to promote erasure of fear memory and prevent the onset of post-traumatic stress disorder. The composition according to any one of claims 1 to 4, which is a pharmaceutical, quasi-drug, or food or drink that is orally ingested or administered.