JP7724269B2 - Composition for inhibiting increases in brain glutamate concentration - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies. Posting date: February 28, 2019. Posting address: http://pharmacology.main.jp/92nenkai/pdf/pdf/09.pdf. Posting content: Abstract of the 92nd Annual Meeting of the Japanese Pharmacological Society (presentation number: 1-P-031).

The present invention relates to a composition for inhibiting increases in brain glutamate levels, etc.

It is known that age-related brain aging causes changes in the structure and function of the nervous system.
A typical example of morphological changes in the brain due to aging is brain atrophy. For example, it is known that the hippocampus, which controls memory function, shrinks with age and its weight decreases (Non-Patent Document 1).
Although brain atrophy is not necessarily a pathological condition, it can be an indicator of the degree of brain aging. It is also known that the concentration of glutamate in the brain increases in the hippocampus and other areas with age (Non-Patent Document 2). Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and is involved in higher brain functions such as cognition, memory, and learning. However, excessive extracellular glutamate is known to have a neuronal-damaging effect called glutamate excitotoxicity.

Golomb J, Arch Neurol. 1993 Sep; 50(9):967-73 Brewer GJ, Exp Gerontol. 2000 Dec; 35(9-10):1165-83

An object of the present invention is to provide a novel material capable of suppressing an increase in intracerebral glutamic acid concentration and a decrease in brain weight, and a composition containing the same.

The present inventors have now found that, compared with young mice, aged mice experience a decrease in hippocampal weight and an increase in glutamate levels in the hippocampus, whereas aged mice fed WY peptide suppress the decrease in hippocampal weight and the increase in glutamate levels in the hippocampus. The present invention is based on these findings.

That is, the present invention provides the following inventions.
[1] A composition for suppressing an increase in brain glutamate concentration and/or a decrease in brain weight, and an agent for suppressing an increase in brain glutamate concentration and a decrease in brain weight, comprising as an active ingredient a peptide having the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof.
[2] The composition and agent described in [1] above, wherein the increase in brain glutamate concentration and the decrease in brain weight occur in the hippocampus.
[3] The composition and agent described in [1] or [2] above, wherein the increase in brain glutamate concentration and the decrease in brain weight are due to aging.
[4] The composition and agent according to any one of [1] to [3] above, for administration to middle-aged and elderly people.
[5] The composition and agent according to any one of [1] to [4] above, for use in neuroprotection.
[6] The above-mentioned [1] to [5] for use in suppressing neuronal loss and/or promoting neurogenesis.
] The composition and agent described in any one of the above.
[7] The composition and agent according to any one of [1] to [6] above, which is in the form of a food product.
[8] A neuroprotective composition and a neuroprotective agent comprising, as an active ingredient, a peptide having the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof.
[9] A composition for suppressing neurological deficit and an agent for suppressing neurological deficit, comprising, as an active ingredient, a peptide having the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof.
[10] A composition for promoting neurogenesis and a neurogenesis-promoting agent, comprising, as an active ingredient, a peptide having the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof.

The compositions of [1] and [8] to [10] above may be referred to herein as "compositions of the present invention," and the preparations of [1] and [8] to [10] above may be referred to herein as "preparations of the present invention."

The dipeptide WY, which is the active ingredient of the compositions and preparations of the present invention, can be used as a functional material that exhibits the function of suppressing a decrease in hippocampal weight and an increase in glutamate levels in the hippocampus, and is advantageous in that it can be used as a functional material that is safe for mammals, including humans.

FIG. 1 shows the effect of WY peptide intake on hippocampal weight in aged mice. FIG. 2 shows the effect of WY peptide intake on glutamate levels in the hippocampus of aged mice.

Specific Description of the Invention

The compositions and agents of the present invention contain a peptide having the amino acid sequence of WY (hereinafter, sometimes referred to as "the peptide of the present invention") as an active ingredient.
The term "peptide having an amino acid sequence" means a peptide whose sequence is specified by the amino acid sequence.

The peptides of the present invention may be obtained by chemical synthesis or by chemical or enzymatic degradation of proteins or polypeptides derived from milk, soybeans, wheat, eggs, livestock meat, fish, seafood, etc. Specifically, the peptide sequences of the present invention are contained in at least milk whey and casein, bovine serum albumin (BSA), soybean glycinin, wheat glutenin, egg yolk ribovitellin, egg white ovalbumin, ovotransferrin, lysozyme, and chicken protein collagen. Therefore, these foods or food materials can be subjected to acid hydrolysis, protease enzyme treatment, or microbial fermentation to produce the peptides. The compositions and preparations of the present invention may be prepared from the above-mentioned foods or food materials as they are, or may be used as concentrates or as dried powders prepared by spray drying, freeze-drying, or other methods. Furthermore, they may be purified to any degree, such as by removing impurities, salts, enzymes, etc., as needed.

The amino acids constituting the peptide of the present invention may be composed of only L-amino acids, only D-amino acids, or a mixture of both. Furthermore, the peptide may be composed of only naturally occurring amino acids, or only modified amino acids in which an arbitrary functional group, such as phosphorylated or glycosylated amino acids, is bound to an amino acid, or a mixture of both. Furthermore, when the peptide contains two or more asymmetric carbon atoms, it may be an enantiomer, a diastereomer, or a mixture of both.

Furthermore, the peptides of the present invention may be in the form of a free base, a pharmaceutically acceptable salt thereof, or a solvate thereof, and the compositions and pharmaceutical preparations of the present invention contain any of these, or a mixture of all or part of these. Pharmaceutically acceptable salts include acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloride, sulfate, and phosphate, and organic acid salts such as acetate, maleate, fumarate, citrate, and methanesulfonate. Pharmaceutically acceptable metal salts include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt.
Pharmaceutically acceptable ammonium salts include salts of ammonium, tetramethylammonium, etc. Pharmaceutically acceptable organic amine addition salts include addition salts of morpholine, piperidine, etc.

The composition and preparation of the present invention can be provided as the peptide of the present invention alone or as a material containing the peptide of the present invention (e.g., a food ingredient). The composition and preparation of the present invention can also be provided by mixing the peptide of the present invention or a material containing the peptide of the present invention with other ingredients (e.g., a food ingredient, a food additive, a pharmaceutical additive). The content of the peptide of the present invention in the composition and preparation of the present invention depends on the purpose, use, form, dosage form, symptoms,
The content can be arbitrarily determined depending on factors such as body weight, and the present invention is not limited thereto, but the content can be 0.00001 to 50% by mass (solid content equivalent) relative to the total amount, and more preferably 0.0001 to 10% by mass (solid content equivalent). In the present invention, the agent of the present invention can consist of the peptide of the present invention, and the composition of the present invention can contain the peptide of the present invention and other ingredients.

The content of the peptide of the present invention was measured by liquid chromatography tandem mass spectrometry (LCMS).
Those skilled in the art can easily set the conditions, and it goes without saying that a standard peptide of a purity suitable for LC/MS/MS measurement should be used during the measurement. For example, AQUA peptide manufactured by SIGMA ALDRICH can be used.

Food materials containing the peptide of the present invention include enzymatic hydrolysates of proteins, preferably enzymatic hydrolysates of whey proteins (hereinafter, sometimes referred to as "whey hydrolysates"). Methods for producing whey hydrolysates containing the peptide of the present invention are known, and can be produced, for example, according to the description in International Publication No. 2017/086303. Alternatively, commercially available whey enzymatic hydrolysates containing the peptide of the present invention may be used, for example, LE80G.
F-US (manufactured by DMV) and HW-3 (manufactured by Megmilk Snow Brand) may also be used as the peptide of the present invention.

When the peptide of the present invention is contained in the composition and agent of the present invention as a whey protein hydrolysate, the peptide of the present invention can be contained in a proportion of 0.001 to 10% by mass (solid content equivalent) relative to the whey protein hydrolysate, more preferably 0.01 to 5% by mass.
The whey protein hydrolysate can be contained in an amount of 0.001 to 95% by mass (solid content equivalent), preferably 0.01 to 50% by mass (solid content equivalent), relative to the composition and agent of the present invention.

As shown in the Examples below, the peptide of the present invention has the effect of suppressing a decrease in hippocampal weight and an increase in glutamate content in the hippocampus. Therefore, the peptide of the present invention can be used as an active ingredient of a composition or agent for suppressing an increase in intracerebral glutamate concentration or a decrease in brain weight.

In the present invention, the decrease in brain weight preferably occurs in the hippocampus, and the composition and agent of the present invention can be used for suppressing the decrease in hippocampal weight. Also, in the present invention, the increase in brain glutamate concentration and the decrease in brain weight are preferably caused by aging, and the composition and agent of the present invention can be used for suppressing the increase in brain glutamate concentration due to aging and the decrease in brain weight due to aging.

It is known that hippocampal weight decreases and glutamate concentration in the brain increases with age (Non-Patent Documents 1 and 2). Therefore, the compositions and preparations of the present invention can be preferably administered to middle-aged and elderly people (e.g., those aged 45 or older, those aged 55 or older, and those aged 65 or older). It is also known that drinking alcohol and smoking promote brain atrophy and decrease hippocampal weight (Anya Topiwala, BMJ 2017;357:j2353 doi: 10.1136/bmj.j2353
(Published 6 June 2017) and KUBOTA, Tohoku J Exp Med. 1987 Dec;153(4):303-11)
Therefore, the compositions and preparations of the present invention can be preferably intended for consumption by drinkers or smokers.

The composition and agent of the present invention suppress an increase in the concentration of glutamate in the brain. Here, glutamate is a major excitatory neurotransmitter in the mammalian central nervous system.
While glutamate is involved in higher brain functions such as cognition, memory, and learning, excessive extracellular glutamate is known to have a neuronal damage effect called glutamate excitotoxicity. Excessive excitation of neurons by glutamate causes excessive intracellular calcium influx, resulting in cell death. Neuronal death due to excitotoxicity is not only associated with acute neurological disorders such as cerebral ischemia, brain trauma, epilepsy, and hepatic encephalopathy, but also with Alzheimer's disease and amyotrophic lateral sclerosis (ALS).
), and is attracting attention as a common mechanism of neuronal cell death in chronic neurological disorders such as Huntington's disease (Tanaka, CLINICAL NEUROSCIENCE, 35: 1412-1416(2017)). Similarly, increased brain glutamate levels are thought to be associated with psychiatric disorders such as schizophrenia, depression, autism, and obsessive-compulsive disorder (Tanaka, J. Pharmacol. J., 1421-6(2013)).

Therefore, the peptides of the present invention, which suppress an increase in brain glutamate concentration, can be used for neuroprotection (particularly neuroprotection from excitotoxicity), and the nerves to be protected are preferably cranial nerves. That is, according to the present invention, a neuroprotective composition comprising the peptide of the present invention and a neuroprotective agent comprising the peptide of the present invention are provided. The peptides of the present invention can also be used as an active ingredient in an agent for treating, preventing, or ameliorating a cranial neurodegenerative disease (e.g., epilepsy). That is, according to the present invention, a composition for treating, preventing, or ameliorating a cranial neurodegenerative disease comprising the peptide of the present invention and an agent for treating, preventing, or ameliorating a cranial neurodegenerative disease comprising the peptide of the present invention are provided.

The compositions and agents of the present invention also inhibit a decrease in brain weight (particularly hippocampal weight). It is known that aging causes the loss of brain neurons, which is a factor in the decrease in brain weight (Takekawa, Dokkyo Journal of Medical Sciences 35(3):203-208(2008)).
In addition, while it was previously believed that cranial nerves were not regenerated, it has recently been revealed that cranial nerves are continuously regenerated, and that neural stem cells in the subventricular zone and dentate gyrus of the hippocampus in particular actually undergo continuous neurogenesis within the ventricles (Sawamoto, Clin Neurol 49:830-833(2009)).
That is, it can be said that the decrease in brain weight due to aging is caused by the loss of cranial nerves and the inhibition of neurogenesis. Therefore, the peptides of the present invention, which inhibit the decrease in brain weight, can be used for neuroprotection (preferably cranial nerve protection, more preferably hippocampal neuroprotection), and can be used in particular to inhibit neuronal loss and/or promote neurogenesis. That is, according to the present invention, a neuroprotective composition comprising the peptide of the present invention and a neuroprotective agent comprising the peptide of the present invention are provided. The present invention also provides a composition for inhibiting neuronal loss comprising the peptide of the present invention and an agent for inhibiting neuronal loss comprising the peptide of the present invention. The present invention also provides a composition for promoting neurogenesis comprising the peptide of the present invention and an agent for promoting neurogenesis comprising the peptide of the present invention.

The compositions and agents of the present invention can be provided in the form of pharmaceuticals (e.g., pharmaceutical compositions), quasi-drugs, foods (e.g., food compositions), feeds (including pet foods), etc., and can be implemented according to the following description.

The peptides of the present invention can be orally administered to humans and non-human animals. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients,
Examples of such additives include binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

When the peptide of the present invention is provided as a food, it can be provided as it is, or it can be provided by being contained in a food. For example, when the peptide of the present invention is provided as a food, an enzymatic hydrolysate of a protein such as a whey hydrolysate containing the peptide of the present invention can be provided as it is, or it can be provided by being contained in a food. The food provided in this way is a food containing an effective amount of the peptide of the present invention. In this specification, "containing an effective amount" of the peptide of the present invention means a content such that the peptide of the present invention is ingested in the range described below when the amount normally consumed in each food is ingested. Furthermore, "food" includes health foods,
The term "functional foods,""nutritionalfoods,""health claim foods" (e.g., foods for specified health uses, foods with nutrient functions, foods with functional claims), "special dietary foods" (e.g., foods for infants, foods for pregnant women, foods for sick people), and supplements are included. When the peptides of the present invention are to be ingested by animals other than humans, the foods referred to in the present invention are, of course, used as feed.

Because the peptides of the present invention have the above-mentioned inhibitory effects on increases in brain glutamate concentration and decreases in brain weight, they can be incorporated into daily foods or provided as supplements. That is, the compositions and agents of the present invention can be provided in the form of food. In this case, the compositions and agents of the present invention can be provided in a unit packaged form in which the amount to be ingested per meal is predetermined. Examples of unit packaged forms per meal include packs, wrappings, cans, bottles, etc., in which a fixed amount is specified. To better exert the various effects of the compositions and agents of the present invention, the intake amount per meal can be determined according to the intake amount of the peptides of the present invention per serving, as described below. The foods of the present invention may be provided with instructions regarding the intake amount on the packaging or with a document or the like containing such instructions.

The predetermined intake amount per serving in the unit package form may be the effective daily intake amount, or the effective daily intake amount divided into two or more (preferably two or three) intake amounts. Thus, the unit package form of the composition and agent of the present invention may contain the peptide of the present invention at the daily human intake amount described below, or may contain the peptide of the present invention in an amount of one-half to one-sixth of the daily human intake amount described below. For convenience of intake, the composition and agent of the present invention are preferably provided in a "unit package form per serving," in which the intake amount per serving is the effective daily intake amount.

The form of the "food" is not particularly limited, and may be, for example, a drink, a semi-liquid or gel form, or a solid or powder form. Examples of "supplements" include tablets produced by adding an excipient, a binder, etc. to the peptide of the present invention, kneading the mixture, and then tableting, and capsules enclosed in capsules.

The foods provided by the present invention are not particularly limited as long as they contain the peptide of the present invention, and examples thereof include non-alcoholic beverages such as soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, livestock milk such as cow's milk, soy milk, dairy drinks, drink-type yogurt, drink-type or stick-type jellies, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water, near-water, and non-alcoholic beer-flavored drinks; carbohydrate-containing foods and beverages such as rice, noodles, bread, and pasta; dairy products such as cheeses, hard or soft yogurt, fresh cream made from livestock milk or other fats and oils, and ice cream; Western confectioneries such as cookies, cakes, and chocolates; Japanese confectioneries such as manju and yokan; tablet confectioneries (refreshing confectioneries) such as ramune (ramune soda), candies, gums, frozen desserts and ice creams such as jelly and pudding, and snacks;
Alcoholic beverages such as whiskey, bourbon, spirits, liqueur, wine, fruit wine, Japanese sake, Chinese sake, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, happoshu, other miscellaneous alcoholic beverages, chuhai, etc.; processed foods using eggs, processed seafood and meat (including liver and other offal) products (including delicacies), processed foods such as miso soup and other soups, miso, soy sauce, furikake,
Other examples include condiments such as seasonings, and liquid foods such as thick liquid foods. Mineral water includes both sparkling and non-sparkling mineral water. The foods provided by the present invention also include both food manufacturing ingredients and food additives.

Tea beverages include all types of fermented tea, semi-fermented tea, and non-fermented tea, such as black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted green tea, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal teas (e.g., mint tea and jasmine tea).

Examples of fruits used in fruit juice beverages and fruit and vegetable juice beverages include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums, while examples of vegetables used in vegetable juice beverages and fruit and vegetable juice beverages include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

The intake amount of the peptide of the present invention can be determined depending on the recipient's sex, age, body weight, symptoms, administration time, dosage form, administration route, and drugs to be combined, etc. The daily intake amount (in terms of dry mass) of the peptide of the present invention for adults aimed at suppressing an increase in intracerebral glutamate concentration and a decrease in brain weight is, for example, 0.001 to 10,000 mg (preferably 1 to 1,000 mg).
g). The above-mentioned intake amount of the peptide of the present invention and the following intake timing and intake period apply whether the peptide of the present invention is used for non-therapeutic purposes or therapeutic purposes, and in the case of therapeutic purposes, "intake" can be read as "administration." The peptide of the present invention can also be administered to mammals other than humans (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, sea lions, etc.), and the intake amount, intake timing, and intake period can be determined with reference to the description for humans.

The ingestion period of the peptide of the present invention can be at least 3 days (preferably 1 or 2 weeks, more preferably 1 month, 3 months, or 5 months) at the above-mentioned daily dose. The ingestion interval of the peptide of the present invention can be once every 3 days, once every 2 days, or once a day at the above-mentioned daily dose.

The compositions, agents, and foods of the present invention may be labeled to indicate that they have the effect of inhibiting an increase in brain glutamate concentration and/or the effect of inhibiting a decrease in brain weight. In this case, to make the labeling easier for consumers to understand, the compositions, agents, and foods of the present invention may be labeled with some or all of the following labels. It goes without saying that in the present invention, "inhibition of an increase in brain glutamate concentration" and "inhibition of a decrease in brain weight" are used in a sense that includes the following labels.
・For those concerned about brain aging ・For brain anti-aging ・For those concerned about brain atrophy

Another aspect of the present invention provides a method for inhibiting an increase in brain glutamate concentration and a method for inhibiting a decrease in brain weight, comprising administering to a subject in need thereof an effective amount of a peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition containing the same. The present invention also provides a method for treating, preventing, or ameliorating a neurodegenerative disease, comprising administering to a subject in need thereof an effective amount of a peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition containing the same. The present invention also provides a method for neuroprotection, a method for inhibiting neuronal loss, and a method for promoting neurogenesis, comprising administering to a subject in need thereof an effective amount of a peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition containing the same. The methods of the present invention can be carried out according to the description of the compositions and agents of the present invention.

According to yet another aspect of the present invention, there is provided a method for producing an agent for suppressing an increase in brain glutamate concentration, comprising:
The present invention provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, as an agent for suppressing an increase in brain glutamate concentration, or in a method of suppressing an increase in brain glutamate concentration of the present invention. The present invention also provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of an agent for suppressing a decrease in brain weight, or in a method of suppressing a decrease in brain weight of the present invention. The present invention also provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of an agent for the treatment, prevention, or amelioration of a neurodegenerative disease, or as an agent for the treatment, prevention, or amelioration of a neurodegenerative disease, or in a method of the present invention for the treatment, prevention, or amelioration of a neurodegenerative disease. The present invention also provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, as a neuroprotective agent for the manufacture of a neuroprotective agent, or in a neuroprotective method of the present invention. The present invention also provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, as an agent for suppressing neurological deficit, or in a method of suppressing neurological deficit of the present invention, for the manufacture of an agent for the treatment, prevention, or amelioration of a neurological deficit. The present invention also provides use of the peptide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a neurogenesis-promoting agent, as a neurogenesis-promoting agent, or in the neurogenesis-promoting method of the present invention. The use of the present invention can be carried out according to the description of the composition and agent of the present invention.

According to yet another aspect of the present invention, there is provided a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in suppressing an increase in brain glutamate concentration. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in suppressing a decrease in brain weight. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in treating, preventing, or ameliorating a neurodegenerative disease. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in neuroprotection. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in suppressing neuronal loss. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in promoting neurogenesis.
or a pharmaceutically acceptable salt or solvate thereof. The present invention also provides a peptide having the amino acid sequence of WY, or a pharmaceutically acceptable salt or solvate thereof, for use in the methods of the present invention. The above-mentioned peptide having the amino acid sequence of WY and its pharmaceutically acceptable salt and solvate can be prepared according to the description of the compositions and agents of the present invention.

The methods and uses of the present invention may be for use in mammals, including humans,
Both therapeutic and non-therapeutic uses are contemplated. As used herein, "non-therapeutic" means not including surgical, therapeutic, or diagnostic procedures on humans (i.e., medical procedures on humans), and specifically does not include methods of surgical, therapeutic, or diagnostic procedures on humans performed by a physician or a person under the supervision of a physician.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

Example 1: Effect of WY peptide on the central nervous system (1) Study outline: Aged mice were compared in terms of brain condition (hippocampal weight and glutamate concentration in the hippocampus) between groups that ingested the test food and those that did not. They were also compared with a group of young mice that did not ingest the test food. WY peptide was used as the test food.

(2) Mice C57BL/6J male mice (Charles River Japan, sometimes simply referred to as "the mice" in this specification) were used. According to the materials of Charles River Japan (http://www.charlesriverjapan.com/),
s://www.crj.co.jp/cms/cmsrs/img/usr/top/B6-Aged.pdf), this mouse was developed by the Jackson Laboratory and is widely used in aging research. Observation of 32 of these mice revealed that some died at 100 weeks of age, and all died by 170 weeks of age (see Mouse Phenome Database (https://www.phenome.jax.org/)). The correspondence between the lifespan of this mouse and that of humans is such that 20-30 years of age in humans corresponds to 3-6 months of age in this mouse (mature individuals), 38-47 years of age in humans corresponds to 10-14 months of age in this mouse (middle-aged individuals), and 56-69 years of age in humans corresponds to 170 weeks of age in this mouse (middle-aged individuals).
The ages correspond to the ages of the present mice, 18 to 24 months (aged individuals). In this study, 62-week-old present mice were used as the aged mice, and 7-week-old present mice were used as the young mice after one month of acclimation. The aged mice were divided into two groups: an "aged WY intake group" (9 mice) that was given the test food (WY peptide), and an "aged non-intake group" (12 mice) that was not given the test food.
The young mice were divided into groups to avoid weight imbalance. The young mice were assigned to a "young non-intake group" (15 mice) that was not allowed to ingest the test food.

(3) Administration of test food (WY peptide) The elderly WY intake group received 0.05% WY peptide (manufactured by Bachem) in dry weight equivalent.
The rats were allowed to freely consume purified feed (AIN-93M, Oriental Yeast Co., Ltd.) containing (w/w) ethanol.
The old non-fed group and the young non-fed group were given AIN-93M containing no WY peptide ad libitum for a period of 5 months.

(4) Measurement After the intake period, the hippocampus was removed and its weight was measured. The glutamate concentration in the hippocampus was also quantified. The glutamate concentration was quantified according to the method published by Eicom Co., Ltd. (https://www.eicom.co.jp/).
comusa.com/hplc-ecd/applications/glutamate-gaba-12-minute-analysis/). Specifically, the hippocampus was homogenized by adding 10 times the weight of the hippocampus (Wako Pure Chemical Industries, Ltd.) of methanol, and the homogenized sample was filtered through a 0.22 μm filter (Millipore Corporation). The resulting sample was used as an extracted sample. Next, the extracted sample was mixed with OPA solution (4 mM o-phthalaldehyde-2
The sample was mixed with 0.5 mol/L carbonate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) containing 2-mercaptoethanol (OPA-2ME, manufactured by Wako Pure Chemical Industries, Ltd.) at a ratio of 4:1 (v/v), and allowed to react at room temperature for 150 seconds to derivatize the sample. The sample was then introduced into HPLC, and the glutamic acid concentration was quantified. The concentration was converted using homotaurine (Acros organi
The HPLC conditions were as follows:

<HPLC conditions>
Measurement equipment: ECD detector type high performance liquid chromatograph (HPLC-ECD, HTEC-5
10, manufactured by Eicom Co., Ltd.
Column: EICOMPAK FA-3ODS (φ3 mm x 75 mm, manufactured by EICOMPAK)
Precolumn: CA-ODS precolumn (φ3 x 4 mm, manufactured by Eicom Co., Ltd.)
·solvent:
Moving layer A:
100 mM phosphate buffer (pH 6.0) + 7% (v/v) methanol (Wako Pure Chemical Industries, Ltd.)
13% acetonitrile (Wako Pure Chemical Industries, Ltd.)
100 mM phosphate buffer (pH 6.0)
Mobile phase B: 50% acetonitrile (manufactured by Wako Pure Chemical Industries, Ltd.)
Gradient conditions: Mobile phase A: 0 to 12 minutes (100%), Mobile phase B: 12 to 15 minutes (1
00%)

(5) Statistical Processing The measured values for each group were expressed as the mean ± standard deviation. The test was performed by Tukey-Kramer, and a significance level of P<0.05% was considered to indicate a significant difference between the two groups.

(6) Results The results are shown in Figures 1 and 2. From the results in Figure 1, it was confirmed that the hippocampal weight of the old non-ingesting group was significantly reduced compared to that of the young non-ingesting group. On the other hand, it was confirmed that the hippocampal weight of the old WY-ingesting group was significantly increased compared to the old non-ingesting group, and the difference with the young non-ingesting group was not significant. From the results in Figure 2, it was confirmed that the glutamate concentration in the hippocampus of the old non-ingesting group was significantly higher compared to the young non-ingesting group. On the other hand, it was confirmed that the glutamate concentration of the old WY-ingesting group was significantly lower compared to the old non-ingesting group, and the difference with the young non-ingesting group was not significant. From these results, it was confirmed that the intake of WY peptide suppresses the decrease in hippocampal weight associated with aging and also suppresses the increase in glutamate concentration in the brain associated with aging. Therefore,
It was suggested that ingestion of WY peptide could suppress neurodegenerative diseases associated with aging, etc.

Claims (8)

A composition for use in improving symptoms of a psychiatric disorder caused by glutamate excitotoxicity due to an increase in brain glutamate concentration, comprising as an active ingredient a dipeptide consisting of the amino acid sequence of WY or a pharmaceutically acceptable salt or solvate thereof, wherein the psychiatric disorder is associated with an increase in brain glutamate concentration and/or a decrease in hippocampal weight . The composition according to claim 1, wherein the psychiatric disorder is one or more selected from the group consisting of schizophrenia, depression, autism, and obsessive-compulsive disorder. The composition of claim 1 or 2, wherein the increase in brain glutamate concentration occurs in the hippocampus. The composition described in any one of claims 1 to 3, wherein the increase in brain glutamate concentration is due to aging. A composition for protecting brain neurons from glutamate excitotoxicity caused by increased glutamate concentration in the brain , comprising as an active ingredient a dipeptide consisting of the amino acid sequence WY or a pharmaceutically acceptable salt or solvate thereof. The composition according to any one of claims 1 to 5 , intended for ingestion by middle-aged and elderly people. The composition according to any one of claims 1 to 6 for use in suppressing neuronal loss and/or promoting neurogenesis. The composition according to any one of claims 1 to 7, which is a food composition or a food composition for brain neuroprotection for use in improving symptoms of a psychiatric disorder caused by glutamate excitotoxicity by increasing glutamate concentration in the brain, wherein the psychiatric disorder is associated with an increase in glutamate concentration in the brain and/or a decrease in hippocampal weight .