Brain function regulators and foods and beverages containing them

Abstract

A brain function regulator that comprises a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, a salt thereof or a chemical modification of the same.

Description

[Technical field]

[0001] The present invention relates to a brain function regulating agent, and a food or drink containing the same. [Background technology]

[0002] Collagen hydrolysates (hereinafter also referred to as "collagen peptide mixtures") are known to exhibit various physiological activities in living organisms. Recently, it has been reported that collagen peptide mixtures also exhibit physiological activities on brain nerve cells and have the effect of improving brain functions such as memory (hereinafter also referred to as "brain function improving effect"). For example, JP 2010-105996 A (Patent Document 1) discloses a neurogenesis promoter containing a total of nine collagen-derived peptides, including a peptide represented by Gly-Pro-Arg. JP 2011-111440 A (Patent Document 2) discloses that a collagen-derived peptide represented by Gly-Pro-Ala has a neurogenesis promoting effect. Non-Patent Document 1 reports that administration of a fish-derived collagen peptide mixture to an aging model mouse increases the expression level of BDNF (brain-derived neurotrophic factor) in the hippocampus, thereby enhancing spatial learning memory ability and passive avoidance ability. Non-Patent Document 2 reports that in an experiment using rats, a collagen-derived dipeptide represented by Pro-Hyp was transferred to cerebrospinal fluid and exerted an antidepressant effect. Non-Patent Document 3 reports that collagen hydrolysate promoted hippocampal neurogenesis. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2010-105996 A [Patent Document 2] JP 2011-111440 A [Non-patent literature]

[0004] [Non-Patent Document 1] Pei Xinrong et al., "Preventive Effect of Marine Collagen Peptide on Learning and Memory Impairment in SAMP8 Mice", Food and Fermentation Industries, 2009, Vol 135(07), p.1-5 [Non-Patent Document 2] Nagai et al., "Identification of collagen-derived antidepressant peptides and their transfer to cerebrospinal fluid," Abstracts of the 2017 Annual Meeting of the Agricultural Chemical Society, March 5, 2017, p.997 [Non-Patent Document 3] C. Kakoi et al., "Collagen peptides enhance hippocampal neurogenesis and reduce anxiety related behavior in mice", Biomedical Research 33(5), 2012, p.273-279 Summary of the Invention [Problem to be solved by the invention]

[0005] The brain function improving effect or neurogenesis action disclosed in Non-Patent Document 1 and Non-Patent Document 3 is not specified by which collagen-derived peptide. The neurogenesis promoting action disclosed in Patent Document 1 is not specified by which of the nine collagen-derived peptides. The neurogenesis promoting action disclosed in Patent Document 2 is a finding obtained from an experiment using PC12 cells derived from rat adrenal pheochromocytoma, and is not a finding obtained from an experiment targeting brain nerve cells. Furthermore, although Pro-Hyp disclosed in Non-Patent Document 2 suggests that it has a neurogenesis action in the hippocampal dentate gyrus, it has not been confirmed whether Pro-Hyp improves brain function by differentiating and growing brain nerve cells. Therefore, a collagen-derived peptide that has a brain function improving effect by directly acting on brain nerve cells without the intervention of in vivo signaling substances such as hormones is not yet known, and its development is eagerly desired.

[0006] In view of the above situation, the present invention aims to provide a brain function regulating agent containing a peptide or the like that has at least one of the effects of improving brain function or preventing brain function decline by directly acting on brain nerve cells, and a food or beverage containing the same. [Means for solving the problem]

[0007] In developing a brain function regulator containing a peptide or the like that acts directly on brain nerve cells to improve brain function or prevent brain function decline, the inventors focused on Glu-Hyp-Gly (glutamic acid-hydroxyproline-glycine, hereinafter also referred to as "EOG" using the abbreviation of one letter for each amino acid) among the amino acid sequences of peptides contained in a collagen peptide mixture. As a result of acting a collagen peptide mixture containing a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, specifically a tripeptide represented by Glu-Hyp-Gly, on brain nerve cells, they found that these promote the differentiation of brain nerve cells, thereby improving brain function and preventing brain function decline, and thus arrived at the present invention. Specifically, the present invention is as follows.

[0008] The brain function regulating agent according to the present invention contains a peptide comprising an amino acid sequence represented by Glu-Hyp-Gly, or a salt thereof, or a chemically modified form thereof.

[0009] The above peptide, its salt, or its chemically modified form is preferably a tripeptide represented by Glu-Hyp-Gly, or its salt, or its chemically modified form.

[0010] Preferably, the peptide is derived from collagen. The above peptide preferably has a weight-average molecular weight of 315 or more and 10,000 or less.

[0011] The brain function regulating agent is preferably an agent for improving brain function or an agent for preventing brain function decline. The brain function regulating agent preferably has an effect of promoting differentiation of brain nerve cells.

[0012] The brain function regulating agent preferably has an effect of inhibiting cell death of brain nerve cells. The brain function regulating agent preferably has an effect of improving memory or cognitive function.

[0013] The food and drink according to the present invention contains the brain function regulating agent. Effect of the Invention

[0014] According to the present invention, it is possible to provide a brain function regulating agent containing a peptide or the like that has at least one of the effects of improving brain function or preventing a decline in brain function by directly acting on brain nerve cells, and a food or beverage containing the same. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a graph showing the amount of EOG in the plasma (sample a) of male mice at a predetermined time after EOG was forcibly orally administered to the male mice. [Diagram 2] FIG. 2 is a graph showing the amount of EOG in the cerebrospinal fluid (sample b) of male mice at a predetermined time after EOG was forcibly orally administered to the male mice. [Diagram 3] FIG. 3 is a graph showing the amount of EOG in the brain (sample c) of a male mouse at a predetermined time after EOG was forcibly orally administered to the male mouse. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, the embodiment of the present invention will be described in more detail. In this specification, the expression in the form of "A to B" means the upper and lower limits of a range (i.e., A or more and B or less), and when no unit is described for A and only a unit is described for B, the unit of A and the unit of B are the same.

[0017] [Brain function regulator] The brain function regulating agent according to the present invention contains a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, or a salt thereof, or a chemically modified version thereof. The brain function regulating agent having such characteristics directly acts on brain nerve cells to promote the differentiation of brain nerve cells, thereby exerting an effect of improving brain function and an effect of preventing a decline in brain function.

[0018] <A peptide containing an amino acid sequence represented by Glu-Hyp-Gly, a salt thereof, or a chemically modified form thereof> As described above, the brain function regulator contains a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, a salt thereof, or a chemically modified form thereof. In this specification, "amino acid" means L-amino acid unless otherwise specified. "A peptide containing an amino acid sequence represented by Glu-Hyp-Gly" means that the amino acid sequence represented by "Glu-Hyp-Gly (glutamic acid-hydroxyproline-glycine)" is contained in the amino acid sequence constituting the peptide at one or more positions.

[0019] In the above brain function regulator, the peptide, its salt, or its chemically modified form is preferably a tripeptide represented by Glu-Hyp-Gly, a salt thereof, or a chemically modified form thereof. In this case, the brain function regulator can more remarkably exhibit the action of promoting the differentiation of brain nerve cells.

[0020] The "salt" of the above peptide can be formed, for example, as inorganic acid salts such as hydrochloride, sulfate, phosphate, organic acid salts such as methanesulfonate, benzenesulfonate, succinate, oxalate, inorganic base salts such as sodium salt, potassium salt, calcium salt, and organic base salts such as triethylammonium salt.

[0021] The "chemically modified form" of the above peptide refers to a peptide in which the free functional groups of the amino acid residues as the constituent units are chemically modified. Chemical modification can be performed, for example, on the hydroxyl group of hydroxyproline, the amino group of the amino acid on the N-terminal (amino terminal) side, and the carboxyl group of the amino acid on the C-terminal (carboxyl terminal) side. Specific means of chemical modification and their treatment conditions are applied with conventionally known peptide chemical modification techniques. By such chemical modification, the chemically modified form of the above peptide can exhibit effects such as improving solubility from weakly acidic to neutral and improving compatibility with other active ingredients.

[0022] For example, for a tripeptide Glu-Hyp-Gly, the hydroxyl group in hydroxyproline can be chemically modified by O-acetylation. This O-acetylation can be carried out, for example, by the action of acetic anhydride in an aqueous or non-aqueous solvent. The carboxyl group in glycine can be chemically modified by esterification, amidation, etc. The esterification can be carried out by suspending the peptide in methanol and then bubbling dry hydrogen chloride gas through the suspension. The amidation can be carried out by the action of carbodiimide, etc. on the peptide.

[0023] Furthermore, the free amino groups in the tripeptide can be chemically modified by methylation, and the free hydroxyl groups in the tripeptide can be chemically modified by at least one of phosphorylation and sulfation.

[0024] The peptide is preferably derived from collagen. In this case, the collagen as a raw material can be obtained by carrying out a conventionally known degreasing or demineralizing treatment, extraction treatment, etc. on the hide, skin, bone, cartilage, tendon, etc. of animals such as cows, pigs, sheep, chickens, ostriches, etc., or the bones, skin, scales, etc. of fish. Furthermore, gelatin can also be used as a raw material for the peptide. Gelatin can be obtained by treating the collagen obtained as described above with a conventionally known method such as hot water extraction. Commercially available collagen and gelatin can also be used as raw materials.

[0025] The peptide can be obtained by hydrolyzing both or either of the collagen and gelatin with a combination of two or more types of endo-type proteases and exo-type proteases. The peptide is obtained as a collagen peptide mixture mixed with other collagen peptides by the hydrolysis, and this collagen peptide mixture itself and a partially purified mixture thereof can be used as the brain function regulator of the present invention. Furthermore, by further purifying the collagen peptide mixture, a peptide containing an amino acid sequence represented by Glu-Hyp-Gly can be obtained with high purity. When the peptide is derived from collagen, it is preferable to obtain it by using a method of treating collagen or gelatin with enzymes in two stages, which will be described later.

[0026] Furthermore, the weight-average molecular weight of the peptide is preferably 315 or more and 10,000 or less. The weight-average molecular weight of the peptide is more preferably 315 or more and 5,000 or less, and more preferably 315 or more and 2,000 or less. When the weight-average molecular weight of the peptide is within the above-mentioned range, the brain function regulator exhibits a more prominent effect of promoting the differentiation of brain nerve cells, and can therefore obtain a more sufficient effect of improving brain function and preventing brain function decline. When the weight-average molecular weight of the peptide is more than 10,000, the brain function regulator may have an insufficient effect of improving brain function and preventing brain function decline.

[0027] The weight-average molecular weight of the above peptide can be determined by carrying out size exclusion chromatography (SEC) under the following measurement conditions. Equipment: High performance liquid chromatography (HPLC) (Tosoh Corporation) Column: TSKGel® G2000SW XL Column temperature: 40℃ Eluent: 45% by mass acetonitrile (containing 0.1% by mass TFA) Flow rate: 1.0mL / min Injection volume: 10μL Detection: UV214nm Molecular weight markers: The following five types are used Cytochrom C Mw:12000 Aprotinin Mw:6500 Bacitracin Mw:1450 Gly-Gly-Tyr-Arg Mw: 451 Gly-Gly-Gly Mw: 189.

[0028] Specifically, about 0.2 g of a sample (collagen peptide mixture) containing the above peptide is added to about 100 ml of distilled water, stirred, and then filtered using a 0.2 μm filter to prepare a sample (subject) for measuring the weight-average molecular weight. The subject is subjected to the above-mentioned size exclusion chromatography to determine the weight-average molecular weight of the above peptide. When the above peptide is a tripeptide represented by Glu-Hyp-Gly, its molecular mass can be used as the weight-average molecular weight.

[0029] <Method of manufacturing brain function regulator> The peptide containing the amino acid sequence represented by Glu-Hyp-Gly contained in the brain function regulator can be obtained by using a conventionally known liquid-phase or solid-phase peptide synthesis method, or a method of hydrolyzing collagen or gelatin. For example, from the viewpoint of efficiency, the above peptide is preferably produced by using a chemical synthesis method using amino acids described below, or a method of enzymatically treating collagen or gelatin in two stages described below. Furthermore, the above peptide can also be produced by a method of enzymatically treating collagen or gelatin only with a secondary enzyme without the primary enzyme, or a method of simultaneously enzymatically treating collagen or gelatin with a primary enzyme and a secondary enzyme, instead of the method of enzymatically treating collagen or gelatin in two stages.

[0030] (chemical synthesis method) The above peptides can be obtained by using a general peptide synthesis method. Known peptide synthesis methods include solid-phase synthesis and liquid-phase synthesis. Known solid-phase synthesis methods include the Fmoc method and the Boc method. The above peptides can be obtained by using either the Fmoc method or the Boc method. Hereinafter, as an example of the solid-phase peptide synthesis method, a method for synthesizing a tripeptide represented by Glu-Hyp-Gly will be described.

[0031] First, polystyrene polymer gel beads with a diameter of about 0.1 mm whose surface is modified with amino groups are prepared as a solid phase. Diisopropylcarbodiimide is separately prepared as a condensation agent. Next, the amino group of glycine, which is the amino acid at the C-terminus (carboxyl terminus) side in the above amino acid sequence, is protected with an Fmoc (fluorenyl-methoxy-carbonyl) group, and the carboxyl group of the glycine is peptide-bonded to the amino group of the solid phase by a dehydration reaction using the above condensation agent. Furthermore, the solid phase is washed with a solvent to remove the remaining condensation agent and amino acid, and then the protecting group of the amino group of glycine peptide-bonded to the solid phase is removed (deprotected).

[0032] Next, hydroxyproline with its amino group protected by an Fmoc group is prepared, and the carboxyl group of this hydroxyproline and the deprotected amino group of the glycine are peptide-bonded using the condensing agent. Thereafter, the amino group of the hydroxyproline is deprotected in the same manner, glutamic acid is prepared with an Fmoc group, and the glutamic acid and the hydroxyproline are peptide-bonded to synthesize a tripeptide represented by Glu-Hyp-Gly on the solid phase. Finally, the amino group of the glutamic acid is deprotected, and a solution containing trifluoroacetic acid is added at room temperature and shaken for a certain period of time to separate the tripeptide from the solid phase. This allows the tripeptide to be produced.

[0033] (Production method using collagen or gelatin) As an example of a method for producing the above-mentioned peptide, a method for producing a tripeptide represented by Glu-Hyp-Gly (hereinafter also referred to as a "specific peptide") by enzymatically treating collagen or gelatin in two stages will be described below.

[0034] Here, "two-stage enzyme treatment" of collagen or gelatin means the following. That is, after performing a first enzyme treatment by a conventionally known method for cleaving the peptide bond of collagen or gelatin, a second enzyme treatment is performed by an enzyme having aminopeptidase N activity, an enzyme having both aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, or a combination of an enzyme having aminopeptidase N activity and an enzyme having prolyl tripeptidyl aminopeptidase activity. By performing the first enzyme treatment, a collagen peptide mixture precursor can be obtained. Furthermore, by performing the second enzyme treatment, a collagen peptide mixture containing the above-mentioned specific peptides can be obtained from the collagen peptide mixture precursor. The method of performing two-stage enzyme treatment of collagen or gelatin will be described in more detail below.

[0035] -First enzyme treatment- The enzyme used in the first enzyme treatment should not be particularly limited as long as it is an enzyme capable of cleaving the peptide bond of collagen or gelatin, and any proteolytic enzyme can be used. Specifically, collagenase, thiol protease, serine protease, acid protease, alkaline protease, metal protease, etc. can be mentioned, and one selected from these groups may be used alone, or two or more may be used in combination. As the thiol protease, plant-derived chymopapain, papain, bromelain, ficin, animal-derived cathepsin, calcium-dependent protease, etc. can be used. As the serine protease, trypsin, cathepsin D, etc. can be used. As the acidic protease, pepsin, chymotrypsin, etc. can be used. As the enzyme used in the first enzyme treatment, when considering that the brain function regulator according to the present invention is used in medicine, specified health food, etc., it is preferable to use an enzyme other than the enzyme derived from a pathogenic microorganism.

[0036] The amount of enzyme in the first enzyme treatment is preferably 0.1 to 5 parts by mass of the enzyme per 100 parts by mass of collagen or gelatin. The treatment temperature in the first enzyme treatment is preferably 30 to 65°C, and the treatment time is preferably 10 minutes to 72 hours. The weight-average molecular weight of the collagen peptide mixture precursor obtained by the first enzyme treatment is preferably 500 to 10,000, more preferably 500 to 5,000, and even more preferably 500 to 2,000. If the weight-average molecular weight is within the above range, it can be said that peptides with appropriate molecular weights are sufficiently produced. After the first enzyme treatment, the enzyme can be inactivated as necessary. In this case, the inactivation temperature is preferably 70 to 100°C. The weight-average molecular weight of the collagen peptide mixture precursor can be determined by the method using SEC described above.

[0037] -Secondary enzyme treatment- Examples of the enzyme used in the secondary enzyme treatment include an enzyme having aminopeptidase N activity, an enzyme having both aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, or a combination of an enzyme having aminopeptidase N activity and an enzyme having prolyl tripeptidyl aminopeptidase activity. Here, in the present specification, the term "enzyme having aminopeptidase N activity" refers to a peptidase that has the function of releasing an amino acid from the N-terminus of a peptide chain, and acts when an amino acid other than proline or hydroxyproline is present at the second position from the N-terminus. In the present specification, the term "enzyme having prolyl tripeptidyl aminopeptidase activity" refers to a peptidase that releases only the three amino acid residues at the N-terminus from a peptide in which the third position from the N-terminus is proline or hydroxyproline. When considering that the brain function regulator according to the present invention is used in medicines, foods for specified health uses, etc., it is preferable to use enzymes other than those derived from pathogenic microorganisms in the secondary enzyme treatment.

[0038] Examples of the enzymes having aminopeptidase N activity include aminopeptidase N (EC3.4.11.2.; T. Yoshimoto et al., Agric. Biol. Chem., 52:217-225(1988)). Examples of the enzymes having aminopeptidase N activity derived from the genus Aspergillus include. Examples of the enzymes having prolyl tripeptidyl aminopeptidase activity include prolyl tripeptidyl aminopeptidase (EC3.4.14.; A. Banbula et al., J. Biol. Chem., 274:9246-9252(1999)).

[0039] By carrying out the secondary enzyme treatment, a collagen peptide mixture containing peptides not contained in the collagen peptide mixture precursor can be obtained. Specifically, a collagen peptide mixture containing the specific peptide can be obtained.

[0040] The amount of enzyme in the secondary enzyme treatment is preferably 0.01 to 5 parts by mass of the enzyme per 100 parts by mass of the collagen peptide mixture precursor. The treatment temperature in the secondary enzyme treatment is preferably 30 to 65°C, and the treatment time is preferably 1 to 72 hours. The weight-average molecular weight of the collagen peptide mixture obtained by the secondary enzyme treatment is preferably 315 to 10,000, more preferably 315 to 5,000, and even more preferably 315 to 2,000. The weight-average molecular weight of the collagen peptide mixture can also be determined by the method using the SEC described above.

[0041] The secondary enzyme treatment is carried out mainly for the purpose of producing the above-mentioned specific peptide. For this reason, it is preferable to adjust the amount of enzyme, treatment temperature, treatment time and pH in the secondary enzyme treatment so that the peptides contained in the collagen peptide mixture precursor are not excessively hydrolyzed. This makes it preferable to make the collagen peptide mixture fall within the above-mentioned range of weight-average molecular weight. After the secondary enzyme treatment, it is necessary to inactivate the enzyme. In this case, the inactivation temperature is preferably, for example, 70 to 100°C. Furthermore, it is preferable to perform a sterilization treatment at 120°C for several seconds or more. It is also possible to apply heat of 200°C or more to this and spray dry it.

[0042] In the secondary enzyme treatment, in addition to the above-mentioned enzyme having aminopeptidase N activity and enzyme having prolyl tripeptidyl aminopeptidase activity, enzymes having different activities can be used, and two or more enzymes having different activities can be used in combination. This makes it possible to decompose and remove by-products. In this case, the enzyme to be used is preferably selected appropriately depending on the type of collagen used as the raw material and the type of enzyme used in the primary enzyme treatment. Examples of the above-mentioned different activities include dipeptidase activities such as prolidase activity and hydroxyprolidase activity. This makes it possible to decompose and remove by-products such as dipeptides.

[0043] Furthermore, aminopeptidase N activity is basically an activity that releases amino acids one by one on the N-terminal side. Therefore, when the collagen peptide mixture precursor obtained by the first enzyme treatment contains peptides with extremely large molecular weights, the processing time will be significantly extended if the second enzyme treatment is performed only with an enzyme having aminopeptidase N activity. To deal with such cases, for example, prolyl oligopeptidase, an endopeptidase that has the activity of hydrolyzing the carboxyl group side of proline (prolidase activity), can be used in the second enzyme treatment. This allows the second enzyme treatment to be performed efficiently.

[0044] In the method of treating collagen or gelatin with enzymes in two stages, a peptide with a relatively large molecular weight can be produced by the first enzyme treatment. This peptide can have an amino acid sequence represented by, for example, [X1-Gly-X2-Glu-Hyp-Gly] (X1 and X2 ≠ Hyp). In the subsequent second enzyme treatment, an enzyme having aminopeptidase N activity acts on the peptide represented by [X1-Gly-X2-Glu-Hyp-Gly], and X1 at the N-terminus is liberated to obtain a peptide having an amino acid sequence represented by [Gly-X2-Glu-Hyp-Gly]. Next, an enzyme having aminopeptidase N activity acts twice on the peptide represented by [Gly-X2-Glu-Hyp-Gly], and glycine and X2 are liberated to obtain a peptide represented by [Glu-Hyp-Gly]. From the above, a specific peptide can be produced.

[0045] -Purification of collagen peptide mixture- By carrying out the above-mentioned two-stage enzyme treatment, a collagen peptide mixture containing a specific peptide can be produced. The above-mentioned collagen peptide mixture also contains peptides other than the specific peptide, i.e., peptides other than the tripeptide represented by Glu-Hyp-Gly, so it is preferable to purify it as necessary. In this case, the purification method can be a conventionally known method, for example, ultrafiltration, size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, and other various liquid chromatography methods can be used.

[0046] Specifically, the collagen peptide mixture can be purified by the following procedure: About 2 g / 10 mL of the collagen peptide mixture is loaded onto an ion exchange column (e.g., trade name: "Toyopearl (registered trademark) DEAE-650", manufactured by Tosoh Corporation), and the first void volume fraction eluted with distilled water is collected. Next, the first void volume fraction is loaded onto a column having an ion exchange group opposite to that of the ion exchange column (e.g., trade name: "Toyopearl (registered trademark) SP-650", manufactured by Tosoh Corporation), and the second void volume fraction eluted with distilled water is collected.

[0047] Next, the second void volume fraction is loaded onto a gel filtration column (e.g., Sephadex LH-20, manufactured by GE Healthcare Japan, Inc.) and eluted with 30% by weight aqueous methanol to recover a fraction containing the specific peptide. Finally, this fraction is fractionated using a high performance liquid chromatography (HPLC) equipped with a reverse phase column (e.g., μBondasphere 5μC18 300Å column, manufactured by Waters Corporation) with a linear concentration gradient of 32% or less acetonitrile aqueous solution containing 0.1% by weight trifluoroacetic acid, thereby obtaining a specific peptide with high purity.

[0048] <Agents for improving brain function or preventing brain function decline> The brain function regulator according to the present invention is preferably a brain function improving agent or a brain function decline preventing agent. The brain function regulator contains a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, or a salt thereof, or a chemically modified form thereof, and can exhibit a brain function differentiation promoting effect by directly acting on brain nerve cells, as described in [Evaluation Test 1] and [Evaluation Test 2] below. This allows the brain function regulator to have a brain function improving effect and a brain function decline preventing effect. Therefore, the brain function regulator can be used as a brain function improving agent for treating patients with decreased brain function. The brain function regulator can also be used as a brain function decline preventing agent for the purpose of preventing brain function decline due to aging, etc.

[0049] The brain function regulator preferably has a differentiation promoting effect on brain nerve cells. Furthermore, the brain function regulator preferably has a cell death inhibiting effect on brain nerve cells. It is also preferable that the brain function regulator has an effect of improving memory or cognitive function. As described below in [Evaluation Test 1] and [Evaluation Test 2], the brain function regulator can exhibit a differentiation promoting effect on brain nerve cells by acting directly on the brain nerve cells. From [Evaluation Test 1] and [Evaluation Test 2] described below, the brain function regulator can also be considered to have a cell death inhibiting effect on brain nerve cells. Through these actions, the brain function regulator can exert an effect of improving memory or cognitive function.

[0050] The brain function regulator can be administered orally or parenterally in various forms. For oral administration, the form can be, for example, tablets, granules, capsules, powders, liquids, suspensions, emulsions, etc. Furthermore, the brain function regulator in the above-mentioned form can be mixed with food and drink.

[0051] When the brain function regulator is administered parenterally, it can be in the form of injection into the brain, injection, transdermal agent, suppository, nasal drops, inhalant, etc. The preferred forms of the brain function regulator include tablets, granules, capsules, powders, and liquids for direct injection into the brain. The brain function regulator includes, for example, the tripeptide represented by Glu-Hyp-Gly described above, which is rapidly absorbed in the intestinal tract and can be taken by oral administration.

[0052] The dosage of the brain function regulator varies depending on the age, sex, weight, sensitivity difference, administration method, administration interval, type of formulation, etc. of the subject. When the brain function regulator is orally administered, the dosage is preferably 0.0001 to 2500 mg / kg per day for an adult, and more preferably 0.0001 to 500 mg / kg per day. When the brain function regulator is in the form of a tablet, for example, it can be a tablet containing the brain function regulator at 0.001 to 80% by mass per tablet, and when it is in the form of a powder, it can be a powder containing the brain function regulator at 0.001 to 100% by mass. The dosage can be appropriately determined by referring to the dosage when administered parenterally or by administering in other forms of formulation. The brain function regulator can be administered once or in several divided doses per day, or once every one to several days.

[0053] The brain function regulator may contain other active ingredients, carriers for formulation, etc., as appropriate, within the scope that does not adversely affect the effects of the present invention. Examples of other active ingredients include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), astaxanthin, ginkgo leaf extract, arachidonic acid, etc. Furthermore, examples of pharmaceutical acceptable carriers used in formulation into pharmaceutical preparations include diluents, binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, potassium phosphate, sorbitol, glycine), lubricants (magnesium stearate, talc, polyethylene glycol, silica), disintegrants (potato starch) and wetting agents (sodium lauryl sulfate).

[0054] <Use invention> The brain function regulator according to the present invention contains a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, or a salt thereof, or a chemically modified form thereof. The brain function regulator has an unknown attribute of a peptide containing an amino acid sequence represented by Glu-Hyp-Gly, for example a tripeptide represented by Glu-Hyp-Gly, which has a differentiation promoting effect on brain nerve cells, and can obtain at least one of an effect of improving brain function or an effect of preventing brain function decline. In other words, the present invention can be said to be a peptide containing an amino acid sequence represented by Glu-Hyp-Gly for regulating brain function, or a salt thereof, or a chemically modified form thereof.

[0055] [Food and beverages] The food and drink according to the present invention contains the brain function regulator. For example, the tripeptide represented by Glu-Hyp-Gly, which is preferably contained in the brain function regulator, is rapidly absorbed in the intestinal tract as described above, and can be taken by oral administration. Therefore, the food and drink according to the present invention can be taken by mixing it with a meal or drink as a food and drink containing the brain function regulator. Furthermore, the food and drink according to the present invention can be used as a specified health food or a functional food. The concentration of the brain function regulator contained in the food and drink is preferably 0.001 to 100% by mass. EXAMPLES

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0057] [Evaluation test 1: Clinical trial] <Test foods (foods and drinks containing brain function regulators)> The test food used was a collagen peptide mixture containing an amino acid sequence represented by Glu-Hyp-Gly (product name: "Collagen Peptide CP-B", manufactured by Nitta Gelatin Co., Ltd.). During the test period described below, subjects were asked to ingest 5 g of the test food once a day by adding it to a beverage of their choice. The time of ingestion of the beverage was not specified.

[0058] <Study design> The study period was a consecutive 4-week period selected from October 20th to November 21st, 2016. An open-label comparative study before and after intervention was carried out at the Kokoro Research Center, Kyoto University. In accordance with the principles of the World Medical Association's Declaration of Helsinki, all subjects provided informed consent and gave written consent. Approval for this study was obtained from the Ethics Committee of the Kyoto University Kokoro Research Unit on September 18th, 2015 (approval number: 27-P-13).

[0059] In this study, the effects of the above test foods on brain function were evaluated by obtaining Gray Matter Volume-Brain Healthcare Quotient (GM-BHQ) scores, Fractional Anisotropy-Brain Healthcare Quotient (FA-BHQ) scores, Mild Cognitive Impairment (MCI) scores, and Standard Verbal Paired Associative Learning (S-PA) scores.

[0060] <Subject> Thirty healthy subjects aged 49 to 63 years (mean age 56.1 ± 3.6 years) were enrolled in this study. The main criteria for enrollment were: "Those who had not consumed collagen hydrolysates for one month prior to the start of the study," "Those who had no history of major food allergies, including gelatin," and "Those who had no history of neurological or psychiatric disorders, such as cerebral infarction or dementia." In addition, the items listed in section 6 of "https: / / www.acrin.org / Portals / 0 / Protocols / 6684 / ACRIN6684_Amend7_012412_master_ForOnline.pdf" were used as criteria for excluding subjects. During the study period, one subject dropped out due to personal reasons. In addition, five subjects did not take the test to obtain the S-PA score due to personal reasons. Therefore, statistical analysis was performed according to each protocol by 24 subjects for the test to obtain the S-PA score and 29 subjects for the other tests.

[0061] <Evaluation method and results> (MRI scan) Magnetic resonance imaging (MRI) scans were performed on the day before the start of the intake of the test food (hereinafter also referred to as "pre-intervention") and the day after the end of the intake of the test food (29 days after the start of intake, hereinafter also referred to as "post-intervention"). MRI scans before the intervention were performed between October 20 and October 24, 2016, and MRI scans after the intervention were performed between November 18 and November 21, 2016. MRI scans were performed at the Kokoro Research Center, Kyoto University. The specific MRI scan method was as described in Nemoto K et al., "MRI-based Brain Healthcare Quotients: A bridge between neural and behavioral analyses for keeping the brain healthy", PLoS ONE, 2017, 12(10): e0187137 (https: / / doi.org / 10.1371 / journal.pone.0187137).

[0062] The MRI system was a 3T scanner "Verio" from Siemens, Germany, and images were taken using a 32-channel head array coil. For 3D T1-weighted images, the MP-RAGE pulse sequence was used. The parameters were as follows: repetition time (TR) 1900 ms; echo time (TE) 2.52 ms; inversion time (TI) 900 ms; flip angle 9°; matrix size 256 × 256; field of view (FOV) 256 mm; slice thickness 1 mm. Diffusion tensor images (DTI) were acquired by spin-echo echo-planar imaging (SE-EPI) using GRAPPA. The slices for diffusion tensor images were parallel to the OM line.

[0063] The parameters were as follows: repetition time (TR) 14100 ms; echo time (TE) 81 ms; flip angle 90°; matrix size 114 × 114; field of view (FOV) 224 mm; slice thickness 2 mm. The baseline image (b = 0 s / mm 2 ) and b=1000s / mm 2 Thirty axial diffusion images were obtained.

[0064] Gray matter was extracted from the T1-weighted images using the brain function mapping tool SPM12, and the whole-brain GM-BHQ score was calculated using the gray matter volume and intracranial volume. Fractional anisotropy (FA) images were generated from the diffusion tensor images using the software FSL 5.0.11, and the whole-brain FA-BHQ score was calculated based on these.

[0065] The tool used for the statistical processing in the process of calculating the GM-BHQ score and FA-BHQ score here was the medical statistics program "STAT MateIII." Significance was assessed by comparing within groups before and after the intervention using the Wilcoxon rank sum test, and a significance level (P value) of 0.05 or less was determined to indicate significance before and after the intervention. The GM-BHQ score and FA-BHQ score values ​​before and after the intervention, their differences, and P values ​​are shown in Table 1 below. In Table 1, "N" indicates the number of subjects, "Baseline" indicates before the intervention, "Post" indicates after the intervention, and "Δ" indicates the difference before and after the intervention.

[0066] [Table 1]

[0067] According to Table 1, a significant improvement was observed in the FA-BHQ score before and after the intervention. No significant improvement was observed in the GM-BHQ score. Therefore, it was found that the above test food (a collagen peptide mixture containing a tripeptide represented by Glu-Hyp-Gly as a brain function regulator) has the effect of regulating brain function.

[0068] (MCI score) Mild cognitive impairment (MCI) scores were calculated using the cognitive function check scale "Atama no Kenkou Check" (Millenia Corporation). Specifically, subjects were asked to memorize 10 words, and after explanations unrelated to the test, the number of words they could verbally answer was measured. Each subject's MCI score was calibrated by comparing the test results of each subject with those of the same age group based on information such as the subject's gender, age, years of study, and race, and objectively evaluating demographically. This test was conducted on the same day as the MRI scan for each subject.

[0069] (S-PA score) The standard verbal paired associative learning (S-PA) score was developed by the Japanese Society for Higher Brain Dysfunction to grasp human verbal memory. The specific test contents of the S-PA score are as follows. First, a sheet consisting of 10 pairs of related words (semantically related words) and 10 pairs of unrelated words (words with weak semantic association) is prepared. Next, the examiner reads out the 10 pairs of related words written on the sheet and asks the subject to remember them. After that, one of these pairs of words is presented and the subject is asked to answer the other word that was paired with it. Furthermore, the examiner reads out the 10 pairs of unrelated words written on the sheet and asks the subject to remember them. After that, one of these pairs of words is presented and the subject is asked to answer the other word that was paired with it. Finally, the S-PA score of each subject is calculated from the number of correct answers. Note that sheets containing different 10 pairs of related words and 10 pairs of unrelated words are prepared before and after the intervention. This study was also conducted on the same day that each subject had their MRI scan.

[0070] The tool used for the statistical processing in the process of calculating the MCI score and S-PA score was also the aforementioned "STAT MateIII." Significance was assessed by comparing within groups before and after the intervention using the Wilcoxon rank sum test, and a significance level (P value) of 0.05 or less was determined to indicate significance before and after the intervention. The MCI score and S-PA score values ​​before and after the intervention, their differences, and P values ​​are shown in Table 2 below. In Table 2, "N" indicates the number of subjects, "Baseline" indicates before the intervention, "Post" indicates after the intervention, and "Δ" indicates the difference before and after the intervention.

[0071] [Table 2]

[0072] According to Table 2, significant improvements were observed in both MCI scores and S-PA scores before and after the intervention. Considering the results of Table 2 in light of the results of Table 1, it is suggested that the above test foods have an effect of improving memory or cognitive function through their effect of regulating brain function, and thus have an effect of improving brain function. Furthermore, the above results suggest that the test foods also have an effect of preventing brain function decline. Therefore, brain function regulators can be used as brain function improvers or brain function decline preventers.

[0073] (Effects of brain function regulators on the brain functions of individual subjects) The effect of the brain function regulator on the brain function of each subject was examined by examining whether there was a correlation between the increase / decrease in the GM-BHQ score and the FA-BHQ score and the increase / decrease in the MCI score and the S-PA score for each subject. Regarding the above correlation, Spearman's rank correlation coefficient (r) was calculated using each measurement value, and statistical processing was performed before and after the intervention. The tool used for this statistical processing was also the above-mentioned "STAT MateIII". When r was 0.2 or less, it was evaluated as "no correlation", when r was greater than 0.2 and less than 0.4, it was evaluated as "low correlation", when r was greater than 0.4 and less than 0.7, it was evaluated as "correlation", when r was greater than 0.7 and less than 1, it was evaluated as "strong correlation", and when r was 1, it was evaluated as "perfect correlation". The resulting r values ​​are shown in Table 3.

[0074] [Table 3]

[0075] According to Table 3, a correlation was found between increases or decreases in GM-BHQ scores and increases or decreases in MCI scores, and a correlation was found between increases or decreases in FA-BHQ scores and increases or decreases in S-PA scores.

[0076] <Consideration> According to the above, when focusing on the action effect of the brain function regulator containing a specific peptide on the brain function of each subject, it was estimated that the improvement of the FA-BHQ score would improve the S-PA score, and the improvement of the GM-BHQ score would improve the MCI score. Therefore, the brain function regulator can be used as a brain function improver or a brain function decline preventive agent.

[0077] [Evaluation test 2: Cell biological test (in vitro test)] <Sample preparation> All experimental animals were in accordance with the "Standards for the breeding and keeping of laboratory animals" of the Prime Minister's Office and the "Guidelines for animal experiments" of the Josai University Life Science Research Center. 15-25 week-old male and female Wistar rats were purchased from Japan CLEA Co., Ltd. The rats were then temperature 23±2℃, relative humidity 55±10%, lighting cycle 12 hours, light period 7:00~19:00, Solid feed (Product name: "CE-2", manufactured by CLEA Japan, Inc.) and water ad libitum, and were bred under the same conditions to obtain 7-day-old rats. In addition, the above-mentioned Wistar rats aged 3 to 8 days were purchased from CLEA Japan, Inc.

[0078] (Preparation and culture of primary cultured cerebellar granule cells (CGCs)) The Wistar rats were raised under the above conditions until they were 7 to 9 days old, and then the cerebellum was removed from the rats and dispersed by the method described below using each dispersion solution (solution I to solution V) shown in Table 4 to obtain a dispersion solution containing granule cells and other brain nerve cells (hereinafter, these cells are also collectively referred to as "cerebellar granule cells (CGCs)"). These cerebellar granule cells (CGCs) are brain nerve cells. The CGCs contained in the dispersion solution were then cultured. The "solution name," "contained reagents," and "amounts thereof" of the dispersion solution are shown in Table 4. The method for preparing cerebellar granule cells (CGCs) is as follows.

[0079] [Table 4]

[0080] First, 10 rats aged 7 to 9 days were decapitated, and the cerebellum was removed in a clean bench. After removing excess tissue, blood vessels, and serous membrane, the cerebellum was minced with a razor. Liquid I was added to this paste to make a suspension, and the suspension was centrifuged at 1000 rpm for 30 seconds to obtain a first separation solution containing a supernatant and cell populations. After removing the supernatant from this first separation solution, liquid II, which had been preheated to 37°C in a thermostatic bath, was added little by little to the remaining cell population and stirred, and the mixture was transferred to a 50 mL medium bottle and stirred at 37°C for 15 minutes to perform tissue digestion with trypsin.

[0081] Next, the trypsin was inactivated by adding solution IV to the medium bottle, and the medium bottle was centrifuged at 1000 rpm for 30 seconds to obtain a second separation solution containing a supernatant and cell groups. After removing the supernatant from the second separation solution, a small amount of solution III was added to the remaining cell groups using a Pasteur pipette to disperse them. After that, the third separation solution containing a supernatant and cell groups was obtained by leaving the bottle to stand for 15 minutes, and the supernatant was collected in solution V. Furthermore, a small amount of solution III was added to the cell groups remaining in the third separation solution to redisperse them. After that, the bottle was left to stand for 5 to 10 minutes to obtain a fourth separation solution containing a supernatant and cell groups, and the supernatant was collected in the above-mentioned solution V containing the supernatant of the third separation solution.

[0082] The above-mentioned solution V, which contains the supernatant of the third separation solution and the supernatant of the fourth separation solution, was centrifuged at 1000 rpm for 5 minutes to obtain a fifth separation solution containing the supernatant and the cell population. After removing the supernatant from this fifth separation solution, a basal medium (BME: Basal Medium Eagle, manufactured by Sigma-Aldrich) was added to the remaining cell population and stirred to obtain a mixture containing cerebellar granule cells (CGCs) and BME. Then, this mixture was stained with 0.4% by mass of trypan Blue solution, and the number of stained cells and their viability were measured.

[0083] Next, the cell density of viable cells was adjusted to 1.2×10 5 K is calculated to be 1.5 cells / mL. + 5 mM serum (+) BME was added and suspended to obtain a culture sample. 0.766 mL of this culture sample was seeded onto a 12-well plate previously coated with 50 μg / mL poly-L-lysin (PLL) and cultured at 37°C in an incubator with 5% CO2 (this day was designated "culture day 0").

[0084] On the first day of culture, cytosine β-D-arabinofuranoside (AraC) was added to each well at a final concentration of 10 μM in order to maintain only CGCs and suppress the proliferation of non-neuronal cells. Furthermore, KCl was added to the control sample described below at a final concentration of 25 mM, and to the other samples (samples to which peptides or amino acids shown in Tables 5 to 7 described below were added), and was added at a final concentration of 15 mM.

[0085] Furthermore, on the 1st and 4th days of the culture, the peptides or amino acids shown in Tables 5 to 7 below were added to the final concentrations shown in Tables 5 to 7, respectively, to prepare each sample.

[0086] On the seventh day of culture, the cell viability of CGC was measured using the MTT assay to examine whether each sample had a differentiation-promoting effect on CGC. Specifically, the percentage of the number of viable cells in each sample relative to the number of viable cells in the control sample with a KCl concentration of 25 mM was calculated, and the percentage was statistically processed to evaluate the significance of the differentiation-promoting effect on CGC. The significance was evaluated by performing Smirnov-Grubbs (two-sided test) using software (product name: "Excel Statistics (Ver2.1)" manufactured by Social Information Services Co., Ltd.) as a statistical process, and the significance level (P value) was set to 0.05 and rejected. Then, Student's t-test was performed to determine the significance. The results are shown in Tables 5 to 7. The samples marked with "* (asterisk)" in Tables 5 to 7 were determined to have a differentiation-promoting effect on CGC (significant).

[0087] In Table 5, "Control (K + )" refers to a control sample. "NMDA" (trade name: "M3262", Sigma-Aldrich) refers to a sample to which N-methyl-D-aspartic acid was added, and "BDNF" (trade name: "SRP3014", Sigma-Aldrich) refers to a sample to which brain-derived neurotrophic factor was added. "NMDA" and "BDNF" are known to be involved in the differentiation of brain nerve cells, and both serve as reference examples in this test.

[0088] Furthermore, for the peptides shown in Tables 5 to 7, abbreviations are used to represent amino acids with one letter. C stands for cyclic, H stands for histidine, P stands for proline, G stands for glycine, O stands for hydroxyproline, E stands for glutamic acid, and A stands for alanine. That is, in Table 5, "C-HP" means a sample to which a cyclic dipeptide consisting of histidine-proline (manufactured by PH Japan Co., Ltd.) has been added, "C-GP" means a sample to which a cyclic dipeptide consisting of glycine-proline (manufactured by PH Japan Co., Ltd.) has been added, and "TRH" means a sample to which thyrotropin-releasing hormone (manufactured by PH Japan Co., Ltd.) has been added. These are, Blood-brain barrierIt is known that the β-glutamate passes through the β-glutamate membrane, and all of these are comparative examples. In Table 5, "glutamic acid" refers to a sample to which glutamic acid (trade name: "G1251", manufactured by Sigma-Aldrich Co.), which is known to have an antioxidant effect, was added. "Glutathione" refers to a sample to which glutathione (trade name: "G6013", manufactured by Sigma-Aldrich Co.), which has a similar chemical structure to Glu-Hyp-Gly (EOG), was added. "GPO" refers to a sample to which a tripeptide consisting of glycine-proline-hydroxyproline (manufactured by PH Japan Co., Ltd.) was added. "Glutamic acid", "glutathione", and "GPO" are all comparative examples.

[0089] In Table 6, "C-PO" refers to a sample to which a cyclic dipeptide consisting of proline-hydroxyproline (manufactured by PH Japan Co., Ltd.) has been added, "C-OG" refers to a sample to which a cyclic dipeptide consisting of hydroxyproline-glycine (manufactured by PH Japan Co., Ltd.) has been added, and "C-EO" refers to a sample to which a cyclic dipeptide consisting of glutamic acid-hydroxyproline (manufactured by PH Japan Co., Ltd.) has been added. In Table 6, "PO" refers to a sample to which a dipeptide consisting of proline-hydroxyproline (trade name: "G-3025", manufactured by BACHEM) has been added, "OG" refers to a sample to which a dipeptide consisting of hydroxyproline-glycine (trade name: "G-2365", manufactured by BACHEM) has been added, and "AO" refers to a sample to which a dipeptide consisting of alanine-hydroxyproline (manufactured by PH Japan Co., Ltd.) has been added. With regard to "PO", Blood-brain barrier It is known that the fluoride passes through the fluoride-containing polymer. "C-PO", "C-OG", "C-EO", "PO", "OG" and "AO" are all comparative examples.

[0090] In Table 7, "GP" refers to a sample to which a dipeptide consisting of glycine-proline (product name: "G-3015", manufactured by BACHEM) was added, and "EO" refers to a sample to which a dipeptide consisting of glutamic acid-hydroxyproline (manufactured by PH Japan Co., Ltd.) was added. Both "GP" and "EO" are comparative examples. "EOG" refers to a sample to which a tripeptide represented by Glu-Hyp-Gly (manufactured by PH Japan Co., Ltd.) was added, and is an example in this test.

[0091] [Table 5]

[0092] [Table 6]

[0093] [Table 7]

[0094] <Consideration> According to Tables 5 to 7, EOG, like the reference example in which NMDA and BDNF were added, was found to have the effect of promoting the differentiation of CGCs by directly acting on CGCs. Tables 5 to 7 also suggest that EOG has the effect of suppressing the cell death of CGCs.

[0095] <Considerations obtained from evaluation test 1 and evaluation test 2> According to the above-mentioned evaluation test 1 and evaluation test 2, the brain function regulator according to the present invention and the food and drink containing it can promote the differentiation of brain nerve cells by directly acting on the brain nerve cells. Due to such action, the brain function regulator and the food and drink containing it can improve memory or cognitive function. Therefore, the brain function regulator can be used as a brain function improving agent or a brain function decline preventing agent.

[0096] [Evaluation test 3: In vitro test to analyze the effective concentration of EOG] The effective concentration of EOG capable of promoting differentiation of CGCs was examined by using the same method as in Evaluation Test 2, except that EOG was added on the first and fourth days of culture to the final concentrations shown in Table 8. The results are shown in Table 8.

[0097] [Table 8]

[0098] <Consideration> According to Table 8, EOG can exert a significant differentiation promoting effect on CGCs when its concentration is 7 μM or more.

[0099] [Evaluation test 4: Evaluation test to analyze the delivery of orally ingested EOG to the brain parenchyma] <Sample preparation> All experimental animals were in accordance with the "Standards for the breeding and storage of experimental animals" of the Prime Minister's Office. Two 8-week-old ddy male mice were purchased from Sankyo Labo Service Co., Ltd. The male mice were then acclimatized for one week to become 9-week-old male mice, and then fasted for 12 hours. Next, 20 mg of EOG (Glu-Hyp-Gly, manufactured by PH Japan Co., Ltd., purity 95% or higher) was forcibly orally administered to the male mice in a 250 μL aqueous solution to prepare two male mice for analysis of the delivery of EOG to the brain parenchyma.

[0100] An anesthetic was subcutaneously injected into the male mice 10 minutes before oral administration (0 hours), 1 hour, 2 hours, and 4 hours after oral administration. The anesthetic was called triple-mix anesthesia, and contained 0.3 mg / kg medetomidine hydrochloride, 4 mg / kg midazolam, and 4 mg / kg butorphanol tartrate. 10 minutes after the injection of the anesthetic, 0.5 to 7 μL of cerebrospinal fluid was collected from each of the male mice, 20 to 80 μL of blood was collected from the tail vein, and 0.28 to 0.33 mg of a portion of the brain parenchyma (hereinafter also simply referred to as "brain") was extracted.

[0101] The blood was first centrifuged at 4°C and a centrifugal acceleration of 20400G for 10 minutes to obtain plasma, which was temporarily stored at -80°C. Next, the plasma was mixed with ethanol in an amount three times the volume of the plasma, and then centrifuged at 4°C and a centrifugal acceleration of 1000G for 10 minutes to obtain a supernatant. Then, ammonium bicarbonate adjusted to 50 mM was added to the supernatant in an amount four times the volume of the supernatant and mixed. The supernatant containing the ammonium bicarbonate was further filtered through a 0.2 μm filter (manufactured by Sartorius Co., Ltd.) to obtain each sample for measuring the amount of EOG in the plasma of the male mice (each sample before oral administration (0 hours), 1 hour, 2 hours, and 4 hours after oral administration. These are collectively referred to as "sample a" below).

[0102] The cerebrospinal fluid was mixed with ethanol in an amount three times the volume of the cerebrospinal fluid, and then centrifuged at 4°C, a centrifugal acceleration of 1000G, and for 10 minutes. Then, 50 μL of ammonium bicarbonate adjusted to 50 mM was added to the centrifuged cerebrospinal fluid and mixed. The cerebrospinal fluid containing the ammonium bicarbonate was further filtered with a 0.2 μm filter (manufactured by Sartorius Co., Ltd.) to obtain samples for measuring the amount of EOG in the cerebrospinal fluid of the male mice (samples before oral administration (0 hours), 1 hour, 2 hours, and 4 hours after oral administration. These are collectively referred to as "sample b" below).

[0103] The brain was washed with PBS (phosphate buffered saline), wrapped in aluminum foil, frozen in liquid nitrogen, and then frozen and stored in a freezer (-80°C). The brain was then thawed, homogenized with an equal amount of PBS to the brain, and mixed with acetonitrile in an amount three times the weight of the brain, followed by centrifugation at 4°C, a centrifugal acceleration of 1000G, and 10 minutes to obtain a suspension. Next, ammonium bicarbonate adjusted to 50 mM was added to the suspension in an amount twice the liquid volume of the suspension and mixed. The suspension containing the ammonium bicarbonate was further filtered with a 0.2 μm filter (manufactured by Sartorius Co., Ltd.) to obtain each sample for measuring the amount of EOG in the brain (each sample before oral administration (0 hours), 1 hour, 2 hours, and 4 hours after oral administration. These are collectively referred to as "sample c" below).

[0104] The amount of EOG in the above samples a, b, and c was quantitatively analyzed by LC-MS / MS under the conditions described below. Since each sample was appropriately diluted, the actual amount of EOG was calculated by correcting using the following formula. Actual EOG amount (μM) = analytical value (nM) × dilution conversion factor / 1000 (unit conversion).

[0105] The results are shown in Figures 1, 2 and 3. Figure 1 is a graph showing the amount of EOG in the plasma (sample a) of a male mouse after a predetermined time has elapsed since EOG was forcibly orally administered to the male mouse. Figure 2 is a graph showing the amount of EOG in the cerebrospinal fluid (sample b) of a male mouse after a predetermined time has elapsed since EOG was forcibly orally administered to the male mouse. Figure 3 is a graph showing the amount of EOG in the brain (sample c) of a male mouse after a predetermined time has elapsed since EOG was forcibly orally administered to the male mouse. The amounts of EOG shown in Figures 1 to 3 are average values ​​obtained from two male mice.

[0106] The quantitative analysis by LC-MS / MS was carried out under the following conditions. HPLC equipment: ACQUITY UPLC H-Class Bio (Waters Corporation) Column: Hypersil GOLD PFP 2.1 x 150 mm, 5 μm (Thermo Fisher Scientific. Inc.) Column temperature: 40°C (linear gradient) Mobile phase: (A) Aqueous solution containing 0.2% formic acid and 2 mM ammonium acetate (B) 100% methanol (Gradient settings) Time (min) Flow rate (μL / min) Mass % of mobile phase (A) Initials 200 98 3.50 200 98 3.51 400 5 7.00 400 5 7.10 200 98 17.00 200 98 Injection volume: 0.5 μl.

[0107] MS / MS device: "Xevo TQ-XS", manufactured by Waters Corporation Ionization method: Positive ESI Capacitor (kV):1 Desolvation temperature(℃):500 Source temperature(℃):150 MRM conditions: Peptide (abbreviation) precursor ion (m / z) product ion (m / z) Glu-Hyp-Gly(EOG) 318 225.

[0108] <Consideration> According to Figures 1 to 3, in male mice to which EOG was forcibly orally administered, it is understood that EOG migrates into the blood and then into the cerebrospinal fluid. It is understood that EOG that has migrated into the cerebrospinal fluid also migrates into brain tissue in a short time of about 1 hour after oral administration. From the above, it was suggested that EOG ingested orally is delivered to brain tissue via the blood, and a portion of it is delivered to brain tissue via the cerebrospinal fluid.

[0109] Although the embodiments and examples of the present invention have been described above, it is intended from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0110] The embodiments and examples disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present invention is defined by the claims, not the above description, and is intended to include all modifications within the meaning and scope of the claims.

Claims

1. The present invention comprises a peptide having an amino acid sequence represented by Glu-Hyp-Gly or a salt thereof, The peptide or its salt is a brain function regulating agent which is a tripeptide represented by Glu-Hyp-Gly or its salt, The brain function regulator is for improving memory or cognitive function by directly acting on brain nerve cells through the blood-brain barrier to promote the differentiation of the brain nerve cells, The brain nerve cells are cerebellar granule cells, and the cerebellar brain function regulator.

2. The brain function regulating agent according to claim 1 , wherein the concentration of the brain function regulating agent is 7 μM or more.

3. The brain function regulating agent according to claim 1 or 2, wherein the peptide is derived from collagen.

4. The brain function regulating agent according to any one of claims 1 to 3, which has an effect of inhibiting cell death of brain nerve cells.

5. A brain function regulating agent according to any one of claims 1 to 3, It is for improving memory or cognitive function by promoting the differentiation of brain nerve cells by directly acting on the brain nerve cells through the blood-brain barrier, The food or beverage, wherein the brain nerve cells are cerebellar granule cells.

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