Brain function modulators

Abstract

A brain function regulator containing a peptide, a salt thereof or a chemically modified product thereof, wherein the peptide comprises an amino acid sequence represented by Glu-Hyp-Gly.

Description

Technical Field

[0001] The present invention relates to a brain function regulator. Background Art

[0002] It is known that collagen hydrolysate (hereinafter also referred to as "collagen peptide mixture") exhibits a variety of physiological activities on living organisms. Recently, it has been reported that collagen peptide mixtures exhibit physiological activities for brain nerve cells, etc., and have an improving effect on brain functions (such as memory) (hereinafter, also referred to as "brain function improvement effect"). For example, Japanese Patent Laid-Open No. 2010-105996 (PTL1) discloses a neurogenesis promoter comprising a total of nine collagen-derived peptides, including peptides represented by Gly-Pro-Arg. Japanese Patent Laid-Open No. 2011-111440 (PTL2) discloses that peptides derived from collagen and represented by Gly-Pro-Ala have the effect of promoting neurogenesis. NPL1 reported that applying a fish-derived collagen peptide mixture to aged model mice increased the expression level of BDNF (brain-derived neurotrophic factor) in the hippocampus, thereby enhancing spatial learning and memory ability and passive avoidance ability. NPL2 reported that, in an experiment using rats, a collagen-derived dipeptide represented by Pro-Hyp exhibited an antidepressant effect when transferred into the cerebrospinal fluid. NPL3 reported that collagen hydrolysate promoted neurogenesis in the hippocampus.

[0003] Citation List

[0004] Patent Literature

[0005] PTL 1: Japanese Patent Laying-Open No.2010-105996

[0006] PTL 2: Japanese Patent Laying-Open No.2011-111440

[0007] Non-patent literature

[0008] NPL1: Pei Xinrong et al., "Preventive Effect of Marine Collagen Peptide onLearning and Memory Impairment in SAMP8 Mice", Food and Fermentation Industries, 2009, Vol 135(07), p.1-5

[0009] NPL2: Nagai et al., "Identification of Antidepressant Collagen Peptide and the Penetration into Cerebrospinal Fluid", Proceedings of 2017Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, 5March, 2017, p.997 NPL3: 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

[0010] Technical issues

[0011] NPL1 and NPL3 do not specify which collagen-derived peptides produce the disclosed brain function improvement effect or neurogenesis effect. PTL1 does not specify which of the nine collagen-derived peptides have the disclosed effect of promoting neurogenesis. The effect of promoting neurogenesis disclosed in PTL2 is a discovery obtained from an experiment using PC12 cells derived from rat adrenal pheochromocytoma, rather than a discovery obtained from an experiment targeting brain nerve cells. In addition, Pro-Hyp disclosed in NPL2 shows a neurogenesis effect in the dentate gyrus of the hippocampus, but it has not yet been confirmed whether Pro-Hyp can differentiate and grow brain nerve cells to improve brain function. Therefore, it is not known that collagen-derived peptides can produce the effect of improving brain function by directly acting on brain nerve cells without the need for signal transducers (such as hormones) in the body, and therefore it is expected to develop such peptides.

[0012] In view of the above circumstances, an object of the present invention is to provide a brain function regulator comprising a peptide or the like, which produces at least one of an effect of improving brain function and an effect of preventing brain function decline by directly acting on brain nerve cells.

[0013] Solution to the Problem

[0014] In developing a brain function regulator comprising peptides and the like that produces at least one of an effect of improving brain function and an effect of preventing brain function decline by directly acting on brain nerve cells, the present inventors focused on Glu-Hyp-Gly (glutamic acid-hydroxyproline-glycine, hereinafter referred to as "EOG", where each amino acid is represented by one character) in the amino acid sequence of the peptide contained in the collagen peptide mixture. The present inventors found that when acting on brain nerve cells, the collagen peptide mixture promotes the differentiation of brain nerve cells, thereby producing an effect of improving brain function and an effect of preventing brain function decline, and thus completed the present invention, wherein the collagen peptide mixture contains a peptide comprising an amino acid sequence represented by Glu-Hyp-Gly, in particular a tripeptide represented by Glu-Hyp-Gly, and the like. Specifically, the present invention is as described below.

[0015] The brain function regulator according to the present invention comprises a peptide comprising an amino acid sequence represented by Glu-Hyp-Gly, or a salt thereof or a chemically modified product thereof.

[0016] Preferably, the peptide, a salt thereof, or a chemically modified product thereof is a tripeptide represented by Glu-Hyp-Gly, a salt thereof, or a chemically modified product thereof.

[0017] Preferably, the peptide is derived from collagen.

[0018] Preferably, the weight average molecular weight of the peptide is 315 or more and 10,000 or less.

[0019] Preferably, the brain function regulator is a brain function improving agent or a brain function decline preventing agent.

[0020] Preferably, the brain function regulator has the effect of promoting the differentiation of brain nerve cells.

[0021] Preferably, the brain function regulator has an effect of inhibiting brain nerve cell death.

[0022] Preferably, the brain function regulator has an effect of improving memory or cognitive function.

[0023] Beneficial effects of the present invention

[0024] According to the present invention, there can be provided a brain function regulator comprising a peptide or the like, which produces at least one of an effect of improving brain function and an effect of preventing brain function decline by directly acting on brain nerve cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the amount of EOG in the plasma (sample a) of male mice after a predetermined period of time has passed after EOG was gavage-administered to the male mice.

[0026] Figure 2 This is a graph showing the amount of EOG in the cerebrospinal fluid (sample b) of male mice after a predetermined period of time has passed since EOG was gavage-administered to the male mice.

[0027] Figure 3 This is a graph showing the amount of EOG in the brain (sample c) of male mice after a predetermined period of time has passed after EOG was gavage-administered to the male mice. DETAILED DESCRIPTION

[0028] Hereinafter, embodiments of the present invention will be described in more detail. As used herein, the description in the form of "A to B" represents the upper and lower limits of a range (i.e., above A and below B), and when no unit is described for A and only a unit is described for B, the unit of A is the same as the unit of B.

[0029] [Brain function regulator]

[0030] The brain function regulator according to the present invention comprises a peptide, a salt thereof or a chemically modified product thereof, wherein the peptide comprises an amino acid sequence represented by Glu-Hyp-Gly. The brain function regulator having such characteristics can promote the differentiation of brain nerve cells by directly acting on brain nerve cells, thereby producing an effect of improving brain function and preventing brain function decline.

[0031] <Peptide containing an amino acid sequence represented by Glu-Hyp-Gly, a salt thereof, or a chemically modified product thereof>

[0032] As described above, the brain function regulator comprises a peptide, a salt thereof or a chemically modified product thereof, wherein the peptide comprises an amino acid sequence represented by Glu-Hyp-Gly. Unless otherwise indicated, the term "amino acid" used herein refers to "L-amino acid". The term "peptide comprising an amino acid sequence represented by Glu-Hyp-Gly" means that the amino acid sequence constituting such a peptide comprises one or more amino acid sequences represented by "Glu-Hyp-Gly (glutamic acid-hydroxyproline-glycine)".

[0033] In the brain function regulator, the peptide, its salt or chemically modified product thereof is preferably a tripeptide represented by Glu-Hyp-Gly, its salt or chemically modified product thereof. Preferably, the brain function regulator can more significantly promote the differentiation of brain nerve cells.

[0034] The term "salt" of the peptide may be, for example, an inorganic acid salt such as hydrochloride, sulfate or phosphate; an organic acid salt such as methanesulfonate, benzenesulfonate, succinate or oxalate; an inorganic base salt such as sodium salt, potassium salt or calcium salt; an organic base salt such as triethylammonium salt.

[0035] The term "chemically modified product" of the peptide is a peptide in which the free functional groups of the amino acid residues as the structural unit are chemically modified. Chemical modification can be performed, for example, on the hydroxyl group of hydroxyproline, on the amino group of the amino acid on the N-terminal (amino terminal) side, and on the carboxyl group of the amino acid on the C-terminal (carboxyl terminal) side. The specific method and treatment conditions for chemical modification follow the known conventional techniques for chemical modification of peptides. The chemically modified product of the peptide obtained by such chemical modification can enhance solubility under weakly acidic to neutral conditions, enhance compatibility with other active ingredients, etc.

[0036] For example, the tripeptide of Glu-Hyp-Gly can be O-acetylated, which is a chemical modification of the hydroxyl group in hydroxyproline. The O-acetylation can be performed by applying acetic anhydride to the peptide in an aqueous solvent or a non-aqueous solvent. Esterification, amidation, etc. can be performed, which is a chemical modification of the carboxyl group in glycine. The esterification can be performed by suspending the peptide in methanol and then passing dry hydrogen chloride gas through the resulting suspension. The amidation can be performed by applying a carbodiimide or the like to the peptide.

[0037] Methylation can be performed, which is a chemical modification of the free amino groups in the tripeptide. At least one of phosphorylation and sulfation can be performed, which is a chemical modification of the hydroxyl groups in the tripeptide.

[0038] Preferably, the peptide is derived from collagen. In this article, the collagen as a raw material can be obtained in the following manner: for example, the skin, dermis, bone, cartilage, tendon, etc. of animals (representatively, cattle, pigs, sheep, chickens or ostriches) or the bones, skin, scales, etc. of fish are subjected to known conventional defatting or decalcification treatment, extraction treatment, etc. In addition, gelatin can be used as the raw material of the peptide. The gelatin can be obtained by treating the collagen thus obtained by known conventional methods (e.g., extraction with hot water). For the collagen and gelatin, commercially available products can be used as raw materials.

[0039] The peptide can be obtained by hydrolyzing the collagen and / or the gelatin with two or more endoproteases and exoproteases in combination. The peptide can be obtained as a collagen peptide mixture, which exists together with other collagen peptides due to hydrolysis, but any of the collagen peptide mixture itself and the mixture obtained by partially purifying the collagen peptide mixture can be used as a brain function regulator according to the present invention. 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 preferably obtained by using a method as described below in which collagen or gelatin is subjected to a two-stage enzyme treatment.

[0040] In addition, the weight average molecular weight of the peptide is preferably 315 or more and 10000 or less. The weight average molecular weight of the peptide is more preferably 315 or more and 5000 or less, more preferably 315 or more and 2000 or less. When the weight average molecular weight of the peptide is within the above range, the brain function regulator can more significantly promote the differentiation of brain nerve cells, thereby producing a more sufficient effect of improving brain function and preventing brain function decline. When the weight average molecular weight of the peptide is greater than 10000, the brain function regulator may have insufficient brain function improvement effect and brain function decline prevention effect.

[0041] The weight average molecular weight of the peptide can be determined by performing size exclusion chromatography (SEC) under the following measurement conditions.

[0042] Instrument: High Performance Liquid Chromatography (HPLC) (manufactured by TOSOH CORPORATION)

[0043] Column: TSKGel (registered trademark) G2000SW XL

[0044] Column temperature: 40°C

[0045] Eluent: 45 mass% acetonitrile (containing 0.1 mass% TFA)

[0046] Flow rate: 1.0 mL / min

[0047] Injection volume: 10 μL

[0048] Detection: UV 214 nm

[0049] Molecular weight markers: The following five types are used

[0050] Cytochrome C Mw: 12,000

[0051] Aprotinin Mw: 6,500

[0052] Bacitracin Mw: 1,450

[0053] Gly-Gly-Tyr-Arg Mw:451

[0054] Gly-Gly-Gly Mw:189

[0055] Specifically, a sample containing about 0.2 g of peptide (collagen peptide mixture) is added to about 100 ml of distilled water, the mixture is stirred, and then filtered with a 0.2 μm filter to prepare a sample for measuring the weight average molecular weight (measurement sample). The weight average molecular weight of the peptide can be determined by performing size exclusion chromatography on the measurement sample. When the peptide is a tripeptide represented by Glu-Hyp-Gly, its molecular weight can be a weight average molecular weight.

[0056] <Method for preparing brain function regulator>

[0057] The peptide contained in the brain function regulator and comprising an amino acid sequence represented by Glu-Hyp-Gly can be obtained by using a known conventional liquid phase or solid phase peptide synthesis method or a method including hydrolyzing collagen or gelatin. For example, from the viewpoint of efficiency, it is preferred to prepare the peptide by using a chemical synthesis method using amino acids as described below, or a method including two-stage enzyme treatment of collagen or gelatin as described below. In addition, the peptide can be produced by using a method including enzyme treatment with only a secondary enzyme and omitting a primary enzyme, or a method including enzyme treatment with a primary enzyme and a secondary enzyme at the same time, rather than using a method including two-stage enzyme treatment of collagen or gelatin.

[0058] (Chemical Synthesis Method)

[0059] The peptide can be obtained by using a conventional peptide synthesis method. As a peptide synthesis method, a solid phase synthesis method and a liquid phase synthesis method are known. As a solid phase synthesis method, an Fmoc method and a Boc method are known. The peptide can be obtained by using any one of the Fmoc method and the Boc method. Hereinafter, a method for synthesizing a tripeptide represented by, for example, Glu-Hyp-Gly will be described, which is a solid phase peptide synthesis method.

[0060] First, beads of polystyrene polymer gel having a diameter of about 0.1 mm and a surface modified with amino groups are provided as solid phases. In addition, diisopropylcarbodiimide is provided as a condensing agent. Next, the amino group of glycine is protected with Fmoc (fluorenyl-methoxy-carbonyl), which is an amino group on the C-terminal (carboxyl terminal) side of the amino acid sequence, and the carboxyl group of the above-mentioned glycine is peptide-bonded to the amino group on the above-mentioned solid phase by a dehydration reaction using a condensing agent. In addition, the solid phase is washed with a solvent to remove the residual condensing agent and amino acid, and then the protective group (deprotection) of the amino group of the glycine that is peptide-bonded to the solid phase is removed.

[0061] Subsequently, hydroxyproline in which the amino group is protected by an Fmoc group is provided, and the carboxyl group of the hydroxyproline is peptide-bonded to the deprotected amino group of glycine by using a condensing agent. Afterwards, in the same manner as above, the amino group of the hydroxyproline is deprotected, glutamic acid protected by an Fmoc group is provided, and a reaction is performed to peptide-bind glutamic acid to the hydroxyproline, thereby synthesizing a tripeptide represented by Glu-Hyp-Gly on a solid phase. Finally, the amino group of the glutamic acid is deprotected, a solution containing trifluoroacetic acid is added at room temperature, and the mixture is shaken for a fixed time to separate the tripeptide from the solid phase. In this way, the tripeptide can be produced.

[0062] (Production method using collagen and gelatin)

[0063] Hereinafter, a method for preparing a tripeptide represented by Glu-Hyp-Gly (hereinafter also referred to as a "specific peptide") by subjecting collagen or gelatin to two-stage enzyme treatment will be described as an example of a method for preparing the peptide.

[0064] The term "two-stage enzyme treatment (collagen or gelatin)" refers to the following. That is, the primary enzyme treatment is carried out by a known conventional method for breaking the peptide bonds of collagen or gelatin, and then the secondary enzyme treatment is carried out with 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 carrying out the primary enzyme treatment, a collagen peptide mixture precursor can be obtained. By further carrying out the secondary enzyme treatment, a collagen peptide mixture containing specific peptides can be obtained from the collagen peptide mixture precursor. The method for two-stage enzyme treatment of collagen or gelatin will be described in more detail below.

[0065] -Primary enzyme treatment-

[0066] The enzyme used in the primary enzyme treatment is not particularly limited as long as it is an enzyme that can destroy the peptide bonds of collagen or gelatin, and any proteolytic enzyme can be used. Specifically, examples thereof include collagenase, thiol protease, serine protease, acidic protease, alkaline protease and metalloprotease. One selected from the group consisting of these enzymes can be used alone, or two or more thereof can be used in combination. As thiol protease, chymopapain, papain, bromelain and ficin derived from plants, tissue proteases and calcium-dependent proteases derived from animals, etc. can be used. As serine protease, trypsin, cathepsin D, etc. can be used. As acidic protease, pepsin, chymotrypsin, etc. can be used. Considering that brain function regulators are used in medicines, etc., preferably, those enzymes other than enzymes derived from pathogenic microorganisms are used as the enzymes used in the primary enzyme treatment.

[0067] Based on 100 parts by mass of collagen or gelatin, the amount of the enzyme in the primary enzyme treatment is, for example, preferably 0.1 to 5 parts by mass of the above enzyme. Preferably, the treatment temperature and treatment time in the primary enzyme treatment are 30 to 65 ° C and 10 minutes to 72 hours, respectively. The weight average molecular weight of the collagen peptide mixture precursor obtained by the primary enzyme treatment is preferably 500 to 10000, more preferably 500 to 5000, and even more preferably 500 to 2000. It can be said that when the weight average molecular weight is within the above range, peptides with appropriate molecular weight can be appropriately produced. If necessary, the enzyme can be inactivated after the primary enzyme treatment. In this case, the inactivation temperature is, for example, preferably 70 to 100 ° C. The weight average molecular weight of the collagen peptide mixture precursor can be determined by using the SEC method.

[0068] -Secondary enzyme treatment-

[0069] Examples of enzymes used in the secondary enzyme treatment include enzymes with aminopeptidase N activity, enzymes with aminopeptidase N activity and prolyl tripeptidyl aminopeptidase activity, or enzymes with aminopeptidase N activity and enzymes with prolyl tripeptidyl aminopeptidase activity. As used herein, the term "enzyme with aminopeptidase N activity" is a peptidase having the function of releasing amino acids from the N-terminal side of a peptide chain, wherein when an amino acid other than proline or hydroxyproline is present in the second position of the N-terminal side, the enzyme works. As used herein, the term "enzyme with prolyl tripeptidyl aminopeptidase activity" is a peptidase that releases only three amino acid residues from the N-terminal side of a peptide, and the peptide has proline or hydroxyproline at the third position of the N-terminal side. Considering that brain function modulators are used in medicines, etc., preferably, enzymes other than enzymes derived from pathogenic microorganisms are used as enzymes used in the secondary enzyme treatment.

[0070] Examples of enzymes having aminopeptidase N activity include aminopeptidase N (EC 3.4.11.2.; T. Yoshimoto et al., Agric. Biol. Chem., 52: 217-225 (1988)), and enzymes having aminopeptidase N activity derived from Aspergillus. Examples of enzymes having prolyl tripeptidyl aminopeptidase activity include prolyl tripeptidyl aminopeptidase (EC 3.4.14.; A. Banbula et al., J. Biol. Chem., 274: 9246-9252 (1999)).

[0071] By performing 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 specific peptides can be obtained.

[0072] Based on 100 parts by mass of the collagen peptide mixture precursor, the amount of the enzyme in the secondary enzyme treatment is, for example, preferably 0.01 to 5 parts by mass of the above enzyme. Preferably, the treatment temperature and treatment time in the secondary enzyme treatment are 30 to 65°C and 1 to 72 hours, respectively. 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 be determined by using the SEC method.

[0073] The secondary enzyme treatment is mainly carried out to produce specific peptides. Therefore, it is preferred 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. Therefore, the weight average molecular weight of the collagen peptide mixture is preferably within the above range. It is necessary to inactivate the enzyme after the secondary enzyme treatment. In this case, the inactivation temperature is preferably 70 to 100°C, for example. In addition, it is preferred to perform a sterilization treatment at 120°C for several seconds or longer. In addition, the collagen peptide mixture can be spray-dried by heating at 200°C or higher.

[0074] In the secondary enzyme treatment, not only can use the enzyme with aminopeptidase N activity and the enzyme with prolyl tripeptidyl aminopeptidase activity, but also can use enzymes with different activities, and can use two or more enzymes with different activities respectively in combination.Therefore, by-products can be digested and removed.Preferably, according to the type of collagen used as raw material and the type of enzyme used in the primary enzyme treatment, the enzyme used in this case is appropriately selected.The example of different activities includes dipeptidase activity, such as proline peptidase (prolidase) activity and hydroxyproline peptidase (hydroxyprolidase) activity.Therefore, by-products (such as dipeptides) can be digested and removed.

[0075] In addition, aminopeptidase N activity is basically an activity that causes the amino acids on the N-terminal side to be released one by one. Therefore, in the case where the collagen peptide mixture precursor obtained by the primary enzyme treatment contains a very large peptide of molecular weight, when the secondary enzyme treatment is carried out only with an enzyme having aminopeptidase N activity, the duration of the secondary enzyme treatment is significantly increased. In order to cope with this situation, for example, a prolyl oligopeptidase can be used in the secondary enzyme treatment, and the proline oligopeptidase is an endopeptidase with an activity (proline peptidase activity) that causes the hydrolysis of proline on the carboxyl side. Therefore, the secondary enzyme treatment can be effectively carried out.

[0076] In a method comprising two-stage enzyme treatment of collagen or gelatin, the primary enzyme treatment is capable of producing a peptide having a relatively large molecular weight. The peptide may have, for example, an amino acid sequence represented by [X1-Gly-X2-Glu-Hyp-Gly] (X1 and X2≠Hyp). In the subsequent secondary enzyme treatment, an enzyme having aminopeptidase N activity acts on a peptide represented by [X1-Gly-X2-Glu-Hyp-Gly], thereby releasing X1 at the N-terminus 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 a peptide represented by [Gly-X2-Glu-Hyp-Gly], thereby releasing glycine and X2 to obtain a peptide represented by [Glu-Hyp-Gly]. In this way, a specific peptide can be produced.

[0077] -Purification of collagen peptide mixture-

[0078] By performing two-stage enzyme treatment as described above, a collagen peptide mixture containing a specific peptide can be prepared. Since the collagen peptide mixture contains peptides other than the specific peptide, that is, peptides other than the tripeptide represented by Glu-Hyp-Gly, it is preferred to purify the collagen peptide mixture if necessary. As a purification method in this case, known conventional methods can be used, examples of which include ultrafiltration and various types of liquid chromatography, such as size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and affinity chromatography.

[0079] Specifically, the collagen peptide mixture can be purified according to the following steps. That is, about 2g / 10ml of the collagen peptide mixture is loaded into an ion exchange column (e.g., "TOYOPEARL" (registered trademark) DEAE-650" (trade name) manufactured by TOSOH CORPORATION), and then the first pore volume fraction eluted with distilled water is collected. Subsequently, the first pore volume fraction is loaded into a column having an ion exchange group opposite to the ion exchange group of the above-mentioned ion exchange column (e.g., "TOYOPEARL" (registered trademark) SP-650 manufactured by TOSOH CORPORATION), and then the second pore volume fraction eluted with distilled water is collected.

[0080] Next, the second pore volume fraction is loaded into a gel filtration column (e.g., "SEPHADEX LH-20" (trade name) manufactured by GE Healthcare Japan Corporation), and eluted with a 30% by mass methanol aqueous solution to collect a fraction containing a specific peptide. Finally, the fraction is eluted using a reverse phase column (e.g., "μBondasphere 5μC18 The fractions were fractionated by high performance liquid chromatography (HPLC) using a HPLC Column (trade name) according to a linear concentration gradient of a 32 mass % or less acetonitrile aqueous solution containing 0.1 mass % of trifluoroacetic acid. In this way, the specific peptide can be obtained with high purity.

[0081] <Brain function improving agent or brain function decline preventing agent>

[0082] The brain function regulator of the present invention is preferably a brain function improver or a brain function decline preventive. The brain function regulator contains a peptide, a salt thereof or a chemically modified product thereof, and the peptide comprises an amino acid sequence represented by Glu-Hyp-Gly, and, as described in the following [Evaluation Test 1] and [Evaluation Test 2], can promote the differentiation of brain nerve cells by directly acting on brain nerve cells. In this way, the brain function regulator can produce a brain function improvement effect and a brain function decline preventive effect. Therefore, the brain function regulator can be used as a brain function improver for treating patients with brain function decline. The brain function regulator can also be used as a brain function decline preventive to prevent brain function decline caused by aging, etc.

[0083] Preferably, the brain function regulator has the effect of promoting the differentiation of brain nerve cells. In addition, preferably, the brain function regulator has the effect of inhibiting the death of brain nerve cells. The brain function regulator also preferably has a memory or cognitive function improvement effect. As described in the following [Evaluation Test 1] and [Evaluation Test 2], the brain function regulator can show the effect of promoting the differentiation of brain nerve cells by directly acting on brain nerve cells. According to the following [Evaluation Test 1] and [Evaluation Test 2], it can be considered that the brain function regulator has the effect of inhibiting the death of brain nerve cells. Through these effects, the brain function regulator can show memory or cognitive function improvement effects.

[0084] The brain function regulator can be administered orally or parenterally in various forms. For these forms, the brain function regulator can take the following dosage forms when administered orally, such as tablets, granules, capsules, powders, liquids, suspensions and emulsions.

[0085] When administered parenterally, the brain function regulator can take the following dosage forms, such as preparations injected into the brain, injections, transdermal preparations, suppositories, nasal preparations and inhalants. Preferred dosage forms of the brain function regulator include tablets, granules, capsules, powders and liquids directly injected into the brain. The brain function regulator contains a tripeptide such as Glu-Hyp-Gly, and the tripeptide is rapidly absorbed in the intestinal tract and can therefore be taken orally.

[0086] The dosage of the brain function regulator varies according to the age, sex, weight and sensitivity difference of the subject, the method of administration, the interval of administration, the type of preparation, etc. When the brain function regulator is orally administered, the dosage of each adult is, for example, preferably 0.0001 to 2500 mg / kg, more preferably 0.0001 to 500 mg / kg. When the dosage form of the brain function regulator is, for example, a tablet, the amount of the brain function regulator contained in the tablet can be 0.001 to 80% by mass per tablet, and when the dosage form of the brain function regulator is, for example, a powder, the amount of the brain function regulator contained in the powder can be 0.001 to 100% by mass. When the brain function regulator is administered parenterally or by another form of preparation, the dosage can be appropriately determined with reference to the dosage of oral administration. The brain function regulator can be administered once a day or several times, or once a day or once every few days.

[0087] The brain function regulator can suitably include other active ingredients, formulation carriers, etc., as long as the effect of the present invention is not adversely affected. The example of other active ingredients includes docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), astaxanthin, ginkgo leaf extract and arachidonic acid. In addition, the example of the pharmaceutically acceptable carrier for being prepared into pharmaceutical preparations includes diluent, adhesive (syrup, gum arabic, gelatin, sorbitol, tragacanth and polyvinyl alkanoate), excipient (lactose, sucrose, corn starch, potassium phosphate, sorbitol and glycine), lubricant (magnesium stearate, talc, polyethylene glycol and silicon dioxide), disintegrant (potato starch) and wetting agent (sodium lauryl sulfate).

[0088] <Use invention>

[0089] The brain function regulator according to the present invention comprises a peptide, a salt thereof or a chemically modified product thereof, and the peptide comprises an amino acid sequence represented by Glu-Hyp-Gly. The brain function regulator has the effect of promoting the differentiation of brain nerve cells, and is an unknown property of a peptide (such as a tripeptide represented by Glu-Hyp-Gly) comprising an amino acid sequence represented by Glu-Hyp-Gly, therefore, at least one of a brain function improvement effect and a brain function decline prevention effect can be obtained. In other words, the present invention is a peptide (which comprises an amino acid sequence represented by Glu-Hyp-Gly) for regulating brain function, a salt thereof or a chemically modified product thereof.

[0090] Example

[0091] Hereinafter, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

[0092] [Evaluation Test 1: Clinical Test]

[0093] <Test products (products containing brain function regulators)

[0094] As a test product, a collagen peptide mixture containing an amino acid sequence represented by Glu-Hyp-Gly ("Collagen Peptide CP-B" (trade name) manufactured by Nitta Gelatin Inc.) was used. During the test period described below, 5 g of the test product added to a beverage was administered to the subject once a day. The administration time of the beverage was not specified.

[0095] <Test Design>

[0096] The test period was selected for four consecutive weeks from October 20 to November 21, 2016. The open-label intervention before and after comparison test was conducted at the Kokoro Research Center of Kyoto University. In this test, the informed consent procedure was carried out for all subjects in accordance with the spirit of the World Medical Association's Declaration of Helsinki, and written consent was obtained from all subjects. The Ethics Review Committee of the Advanced Research Unit of Psychology, Kyoto University approved this test on September 18, 2015 (approval number 27-P-13).

[0097] In this test, the gray matter volume-brain health quotient (GM-BHQ) score, the fractional anisotropy-brain health quotient (FA-BHQ) score, the mild cognitive impairment (MCI) score, and the standard verbal paired associate learning (S-PA) score were obtained to evaluate the effects of the test products on brain function.

[0098] <Subject>

[0099] In this test, the subjects were 30 healthy people aged between 49 and 63 years old (mean age: 56.1±3.6 years old). The main criteria for the eligibility of the subjects were as follows: "People who have not taken collagen hydrolysate within one month before the start of the test", "People who have no history of major food allergies including gelatin allergy", and "People who have no history of neurological diseases (such as cerebral infarction and dementia) and psychological diseases". In addition, people who belong to the items described in Section 6 of "https: / / www.acrin.org / Portals / 0 / Protocols / 6684 / ACRIN6684_Amend7_012412_mas ter_ForOnline.pdf" were excluded from the subjects. During the test, one subject withdrew for personal reasons. In addition, five subjects did not undergo the test to obtain the S-PA score due to personal reasons. Therefore, statistical analysis was performed on 24 subjects who underwent the test to obtain the S-PA score and 29 subjects who underwent other tests according to the test protocol.

[0100] <Evaluation methods and evaluation results>

[0101] (MRI Scan)

[0102] Magnetic resonance imaging (MRI) scans were performed one day before the start of administration of the test product (hereinafter, also referred to as "pre-intervention") and one day after the end of administration of the test product (29th day after the start of administration; hereinafter also referred to as "post-intervention"). The MRI imaging before the intervention was performed between October 20 and October 24, 2016, and the MRI imaging after the intervention was performed between November 18 and November 21, 2016. The MRI imaging was performed at the Kokoro Research Center, Kyoto University. The specific MRI scanning method followed the method described in "Nemoto K et al., "MRI-based Brain Healthcare Quotients: Abridge between neural and behavioral analyses for keeping the brain healthy", PLoS ONE, 2017, 12(10): e0187137 (https: / / doi.org / 10.1371 / journal.pone.0187137)".

[0103] As the MRI equipment, a 3T scanner "Verio" of Siemens AG, Germany was used, and MRI imaging was performed with a 32-channel head array coil. For three-dimensional T1-weighted images, the MP-RAGE pulse sequence was used. The parameters are as follows. Repetition time (TR): 1900 milliseconds; echo time (TE): 2.52 milliseconds; inversion time (TI): 900 milliseconds; flip angle: 9°; matrix size: 256×256; field of view (FOV): 256 mm; layer thickness: 1 mm. Diffusion tensor imaging (DTI) was acquired by spin echo-echo planar imaging (SE-EPI) using GRAPPA. The layer of diffusion tensor imaging was parallel to the OM line.

[0104] 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. A baseline image (b=0 s / mm 2 ) and 30 diffusion directions, where b = 1000s / mm 2 .

[0105] Gray matter was extracted from T1-weighted images using SPM 12, a brain functional mapping tool, and the GM-BHQ score of the whole brain was calculated based on the gray matter volume and cranial volume. Fractional anisotropy (FA) images were generated from diffusion tensor imaging using the software FSL 5.0.11, and the FA-BHQ score of the whole brain was calculated based on the FA images.

[0106] Here, the tool used for statistical analysis performed in the process of calculating the GM-BHQ score and the FA-BHQ score is the medical statistics program "STAT Mate III". In order to evaluate the significance, the intra-group comparison before and after the intervention was performed according to the Wilcoxon rank sum test, and it was determined that the significance existed when the significance level (P value) was 0.05 or less. Table 1 below shows the values ​​before and after the intervention for each of the GM-BHQ score and the FA-BHQ score, the difference between these values, and the Pvalue (P value). In Table 1, the character "N" represents the number of subjects, the term "baseline" represents the value before the intervention, the term "after" represents the value after the intervention, and the symbol "Δ" represents the difference before and after the intervention.

[0107] [Table 1]

[0108]

[0109] *Based on Wilcoxon rank sum test

[0110] As shown in Table 1, the FA-BHQ score after the intervention was significantly improved compared to the FA-BHQ score before the intervention. The GM-BHQ score did not improve significantly. Therefore, the test product (a collagen peptide mixture containing a tripeptide represented by Glu-Hyp-Gly as a brain function regulator) was shown to have a brain function regulating effect.

[0111] (MCI score)

[0112] Mild cognitive impairment (MCI) score is calculated by using "About MCI Screen (Millennia Corporation manufacture)" as a cognitive function examination scale. Specifically, the subject learned 10 words, and the number of words (in the 10 words) that the subject can report verbally after listening to comments unrelated to the test was determined. According to information such as sex, age, years of study and race, the test results of each subject were compared with those of the same age group, and demographic assessment was performed in an objective manner to correct the MCI score of each subject. The test was performed on the same day that each subject was scanned with an MRI.

[0113] (S-PA) score

[0114] The Japanese Association for Advanced Brain Dysfunction has developed a standard verbal paired associative learning (S-PA) score for understanding human verbal memory. The specific steps of the test of the S-PA score are as follows. First, a sheet of paper with 10 pairs of related paired words (semantically related words) and 10 pairs of irrelevant paired words (semantically almost irrelevant words) is provided. Next, the examiner verbally reads the 10 pairs of related paired words described on the paper and is learned by the subject. Thereafter, the examiner shows one word in each pair of paired words, and the subject reports the words paired with the displayed words. Next, the examiner verbally reads the 10 pairs of irrelevant paired words described on the paper and is learned by the subject. Thereafter, the examiner shows one word in each pair of paired words, and the subject reports the words paired with the displayed words. Finally, the S-PA score of each subject is calculated according to the number of words correctly reported by the subject. The 10 pairs of related paired words and 10 pairs of irrelevant paired words described on the sheet provided before the intervention are different from those described on the sheet provided after the intervention. The test was performed on the same day when each subject was scanned with MRI.

[0115] Here, "STAT Mate III" is also used as a tool for statistical analysis in the process of calculating MCI scores and S-PA scores. In order to evaluate significance, the intra-group comparison before and after the intervention was performed according to the Wilcoxon rank sum test, and it was determined that when the significance level (P value) was 0.05 or lower, there was significance. Table 2 below shows the values ​​before and after the intervention for each of the MCI score and S-PA score, the difference between these values, and the Pvalue (P value). In Table 2, the character "N" represents the number of subjects, the term "baseline" represents the value before the intervention, the term "after" represents the value after the intervention, and the symbol "Δ" represents the difference before and after the intervention.

[0116] [Table 2]

[0117]

[0118] *Based on Wilcoxon rank sum test

[0119] As shown in Table 2, compared with the scores before the present invention, the MCI scores and S-PA scores after the intervention were significantly improved. When the results in Table 1 are reviewed, the results in Table 2 show that the test product has an improvement in memory or cognitive function through its brain function regulation, thereby producing an improvement in brain function. In addition, the above results show that the test product also has the effect of preventing brain function from declining. Therefore, the brain function regulator can be used as a brain function improver or a brain function decline preventive.

[0120] (Effects of brain function modulators on brain function in individual subjects)

[0121] Whether the increase or decrease of GM-BHQ score and the increase or decrease of FA-BHQ score are correlated with the increase or decrease of MCI score and the increase or decrease of S-PA score in individual subjects was examined to study the effect of the brain function modulator on the brain function of the individual subjects. For correlation, the Spearman rank correlation coefficient (r) was calculated based on the measured values, and the values ​​obtained before and after the intervention were statistically processed. For statistical processing, "STAT Mate III" was used. It has been determined that when r is 0.2 or less, "there is no correlation"; when r is greater than 0.2 and less than or equal to 0.4, "the correlation is low"; when r is greater than 0.4 and less than or equal to 0.7, "there is a correlation"; when r is greater than 0.7 and less than 1, "there is a strong correlation"; and when r is 1, "there is a complete correlation". Table 3 shows the value of r as the evaluation result.

[0122] [Table 3]

[0123]

[0124]

[0125] #: P < 0.05, based on Spearman rank correlation coefficient (r)

[0126] As shown in Table 3 , there was a correlation between an increase or decrease in the GM-BHQ score and an increase or decrease in the MCI score, and there was a correlation between an increase or decrease in the FA-BHQ score and an increase or decrease in the S-PA score.

[0127] <Discussion>

[0128] Based on the above results, it can be inferred that the brain function regulator containing a specific peptide has an effect on the brain function of an individual subject, so that the brain function regulator can improve the FA-BHQ score and thus improve the S-PA score, and can also improve the GM-BHQ score and thus improve the MCI score. Therefore, the brain function regulator can be used as a brain function improver or a brain function decline preventive.

[0129] [Evaluation Test 2: Cell Biology Test (In vitro Test)

[0130] <Sample Preparation>

[0131] All animal experiments were in accordance with the "Standards for the Care and Care of Laboratory Animals" of the Cabinet Administration General Administration and the "Animal Experiment Guidelines" of the Life Science Research Center of Josai University. Male and female Wistar rats aged 15 to 25 weeks were provided by purchase from CREA Japan, Inc. Thereafter, the rats were kept under the following conditions: the temperature was 23±2°C, the relative humidity was 55±10%, the lighting cycle was 12 hours, the lighting cycle started at 7:00 and ended at 19:00, and the rats were fed solid feed ("CE-2" (trade name) manufactured by CREA Japan, Inc) and allowed free access to drinking water. Under the same conditions as above, the rats were mated to obtain 7-day-old rats. In addition, 3- to 8-day-old Wistar rats were provided by purchase from CREA Japan, Inc.

[0132] (Preparation and culture of primary cultured cerebellar granule cells (CGC))

[0133] Wistar rats were raised under the above conditions to an age of 7 to 9 days, and then the cerebellum was cut from these rats and dispersed by the following method using the dispersion solutions (liquids I to V) shown in Table 4. In this way, the dispersion contains granule cells and other brain nerve cells from the cerebellum (hereinafter, these cells are also collectively referred to as "cerebellar granule cells (CGC)"). The cerebellar granule cells (CGC) are brain nerve cells. Afterwards, the CGC contained in the dispersion is cultured. Table 4 shows the "solution name", "agents included" and "amounts" of the dispersion solution. The method for preparing the cerebellar granule cells (CGC) is as follows.

[0134] [Table 4]

[0135]

[0136] First, ten of the 7 to 9-day-old rats were decapitated, and the cerebellum was cut off on a clean workbench, and then formed into a minced paste with a razor after removing unnecessary tissues, blood vessels, chorion, etc. Liquid I was added to the paste to obtain a suspension, and the suspension was centrifuged at 1000 rpm for 30 seconds to obtain a first separation liquid containing a supernatant and a cell mass. The supernatant was removed from the first separation liquid, and then Liquid II preheated to 37° C. in a constant temperature bath was added little by little to the remaining cell mass, and the mixture was stirred, moved to a 50 mL medium-sized bottle, and further stirred at 37° C. for 15 minutes to perform tissue digestion with trypsin.

[0137] Next, liquid IV is added to the medium-sized bottle to inactivate trypsin, and the mixture is centrifuged at 1000rpm for 30 seconds to obtain the second separation liquid comprising supernatant and cell mass. From the second separation liquid, remove supernatant, then with Pasteur pipette, a small amount of liquid III is added to the remaining cell mass, so that the cell mass is dispersed. After this, the mixture was left standstill for 15 minutes to obtain the 3rd separation liquid comprising supernatant and cell mass, and the supernatant was taken out into liquid V. A small amount of liquid III is added to the cell mass remaining in the 3rd separation liquid so that the cell mass is dispersed again. After this, the mixture was left standstill for 5 to 10 minutes to obtain the 4th separation liquid containing supernatant and cell mass, and the supernatant was taken out into liquid V containing the supernatant of the 3rd separation liquid.

[0138] The liquid V containing the supernatant of the third separation liquid and the supernatant of the fourth separation liquid was centrifuged at 1000 rpm for 5 minutes to obtain a fifth separation liquid containing a supernatant and a cell mass. The supernatant was removed from the fifth separation liquid, and then a basal medium (BME: basal medium Eagle manufactured by Sigma-Aldrich Co. LLC) was added to the remaining cell mass, and the mixture was stirred to obtain a mixed solution containing cerebellar granule cells (CGC) and BME. Thereafter, the mixed solution was stained with a trypan blue solution having a concentration of 0.4 mass%, and the number of stained cells and their viability were measured.

[0139] Next, K + 5mM serum (+) BME with a cell density of 1.2×10 5 The culture sample was inoculated in a 12-well plate pre-coated with poly-L-lysine (PLL) at 0.766 mL / well, with a sample concentration of 50 μg / mL, and cultured in an incubator at 37° C. and a CO2 concentration of 5% by volume (“Day 0 after the start of culture”).

[0140] On the first day after the start of culture, cytosine β-D-arabinofuranoside (AraC) was added to each well to a final concentration of 10 μM to inhibit the growth of non-neuronal cells and maintain the growth of CGCs only. In addition, in the following control samples, KCl was added to a final concentration of 25 mM. In other samples (samples containing peptides or amino acids shown in Tables 5 to 7 below), KCl was added and the final concentration was adjusted to 15 mM.

[0141] Furthermore, on the 1st day and the 4th day after the start of the culture, the peptides or amino acids shown in Tables 5 to 7 were added to the final concentrations shown in Tables 5 to 7, respectively, to prepare samples.

[0142] On the 7th day after starting to culture, the viability of CGC was measured by MTT test to test whether each sample had a differentiation-promoting effect on CGC. Specifically, the percentage ratio of the number of live cells in each sample to the number of live cells in the control sample with a KCl concentration of 25mM was determined, and statistical processing was performed to evaluate the significance of the differentiation-promoting effect on CGC. Software ("BellCurve forExcel (version 2.1)" (trade name) manufactured by "Social Survey Research Information Co., Ltd.) was used for significance evaluation and statistical processing, Smirnov-Grubbs (two-sided test) was performed, and the significance level (P value) of rejection (refusal) was set to 0.05. Thereafter, a student's t-test (t-test) was performed to evaluate significance. Tables 5 to 7 show the results. Samples with "* (asterisk)" in Tables 5 to 7 were determined to have a differentiation-promoting effect (with significance) on CGC.

[0143] Here, in Table 5, "Control (K+)" refers to a control sample. "NMDA" ("M3262" (trade name) manufactured by Sigma-Aldrich Co. LLC) refers to a sample containing N-methyl-D-aspartic acid, and "BDNF" ("SRP3014" (trade name) manufactured by Sigma-Aldrich Co. LLC) refers to a sample containing brain-derived neurotrophic factor. "NMDA" and "BDNF" are known to be involved in the differentiation of brain nerve cells, and are used as reference examples in this test, respectively.

[0144] In addition, for the peptides shown in Tables 5 to 7, abbreviations are used in which amino acids are represented by one character. C represents a cyclic structure, H represents histidine, P represents proline, G represents glycine, O represents hydroxyproline, E represents glutamic acid, and A represents alanine. That is, in Table 5, "C-HP" refers to a sample containing a cyclic dipeptide consisting of histidine-proline (manufactured by PH Japan Co., Ltd.), "C-GP" refers to a sample containing a cyclic dipeptide consisting of glycine-proline (manufactured by PH Japan Co., Ltd.), and "TRH" refers to a sample containing thyrotropin-releasing hormone (manufactured by PH Japan Co., Ltd.). These samples are known to pass through the blood-brain barrier, and each is used as a comparative example. In Table 5, "glutamic acid" refers to a sample containing glutamic acid known to have an antioxidant effect ("G1251" (trade name) manufactured by Sigma-Aldrich Co. LLC). "Glutathione" refers to a sample containing glutathione having a chemical structure similar to Glu-Hyp-Gly (EOG) ("G6013" (trade name) manufactured by Sigma-Aldrich Co. LLC). "GPO" refers to a sample containing a tripeptide consisting of glycine-proline-hydroxyproline (manufactured by PH Japan Co., Ltd.). "Glutamic acid", "glutathione" and "GPO" were each used as a comparative example.

[0145] In Table 6, "C-PO" refers to a sample containing a cyclic dipeptide composed of proline-hydroxyproline (manufactured by PH Japan Co., Ltd.), "C-OG" refers to a sample containing a cyclic dipeptide composed of hydroxyproline-glycine (manufactured by PH Japan Co., Ltd.), and "C-EO" refers to a sample containing a cyclic dipeptide composed of glutamic acid-hydroxyproline (manufactured by PH Japan Co., Ltd.). In Table 6, "PO" refers to a sample containing a dipeptide composed of proline-hydroxyproline ("G-3025" (trade name) manufactured by BACHEM Co.), "OG" refers to a sample containing a dipeptide composed of hydroxyproline-glycine ("G-2365" (trade name) manufactured by BACHEM Co.), and "AO" refers to a sample containing a dipeptide composed of alanine-hydroxyproline (manufactured by PH Japan Co., Ltd.). It is known that "PO" can cross the blood-brain barrier. “C-PO”, “C-OG”, “C-EO”, “PO”, “OG”, and “AO” were used as comparative examples, respectively.

[0146] In Table 7, "GP" refers to a sample containing a dipeptide composed of glycine-proline ("G-3015" (trade name) manufactured by BACHEM Co.), and "EO" refers to a sample containing a dipeptide composed of glutamic acid-hydroxyproline (manufactured by PH Japan Co., Ltd.). "GP" and "EO" are used as comparative examples, respectively. "EOG" refers to a sample containing a tripeptide represented by Glu-Hyp-Gly (manufactured by PH Japan Co., Ltd.), and is used as an example in this test.

[0147] [Table 5]

[0148]

[0149]

[0150] [Table 6]

[0151]

[0152]

[0153] [Table 7]

[0154]

[0155]

[0156] <Discussion>

[0157] From Tables 5 to 7, it is clear that EOG has an effect of promoting CGC differentiation by directly acting on CGCs, as in the case of the reference example in which NMDA and BDNF were added. Tables 5 to 7 show that EOG has a CGC death suppressive effect.

[0158] <Discussion from Assessment Tests 1 and 2>

[0159] Evaluation tests 1 and 2 show that the brain function regulator according to the present invention can promote the differentiation of brain nerve cells by directly acting on brain nerve cells. Through this effect, the brain function regulator can show memory or brain function improvement. Therefore, the brain function regulator can be used as a brain function improver or a brain function decline preventive.

[0160] [Evaluation Test 3: In vitro test for analyzing the effective concentration of EOG]

[0161] The effective concentration of EOG that can exhibit a differentiation promoting effect on CGCs was examined by using the same method as in Evaluation Test 2 (except that EOG was added to the final concentration shown in Table 8 on Day 1 and Day 4 after the start of culture). Table 8 shows the results.

[0162] [Table 8]

[0163]

[0164]

[0165] <Discussion>

[0166] As shown in Table 8, when the concentration of EOG was 7 μM or higher, EOG could exhibit a significant differentiation promoting effect on CGCs.

[0167] [Evaluation Test 4: Evaluation test for analyzing the deliverability of oral EOG to the brain parenchyma]

[0168] <Sample Preparation>

[0169] All experimental animals were handled in accordance with the "Standards for the Breeding and Care of Laboratory Animals" of the Cabinet General Administration. Two 8-week-old ddy male mice were purchased from Sankyo Labo Service Corporation, Inc. After that, the male mice were adapted for 1 week to grow into 9-week-old male mice, and then fasted for 12 hours. Subsequently, 250 μL of an aqueous solution containing 20 mg EOG (Glu-Hyp-Gly manufactured by PH Japan Co., Ltd., purity: 95% or more) was forcibly orally administered to the male mice to prepare two male mice for analyzing the delivery ability of EOG to the brain parenchyma.

[0170] The anesthetic was subcutaneously injected into the male mice 10 minutes before oral administration (0 hours) and 1 hour, 2 hours and 4 hours after oral administration. The anesthetic is called a triple anesthetic, containing 0.3 mg / kg of medetomidine hydrochloride, 4 mg / kg of midazolam and 4 mg / kg of butorphanol tartrate. 10 minutes after the injection of the anesthetic, 0.5 to 7 μL of cerebrospinal fluid was drawn from each male mouse, 20 to 80 μL of blood was drawn from the tail vein, and 0.28 to 0.33 mg of a portion of the brain parenchyma (hereinafter also referred to as "brain") was stimulated.

[0171] First, the blood was centrifuged at 4°C with a centrifugal acceleration of 20400G for 10 minutes to obtain plasma, and the plasma was temporarily stored at -80°C. Subsequently, the plasma was mixed with ethanol three times its volume, and the mixture was centrifuged at 4°C with a centrifugal acceleration of 1000G for 10 minutes to obtain a supernatant. After that, ammonium bicarbonate prepared as 50mM was added to the supernatant in an amount four times the volume of the supernatant, and then mixed. In addition, the supernatant containing ammonium bicarbonate was filtered with a 0.2μm filter (manufactured by Sartorius AG) to obtain samples for measuring the amount of EOG in the plasma of the male mice (samples before oral administration (0 hours) and 1 hour, 2 hours and 4 hours after oral administration; hereinafter, these samples are also collectively referred to as "sample a").

[0172] The cerebrospinal fluid was mixed with ethanol three times its volume, and then the mixture was centrifuged at 4°C for 10 minutes at a centrifugal acceleration of 1000G. After that, ammonium bicarbonate prepared as 50mM was added to the centrifuged cerebrospinal fluid in an amount of 50μL, and the mixture was stirred. In addition, the cerebrospinal fluid containing ammonium bicarbonate was filtered with a 0.2μm filter (manufactured by Sartorius AG) to obtain samples for measuring the amount of EOG in the cerebrospinal fluid of the male mice (samples before oral administration (0 hours) and after 1 hour, 2 hours, and 4 hours after oral administration; hereinafter, these samples are also collectively referred to as "sample b").

[0173] The brain was washed with PBS (phosphate buffered saline), then wrapped in aluminum foil, then frozen in liquid nitrogen, and then stored in a refrigerator (-80°C) in a frozen state. Thereafter, the brain was thawed, homogenized with an equal amount of PBS to the brain, and then mixed with acetonitrile in an amount three times the weight of the brain, and then the mixture was centrifuged at 4°C for 10 minutes at a centrifugal acceleration of 1000G to obtain a suspension. Thereafter, ammonium bicarbonate prepared as 50mM was added to the suspension in an amount twice the volume of the suspension, and then mixed. In addition, the suspension containing ammonium bicarbonate was filtered with a 0.2μm filter (manufactured by Sartorius AG) to obtain samples for measuring the amount of EOG in the brain (samples before oral administration (0 hours) and 1 hour, 2 hours, and 4 hours after oral administration; hereinafter, these samples are also collectively referred to as "sample c").

[0174] The amount of EOG in samples a, b and c was quantitatively analyzed by LC-MS / MS under the following conditions. Since the samples were appropriately diluted, corrections were made using the following calculation formula to determine the actual amount of EOG.

[0175] Actual amount of EOG (μM) = analyzed value (nM) × dilution conversion factor / 1000 (unit conversion)

[0176] The results are as follows Figure 1 , 2 and 3. Figure 1 This is a graph showing the amount of EOG in the plasma (sample a) of male mice after a predetermined period of time has passed since EOG was forcibly orally administered to male mice. Figure 2 This is a graph showing the amount of EOG in the cerebrospinal fluid (sample b) of male mice after a predetermined period of time has passed since EOG was forcibly orally administered to the male mice. Figure 3 This is a graph showing the amount of EOG in the brain (sample c) of a male mouse after a predetermined time has passed since EOG was forcibly orally administered to the male mouse. Figures 1 to 3 Each of the EOG quantities shown is the average of two male mice.

[0177] Quantitative analysis by LC-MS / MS was performed under the following conditions.

[0178] HPLC instrument: ACQUITY UPLC H-Class Bio (manufactured by Waters Corporation) Column: HypersilGOLD PFP 2.1×150 mm, 5 μm (Thermo Fisher Scientific. Inc.

[0179] manufacture)

[0180] Column temperature: 40°C (linear gradient)

[0181] Mobile phase: (A) aqueous solution containing 0.2% formic acid and 2 mM ammonium acetate

[0182] (B) 100% methanol

[0183] (Gradient setting)

[0184]

[0185] Injection volume: 0.5 μL

[0186] MS / MS instrument: "Xevo TQ-XS", manufactured by Waters Corporation

[0187] Ionization method: positive ESI

[0188] Capillary voltage(kV):1

[0189] Desolvation temperature (℃): 500

[0190] Source temperature (℃): 150

[0191] MRM conditions:

[0192] Peptide (abbreviation) Precursor ion (m / z) Product ion (m / z)

[0193] Glu-Hyp-Gly(EOG) 318 225

[0194] <Discussion>

[0195] according to Figure 1-3 , it is understood that EOG forcibly orally administered to male mice was transferred to the blood and then to the cerebrospinal fluid. It is understood that EOG transferred to the cerebrospinal fluid was also transferred to the brain tissue within a short period of about 1 hour after oral administration. This suggests that the orally administered EOG is transferred via the blood, and therefore a portion of EOG is delivered to the brain tissue via the cerebrospinal fluid.

[0196] Although the embodiments and examples of the present invention have been described above, the configurations of the above-described embodiments and examples may be appropriately combined as originally conceived.

[0197] The embodiments and examples disclosed herein should be considered illustrative rather than limiting in any way.The scope of the invention is given by the appended claims rather than the foregoing description, and all changes that fall within the scope of the appended claims and their equivalents are embraced therein.

Claims

1. Use of a peptide, a salt thereof or a chemically modified product thereof in the preparation of a brain function regulator for improving memory or cognitive function, wherein the peptide is a tripeptide represented by Glu-Hyp-Gly, wherein the concentration of Glu-Hyp-Gly contained in the brain function regulator is 7 μM or higher, and wherein the brain function regulator has a brain nerve cell differentiation promoting effect.

2. The use according to claim 1, wherein the peptide is derived from collagen.

3. The use according to claim 1 or 2, wherein the brain function regulator is a brain function improver or a brain function decline preventer.

4. The use according to claim 1 or 2, wherein the brain function regulator has an inhibitory effect on brain nerve cell death.

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