JP2016193865A - Compositions for microglia phagocytosis activity acceleration and prediction method of microglia phagocytosis activity-enhancing action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide compositions for use in acceleration of microglia phagocytosis activity and methods for predicting the magnitude of the microglia phagocytosis activity-enhancing action of a test substance, based on the finding of dipeptide excellent in the effect of accelerating microglia phagocytosis activity.SOLUTION: A composition for microglia phagocytosis activity acceleration contains dipeptide, i.e. the following (1) and/or (2), or a pharmaceutically acceptable salt or solvate thereof. Whether the dipeptide contained in a test substance satisfies the following conditions (1) and/or (2) is determined. (1) The charge of N-terminal amino acid is 5.99 or less. (2) The ORAC value is 0.734 or more.SELECTED DRAWING: None

Description

  The present invention relates to a composition used for enhancing the phagocytic activity of microglia and a method for predicting the magnitude of the microglia phagocytic activity enhancing action of a test substance.

  Microglia, which exists as the only immune cell in the brain, occupies 10% of the brain and has essential functions for maintaining homeostasis in the brain, such as phagocytosis of waste products and repair of damaged tissues. It is clear to have. It has also been reported that microglia contribute to maintenance, enhancement, and improvement of cognitive function, whereas excessive activation of microglia can cause inflammatory conditions and cause cognitive decline (non-patented). Literatures 1-4). Inflammatory cytokines such as TNF-α and chemokines produced by excessively activated microglia are reported to be closely related to the pathology of pain, depression and chronic fatigue (Non-Patent Documents 5 to 10). . Therefore, controlling the activity of microglia is important in preventing and treating various diseases related to the brain. Particularly in dementia, waste products such as amyloid β that accumulate in the brain have caused a decline in cognitive function, and it can be expected to cope with dementia by activating the phagocytic removal ability of waste products of microglia. .

  With regard to the agent for microglia and amyloid β, for example, Patent Document 1 discloses pyrimidine derivatives that show activity of suppressing the activation of mouse peritoneal macrophages used as model cells of microglia, Alzheimer's disease, Parkinson's disease, It is described as an active ingredient of drugs for central diseases such as Huntington's disease and Pick's disease.

  In addition, Patent Document 2 uses an α-helical peptide derivative, which is an ApoE fragment, which shows an activity of suppressing activation of mouse microglia by stimulation with lipopolysaccharide (LPS), for the treatment or prevention of central nervous system diseases such as Alzheimer. It is described.

  Patent Document 3 describes the use of a humanized antibody against beta-amyloid peptide for the treatment, prevention and / or delay of progression of Alzheimer's disease.

  Patent Document 4 discloses that a chimeric antibody or a humanized antibody that specifically binds to an epitope within residues 1-10 of amyloid β is used to prevent or treat Alzheimer's disease or Down's syndrome or cognitive impairment. Is described.

  Patent Document 5 discloses that the ability of microglia to treat amyloid β oligomers can be enhanced by an oligodeoxynucleotide having a CpG motif without inducing the production of neurotoxic factors glutamic acid, nitric oxide, and TNF-α. And application of this to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease.

  Patent Document 6 describes that lipoprotein lipase has an effect of promoting uptake of amyloid β by mouse primary cultured astrocytes, and uses it for the prevention and treatment of Alzheimer's disease.

Wake, H., et al., 2009, J. Neurosci., Vol. 29 (13), pp. 3974-3980 Yoneyama, Masaki, Tomita, Kiyoichi, 2013, Journal of Japanese Pharmacology, 142, 17-21 Welberg, L., 2014, Nat. Rev. Neurosci., Vol.15, pp.68-69 Brown, G.C. & Neher, J.J., 2014, Nat. Rev. Neurosci. Vol.15, pp.209-216 Berta, T., et al., 2014, J. Clin. Invest., Vol.124 (3), pp.1173-1186 Riazi, K., et al., 2008, Proc. Natl. Acad. Sci. USA, Vol.105 (44), pp.17151-17156 Grinberg, Y.Y., et al., 2013, J. Neurochem., Vol. 126 (5), pp. 662-672 Couch, Y., et al., 2013, Brain Behav. Immun., Vol.29, pp.139-146 Yasui, M., et al., 2014, Glia, doi: 10.1002 / glia.22687 Nakatomi, Y., et al., 2014, J. Nucl. Med., Vol.55, pp.945-950

Japanese Patent No. 4398724 Japanese Patent No. 5052345 Japanese Patent No. 550030 Japanese Patent No. 5580504 Japanese Patent No. 5481762 Japanese Patent No. 5629896

  However, there have been few reports on the effectiveness of dipeptides as active ingredients that enhance the phagocytic activity of microglia.

  Therefore, an object of the present invention is to find a dipeptide excellent in the effect of enhancing the phagocytic activity of microglia, and based on the knowledge, the composition used for enhancing the phagocytic activity of microglia, and the microglia phagocytic activity of the test substance The object is to provide a method for predicting the magnitude of the enhancing action.

  In order to achieve the above object, the present inventors have intensively researched and found that a specific dipeptide is excellent in the effect of enhancing the phagocytic activity of microglia, and have completed the present invention.

  That is, the first of the present invention is a composition for enhancing microglia phagocytic activity, which contains a dipeptide that satisfies the following conditions (1) and / or (2), or a pharmaceutically acceptable salt or solvate thereof: It is to provide.

(1) The charge of the N-terminal amino acid is 5.99 or less. (2) The ORAC value is 0.734 or more. WR, RN, AQ, VW, WQ, AY, HI, AM, AV, QK, VI , NY, AD, WM, WH, EK, AP, MV, WN, AI, GY, MT, VK, PA, RM, NF, DT, AL, GA, PF, NP, VA, YD, AE, AF, YW , FG, EA, GN, KG, FL, AH, AG, ND, AS, FS, WP, TP, AR, AA, WL, TA, YA, LG, TY, NN, VL, RT, QD, YQ, WT , AW, GF, ME, DS, AK, WI, DV, WK, DH, DF, KF, YE, PT, KR, YM, DW, FQ, DY, YF, ML, WD, KV, LA, EI, MI , NA, QA, GI, FN, VY, FR, MH, MW, IT, GP YN, HL, NK, ES, MK, MR, FY, FF, LF, IR, DP, LD, WE, PN, AT, YG, DN, MS, KT, LW, WA, PP, DM, PK, WW, The present invention provides a composition for enhancing microglial phagocytic activity comprising a dipeptide selected from KH, NI, PS, PW, FW, GD, WV, QV, and GE.

  The composition of the present invention is preferably used for preventing accumulation of amyloid in the brain or suppressing a state caused by accumulation of amyloid in the brain.

  Moreover, it is preferable that it is used in order to prevent or treat dementia.

  Moreover, it is preferable for maintenance, improvement, and / or improvement of brain function.

  On the other hand, the second of the present invention is a method for predicting the magnitude of the microglia phagocytic activity enhancing action of a test object, wherein the dipeptide contained in the test object is the following (1) and / or (2) The method includes the step of determining whether or not a condition is satisfied.

(1) The N-terminal amino acid has a charge of 5.99 or less. (2) The ORAC value is 0.734 or more. In the method of the present invention, the dipeptide is added to the microglial cells cultured by adding the test dipeptide. It is preferable to further include a step of determining whether or not the test for measuring the amount of amyloid β incorporated satisfies the condition that the amount is 1.215 times or more that of the control.

  According to the present invention, a dipeptide excellent in the effect of enhancing the phagocytic activity of microglia is found, the composition used for enhancing the phagocytic activity of microglia, and the magnitude of the microglia phagocytic activity enhancing action of the test substance. Can be provided.

4 is a histogram arranged in order from the top of the index value of enhanced phagocytic activity evaluated in Test Example 1. FIG. 2a is a chart showing the results of performing t-test with the N-terminal charge of dipeptides by dividing the upper and lower 168 sequences of the index value of enhanced phagocytic activity evaluated in Test Example 1 into two groups, and FIG. FIG. 2c is a chart showing the results of t-test with the N-terminal charge of a dipeptide divided into two groups of the upper and lower 50 sequences of the index value of enhanced phagocytic activity evaluated in Test Example 1. FIG. It is a graph which shows the result of having divided the upper and lower 168 arrangement | sequence of the index value of the evaluated phagocytic activity evaluation into 2 groups, and performing the t test by ORAC value.

  In the composition of the present invention, a dipeptide that satisfies the following conditions (1) and / or (2), which is excellent in the effect of enhancing the phagocytic activity of microglia, is used.

(1) The charge of the N-terminal amino acid is 5.99 or less. (2) The ORAC value is 0.734 or more. Here, the charge of the amino acid is a value inherent to the amino acid (for example, Zimmerman, JM, Eliezer, N. & Simha, R. "The characterization of amino acid sequences in proteins by statistical methods." J. Theor. Biol. 21, 170-201 (1968)).

  The ORAC value (Oxygen Radical Absorbance Capacity) is one of the indexes that measure the antioxidant function of foodstuffs and health foods that are being adopted in public institutions in recent years. . Also called TEAC (Trolox Equivalents Antioxidant Capacity). The ORAC value can be measured by a commercially available kit or the like.

  Examples of the dipeptide satisfying the above (1) include WR, VW, WQ, QK, VI, NY, WM, WH, EK, MV, WN, GY, MT, VK, NF, DT, GA, NP, VA, YD, YW, FG, EA, GN, FL, ND, FS, WP, TP, WL, TA, YA, LG, TY, NN, VL, QD, YQ, WT, GF, ME, DS, WI, DV, WK, DH, DF, YE, YM, DW, FQ, DY, YF, ML, WD, LA, EI, MI, NA, QA, GI, FN, VY, FR, MH, MW, GP, YN, NK, ES, MK, MR, FY, FF, LF, DP, LD, WE, YG, DN, MS, LW, WA, DM, WW, NI, FW, GD, WV, QV, GE, LY, GW, NG, NM, NH, VP, YP, VS, TG, YY, SS, DK, S , VD, NQ, SF, DA, FE, LV, TF, MQ, VH, NW, VM, QW, SM, and the like.

  Examples of the dipeptide satisfying the above (2) include WR, VW, WQ, AY, NY, WM, WH, WN, GY, YW, WP, WL, TY, YQ, WT, AW, WI, YM, DW, DY, YF, WD, MW, FY, WE, LW, WA, WW, PW, FW, WV, LY, GW, YY, MQ, NW, PY, QW, and the like.

  Examples of dipeptides satisfying the above (1) and (2) include WR, VW, WQ, NY, WM, WH, WN, GY, YW, WP, WL, TY, YQ, WT, WI, YM. , DW, DY, YF, WD, MW, FY, WE, LW, WA, WW, FW, WV, LY, GW, YY, MQ, NW, QW, and the like. Two or more dipeptides may be used in combination.

  In this specification, amino acid abbreviations are represented by A for alanine, D for aspartic acid, E for glutamic acid, F for phenylalanine, G for glycine, H for histidine, I for isoleucine, K for lysine, L for leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, Y is tyrosine, and C is cysteine.

  The amino acid constituting the dipeptide used in the composition of the present invention may be composed only of the L-form amino acid, may be composed only of the D-form amino acid, or any of the dipeptides in which both are mixed. It may be. Further, it may be composed only of naturally occurring amino acids, may be composed only of modified amino acids in which any functional group is bonded to amino acids, or may be any of dipeptides in which both are mixed. Good. When the dipeptide contains two or more asymmetric carbons, it may be an enantiomer, a diastereomer, or a dipeptide in which both are mixed.

  The dipeptide used in the composition of the present invention may be a pharmaceutically acceptable salt or solvate thereof. For example, pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloride, sulfate and phosphate, organic salts such as acetate, maleate, fumarate, citrate and methanesulfonate. Examples include acid salts. Examples of the pharmaceutically acceptable metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the pharmaceutically acceptable ammonium salt include salts such as ammonium and tetramethylammonium. Examples of pharmaceutically acceptable organic amine addition salts include addition salts of morpholine, piperidine and the like.

  Dipeptides used in the composition of the present invention may be those obtained by chemical synthesis, chemically or from protein or polypeptide raw materials derived from milk, soybeans, wheat, eggs, livestock meat, fish meat, seafood, etc. You may use what was obtained by decomposing | disassembling enzymatically. Specifically, for example, any dipeptide sequence used in the composition of the present invention is at least alpha protein casein, beta casein, kappa casein, lactoglobulin, lactalbumin, immunoglobulin, lactoperoxidase, lactoferrin, BSA. , Soy protein glycinin, conglycinin, wheat protein gliadin, glutenin, egg yolk protein ribovitellin, egg white protein ovalbumin, ovomucoid, ovotransferrin, mucin, lysozyme, chicken protein collagen, and so on The above-mentioned dipeptide can be prepared by subjecting foods and food raw materials containing bismuth to acid hydrolysis and enzyme treatment. The preparation may be used as it is, or may be used after further purification of any degree.

  The composition of the present invention contains at least the above-mentioned dipeptide, and is used for enhancing the phagocytic activity of microglia, thereby preventing the accumulation of amyloid in the brain or suppressing the state caused by the accumulation. can do. Thus, for example, it is suitably used for preventing or treating dementia.

  Moreover, it is suitably used for maintaining, improving and / or improving brain function including at least any of the following contents.

・ Improve motivation ・ Improve vitality ・ Improve motivation ・ Improve motivation ・ Improve cognitive function ・ Improve memory retention ・ Improve confidence ・ Improve curiosity ・ Recover depressed feelings・ Encourage feelings ・ Encourage feelings ・ Suppress the decline in memory associated with aging In addition, according to the findings of this specification, a microglia phagocytic activity enhancer comprising the above-mentioned dipeptide as an active ingredient Also provided are a method for enhancing the phagocytic activity of microglia (excluding medical practice for humans) that causes a human or animal to ingest a composition containing the dipeptide, and the use of the dipeptide for enhancing the phagocytic activity of microglia. .

  There is no restriction | limiting in particular about the usage form of the composition of this invention. For example, it can be used as a pharmaceutical composition, an additive used for blending in the pharmaceutical composition, a food composition, an additive used for blending in the food composition, or the like. Moreover, you may use for animals, such as not only a human but pets and livestock. In order to effectively act the dipeptide on humans and animals, the form containing 0.00001 to 100% by mass of the dipeptide is preferable, and the form containing 0.0001 to 100% by mass is more preferable. , 0.001 to 100% by mass is most preferable. The composition of the present invention may be contained together with a component that reduces amyloid β, or a compound or composition having an effect of maintaining or improving cognitive function, or may be administered or ingested in combination. Good. Examples of components that reduce amyloid β include anti-amyloid β antibodies, and compounds or compositions that are known to have effects such as maintenance and improvement of cognitive function, such as docosahexaenoic acid (DHA) and ginkgo biloba extract. It is done.

  The pharmaceutical composition can be produced by mixing at least one of the above-mentioned dipeptides as an active ingredient and mixing a carrier, an excipient, a binder, a diluent and the like. It can be administered orally or parenterally, and when administered orally, it may be in any form such as granules, powders, tablets, pills, capsules, syrups and the like. Examples of parenteral dosage forms include, but are not limited to, external preparations such as injections, drops, and nasal preparations.

  When used as a medicine, the dosage varies depending on the dosage form, patient age, body weight, nature or severity of symptoms to be treated, etc., but preferred oral dosage per day for adults per day The first is the above-mentioned dipeptide 0.002 to 40 g, the second is the above-mentioned dipeptide 0.002 to 20 g, and the third is the above-mentioned dipeptide 0.002 to 2 g. 4 is 0.02 to 40 g of the dipeptide, 5 is 0.02 to 20 g of the dipeptide, 6 is 0.02 to 2 g of the dipeptide, and 7 is 0.2 to 0.2 g of the dipeptide. ˜40 g, eighth is the above dipeptide 0.2-20 g, and ninth is the above dipeptide 0.2-2 g. In addition, for example, in the case of parenteral administration such as intravenous administration, the range of preferable dosage per day for an adult for normal intravenous administration is given. First, the dipeptide is 0.0002 to 4 g. The second is the above-mentioned dipeptide 0.0002-2g, the third is the above-mentioned dipeptide 0.0002-0.2g, the fourth is the above-mentioned dipeptide 0.002-4g, and the fifth is the above-mentioned Dipeptide 0.002 to 2 g, sixth is the above dipeptide 0.002 to 0.2 g, seventh is the above dipeptide 0.02 to 4 g, and eighth is the above dipeptide 0.02 to 2 g. Ninth, the above-mentioned dipeptide is 0.02 to 0.2 g. Of course, regarding these doses, an optimal form may be appropriately selected according to various conditions.

  The form of the food composition is not particularly limited, and may be liquid, semi-liquid, or solid, and includes a beverage-like form. In addition, health foods (specific insurance foods, nutritional functional foods, functional labeling foods), diseases, etc., including so-called health foods, functional foods, dietary supplements, supplements, and foods with a disease risk reduction label. Consumer food is also included.

  Non-alcoholic beverages when the food composition is applied to non-alcoholic beverages include, for example, mineral water, near water, sports drinks, tea beverages, milk beverages, coffee beverages, fruit juice beverages, vegetable juice beverages, fruit juices and Although vegetable juice drink, carbonated drink, etc. are mentioned, it is not limited to these. It may be a beer beverage having an alcohol content of less than 1%, such as non-alcohol beer. Mineral water includes both foaming and non-foaming mineral water.

  The tea drink in the non-alcoholic drinks refers to drinks for decocting tea leaves (tea leaves) that are evergreen trees of the camellia family, or leaves or cereals of plants other than tea trees, such as fermented tea, semi-fermented tea, And non-fermented tea are both included. Specific examples of tea beverages include Japanese tea (for example, green tea, barley tea), black tea, herbal tea (for example, jasmine tea), Chinese tea (for example, Chinese green tea, oolong tea), hoji tea, and the like. Milk beverages refer to beverages made mainly from raw milk, milk, etc. or foods made from these raw materials. In addition to those made from milk itself, for example, nutrient-enriched milk, flavored milk, and sugar-decomposed milk And the like using processed milk as a raw material.

  Examples of fruits used in fruit juice-containing beverages and fruit and vegetable juice-containing beverages include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acais, and blueberries. Examples of vegetables used in vegetable juice-containing beverages, fruit juices, and vegetable juice-containing beverages include tomato, carrot, celery, pumpkin, celery, and cucumber.

When used as a food composition, in the normal intake form, if the range of preferable intake amount per day per adult is given, the first is the above-mentioned dipeptide 0.002 to 40 g, the second is the above-mentioned dipeptide 0 0.002 to 20 g, the third is 0.002 to 2 g of the dipeptide, the fourth is 0.02 to 40 g of the dipeptide, the fifth is 0.02 to 20 g of the dipeptide, 6 is 0.02 to 2 g of the dipeptide, 7th is 0.2 to 40 g of the dipeptide, 8 is 0.2 to 20 g of the dipeptide, and 9 is 0.2 to g of the dipeptide. ~ 2g. Moreover, in order to make the said dipeptide act effectively, it is preferable that it is a form containing the said dipeptide 0.00001-100 mass%, It is more preferable that it is a form containing 0.0001-100 mass%, 0 0.001 to 100% by mass is more preferable, 0.01 to 90% by mass is still more preferable, and 0.1 to 80% by mass is most preferable. . In addition, when the above-mentioned dipeptide is contained in a container that is subdivided into one serving or one serving, the first is the above-mentioned dipeptide 0.002 to 40 g if the range of the content in the usual blending form is given. 2 is 0.002 to 20 g of the dipeptide, 3rd is 0.002 to 2 g of the dipeptide, 4 is 0.02 to 40 g of the dipeptide, and 5 is 0.02 of the dipeptide. ˜20 g, sixth is 0.02 to 2 g of the dipeptide, seventh is 0.2 to 40 g of the dipeptide, eighth is 0.2 to 20 g of the dipeptide, Is 0.2 to 2 g of the above dipeptide. When used as a beverage, it is preferably in a form containing 0.00001 to 10% by mass of the dipeptide, more preferably in a form containing 0.0001 to 5% by mass, and 0.001 to 5% by mass. More preferably, it is a form containing 0.01 to 5% by mass, and still more preferably 0.1 to 1% by mass.

  On the other hand, according to the knowledge of the present specification, there is further provided a method for predicting the magnitude of the microglia phagocytic activity enhancing action of the test object.

  That is, as shown in the Examples described later, the degree of the effect of dipeptides enhancing the phagocytic activity of microglia is related to the charge of the N-terminal amino acid of the dipeptide and the ORAC activity. Therefore, it is possible to predict whether or not the test substance is effective in enhancing the phagocytic activity of microglia based on the predetermined threshold value set with respect to the properties of these dipeptides. Specifically, by determining whether or not the dipeptide contained in the test object satisfies the following conditions (1) and / or (2), It can be predicted that the product is effective in enhancing the phagocytic activity of microglia.

(1) The N-terminal amino acid has a charge of 5.99 or less. (2) The ORAC value is 0.734 or more. In the method, amyloid β is added to the culture solution during microglia culture, Measure the amount of amyloid β taken up into the microglial cells within the period of time, determine the ratio of the uptake amount of the test dipeptide added and the control not added, set a predetermined threshold for this ratio in advance, this It is preferable to predict whether or not the test object is useful for enhancing the phagocytic activity of microglia. For example, according to the examples described later, when the ratio is 1.215 times or more of the control, more typically 1.343 times or more, the dipeptide has a high activity of enhancing the phagocytic activity of microglia. Thus, it can be predicted more accurately whether or not a test substance containing the dipeptide is useful for enhancing the phagocytic activity of microglia.

  Since the dipeptide provided by the present invention has a relatively small molecular weight, it easily passes through the brain barrier after ingestion into the body, and easily exerts its effect in the brain. Moreover, when a dipeptide has ORAC activity, the effect of removing the reactive oxygen species which microglia produces can also be exhibited.

  The present invention will be described in detail below with reference to examples, but the present invention is not limited to the description of the following examples.

<Test Example 1>
In order to comprehensively investigate the effects of dipeptides on the phagocytic activity of microglia, the following tests were conducted.

[Dipeptide]
Of the 361 combinations of dipeptides that can be composed of 19 amino acids excluding cysteine (C) among the 20 amino acids constituting the protein, the N-terminal amino acid (first amino acid) and the C-terminal amino acid (second amino acid) As shown in Table 1 below, 336 combinations of dipeptides except 25 types of dipeptides were prepared. When there was a stereoisomer, L form was used.

[Microglia]
Microglia were isolated from mouse brain by magnetic cell separation. Specifically, a brain tissue dispersion was obtained by papain treatment of the brain removed from the mouse, and after stopping the enzyme reaction, the CD11b antibody (Miltenyi Biotec), a pan-microglia marker magnetically labeled with superparamagnetic microbeads. The microglia was isolated by reacting with the product and magnetically separating it.

[Evaluation of phagocytic activity]
The phagocytic activity of microglia was evaluated as follows. That is, the isolated microglia was cultured for 12 hours in a medium to which dipeptide was added at a concentration of 25 μM as a test substance, and then amyloid β labeled with a fluorescent dye (FAM) (“β-Amyloid (1-42), FAM Conjugate (AnaSpec) was added to the medium at a concentration of 500 nM and cultured for 5 hours. The supernatant was removed, the extracellular fluorescence was quenched with trypan blue, the amount of amyloid β incorporated into the microglia was measured as a fluorescence amount with a microplate reader, and the fluorescence amount of the control measured without adding dipeptide was 1 The enhancement of phagocytic activity was evaluated using the ratio as an index. The results are shown in Table 1. FIG. 1 shows a histogram in which the ratio of the measured fluorescence amount to the control, that is, the index value of phagocytic activity enhancement, is arranged in descending order of numerical values.

  As a result, assuming that the index value of phagocytic activity enhancement is within the top 168 (the ratio to the control is 1.215 or more), WR, RN, AQ, VW, WQ, AY, HI, AM, AV, QK, VI, NY, AD, WM, WH, EK, AP, MV, WN, AI, GY, MT, VK, PA, RM, NF, DT, AL, GA, PF, NP, VA, YD, AE, AF, YW, FG, EA, GN, KG, FL, AH, AG, ND, AS, FS, WP, TP, AR, AA, WL, TA, YA, LG, TY, NN, VL, RT, QD, YQ, WT, AW, GF, ME, DS, AK, WI, DV, WK, DH, DF, KF, YE, PT, KR, YM, DW, FQ, DY, YF, ML, WD, KV, LA, EI, MI, NA, QA, GI, FN, VY, FR, MH, MW, IT GP, YN, HL, NK, ES, MK, MR, FY, FF, LF, IR, DP, LD, WE, PN, AT, YG, DN, MS, KT, LW, WA, PP, DM, PK, WW, KH, NI, PS, PW, FW, GD, WV, QV, GE, LY, GW, NG, NM, NH, IK, KP, VP, YP, VS, TG, YY, IS, SS, HD, DK, SR, PD, PM, HK, PE, VD, NQ, SF, IH, PG, PI, DA, FE, LV, TF, MQ, VH, NW, PY, VM, QW, SM are selected, these This dipeptide was found to have a great effect of enhancing the phagocytic activity of microglia.

  Also, WR, RN, AQ, VW, WQ, AY, HI, AM, AV, QK, VI, NY, AD, WM, WH, EK, AP, MV, WN with a ratio of 1.25 or more with the control , AI, GY, MT, VK, PA, RM, NF, DT, AL, GA, PF, NP, VA, YD, AE, AF, YW, FG, EA, GN, KG, FL, AH, AG, ND , AS, FS, WP, TP, AR, AA, WL, TA, YA, LG, TY, NN, VL, RT, QD, YQ, WT, AW, GF, ME, DS, AK, WI, DV, WK , DH, DF, KF, YE, PT, KR, YM, DW, FQ, DY, YF, ML, WD, KV, LA, EI, MI, NA, QA, GI, FN, VY, FR, MH, MW IT, GP, YN, HL, NK, ES, MK, MR, FY, FF, LF IR, DP, LD, WE, PN, AT, YG, DN, MS, KT, LW, WA, PP, DM, PK, WW, KH, NI, PS, PW, FW, GD, WV, QV, GE These dipeptides were selected and found to be even more effective in enhancing the phagocytic activity of microglia.

  Furthermore, WR, RN, AQ, VW, WQ, AY, HI, AM, AV, QK, VI, assuming that the index value of enhanced phagocytic activity is within the top 50 (ratio to control is 1.343 or more) , NY, AD, WM, WH, EK, AP, MV, WN, AI, GY, MT, VK, PA, RM, NF, DT, AL, GA, PF, NP, VA, YD, AE, AF, YW , FG, EA, GN, KG, FL, AH, AG, ND, AS, FS, WP, TP, AR, and AA are selected, and it is found that these dipeptides have a particularly large effect of enhancing the phagocytic activity of microglia. It was.

<Test Example 2>
It was examined whether the dipeptide, which was found to have an effect of enhancing the phagocytic activity of microglia in Test Example 1, was included in the protein sequence used in food. Specifically, it was examined whether the sequences of these dipeptides appeared in the sequences of 34 types of proteins shown in Table 2 below.

  Tables 3 to 10 show the results of the appearance of dipeptide sequences for each of milk casein protein, milk whey protein, and milk BSA. In addition, since γ-Casein and proteose peptone are degradation products of β-Casein, dipeptides that have been confirmed to appear in β-Casein may also appear in γ-Casein and proteose peptone. It is done.

  Tables 11 to 18 show the results of the appearance of dipeptide sequences for soybean glycine, soybean β-conglycinin, wheat gliadin, wheat glutenin, and egg yolk lipoviterin.

  Tables 19 to 26 show the results of appearance of dipeptide sequences for each of egg white ovalbumin, egg white ovotransferrin, egg white ovomucoid, egg white ovomucin, egg white ovomucin, egg white lysotium, and chicken collagen.

  As a result, it was found that the sequence of the dipeptide, which was found to have an effect of enhancing the phagocytic activity of microglia in Test Example 1, was included in any of the 34 types of proteins described above, and acid hydrolysis was performed from the raw material containing those proteins. It was confirmed that it can be prepared by enzyme treatment. It should be noted that dipeptides containing cysteine (abbreviation C) that have not been evaluated for phagocytic activity in Test Example 1 are also included in any protein except CW, and can be prepared from raw materials containing those proteins. confirmed.

<Test Example 3>
In Test Example 1, the influence of the dipeptide on the phagocytic activity of microglia was shown, and the degree varied depending on each dipeptide. Therefore, the relationship with the unique properties of each dipeptide was investigated. Specifically, multiple regression analysis was performed using the presence or absence of an aromatic ring, the charge of the N-terminal amino acid, and the charge of the C-terminal amino acid as explanatory variables, and the index value of enhanced phagocytic activity evaluated in Test Example 1 as a dependent variable. . Moreover, the ORAC value of each dipeptide was measured and the same multiple regression analysis was performed. The ORAC value was measured using “OxiSelect ^™ ORAC Activity Assay Kit” manufactured by Cell Biolabs Inc., and the results were as shown in Table 27 below (the cells in the table are shaded with ORAC). Indicates that the value is 0.734 or more.)

  Table 28 shows the results of the multiple regression analysis.

  As a result of the multiple regression analysis, the index value of enhanced phagocytic activity evaluated in Test Example 1 was found to be related to the N-terminal charge of the dipeptide and the ORAC value. Therefore, the upper and lower 168 sequences of the index value of enhanced phagocytic activity evaluated in Test Example 1 were selected, divided into two groups, and t-test was performed using the N-terminal charge or ORAC value. In addition, the upper and lower 50 sequences of the index value of enhanced phagocytic activity were selected, divided into two groups, and t-test with N-terminal charge was performed. As a result, as shown in FIG. 2, a significant relationship was recognized.

  Specifically, with respect to the charge of the N-terminal amino acid, WR, VW, WQ, QK, VI, NY, WM, WH, EK, MV, WN, GY, among the upper 168 sequences of the index value of increased phagocytic activity, MT, VK, NF, DT, GA, NP, VA, YD, YW, FG, EA, GN, FL, ND, FS, WP, TP, WL, TA, YA, LG, TY, NN, VL, QD, YQ, WT, GF, ME, DS, WI, DV, WK, DH, DF, YE, YM, DW, FQ, DY, YF, ML, WD, LA, EI, MI, NA, QA, GI, FN, VY, FR, MH, MW, GP, YN, NK, ES, MK, MR, FY, FF, LF, DP, LD, WE, YG, DN, MS, LW, WA, DM, WW, NI, FW, GD, WV, QV, GE, LY, GW, NG, NM, NH, V 117 sequence of, YP, VS, TG, YY, SS, DK, SR, VD, NQ, SF, DA, FE, LV, TF, MQ, VH, NW, VM, QW, SM, N-terminal amino acid charge Was below 5.99.

  In addition, among the top 50 sequences of the index values for increased phagocytic activity, WR, VW, WQ, QK, VI, NY, WM, WH, EK, MV, WN, GY, MT, VK, NF, DT, GA, NP , VA, YD, YW, FG, EA, GN, FL, ND, FS, WP, and TP, the N-terminal amino acid had a charge of 5.99 or less.

  As for the ORAC value, WR, VW, WQ, AY, NY, WM, WH, WN, GY, YW, WP, WL, TY, YQ, WT, among the top 168 sequences of the index value of increased phagocytic activity, 38 arrays of AW, WI, YM, DW, DY, YF, WD, MW, FY, WE, LW, WA, WW, PW, FW, WV, LY, GW, YY, MQ, NW, PY, QW are The ORAC value was 0.734 or higher.

  From the above results, when predicting the magnitude of the microglia phagocytic activity enhancing action of a certain test object, whether or not the charge of the N-terminal amino acid of the dipeptide contained therein is 5.99 or less and / or contained therein By determining whether or not the ORAC value of the dipeptide is 0.734 or more, it was revealed that the magnitude of the microglia phagocytic activity enhancing action of the test object can be accurately predicted.

Claims (7)

A composition for enhancing microglia phagocytic activity comprising a dipeptide that satisfies the following conditions (1) and / or (2), or a pharmaceutically acceptable salt or solvate thereof:
(1) The N-terminal amino acid has a charge of 5.99 or less (2) The ORAC value is 0.734 or more A method for predicting the magnitude of the microglia phagocytic activity enhancing action of a test object, wherein the dipeptide contained in the test object satisfies the following condition (1) and / or (2) Determining the method.
(1) The N-terminal amino acid has a charge of 5.99 or less (2) The ORAC value is 0.734 or more   Whether or not the dipeptide satisfies the condition that the amount is 1.215 times or more of the control in a test for measuring the amount of amyloid β taken up into microglia cells cultured with the addition of the test dipeptide The method of claim 1, further comprising determining.   WR, RN, AQ, VW, WQ, AY, HI, AM, AV, QK, VI, NY, AD, WM, WH, EK, AP, MV, WN, AI, GY, MT, VK, PA, RM, NF, DT, AL, GA, PF, NP, VA, YD, AE, AF, YW, FG, EA, GN, KG, FL, AH, AG, ND, AS, FS, WP, TP, AR, AA, WL, TA, YA, LG, TY, NN, VL, RT, QD, YQ, WT, AW, GF, ME, DS, AK, WI, DV, WK, DH, DF, KF, YE, PT, KR, YM, DW, FQ, DY, YF, ML, WD, KV, LA, EI, MI, NA, QA, GI, FN, VY, FR, MH, MW, IT, GP, YN, HL, NK, ES, MK, MR, FY, FF, LF, IR, DP, LD, WE, PN, AT, For enhancing microglia phagocytic activity including dipeptides selected from G, DN, MS, KT, LW, WA, PP, DM, PK, WW, KH, NI, PS, PW, FW, GD, WV, QV, and GE Composition.   The composition according to claim 1 or 4, which is used for preventing accumulation of amyloid in the brain or suppressing a state caused by accumulation of amyloid in the brain.   The composition according to claim 1 or 4, which is used for preventing or treating dementia. The composition according to claim 1 or 4 for maintaining, improving and / or improving brain function.