Agent for suppressing amyloid β aggregation / deposition and agent for suppressing / improving cognitive decline of subject

Abstract

The purpose of the present invention is to provide an agent for suppressing amyloid β aggregation / deposition and an agent for suppressing / improving cognitive decline of a subject both of which are expected to have the effect of treating and / or preventing Alzheimer's disease. Specifically, the present invention pertains to an agent for suppressing amyloid β aggregation / deposition or an agent for suppressing / improving cognitive decline of a subject, the agent comprising an extract of a plant belonging to Myrtaceae.

Description

Agent for inhibiting amyloid-β aggregation / deposition and agent for inhibiting / improving decline in cognitive function in a subject

[0001] The present invention relates to an amyloid beta aggregation and / or deposition inhibitor (hereinafter also referred to as "amyloid beta aggregation / deposition inhibitor") that exhibits a high aggregation / deposition inhibitory effect in a method for evaluating amyloid beta aggregation and / or deposition inhibitory (hereinafter also referred to as "aggregation / deposition inhibitor") activity on organoids, and an agent for inhibiting and / or improving cognitive function decline in a subject (hereinafter also referred to as "cognitive function decline inhibitor / improver").

[0002] Alzheimer's disease (often abbreviated as "AD" herein) is a type of irreversible, progressive central nervous system disorder accompanied by symptoms such as cognitive impairment (dementia), behavioral disorders, and personality changes. The number of dementia patients worldwide is estimated to be over 50 million as of 2019, of which approximately 70% are thought to be AD patients, and the incidence rate is on the rise. The increase in medical expenses and nursing care issues associated with the increase in AD patients have become a major social issue in recent years, placing an economic and psychological burden on countries and patients involved. AD begins with the aggregation and accumulation of amyloid beta protein (often abbreviated as "Aβ" herein) in the patient's brain. Subsequently, tau protein, a microtubule-associated protein, becomes hyperphosphorylated and fibrillated, leading to neuronal destruction and brain atrophy (Non-Patent Documents 1-3). Based on this mechanism of AD pathogenesis, a technology has been developed to evaluate the Aβ aggregation inhibitory effect of a test substance added in vitro and screen candidate compounds for AD treatment. For example, the micro-scale high-throughput screening (hereinafter referred to as "MSHTS") method for amyloid β protein aggregation inhibitors using quantum dot nanoprobes is a screening technology based on a cell-free assay system that can search for candidate compounds that have an amyloid β protein aggregation inhibitory effect in PBS solvent (Non-Patent Document 4).As an example of MSHTS, for example, Patent Document 1 discloses a method, device, and program for evaluating amyloid formation. Specifically, the method discloses a method for determining the amyloid formation inhibitory activity of a test substance, including: an aggregation reaction step in which an amyloidogenic protein such as amyloid β protein is reacted with a fluorescent probe (containing quantum dots as a fluorescent dye, or quantum dots, etc.) capable of binding to amyloid formed by polymerization of the amyloidogenic protein in PBS, in the presence or absence of a test substance; an imaging step in which the fluorescence of the aggregation reaction product obtained in the aggregation reaction step is imaged; a standard deviation calculation step in which a standard deviation is calculated from the luminance values ​​of each pixel included in a region of interest in the fluorescent image imaged in the imaging step; and an activity determination step in which, based on the comparison of the standard deviation of luminance values ​​in the presence of the test substance calculated in the standard deviation calculation step with the standard deviation of luminance values ​​in the absence of the test substance, the test substance is determined to have amyloid formation inhibitory activity if the standard deviation of luminance values ​​in the presence of the test substance is smaller than the standard deviation of luminance values ​​in the absence of the test substance. Furthermore, Patent Document 2 discloses a versatile quantum dot nanoprobe for evaluating the amyloid aggregation properties of proteins and peptides, and a method for evaluating amyloid formation inhibitors using the quantum dot nanoprobe. Specifically, it discloses a quantum dot nanoprobe in which a quantum dot is bound to the N-terminus or C-terminus of an amyloidogenic peptide via cysteine. However, even candidate compounds obtained in cell-free assay systems often do not exhibit inhibitory effects in cell-based assay systems (tests using cells). This is thought to be due to the fact that cell-free assay systems do not necessarily reflect the biological environment. In addition, even perilla extract, for example, which has been shown to have an inhibitory effect on Aβ aggregation / deposition in both cell-free assay systems and conventional cell-based assay systems, has not shown a consistent effect on Aβ aggregation / deposition in the brain, and currently, no reliable treatment for Alzheimer's disease has been established (Non-Patent Document 5).On the other hand, organoids (three-dimensional tissues) are expected to be able to reproduce a physiological environment very similar to that in vivo, including the complex exchange of signals between cells and between cells and the extracellular matrix (ECM) (Non-Patent Document 6).

[0003] International Publication No. 2020 / 138265 Japanese Patent Application Laid-Open No. 2017-007990

[0004] Hardy J. and Selkoe D. J. , 2002, Science, 297(5580):353-356Jack C. R. Jr. , et al. , 2010, Lancet Neurol. 9(1):119-128 Akira Tamaoka, 2017, Journal of Geriatric Dementia Research, Vol. 22, No. 3, p. 19-23Ishigaki et al. , 2013, PLOS ONE, 8(8): e72992 Shimamori et al. , 2023, Foods 12(3):486 Antoni, Burckel, Josset and Noel, Int J Mol Sci. , 2015, 16(3):5517-5527.

[0005] In view of the above-mentioned circumstances, the present invention aims to provide an Aβ aggregation / deposition inhibitor that can also be effective in actual biological tissues. More specifically, the present invention aims to provide an aggregation / deposition inhibitor for aggregating proteins that exhibits high aggregation / deposition inhibitory activity using a screening method that can search for candidate compounds that have an aggregation / deposition inhibitory effect on aggregating proteins such as Aβ in a physiological environment closer to in vivo using organoids. The present invention also aims to provide an agent that suppresses / improves the decline in cognitive function of a subject. Therefore, as a result of intensive research conducted to solve the above problems, it was found that, as a test system using organoids, a labeled aggregating protein, a test substance, and organoids were co-cultured, and the aggregated and / or deposited aggregating proteins on the surface of the organoids or inside the organoids were quantified using the label as an indicator, thereby enabling evaluation of whether the test substance is an aggregation / deposition inhibitor or aggregation / deposition promoting substance against aggregating proteins, leading to the completion of the present invention. That is, the present invention encompasses the following. [1] A method for evaluating the aggregation / deposition inhibitory activity or aggregation / deposition promoting activity of a test substance against an aggregating protein, the method comprising the steps of coexisting / culturing a labeled aggregating protein, the test substance, and an organoid in an aqueous solution, and quantifying the aggregating and / or deposited aggregating protein on the surface of the organoid and / or inside the organoid using the label as an index. [2] The method according to [1], wherein the organoid is selected from the group consisting of brain organoids, cerebellar organoids, inner ear organoids, thyroid organoids, thymus organoids, T cell mature lymphoid organoids, cardiac organoids, lung organoids, liver organoids, pancreatic organoids, kidney organoids, gastric gland organoids, intestinal organoids, epithelial organoids, ovarian organoids, testicular organoids, and fused organoids comprising these organoids.[3] The method of [1] or [2], wherein the aggregating protein is selected from the group consisting of amyloid β protein, tau protein, α-synuclein protein, prion protein, huntingtin protein, amylin protein, apolipoprotein A1, serum amyloid A protein, immunoglobulin light chain, MAP4 protein, β2 microglobulin, TDP-43 protein, and cystatin C protein. [4] The method of any one of [1] to [3], wherein the label is an optical label. [5] The method of [4], wherein the optical label is a quantum dot. [6] The method of any one of [1] to [5], wherein the quantifying step comprises taking an image of the organoid and calculating, in the image obtained by the capturing step, the ratio of the area of ​​the fluorescent region from the label to the surface area of ​​the organoid as the aggregating protein on the surface of the organoid and / or inside the organoid. [7] The method according to any one of [1] to [6], wherein the quantification is carried out over time while the organoids are alive. [8] The method according to any one of [1] to [7], wherein the quantification is carried out for at least 7 days without using genetically modified cells. [9] An amyloid-β aggregation / deposition inhibitor comprising an extract of a plant belonging to the Myrtaceae family.

[10] The aggregation / deposition inhibitor according to [9], which exhibits an inhibitory effect on amyloid-β aggregation and / or deposition in an evaluation system comprising a step of co-culturing labeled amyloid-β, a test substance, and cerebral organoids, and a step of quantifying amyloid-β aggregated on the surface and / or inside the cerebral organoids using the label as an index.

[11] The aggregation / deposition inhibitor according to

[10] , which exhibits an inhibition rate of 50% or more compared to a control group to which no test substance is added in the evaluation system.

[12] The flocculation / deposition inhibitor according to any one of [9] to

[11] , wherein the plant belonging to the Myrtaceae family is guava, Eucalyptus robusta, clove, or allspice.

[13] The flocculation / deposition inhibitor according to

[12] , wherein the extract is an extract obtained by subjecting the pulverized product obtained by pulverizing cloves to solvent extraction.

[14] The aggregation / deposition inhibitor according to

[12] or

[13] , wherein the extract is obtained by subjecting a pulverized product of cloves, the pulverized product having a maximum particle size of 5 mm or less, to solvent extraction.

[15] The aggregation and / or deposition inhibitor according to

[12] to

[14] , wherein the extract is obtained by subjecting a pulverized product of cloves, the pulverized product having a maximum particle size of 0.5 mm or more, to solvent extraction.

[16] The aggregation and / or deposition inhibitor according to

[13] , wherein the extract is obtained by subjecting a pulverized product of cloves, the pulverized product having a particle size range of 0.5 mm to 3 mm, to solvent extraction.

[17] The aggregation / deposition inhibitor according to any one of [9] to

[16] , which inhibits the aggregation and / or deposition of amyloid beta on the surface and / or inside brain tissue cells in a living body.

[18] An agent for inhibiting / improving cognitive decline in a subject, comprising an extract of a plant belonging to the Myrtaceae family.

[19] The agent for preventing / improving cognitive decline according to

[18] , wherein the plant is clove or allspice.

[20] A method for producing an amyloid-β aggregation / deposition inhibitor, the method comprising a step of subjecting a plant belonging to the Myrtaceae family to solvent extraction to obtain an extract.

[21] The method for producing an aggregation / deposition inhibitor according to

[20] , wherein the plant belonging to the Myrtaceae family is guava, Eucalyptus robusta, clove, or allspice.

[22] The method for producing an aggregation / deposition inhibitor according to

[21] , wherein the step of obtaining the extract is a step of subjecting a pulverized product obtained by pulverizing cloves to solvent extraction to obtain the extract.

[23] The method for producing an aggregation / deposition inhibitor according to

[22] , wherein the pulverized product obtained by pulverizing cloves has a maximum particle size of 5 mm or less.

[24] The method for producing an aggregation / deposition inhibitor according to

[22] or

[23] , wherein the pulverized product obtained by pulverizing cloves has a maximum particle size of 0.5 mm or more.

[25] The method for producing an aggregation and / or deposition inhibitor according to

[22] , wherein the maximum particle size of the pulverized product obtained by pulverizing the cloves is in the range of 0.5 mm to 3 mm.

[26] The method for producing an aggregation and / or deposition inhibitor according to any one of

[20] to

[25] , wherein the solvent used in the solvent extraction is an organic solvent, water, or a mixture thereof.

[27] The method for producing the aggregation / deposition inhibitor according to any one of

[20] to

[26] , wherein the solvent used in the solvent extraction is an aqueous ethanol solution.

[28] A method for producing an agent for inhibiting / improving cognitive decline in a subject, the method comprising a step of subjecting a plant belonging to the Myrtaceae family to solvent extraction to obtain an extract.

[29] The method according to

[28] , wherein the plant is clove or allspice.

[30] A composition for inhibiting amyloid-β aggregation / deposition, comprising as an active ingredient the aggregation / deposition inhibitor according to any one of [9] to

[17] .

[31] The aggregation / deposition inhibitor according to

[30] , comprising as active ingredients two or more different aggregation / deposition inhibitors according to any one of [9] to

[17] .

[32] The aggregation / deposition inhibitor according to

[30] or

[31] , which is for treating or preventing Alzheimer's disease or mild cognitive impairment. This specification incorporates the disclosures of Japanese Patent Application No. 2023-146708, from which the present application claims priority.

[0006] 1 is a photograph showing the effect of embryoid body (EB) preparation time on organoid-like tissue formation in Example 1. FIG. 2 is a photograph showing the state of cells infiltrating and filling into a gel in Example 1. FIG. 3 is a graph showing the change in CCL2 (monocyte chemotactic protein) concentration in the culture supernatant during intragel culture in Example 1. FIG. 4 is a photograph showing the state of Aβ aggregation / deposition in organoids over time in Example 2. FIG. 5 is a diagram showing the results of the Aβ aggregation / deposition inhibitory effect of each test sample evaluated in organoids in Example 3. FIG. 6 is a diagram showing the results of the Aβ aggregation / deposition inhibitory effect of each test sample evaluated in organoids in Example 4. FIG. 7 is a diagram showing the results of the Aβ aggregation / deposition inhibitory effect of each test sample evaluated in organoids in Example 5. FIG. 8 is a diagram showing the results of the Aβ aggregation / deposition inhibitory effect of each test sample evaluated in organoids in Example 6.

[0007] The present invention is described in detail below. 1. Aβ Aggregation / Deposition Inhibitor 1-1. Overview The first aspect of the present invention relates to an Aβ aggregation / deposition inhibitor exhibiting aggregation / deposition inhibitory activity against amyloid β. The Aβ aggregation / deposition inhibitor of the present invention is characterized by exhibiting potent Aβ aggregation / deposition inhibitory activity in a method for evaluating the aggregation / deposition inhibitory activity or aggregation / deposition promoting activity of a test substance against an aggregating protein, the method comprising: coexisting / culturing a labeled aggregating protein, a test substance, and an organoid in an aqueous solution; and quantifying the aggregating protein aggregated / deposited on the surface of the organoid and / or inside the organoid using the label as an indicator. The Aβ aggregation / deposition inhibitor of the present invention, discovered using the above evaluation method that is closer to a biological environment using organoids, can also exhibit good Aβ aggregation / deposition inhibitory effects in actual biological tissues. 1-2. Definitions of Terms The following terms used in this specification are defined below. (1) Organoid An organoid is a three-dimensional tissue-like structure that has functions similar to those of individual tissues or organs, formed by the self-organization of cells. Organoids include cerebral organoids, cerebellar organoids, inner ear organoids, thyroid organoids, thymus organoids, T cell mature lymphoid organoids, myocardial organoids, lung organoids, liver organoids, pancreatic organoids, kidney organoids, gastric gland organoids, Foregutt organoids (organoids constituting the oral cavity and stomach), Midgut organoids (organoids constituting the small intestine and ascending colon), Hindgut organoids (organoids constituting parts other than the rectum and ascending colon), intestinal organoids such as Gut organoids, epithelial organoids, ovarian organoids, testicular organoids, etc. Furthermore, fusion organoids, which produce new differentiated cells by fusing these organoids together, can also be mentioned. (2) Aggregating Protein The aggregating protein is a protein that assembles to form an aggregate. The state of the aggregate-forming protein may be liquid or solid, and the type of the aggregating protein is not limited.Examples of the disease-related aggregating proteins include polyglutamic acid, disease-related proteins, and autophagy-related proteins. Examples of the disease-related aggregating proteins include amyloid β protein and tau protein (including phosphorylated tau protein), which are causative proteins of Alzheimer's disease, α-synuclein protein, which is a causative protein of Parkinson's disease, prion protein, which is a causative protein of transmissible spongiform encephalopathy (including Creutzfeldt-Jakob disease, mad cow disease, or prion disease), huntingtin protein, which is a causative protein of Huntington's disease, amylin protein, which is a causative protein of type II diabetes, apolipoprotein A1 (APOA1 protein), which is a causative protein of arteriosclerosis (including cerebral infarction, pulmonary infarction, and myocardial infarction), serum amyloid A protein, which is a causative protein of rheumatoid arthritis, immunoglobulin light chain, which is a causative protein of systemic AL amyloidosis, MAP4 protein, which is a causative protein of myocardial infarction, β2 microglobulin, which is a causative protein of dialysis-related amyloidosis, and TDP-43 protein, which is a causative protein of amyotrophic lateral sclerosis. In particular, amyloid β proteins such as amyloid β40 protein, amyloid β42 protein, amyloid β43 protein, and amyloid β38 protein are suitable. Aggregating proteins (main protein misfolding / aggregating diseases) and diseases (proteopathies) associated with these aggregating proteins are shown in Table 1 below. For example, α-synuclein, TDP-43, prion, cystatin C, amyloid β, etc. are known as aggregating proteins, and are known to cause Parkinson's disease, frontotemporal lobar degeneration, prion disease, chronic kidney disease (CKD), Alzheimer's dementia, diabetic dementia, etc., respectively. Examples of autophagy-related proteins include ubiquitin-like proteins Atg-8 and Atg-12. Aggregating proteins may be natural proteins found in nature, modified proteins artificially mutated or modified natural proteins, or artificial proteins based on artificially designed amino acid sequences. (3) Aggregation / Aggregates. "Aggregation" herein refers to the aggregation of peptide or protein molecules (which may be modified with sugar chains or lipids), and may be water-soluble or water-insoluble. "Aggregates" herein refer to the assembly of two or more aggregating proteins. Aggregates herein also include so-called protein complexes. Aggregates may be homoaggregates composed of the same type of protein or heteroaggregates composed of different types of proteins. (4) Deposition. "Deposition" of aggregating proteins on the surface and / or inside organoids refers to the adhesion of aggregating proteins to the surface and / or inside organoids. The deposition site may be on the surface and / or inside the cells that form the organoids, or may be between the cells and the ECM. (5) Cells In the present invention, the cells that form organoids or the cells from which they are derived will be described below. The cells may be derived from a multicellular organism. Animal-derived cells are preferred, and mammalian-derived cells are more preferred. Examples include rodents such as mice, rats, hamsters, and guinea pigs; domestic or pet animals such as dogs, cats, rabbits, cows, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Human-derived cells are particularly preferred. The type of cell is not limited. Examples include cells derived from biological tissues, cells derived from cells derived from biological tissues, stem cells, or cells differentiated from stem cells. "Biological tissue" refers to various tissues that constitute the living body of an organism. Examples include epithelial tissue, connective tissue, muscle tissue, and nervous tissue. "Stem cells" refer to cells that have the ability to differentiate into various cells and self-replicate. For example, adult stem cells and pluripotent stem cells can be mentioned."Adult stem cells" are stem cells present in each tissue of an adult, which have not yet completed terminal differentiation and have a certain degree of pluripotency. They are also called somatic stem cells or tissue stem cells. Examples include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, melanocyte stem cells, and cancer stem cells. "Pluripotent stem cells" are cells that have the pluripotency (multipotency) to differentiate into all types of cells that make up the body and can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions. Examples include embryonic stem cells (ES cells), embryonic germ stem cells, germline stem cells, and induced pluripotent stem cells (iPS cells). "Differentiated cells" are cells that make up the body and can be cultured in vitro under appropriate conditions. For example, salivary gland mucous cells, salivary gland n1 cells, von Ebner's gland cells of taste buds, mammary gland cells, lacrimal gland cells, auditory canal gland cells, eccrine gland cells, apocrine gland cells, eyelash gland cells, sebaceous gland cells, Bowman's gland cells, Brunner's gland cells, seminal vesicle cells, bulbourethral gland cells, goblet cells, endometrial cells, gastric mucosal cells, parietal cells, pancreatic acinar cells, type II lung pneumocytes, hormone-secreting cells, anterior pituitary gland cells, growth hormone-secreting cells, prolactin-secreting cells, pituitary thyrotrophic cells, adrenocorticotropic hormone-secreting cells, melanocyte-stimulating hormone-producing cells, large neurosecretory neurons, astrocytes, microglia, cardiac muscle cells, atrial muscle cells, oxytocin-secreting cells, vasopressin-secreting cells, serotonin-secreting cells, and the like. These include thyroid cells, thyroid follicular cells, parafollicular cells, parathyroid cells, acidophilic cells, granulosa lutein cells, corneal epithelial cells, hair follicle cells, cone cells, autonomic nerve cells, cholinergic nerve cells, adrenergic nerve cells, peptidergic nerve cells, renal juxtacellular cells, glomerular epithelial cells, hepatocytes, adipocytes, white adipocytes, brown adipocytes, ameloblasts, corneal fibroblasts, tendon fibroblasts, smooth muscle cells, neutrophils, eosinophils, basophils, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, erythroblasts, hepatocytes, T cells, nerve cells, and hormone-secreting cells.Neurons induced to differentiate from iPS cells derived from Alzheimer's disease (AD) patients can also be particularly suitable for the formation of brain organoids. (6) Extracellular Matrix (ECM) In this specification, the term "extracellular matrix" refers to a gel-like substrate capable of embedding cells. The extracellular matrix may be a mixed protein of animal origin, such as Matrigel, or more preferably, a polymer-derived substrate such as collagen, fibronectin, entactin, laminin, vitronectin, or an artificial protein. (7) Culture Medium In the present invention, "culture medium" refers to a liquid or solid substance prepared for cell culture. In principle, it contains the minimum necessary components for cell growth and / or maintenance. The culture medium may be either a basal medium or a specialized cell culture medium. "Basal medium" refers to a medium that serves as the basis for various animal cell culture media. They can be cultured alone, or by adding various culture additives, they can be prepared into media specific to various cells depending on the purpose (special cell culture media). Examples of basal media that can be used include, but are not limited to, Neurobasal (registered trademark) medium, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM / F12 medium). In addition, media used for culturing human iPS cells and human ES cells can also be suitably used. "Special cell culture media" refers to media prepared by adding various supplements to a basal medium as described above to be optimal for culturing specific cells, or media prepared to induce differentiation into specific cells. Examples include media for culturing neural cells, which are commercially available from various manufacturers.Specifically, for example, "M medium" is a neuronal culture medium from Sumitomo Bakelite, prepared by adding serum albumin and culture supernatant from primary astroglial cells cultured in a nutrient medium to a basal medium of DMEM / F12 (5:5) supplemented with insulin and transferrin, and further supplemented with NGF2.5S and BDNF as supplements. This also applies to media for culturing pluripotent stem cells such as human iPS cells and human ES cells. The medium may contain serum or may not contain serum (i.e., serum-free medium). (8) Labeling. As used herein, "labeling" refers to modifying a target substance to identify it. Labeling facilitates and ensures detection or selection of the target substance. Labeling is performed according to the type of target substance. In this specification, since aggregating proteins are the target substance to be detected, the type is protein. Therefore, labeling refers to any labeling method that can directly or indirectly label proteins. Direct labeling includes a labeling method in which a labeling substance is bound to the aggregating protein, or a labeling method in which the aggregating protein is expressed as a fusion protein with a labeled peptide. Indirect labeling includes a method in which the aggregating protein is directly labeled using an antibody or an active fragment thereof that specifically recognizes and binds to the aggregating protein, or indirectly labeled via a secondary antibody. The method of binding a labeling substance is preferred. Labeling substances used for protein labeling include, but are not limited to, optical labels. As used herein, "optical labeling" refers to labeling with a substance that emits visible light, near-infrared light, or near-ultraviolet light, such as a fluorescent substance or a luminescent substance. A "fluorescent substance" is a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to its original ground state. Fluorescent substances include both fluorescent dyes and fluorescent proteins.Examples of "fluorescent dyes" include quantum dots, FITC, Texas Red (registered trademark), Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, Pacific Blue, DyLight 405, DyLight 550, DyLight 650, PE-Cy5 (phycoerythrin-cyanin 5), PE-Cy7 (phycoerythrin-cyanin 7), PE (phycoerythrin), and PerCP (peridinin chlorophyl). Examples of suitable fluorescent dyes include fluorophore (fluorophore ... "Quantum dots" (often abbreviated as "QDs" herein) refer to nanoscale semiconductor crystals that possess optical properties governed by quantum mechanics and emit fluorescence in the visible and near-infrared regions. They typically have a diameter of 2 to 10 nm and are composed of approximately 10 to 50 atoms. They offer a variety of fluorescent colors depending on their particle size, and have excellent properties such as resistance to fluorescent bleaching. As a result, they are increasingly being used as biosensing materials and imaging materials for cells or animals. Examples of quantum dots include semiconductor quantum dots, particularly core-shell CdSe / ZnS quantum dots such as Qdot® 525, Qdot 545, Qdot 565, Qdot 585, Qdot 605, Qdot 655, Qdot 705, and Qdot 800 (all manufactured by Thermo Fisher Scientific). Examples of "fluorescent proteins" include GFP.(9) Test Substances Test substances include, for example, nucleic acids, RNA, peptides, proteins, antibodies, synthetic compounds, cells, cell extracts, cell culture supernatants, exosomes, microbial metabolites, plant extracts, seaweed extracts, etc. (10) Treatment and Prevention As used herein, "treatment" refers to curing a disease, suppressing or preventing the progression or chronicity of disease symptoms, and alleviating, ameliorating, or ameliorating disease symptoms. Furthermore, as used herein, "prevention" refers to suppressing or preventing the onset of a specific disease. (11) Plant Extracts As used herein, "plant extracts" refer to mixtures of multiple components obtained from plants. Typically, these include plant components such as low-molecular-weight compounds and / or soluble components extracted from plants in a solvent, or plant components containing proteins, nucleic acids, lipids, sugars, and low-molecular-weight compounds contained in the juice of plants. All or part of the components contained in a plant extract may be unidentified. The state of the plant extract is not important. The plant may be in a liquid state, a solid state (including a powdered state or a granular state), or a paste state. As used herein, "juice" refers to a liquid component obtained by squeezing a plant. Small pieces of the plant may be included as the solid component, or the liquid component may be obtained by filtering. (12) Plant Body. As used herein, "plant body" refers to the entire plant (whole plant) or a portion of the plant body constituting the plant. The portion of the plant body is not particularly limited. Examples include leaves, stems, buds, leaf sheaths, petioles, corms, rhizomes (including corms, bulbs / bulbs, rhizomes, tubers, etc.), roots (including tuberous roots, aerial roots, etc.), seeds, hypocotyls, fruits, and flower buds. The plant body may be in a raw, fermented, or dried state. The plant body may be in a raw, fermented, or dried state. The plant body may be in a dried state, as appropriate, depending on the form of the plant body. For example, if the plant body is in a powder form, a dried state is preferred. Furthermore, if the plant body is in a juice form, it may be in a raw or fermented state. 1-3. Production of Organoids. An example of organoid production is shown below. Examples of methods for producing organoids include a method comprising the steps of culturing cells to form spheroids, embedding the spheroids obtained in the spheroid formation step in a gel, and culturing the spheroids embedded in the gel in the gel embedding step.According to this method, organoids can be obtained that can be used to select (screen) candidate substances that exhibit aggregation / deposition inhibitory activity or aggregation / deposition promoting activity against aggregating proteins. Each step of the organoid manufacturing method will be explained below. 1-3-1. Spheroid Formation Step The "spheroid formation step" is a step of culturing cells to form spheroids such as embryoid bodies. The shape of the culture vessel used in this step is not particularly limited, and examples include culture vessels with shapes such as dish-shaped, flask-shaped, well-shaped, bag-shaped, and spinner flask-shaped. The seeding density of cells in the culture vessel (cell density at the start of culture) in the medium can be adjusted as appropriate, for example, 0.01 × 10 ５ cells / mL or more, more preferably 0.1 x 10 ５ cells / mL or more, more preferably 1 x 10 ５ cells / mL or more and 20 x 10 ５ cells / mL or less, more preferably 10 x 10 ５ Examples of suitable concentrations include cells / mL or less. The temperature conditions for culture are, for example, 25-40°C, 30-39°C, or 34-38°C. Meanwhile, the culture time is, for example, 10 hours or more, preferably 12 hours or more, particularly preferably 20 hours or more, and 300 hours or less, preferably 200 hours or less, particularly preferably 100 hours or less. If the culture time is too long, exceeding 100 hours, the intercellular bonds become too strong, and the cells may not be properly packed into the gel in the subsequent gel-embedded spheroid culture step. Culture may be static culture or culture under conditions where the medium is flowing (flow culture), but static culture is preferred. In this step, for example, embryoid bodies may be formed during the process of inducing iPS cells into neurons, or embryoid bodies may be formed from iPS cells and then embryoid bodies of neurons may be formed. When embryoid bodies are formed during the process of inducing iPS cells into neurons, iPS cells are first cultured in a neural progenitor cell induction medium (e.g., STEM diff. ＴＭ STEMdiff on Neural Induction Medium ＴＭThe cells are cultured in a medium supplemented with SMADi Neural Induction Supplement (hereinafter referred to as "NIMS medium") and induced to differentiate into neural progenitor cells. At this time, a ROCK inhibitor may be added to the medium. ROCK inhibitors are defined as substances that inhibit the kinase activity of Rho-associated protein kinase (ROCK), and examples of such inhibitors include Y-27632 (4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide or a salt thereof (e.g., dihydrochloride salt)), H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine or a salt thereof (e.g., 2 hydrochloride), Fasudil / HA1077 (1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (e.g., dihydrochloride)), Wf-536 ((+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl)benzamide monohydrochloride), Y39983 (4-[(1R)-1-Aminoethyl]-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), SLx-2119 (2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl] phenoxy]-N-(1-methylethyl)-acetamide), Azabenzimidazole-aminofurazans, DE-1 Examples of ROCK inhibitors include 04, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, and VAS-012. The final concentration of the ROCK inhibitor in the medium is, for example, 5 μM or more, preferably 20 μM or more, and 100 μM or less, preferably 50 μM or less. The obtained neural progenitor cells are then cultured in a neural progenitor cell culture medium (e.g., STEM diff ＴＭThe neural progenitor cells are then cultured in a neural cell culture medium (e.g., a mixed medium of Neuro Basal Medium minus phenol red (Thermo Fisher Scientific) with B-27 supplement (Thermo Fisher Scientific) and Penicillin-Streptomycin Mixed Solution (Nacalai Tesque) (hereinafter referred to as "NbM medium")) for the predetermined culture time described above, and are induced to differentiate into neurons while forming embryoid bodies. A hedgehog protein (e.g., human recombinant sonic hedgehog (SHH, manufactured by Veritas)) is also added to the medium as an essential component. The final concentration of hedgehog protein in the medium is, for example, 1 ng / mL or more, preferably 10 ng / mL or more, and 200 ng / mL or less, preferably 100 ng / mL or less. On the other hand, when forming embryoid bodies from iPS cells and then forming embryoid bodies of neural cells, the iPS cells are cultured in an embryoid body formation medium (e.g., STEM diff ＴＭThe iPS cells are then cultured in a medium containing EB Formation (in the Cerebral Organoid Kit) for the specified culture time described above to form embryoid bodies of iPS cells. The embryoid bodies of iPS cells are then cultured in a neuronal differentiation-inducing medium (e.g., GMEM (Thermo Fisher Scientific) containing 5% KSR (Thermo Fisher Scientific) and AGN193109 (Sigma-Aldrich Japan)) to induce differentiation into neurons, resulting in the production of embryoid bodies of neurons. Whether or not differentiation into neurons has been induced can be determined using neural progenitor cell markers and / or neuronal cell markers as indicators. Examples of neural progenitor cell markers include nestin. Examples of neuronal cell markers include MAP2. Neural progenitor cell markers and / or neuronal cell markers can be detected by any detection method known in the art. Methods for detecting expression markers include, but are not limited to, flow cytometry. When cells are detected in flow cytometry using a fluorescently labeled antibody that emits stronger fluorescence than a negative control (isotype control), the cells are determined to be "positive" for the marker. The percentage of cells that test positive for a fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the positive rate. Any antibody known in the art can be used as the fluorescently labeled antibody, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. Neural progenitor cell markers and / or neuronal markers can be detected by immunostaining. For example, cells or embryoid bodies in a culture vessel are reacted with a primary antibody, followed by a fluorescently labeled secondary antibody, and visually fluorescent cells or embryoid bodies are detected by fluorescence microscopy. For example, the ratio of cells or embryoid bodies stained with a fluorescently labeled secondary antibody to cells or embryoid bodies stained with DAPI (a cell nuclear marker) can be taken as the positive rate.Any antibody known in the art can be used as the fluorescently labeled secondary antibody, including, but not limited to, an antibody labeled with Alexa Fluor 488. For example, when the percentage (ratio) of cells or embryoid bodies positive for neural progenitor cell markers is, for example, 10% or less, preferably 5% or less, and / or the percentage (ratio) of cells or embryoid bodies positive for neuronal markers is 70% or more, preferably 80% or more, it can be determined that neural cells have been significantly differentiated. 1-3-2. Gel Embedding Step: In this step, the spheroids obtained in the spheroid formation step are embedded in a gel. For example, the spheroids are embedded in a gel such as Matrigel. Examples of embedding methods include covering a culture plate with parafilm, creating a depression using a chip attachment, placing the spheroids in the depression, dripping a gel, and allowing the spheroids to stand for, for example, 10 to 720 minutes (preferably 30 to 180 minutes), after which the gel containing the embedded spheroids is recovered and placed in a new culture plate. 1-3-3. In-Gel Culture Step: In this step, the spheroids embedded in the gel embedding step are cultured in a gel. The gel-embedded spheroids are subjected to shaking culture in a culture vessel containing a medium. For example, shaking culture is performed while rotating the spheroids in a bioshaker at 10 to 200 rpm (preferably 30 to 150 rpm). The temperature conditions for the culture are, for example, 25 to 40°C, 30 to 39°C, or 34 to 38°C. On the other hand, the culture time is, for example, 14 days or more, preferably 21 days or more, and 365 days or less, preferably 280 days or less, so that cells can sufficiently infiltrate and fill the gel and form organoids. Furthermore, the maturity of organoids can be determined by quantifying the cell migration protein contained in the culture supernatant during the culture period in which cells infiltrate and fill the gel from the spheroids embedded in the gel. Therefore, preferably, the in-gel culture step includes measuring the concentration of the cell migration protein in the culture supernatant. Examples of cell migration proteins include CCL2 (monocyte chemotactic protein). The concentration of the cell migration protein in the culture supernatant can be measured, for example, by immunological measurement methods such as ELISA.For example, when the CCL2 concentration in the culture supernatant is 500 pg / ml or less, preferably 200 pg / ml or less, it can be determined that cell filling in the gel is complete and organoids have been formed. 1-4. Method for Evaluating the Aggregation / Deposition-Inhibitory or Aggregation / Deposition-Promoting Activity of a Test Substance Against an Aggregating Protein Each step of the method of this embodiment will be explained below. 1-4-1. Coexistence and Culturing Step (hereinafter also referred to as the "Coexistence / Culturing Step") The "coexistence / culturing step" is a step in which a labeled aggregating protein, a test substance, and organoids are allowed to coexist in an aqueous solution, and the organoids are cultured (incubated) in this coexistence. In this step, the labeled aggregating protein is aggregated and / or deposited on the surface and / or inside the organoids by culturing (incubating). Note that "culturing (incubating)" does not necessarily mean increasing the number of cells, but rather simply means maintaining the cells in coexistence for a certain period of time or longer. In a separate control experiment, a labeled aggregating protein and organoids may be coexisted in an aqueous solution (without the test substance), and the organoids may be cultured (incubated) in this coexistence. Examples of aqueous solutions used here include culture medium, physiological saline, Ringer's solution, etc. For example, in a culture vessel (such as a 96-well microplate), a solution containing a labeled aggregating protein diluted with a culture medium or the like and a solution containing a test substance diluted with a culture medium or the like are added to the organoids. Furthermore, a solution containing an unlabeled aggregating protein diluted with a culture medium or the like may be added. These solutions may be added simultaneously or sequentially. The order of addition may be any order. Alternatively, organoids may be cultured (incubated) in the presence of a labeled aggregating protein, and the culture causes the labeled aggregating protein to aggregate and / or deposit on the surface and / or inside the organoids. In this way, a solution containing a test substance can be added to organoids that have aggregated and / or deposited with the labeled aggregating protein, and the labeled aggregating protein, the test substance, and the organoids can be coexisted / cultured.In a culture vessel, for example, for organoids with a diameter of 1 mm or more, the respective concentrations of the aggregating protein and test substance in the aqueous solution (culture medium) include, for example, a final concentration of 5-100 nM (preferably 10-50 nM) for the labeled aggregating protein, a final concentration of 1-100 ng / μL (preferably 10-50 ng / μL) for the test substance, and a final concentration of 5-100 μM (preferably 10-50 μM) for the unlabeled aggregating protein. The conditions for coexistence and culture (incubation) are not particularly limited, as long as the labeled aggregating protein can aggregate and / or deposit on the surface and / or inside the organoid. The temperature is, for example, 25-40°C, 30-39°C, or 34-38°C. Examples of the incubation time include 30 minutes to 1200 hours (50 days), 12 hours to 96 hours (4 days), 24 hours (1 day) to 72 hours (3 days), and 36 hours to 48 hours (2 days). The incubation may be static or may be cultured under conditions where the medium is flowing (flow culture), but static culture is preferred. 1-4-2. Quantification Step The "quantification step" is a step in which, after incubation or while the organoids are still alive (continuing to culture), the amount of aggregating and / or deposited aggregating proteins on the surface and / or inside the organoids obtained in the coculture / culture step is quantified using the label attached to the added aggregating protein as an index. In this step, the aggregation / deposition of aggregating proteins on the surface and / or inside the organoids is quantitatively detected. For example, when the aggregating protein is labeled with an optical labeling substance such as quantum dots, the detection of aggregation / deposition can be performed by fluorescence observation using a fluorescence microscope.If fluorescence is observed on the surface of organoid and / or inside organoid, it can be determined that aggregation / deposition has formed.Then, the fluorescence intensity is quantified, and for example, the presence or absence of aggregation / deposition can be quantitatively detected by comparing the fluorescence intensity value with that of a negative control.Specifically, the quantification step can include a step of taking an image of the organoid, and a step of calculating the ratio of the area of ​​the fluorescent region from the label, such as quantum dots, to the surface area of ​​the organoid, as an index of the fluorescence from the label in the image obtained by the photographing step, as aggregated and / or deposited aggregating proteins on the surface and / or inside the organoid. For example, quantum dot-modified amyloid β protein (e.g., amyloid β) can be administered to brain organoids. ４０ protein) and unlabeled amyloid beta protein (e.g., amyloid beta ４２When a test substance (a test substance) is added, the brain organoid is irradiated with light, and the quantum dots are used to visualize the aggregation / deposition of unlabeled amyloid β protein and the quantum dot-modified amyloid β protein on the surface of the brain organoid and / or inside the brain organoid. Then, the image of the brain organoid is acquired, and then the area of ​​the fluorescent region is calculated from the image analysis of the fluorescence caused by the quantum dots. Since the area of ​​the fluorescent region is positively correlated with the amount of aggregation / deposition of the aggregating protein, for example, by comparing the ratio of the area of ​​the fluorescent region to the surface area of ​​the brain organoid with that of a negative control (for example, in the absence of the test substance) and / or a positive control (for example, in the presence of a known aggregation / deposition inhibitory active substance or aggregation / deposition promoting active substance), the test substance added is evaluated to have aggregation / deposition inhibitory activity or aggregation / deposition promoting activity against the aggregating protein. For example, the image of the organoid is taken, and the organoid area in the image obtained by taking is calculated (organoid surface area). Then, the area of ​​the fluorescent region of the aggregating protein in the image is calculated (aggregating protein deposition area). The aggregation / deposition rate of the aggregating protein for organoids can be calculated by the following formula (1): Aggregating / deposition rate of the aggregating protein for organoids (%) = (aggregating / deposition area of ​​the aggregating protein ÷ organoid surface area) × 100 (1) Furthermore, when this test system is established, by providing a group to which the test sample is also added at the same time, the sample can also quantitatively analyze the aggregation / deposition inhibitory effect of the aggregating protein for organoids. For example, the aggregation / deposition inhibitory function of the aggregating protein possessed by the test substance can be quantified as aggregation / deposition inhibitory rate (%), and can be calculated by the following formula (2): Aggregating protein (in this embodiment, Aβ) aggregation / deposition inhibitory rate (%) = (aggregating protein aggregation / deposition rate (%) in the absence of the test substance) - (aggregating protein aggregation / deposition rate (%) in the presence of the test substance) (2) It can be seen that the larger the aggregation / deposition inhibitory rate (%) calculated by formula (2), the greater the aggregation / deposition inhibitory effect of the test substance. Thus, if the ratio of the area of ​​the fluorescent region to the organoid surface area is lower compared to the negative control, the added test substance can be determined to have aggregation / deposition inhibitory activity.When the ratio of the area of ​​the fluorescent region to the organoid surface area is equal to or less than that of a positive control (for example, in the presence of a known substance with aggregation / deposition inhibitory activity), the added test substance can be determined to have aggregation / deposition inhibitory activity. 1-4-3. Effect Substances evaluated to have aggregation / deposition inhibitory activity by the method of this embodiment are considered to have activity in a state closer to that of a living body, and are therefore expected to be useful as substances / compounds (drugs, supplements, functional foods, health foods, general foods, etc.) for the treatment and prevention of various diseases caused by aggregating proteins, including amyloidosis such as AD and Parkinson's disease. 1-5. Aβ Aggregation / Deposition Inhibitor 1-5-1. Composition (1) Plant Extract The Aβ aggregation / deposition inhibitor of the present invention is composed of a plant extract. The plant extract of the present invention is an extract obtained from a plant body, so its extraction method and state are not important. Examples of extracts include liquid components (so-called extracts) obtained by immersing a plant body in a solvent such as water, an organic solvent (e.g., ethanol), or a mixture thereof at a predetermined temperature for a predetermined time, followed by centrifugation or filtration to remove the plant body; solids (including granules and powders) remaining after removing the solvent from the extract by reducing pressure, evaporation, drying, or the like; and pastes in intermediate states thereof. Other examples include juice obtained by squeezing a plant body, a juice obtained by filtering the juice to remove solid components, or a solid obtained by evaporating or drying the water content. Extraction methods using solvents may be well-known extraction methods. Examples of solvents used for extraction include water, organic solvents, or mixtures of water and a water-soluble organic solvent such as ethanol. When using a mixture of ethanol and water (aqueous ethanol solution), the ethanol concentration of the mixture can be appropriately selected. For example, the ethanol concentration is preferably 65% ​​(w / w%) or more, 75% (w / w%) or more, or 85% (w / w%) or more, and is particularly preferably selected from the range of 85% to 99% (w / w%). When the solvent is water, the solvent temperature during extraction may be in the range of 10 to 100°C, preferably 40 to 99°C, and more preferably 70 to 99°C under 1 atmosphere, or in the range of 100 to 121°C under 1 to 2 atmospheres using a heat-resistant, pressure-resistant sealed vessel such as an autoclave.Furthermore, when the solvent is ethanol, the temperature range is 0 to 78.3°C, preferably 15 to 77°C, and more preferably 20 to 77°C under 1 atmosphere. Furthermore, when the solvent is a mixture of water and ethanol, the conditions for water or ethanol described above can be applied appropriately depending on the ethanol concentration, e.g., 90% (w / w%). The extraction time varies depending on the solvent temperature. Generally, a long time, for example, 1 to 10 days or more, is required when the solvent temperature is low (below 40°C), whereas a short time, for example, 2 minutes to 72 hours, is sufficient when the solvent temperature is high (below 40°C and below the boiling point under 1 atmosphere). When preparing a plant extract by extraction or squeezing, the plant may be used in its intact state, or in its fragmented or powdered form. "Intact plant" refers to the whole plant when the whole plant is used, or to the state of some of its organs, such as flowers, leaves, or roots, when parts of the plant are used. On the other hand, "fragmented" refers to the plant being cut into small pieces by cutting, grating, or the like. The size of the fragments is not particularly limited, but a small size is preferable to improve the extraction efficiency into the solvent and the juicing efficiency. However, a suitable pulverization size (maximum particle size range) can be selected taking into consideration factors such as ease of handling during production and the Aβ aggregation / deposition inhibition rate. For example, a range of 0.15 to 5 mm or 0.2 to 3 mm is sufficient. "Powdered" refers to a powder obtained by pulverizing a plant body in its original state or after freezing, drying, or lyophilization. The size of the particles constituting the powder is also not particularly limited, and may be, for example, granular size, fine powder size, or a mixture thereof. Specifically, the maximum particle size may be 6 mm or less, 5 mm or less, 4 mm or less, or 3 mm or less, and the lower limit of the maximum particle size may be 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, 0.4 mm or more, and preferably 0.5 mm or more. Examples of the maximum particle size range are 0.2 to 6 mm, 0.2 to 5 mm, 0.3 to 4 mm, and 0.5 to 3 mm. The maximum particle size referred to here is a value set by, for example, passing the pulverized material through a sieve, and therefore may include pulverized material smaller than this value.Unlike the juice squeezing method, the extraction method using a solvent is particularly preferred because it has a low contamination rate with impurities such as plant cell fragments and a high concentration of the effective ingredient in the extract, allowing for medicinal effects to be obtained with a small amount. (2) Plants The plants used as raw materials for preparing the plant extract constituting the Aβ aggregation / deposition inhibitor of the present invention are plants belonging to the Myrtaceae family. Plants belonging to the Myrtaceae family are not particularly limited, and include, for example, plants of the genus Eucalyptus, genus Acca, genus Eugenia, genus Pimenta, genus Plinia, genus Myrtaceae, and genus Myrtaceae. Specific examples include adeque, wax berry, myrtaceae, feijoa, jaboticaba, pitanga, guava, Eucalyptus robusta, clove, and allspice. The genus Eucalyptus, genus Pinellia, genus Myrtaceae, and genus Myrtaceae are particularly preferred, with the genus Myrtaceae being particularly preferred. As for species, guava, Eucalyptus robusta, clove, and allspice are preferred. The plant extract may be extracted from the whole plant of the Myrtaceae family, or from a part (organ) of the plant of the Myrtaceae family. Examples of the plant part include leaves, stems, buds, leaf sheaths, petioles, corms, rhizomes, roots, seeds, hypocotyls, fruits, and flower buds. The plant extract can be extracted from any part (organ) of the plant, but it is preferred to extract the plant extract from seeds and fruits of guava, leaves, stems, leaf sheaths, and petioles of Eucalyptus robusta, flower buds of cloves, and fruits of allspice. In particular, in the case of cloves, the extract is preferably obtained by subjecting crushed cloves to solvent extraction so that the maximum particle size is 6 mm or less, 5 mm or less, 4 mm or less, preferably 3 mm or less, and the lower limit of the maximum particle size is 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, 0.4 mm or more, preferably 0.5 mm or more, and the crushed product has a crushed size (maximum particle size range), for example, 0.2 to 6 mm, 0.2 to 5 mm, 0.3 to 4 mm, or 0.5 to 3 mm. In this case, the solvents described above can be used as preferred examples. 1-5-2. Effect The Aβ aggregation / deposition inhibitor of the present invention comprises a plant extract evaluated to exhibit potent Aβ aggregation / deposition inhibitory activity by the method of the above-mentioned embodiment 1-4.Specifically, the plant extract of the present invention exhibits an inhibition rate (or aggregation / deposition inhibition rate in the above-mentioned 1-4) of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, preferably 80% or more, 85% or more, more preferably 90% or more, 95% or more, particularly preferably 96% or more, 97% or more, or 98% or more relative to a control group to which no test substance is added, using the method of the above-mentioned 1-4 aspects. The Aβ aggregation / deposition inhibitor of the present invention, discovered using the above-mentioned evaluation method that is closer to a living body environment using cerebral organoids, can also exhibit a good Aβ aggregation / deposition inhibitory effect in actual living tissue (e.g., the surface and / or interior of living brain tissue cells). Furthermore, the Aβ aggregation / deposition inhibitor of the present invention exhibits an inhibitory / improving effect on the decline of cognitive function in a subject, which is the subject's ability to recognize things via the brain, and can be used as an inhibitor / improver of cognitive decline. 2. Aβ Aggregation / Deposition Inhibitory Composition 2-1. Overview The second aspect of the present invention is a composition for inhibiting Aβ aggregation / deposition. The composition of the present invention comprises the Aβ aggregation / deposition inhibitor described in the first aspect as an active ingredient. The aggregation / deposition inhibitor of the present invention enables the aggregation / deposition inhibitor to be easily handled and applied in a stable state. This reduces the burden and invasiveness of administration to the body, and allows it to be provided in a more easily administered form, for example, as a pharmaceutical composition. 2-2. Composition 2-2-1. Constituent Factors The aggregation / deposition inhibitor composition of this aspect (hereinafter often abbreviated as "the composition") comprises an active ingredient as an essential constituent factor, and a solvent and / or carrier as optional constituent factors. Each constituent factor is described in detail below. (1) Active Component The composition comprises the Aβ aggregation / deposition inhibitor described in the first aspect as an active ingredient. The composition may comprise one or two or more different Aβ aggregation / deposition inhibitors. Furthermore, the composition may selectively contain a known substance (such as a nucleic acid, a polypeptide, or a low-molecular-weight compound) that has a similar inhibitory effect on Aβ aggregation / deposition, other than the Aβ aggregation / deposition inhibitor described in aspect 1. The content of the Aβ aggregation / deposition inhibitor contained in the composition varies depending on the type and / or effective amount of the Aβ aggregation / deposition inhibitor contained therein, the dosage form of the composition, the type of carrier or additive described below, and the type of disease, and may be determined appropriately taking into consideration each of the conditions.As used herein, the term "effective amount" refers to the amount of the Aβ aggregation / deposition inhibitor in the present composition that is required to function as an active ingredient and that causes little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. As used herein, the term "subject" refers to the living body to which the Aβ aggregation / deposition inhibitor or the present composition is applied. Examples include humans, livestock (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, pets (dogs, cats, rabbits, etc.), and laboratory animals (mice, rats, guinea pigs, monkeys, etc.). Humans are preferred (in this case, they are referred to as "subjects"). Furthermore, "subject information" refers to various individual information about the living body to which the composition is applied, such as, in the case of a subject, the overall health status, the progression and severity of any disease or injury, if present, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant medications, and resistance to treatment. The effective amount of the Aβ aggregation / deposition inhibitor in the present composition and the dosage calculated based thereon are ultimately determined by the judgment of a physician or veterinarian, depending on the individual subject's information, etc. (2) Solvent: The present composition may contain a pharmaceutically acceptable solvent. The solvent is an optional component of the present composition and may be added as needed. "Pharmaceutically acceptable" means that the solvent is harmless or low-toxic to living organisms, is generally usable in the formulation technology field, and is preferably usable in pharmaceutical compositions. Examples of solvents include water or aqueous solutions, buffer solutions, emulsifiers, and organic solvents. Examples of pharmaceutically acceptable aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Pharmaceutically acceptable organic solvents include ethanol, etc. (3) Carrier: The present composition may contain a pharmaceutically acceptable carrier. A carrier is an optional component of the composition and may be added as needed.Examples of carriers include suspending agents, diluents, solubilizers, dispersants, surfactants, emulsifiers, soothing agents, stabilizers, preservatives, antiseptics, antioxidants, buffers, and isotonic agents. In addition to the above, if necessary, excipients, fillers, binders, disintegrants, absorption enhancers, bulking agents, wetting agents, humectants, moisturizing agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, and the like commonly used in pharmaceuticals may also be appropriately included. Such carriers are primarily used to facilitate dosage form formation, maintain the dosage form and pharmaceutical efficacy, and make the active ingredient, the Aβ aggregation / deposition inhibitor, less susceptible to degradation in vivo, and may be used appropriately as needed. 2-2-2. Dosage Form and Administration Method The dosage form of the present composition is not particularly limited, as long as it does not inactivate the active ingredients, such as the Aβ aggregation / deposition inhibitor and other known Aβ aggregation / deposition inhibitors, and can exert the pharmacological effects of the active ingredients in vivo after administration. The specific dosage form of the composition may be selected appropriately depending on the administration method and / or formulation conditions. Generally, administration methods can be broadly divided into oral and parenteral administration, and the composition may be formulated in a dosage form suitable for each administration method. For oral administration, dosage forms include solids (including tablets, pills, sublingual tablets, capsules, and drops), granules, powders, and liquids (including oral solutions, suspensions, emulsions, and syrups). Solids may be formulated into coated dosage forms known in the art, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double-layered tablets, and multi-layered tablets, as needed. Oral administration is a preferred method due to its minimal invasiveness and ease of administration. Parenteral administration can be divided into systemic administration and local administration, and local administration can be further subdivided into intratissue administration, transepidermal administration, transmucosal administration, and rectal administration. The composition may also be formulated in a dosage form suitable for each administration method. For example, dosage forms suitable for systemic or intratissue administration include liquid injections. Dosage forms suitable for transepidermal or transmucosal administration include liquids (including liniments, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), powders (including nasal drops and inhalants), pastes, gels, ointments, and plasters. Dosage forms suitable for rectal administration include suppositories.The administration method of the present composition varies depending on the type of target disease and the target application site, as described below. The administration method is not limited as long as the Aβ aggregation / deposition inhibitor, which is the active ingredient of the present composition, ultimately reaches the target application site and exerts its effect. For example, if the target application disease is a neurodegenerative disease, the target application site is mainly the cerebrospinal cord, which is the central nervous system. In this case, the composition may be administered intratissuely by direct administration into the central nervous system, or systemically via the circulatory system. Specific examples include intratissue administration by intracerebrospinal injection, or intracirculatory administration such as intravascular injection (including intravenous injection and intraarterial injection) or intralymphatic injection. Generally, direct administration such as intratissue administration can reliably deliver the active ingredient to the target application site at a high concentration. On the other hand, systemic administration via the circulatory system can deliver the active ingredient to the target application site with minimal invasiveness and without leakage. The method to be selected can be determined appropriately depending on the situation and pathological condition. Injectable preparations can be formulated by appropriately combining the above-mentioned emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and blending them in a unit-dose form required for commonly accepted pharmaceutical practice, and are provided in the form of unit-dose ampoules or multi-dose containers. The specific shape and size of each of the above dosage forms are not particularly limited, as long as they are within the range of dosage forms known in the art. 2-2-3. Target Diseases: The present composition can be used to treat or prevent Alzheimer's disease and mild cognitive impairment caused by Aβ aggregation / deposition. Alzheimer's disease begins with the aggregation and accumulation of amyloid beta protein in neurons in the brain, followed by hyperphosphorylation and fibrillation of the microtubule protein tau protein, which then leads to neuronal destruction and brain atrophy. Therefore, the aggregating proteins that cause Alzheimer's disease can include amyloid beta protein, tau protein, and α-synuclein. The Aβ aggregation / deposition inhibitor contained in this composition can suppress the progression or prevent Alzheimer's dementia by inhibiting Aβ aggregation / deposition. On the other hand, mild cognitive impairment is a cognitive impairment intermediate between healthy individuals and dementia, and is characterized by a mild decline in cognitive function. Most mild cognitive impairment is caused by Alzheimer's dementia.Therefore, the present composition can also be used to treat or prevent mild cognitive impairment. 2-3. Effects The present composition can be effectively used to treat or prevent Alzheimer's disease or mild cognitive impairment. 3. Agent for Preventing / Ameliorating Decline in Cognitive Function of a Subject 3-1. Overview A third aspect of the present invention is an agent for preventing / ameliorating decline in cognitive function of a subject, which is the subject's ability to perceive things via the brain. The agent for preventing / ameliorating decline in cognitive function of a subject of the present invention, like the Aβ aggregation / deposition inhibitor described in the first aspect, contains an extract of a plant belonging to the Myrtaceae family (particularly, clove or allspice) as an active ingredient. 3-2. Composition 3-2-1. Constituent Factors The agent for preventing / ameliorating decline in cognitive function of a subject of this aspect contains an active ingredient as an essential constituent factor, and a solvent and / or carrier as optional constituent factors. The agent for preventing / ameliorating decline in cognitive function of a subject of this aspect can contain an extract of a plant belonging to the Myrtaceae family as an active ingredient, like the Aβ aggregation / deposition inhibitor described in the first aspect. Furthermore, known substances (e.g., nucleic acids, polypeptides, low-molecular-weight compounds) other than extracts of plants belonging to the Myrtaceae family that have similar effects of inhibiting / improving cognitive decline in a subject may be selectively included. The content of extracts of plants belonging to the Myrtaceae family contained in the agent for inhibiting / improving cognitive decline in a subject of this embodiment varies depending on the type and / or effective amount of the extract of plants belonging to the Myrtaceae family included, the dosage form, the type of carrier or additive, and the type of disease, and can be determined appropriately taking into account each condition. The "effective amount," "subject," "solvent," and "carrier" may be similar to those described in the composition of the second embodiment above. 3-2-2. Dosage Form and Administration Method The dosage form (formulation) and administration method of the agent for inhibiting / improving cognitive decline in a subject of this embodiment may be similar to those described in the composition of the second embodiment above. 3-3. Effect The agent for inhibiting / improving cognitive decline in a subject of this embodiment can prevent cognitive decline and improve cognitive function.

[0008] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples. The reagents used in these examples are as follows: <Reagents> STEMdiffＴＭNeural Progenitor Medium (NPM) (STEMCELL / 5833) ・Neuro basal medium minus phenol red (Gibco / 12348017) ・hESC-Qualified Matrigel (hES-matrigel) (Corning / 354277) ・Accutase (STEMCELL / 7920) ・DMEM / F-12 (Gibco / 21041025) ・Y-27632 (Nacalao / 08945-84)・PBS (Gibco / 10010-031) B-27 (Gibco / 17504044) Penicillin-Streptomycin (Nacalai / 26253-84) Amyloid β-Protein human 1-42 (Peptide Institute / 4349-v) 1,1,1,3,3,3-Hexafluoro-2-2propanol (HFIP) (Tokyo Chemical Industry / H0424) Dimethyl sulfoxide (DMSO) (Fujifilm Wako Pure Chemical Industries / 046-21981) QD reagent (described below) Plant material solution (100 mg / mL solution: described below) Physiological saline (saline) (Otsuka Pharmaceutical) 1 mL syringe 0.2 μm filter Preparation of NbM medium 50 mL Neuro NbM medium was prepared by adding 1 mL of B-27 (Gibco / 17504044) and 500 μL of Penicillin-Streptomycin (Nacalai / 26253-84) to basal medium minus phenol red (Gibco / 12348017). Example 1 Preparation of Brain Organoids (1) Cultivation and Passage of iPS Cells Human iPS cells were seeded using HPS08554 (RIKEN BRC) onto cell culture dishes coated with Matrigel (Corning), Vitronectin (Life Technologies Japan), iMatirx (Takara Corporation, T303), or fibronectin (Recombinant HUMAN Fibronectin GMP Protein, CF Summary).The media used were mTeSR1 (STEMCELL Technologies), TeSR2 (STEMCELL Technologies), and Essential 8. ＴＭ (Life Technologies Japan, Inc.) or StemFit was used for maintenance culture. When cultured on Matrigel or Vitronectin, Accutase (Life Technologies Japan, Inc.) or GCDR (STEMCELL Technologies) was used as a cell detachment agent during passaging. When cultured on fibronectin, 0.05% EDTA (ethylenediaminetetraacetic acid) solution was used. When cultured on iMatirx, TrypLE Select (Life Technologies Japan, Inc.) or TrypLE Express was used. Only at the time of cell seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) was added to the medium to a concentration of 10 μM. Medium changes were performed daily or every day. Human iPS cells up to 50 passages were used in the experiments. (2) Preparation of neural-inducible embryoid bodies (EBs) After thawing, human iPS cells that had been passaged at least twice were treated with a detachment agent for 5 to 15 minutes to detach them from the culture substrate, and then dispersed into single cells by pipetting using a pipette. These single cells were then cultured in a STEM diff containing Y-27632 (Wako Pure Chemical Industries, Ltd.) at a final concentration of 10 μM. ＴＭ STEMdiff on Neural Induction Medium ＴＭ The cells were suspended in a medium supplemented with SMADi Neural Induction Supplement (hereinafter referred to as "NIMS medium"), and seeded onto a PLO / Laminin-coated or Matrigel (registered trademark) (Corning (registered trademark)). ２ After confirming cell adhesion, the NIMS medium was completely replaced every day. On the seventh day, the adherent cells were detached with Accutase, and the detached cells were subjected to STEM diff analysis on a PLO / laminin-coated plate or Matrigel. ＴＭThe cells were expanded in Neural Progenitor Medium and passaged three times. The cells were suspended in NbM plus Shh medium (hereinafter referred to as "NbM medium") prepared by mixing 50 mL of Neuro Basal Medium minus phenol red (Thermo Fisher Scientific), 1 mL of B-27 supplement (Thermo Fisher Scientific), and Penicillin-Streptomycin Mixed Solution (Nacalai Tesque) with SHH (human recombinant sonic hedgehog (Veritas)) at a final concentration of 1 ng / mL. 4 × 10 cells were then cultured. ５ A cell suspension was prepared at a cell density of 1000 cells / mL, and 0.1 mL of the suspension was seeded into a U-bottom 96-well plate. After seeding, the plate was incubated in 5% CO for 24 hours. ２ The cells were cultured in mTeSR1 (STEMCELL Technologies) at 37°C to prepare neural induction EBs. Alternatively, human iPS cells cultured in mTeSR1 (STEMCELL Technologies) were treated with GCDR to form single cells, and then 9 × 10 ４ A cell suspension of 0.1 mL / mL was prepared, and the suspension was inoculated into a U-bottom 96-well plate in 0.1 mL portions. ＴＭ The medium was replaced every two days with the EB Formation medium in the Cerebral Organoid Kit, and the cells were cultured in a 5% CO atmosphere. ２The cells were cultured at 37°C for 5 days. The medium was then replaced with Induced Medium, and the EBs formed from iPS cells were cultured for an additional 2 days to induce neural induction, preparing neurally induced EBs. (3) Gel-embedded culture of neurally induced EBs. EBs 2 days after Hedgehog signal addition (Day 2) and 5 days after Hedgehog signal addition (Day 5) were carefully collected using a wide-mouth tip, taking care not to disrupt the EBs, and 12 EBs were embedded in Matrigel. There are several embedding methods, but in this example, parafilm was applied to a 48-well plate, and the required number of wells were created using the tip attachment. 12 EBs were placed in the wells, and excess medium was removed. After that, 15 μL of Matrigel was dropped and left to stand in an incubator for 0.5 to 2 hours. Fresh NbM medium was dropped into a 6-well plate for suspension, and the Matrigel was collected using a tip cut with scissors, being careful not to break it. Each EB embedded in Matrigel was rotated at 90 rpm on a bioshaker while being incubated in 5% CO ２ The EBs were cultured in an environment of 1000 x g and 37°C. During the gel-embedded culture period, half of the NbM medium was replaced every three days. As shown in Figure 1, the formation of organoid-like tissues was confirmed. As a result, good tissues were formed in the EBs on Day 2, whereas in the EBs on Day 5, the cells did not sufficiently infiltrate and fill the gel. (4) Gel-embedded culture and quantification of CCL2 in the culture supernatant. Each EB embedded in Matrigel was rotated at 90 rpm using a bioshaker while being cultured in 5% CO ２The cells were cultured at 37°C. Cells within the gel actively infiltrated and proliferated, filling the gel space. Therefore, media were collected from the initial stage of gel culture (unfilled stage: D0), the growth stage of gel culture (unfilled stage: D7), the cell-filled stage (filled stage: D14), and the cell-filled stage (filled stage: D21), and the cytokine secretion level (CCL2) in the culture supernatant was quantified by ELISA. The results are shown in Figure 2. A correlation was found between the cell infiltration status within the gel (Figure 2A) and the secretion level of CCL2 (monocyte chemotactic protein) (Figure 2B). The level of CCL2 secretion remained high while the cells infiltrated and filled the gel, decreased as filling progressed, and eventually stabilized at a constant level. Thus, measuring the level of CCL2 in the culture supernatant during the organoid-like tissue formation process can confirm whether the gel was filled with cells. Example 2: Preparation of organoids for visualizing deposition of aggregating proteins, and a method for searching for inhibitors of aggregation / deposition of aggregating proteins using the organoids. The tip of a 1000 μL tip was cut with sterilized scissors, and the organoids prepared in Example 1 that had grown to a diameter of 1 mm or more were aspirated without breaking up and transferred to a U-bottom 96-well plate. The medium was then removed from the U-bottom 96-well plate, and (i) quantum dot-modified amyloid β was added. ４０ Protein (QD-Aβ ４０ ) solution 25 μL, (ii) amyloid β42 protein (Aβ ４２ 25 μL of (i) solution, (ii) solution, and (iii) 50 μL of test sample solution (ratio of (i) solution:(ii) solution:(iii) solution is 1:1:2) were dropped into one well, and the plate was incubated in 5% CO ２ and cultured at 37°C for 24 hours (final concentration: QD-Aβ ４０ 30 nM, artificial Aβ ４２ Solution 10 μM, test sample (rosmarinic acid: 50 μM)). Each solution was prepared as follows: (i) QD-Aβ ４０ Preparation of Aβ solution (120 nM) ４０ was modified with quantum dots (QDs) to prepare quantum dot-modified amyloid β protein (QD-Aβ ４０ 125 μL of 8 μM Qdot (QD) ＴＭ605 ITK™ Amino (PEG) Quantum Dots were placed into two 1.5 mL tubes and centrifuged at 10,000 x g for 1 minute at 4°C. Each supernatant was transferred to a centrifugal concentration tube, and 4,500 μL of PBS was added. The tubes were centrifuged at 3,800 x g at 4°C until the total volume of the two tubes was approximately 50 μL or less, and the filtrate was discarded. After refilling with PBS, the tubes were centrifuged again until the total volume reached 50 μL. The resulting QD solution was combined into one tube to a total volume of approximately 180 μL. 20 μL of 10 mM sulfo-EMCS was added, and the tubes were left to stand at room temperature for 1 hour to prepare QD-EMCS. After preparing the QD-EMCS, 20 μL of 100 mM K-glutamic acid was added to deactivate any unreacted N-hydroxysuccinimide groups in the solution, and the mixture was allowed to stand at room temperature for 10 minutes. Two desalting columns were prepared, each containing approximately 800 μL of resin, and centrifuged at 1000 × g and 4 ° C for 1 minute. Each column was loaded with 300 μL of PBSE, and centrifuged at 1000 × g and 4 ° C for 1 minute. This process was repeated twice to prepare the desalting columns. 110 μL of the QD-EMCS was then soaked into the center of each of the two desalting columns, and 15 μL of PBSE was added as a stacker. After centrifugation at 1000 × g and 4 ° C for 2 minutes, the filtrates from the two desalting columns (desalted QD-EMCS) were combined. 20 μL of 1.0 mM Cys-Aβ in DMSO was added to the resulting desalted QD-EMCS, mixed, and allowed to stand at room temperature for 1 hour. Then, 20 μL of 100 mM 2-mercaptoethanol was added to inactivate any unreacted maleimide groups in the solution, and the mixture was allowed to stand at room temperature for 10 minutes. The filtrate was transferred to two centrifugal concentrator tubes (145 μL each), and 4500 μL of water was added. The mixture was then centrifuged at 3800 × g and 4 ° C for 17 minutes. The filtrate was discarded, and the resulting solution was combined into one tube so that the total volume was approximately 140 μL. 300 μL of water was added to a desalting column and centrifuged at 1000 × g and 4 ° C for 1 minute. This process was repeated twice. 70 μL of the filtrate was permeated into the desalting column, and 15 μL of water from the stacker was added. The mixture was centrifuged at 1000×g at 4° C. for 2 minutes to obtain the desired QD-Aβ. ４０ The synthesized QD-Aβ ４０The solution (80 μM) was diluted with NbM medium to 120 nM and subjected to ultrasonic bathing at 25°C and 43 kHz for 5 minutes. (ii) Artificial Aβ ４２ Preparation of Aβ solution (40 μM) １−４２ 500 μL of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) solution was added to a 0.5 mg (Peptide Institute: Cat No. 4394v) reagent bottle, pipetted, sealed with parafilm, and left to stand in a clean bench at room temperature for 1 hour. The lid of the reagent bottle was then opened in the clean bench to volatilize the HFIP. After confirming that all of the HFIP had volatilized, 107 μL of DMSO was added and dissolved over 15 minutes. The solution was dispensed in 10 μL aliquots into tubes and frozen at −80° C. This frozen 1 mM Aβ ４２ After thawing the solution, the solution was pipetted 20 times or more, diluted 25-fold with NbM medium, and diluted to 40 μM Aβ ４２A solution was prepared. (iii) Preparation of Test Sample-Containing Solution Preparation of Small Molecular Weight Compound Solution (100 μM) Rosmarinic acid (hereinafter referred to as "RA") was dissolved in DMSO to prepare a 1 mM RA solution, which was then diluted with NbM medium to prepare a 100 μM RA solution. 1 μL of DMSO solution and / or 150 mM saline was diluted with 19 μL of NbM, and 3 μL of this diluted solution was further extracted and diluted with 72 μL of NbM to prepare a solution. The next day, all of the culture supernatant was removed without aspirating the organoids, and 150 μL of fresh NbM medium was added. The cultured organoids were observed and photographed using a fluorescent microscope. After photography, the organoids were returned to the incubator and cultured, and the following day and thereafter were observed and photographed using a fluorescent microscope in the same manner. Figure 3 shows the time course of Aβ aggregation and deposition observed and photographed. As shown in Figure 3, the organoids for visualizing the deposition of aggregating proteins were able to gradually increase the amount of aggregating protein deposition while keeping the organoids alive, and it was confirmed that the amount of aggregating protein deposition could be observed for at least 8 days. From the above results, it was found that organoids for visualizing the deposition of aggregating proteins, which mimic the rate of accumulation of aggregating proteins in the human brain with aging, can also be produced. [Example 3] Method for quantification of aggregating protein deposition in organoids Using a fluorescence microscope (Keyence BZ-X800), the cultured organoids in Example 2 were photographed in bright field at high resolution with an exposure time of 1 / 500s to 1 / 2000s, or at high sensitivity with an exposure time of 1 / 2000s to 1 / 5000s, with Z stacks taken at 20 μm intervals. When using a x2 objective lens, one image capturing the entire organoid was taken. When using a x4 objective lens, one image centered on the center of the cultured organoid and three images including the outline of the cultured organoid were taken, resulting in a total of four images. To observe the deposition of quantum dot-modified amyloid beta in the cultured organoids, red fluorescence was observed using a TRITC lens at high sensitivity with an exposure time of 1 / 4 s to 1 / 8.5 s, and images were acquired according to the procedure described above. Each of the images acquired above was analyzed as follows. Specifically, the organoid images used in various tests were loaded using the hybrid cell counting function, bright field was selected, and extraction selection was set to OFF.The threshold of the imported bright-field image was set to 200, and the organoid area was calculated (organoid surface area). ４０ / artificial Aβ ４２ Organoid images of organoids cultured without the addition of a test sample; ４０ / artificial Aβ ４２Images of negative control organoids containing DMSO and / or saline as test samples were read into the organoids; and images of organoids containing rosmarinic acid (RA) as test samples were read into the organoids. The black balance level for image processing was adjusted for the fluorescent images of each test. First, the negative control image was read. Images captured with a x2 objective lens were initially set to

[50] , and images captured with a x4 objective lens were initially set to

[70] . This confirmed whether the fluorescent regions of the aggregating proteins deposited on the organoids could be separated from the background. If the fluorescent regions of the aggregating proteins deposited on the organoids could not be separated from the background even at the above black balance levels, the black balance level was adjusted in 5 increments to find the optimal black balance level. The fluorescent image after applying the black balance and the bright-field organoid image were combined, and then fluorescence was selected using the hybrid cell counting function, followed by setting 1 extraction. The detailed brightness mode was selected, the extraction setting range was set to 0-299, and the organoid was selected. The blur filter was unchecked, the threshold was set to 25, and the area of ​​the fluorescent range of the aggregating protein was calculated (aggregating protein deposition area). The aggregation / deposition rate (%) of the aggregating protein on the organoid was calculated using the following formula (1): Aggregating / deposition rate (%) of the aggregating protein on the organoid = (aggregating protein aggregation / deposition area ÷ organoid surface area) × 100 (1). Furthermore, the condition for the above test system to be established was that the aggregation / deposition rate (%) of the aggregating protein must be in the range of 90-100% when DMSO and / or physiological saline were added. Furthermore, when this test system was established, a group to which a test sample was also added was also established, allowing for quantitative analysis of the sample's inhibitory effect on the aggregation / deposition of aggregating proteins on organoids.Specifically, the aggregation / deposition inhibitory function of the test sample against the aggregation / deposition of aggregating proteins could be quantified as the aggregation / deposition inhibition rate (%), which could be calculated using the following formula (2): Aggregation / deposition inhibition rate (%) of aggregating proteins (Aβ in this example) = (aggregation / deposition rate (%) of aggregating proteins in the absence of test substance) - (aggregation / deposition rate (%) of aggregating proteins in the presence of test substance) (2) It can be seen that the greater the aggregation / deposition inhibition rate (%) calculated using formula (2), the greater the aggregation / deposition inhibitory effect of the test sample. This evaluation technique can be used to select drugs, compounds, foods, and substances that can prevent or delay the deposition of aggregating proteins in cells or tissues. The results of the Aβ aggregation / deposition inhibitory effect of each test sample in this example are shown in Figure 4. As shown in Figure 4, the Aβ aggregation / deposition inhibition rate was calculated 24 hours after the addition of each drug: DMSO, saline, and rosmarinic acid. The Aβ aggregation / deposition inhibition rate for cerebral organoids for DMSO was 0.53%, the Aβ aggregation / deposition inhibition rate for cerebral organoids for saline was 0.11%, and the Aβ aggregation / deposition inhibition rate for cerebral organoids for rosmarinic acid was 3.9%. From the above results, it became clear that rosmarinic acid, which is known to have an Aβ aggregation inhibitory effect, is not a drug that has an Aβ aggregation / deposition inhibitory effect on cerebral organoids. [Example 4] Search for amyloid beta (Aβ) aggregation / deposition inhibitors Using the quantitative system for Aβ aggregation / deposition in cerebral organoids constructed as described above, a search was carried out for materials (plant extracts) that have an Aβ aggregation / deposition inhibitory effect. (i) Preparation of Test Sample-Containing Solutions Test samples used were guava seeds and fruit, Eucalyptus robusta leaves, stems, leaf sheaths, and petioles, clove buds, and allspice berries (all Myrtaceae), Lantana umbellata leaves, stems, leaf sheaths, petioles, and petals, sweet Mexican herb leaves, stems, leaf sheaths, and petioles (all Verbenaceae), and Kakido-osu leaves, stems, leaf sheaths, and petioles (all Lamiaceae) (final extract concentration at test: 0.01 mg / well). Each test sample was washed, dried, and crushed to a size of 5 mm x 5 mm or less. 30 mL of 90% EtOH was added to 1 g of the crushed material, and the mixture was extracted by shaking at 150 rpm at 28 °C for 20 hours in the dark.The extract was centrifuged at 3,000 rpm for 20 minutes, filtered through a cotton plug, and then centrifuged and concentrated, followed by lyophilization. The dried product was dissolved in DMSO to prepare various plant extracts at 100 mg / mL, which were then stored at -20°C until use. The plant extract was diluted 25-fold with NbM medium, and 40 μL of the diluted solution was diluted with 960 μL of NbM medium, sterilized by filtration through a 0.2 μm filter, to prepare a solution containing 0.02 mg of material. (ii) Measurement of Aβ Aggregation / Deposition Inhibitory Activity: The inhibitory effect of each plant extract on Aβ aggregation / deposition in cerebral organoids was measured at a final extract concentration of 0.01 mg / well. The cerebral organoids, reagents including QD-Aβ, operating procedures, and methods for image processing and calculation of aggregation / deposition inhibition rates used were as described in Examples 1 to 3. DMSO and rosmarinic acid were used as comparison groups. The results of observation and photography of the inhibitory effect of various test samples on aggregation / deposition of aggregating proteins are shown in Figure 5. Table 2 also shows the Aβ aggregation / deposition inhibition rate (%) 24 hours after the addition of the samples. From the results shown in Figure 5 and Table 2, it was found that the Myrtaceae plant extract has an excellent inhibitory effect on Aβ aggregation / deposition on the surface and / or inside of cerebral organoids. [Example 5] Evaluation of Clove Grind Size The relationship between the grind size of cloves and the Aβ aggregation / deposition inhibition rate was evaluated. (i) Preparation of Clove Grinds Cloves were ground in a mill to obtain the following clove grinds. Grind A: grind size 5mm or less (maximum particle diameter of particles contained in the grinded product 5mm or less) Grind B: grind size 2mm or less (maximum particle diameter of particles contained in the grinded product 2mm or less) Grind C: grind size 0.5mm or less (maximum particle diameter of particles contained in the grinded product 0.5mm or less) In other words, the particle diameter of all particles contained in grind A is 5mm or less, the particle diameter of all particles contained in grind B is 2mm or less, and the particle diameter of all particles contained in grind C is 0.5mm or less. (ii) Preparation of Test Sample-Containing Solutions: 30 mL of 90% EtOH was added to 1 g of each ground material (i) above, and the mixture was extracted by shaking at 150 rpm at 28°C for 20 hours in the dark. The extract was centrifuged at 3,000 rpm for 20 minutes, filtered, concentrated by centrifugation, and then freeze-dried. The dried material was dissolved in DMSO to prepare various plant extracts at 100 mg / mL, which were stored at -20°C until use. The yield of ground material A extract was 13.7%, the yield of ground material B extract was 17.4%, and the yield of ground material C extract was 21.4%. Therefore, the smaller the ground size, the higher the yield. The plant extract was diluted 25-fold with NbM medium, and 40 μL of the diluted solution was diluted with 960 μL of NbM medium and sterilized by filtration through a 0.2 μm filter to prepare a solution containing 0.02 mg of the material. (iii) Measurement of Aβ aggregation / deposition inhibitory activity This was carried out in the same manner as in Example 4. The results of observation and photography of the inhibitory effect of various test samples on the aggregation / deposition of aggregating proteins are shown in Figure 6. The Aβ aggregation / deposition inhibitory rates (%) of various test samples are shown in Table 3. In Figure 6, the areas where red fluorescent quantum dot-modified amyloid beta was deposited exhibit red fluorescence. As shown in Figure 6 and Table 3, it was found that the plant extracts prepared from pulverized material A, pulverized material B, and pulverized material C all possessed high Aβ aggregation / deposition inhibitory activity. This finding indicated that it is preferable to extract plant extracts from pulverized material with a pulverized size of 5 mm or less, 2 mm or less, or 0.5 mm or less, with a maximum particle size of approximately 2 mm being particularly preferable. Interestingly, from a comprehensive perspective, such as extraction recovery rate and Aβ aggregation / deposition inhibition rate, the preferred pulverized size (maximum particle size range) was found to be in the range of 5 mm to 0.5 mm and 3 mm to 0.5 mm. Furthermore, other studies have shown that the 3 mm to 0.5 mm range is particularly preferable. It was found that controlling the pulverized size is extremely important for achieving high extraction recovery rates and high Aβ aggregation / deposition inhibition rates. [Example 6] Evaluation of the effects of plant extracts on preventing cognitive decline and improving cognitive function The effects of allspice extract and clove extract on preventing cognitive decline and improving cognitive function were evaluated using mice. The evaluation schedule is shown in Figure 7. (i) Animal rearing Seven-week-old ddY mice (female) were prepared. This day was designated as the first day after the mice arrived (first day of rearing). For each sample, four mice were randomly assigned to cages, totaling 26 mice. (ii) Aβ for mouse administration ４２ Preparation of Aβ ４２5 mg of Aβ (Peptide Institute: Code 4394) was dissolved in 2.3 mL of hexafluoro-2-propanol (HFIP) and allowed to dissolve at room temperature for 30 minutes. While cooling on ice, 100 μL of the solution was dispensed into tubes and dried. The amount of Aβ per tube was 47,810 pmol = 215.81 μg / 100 μL HFIP / tube. These Aβ tubes were stored in a deep freezer at -60°C. (iii) Preparation before intracerebroventricular administration (ICV administration) A two-stage tuberculin 3m / m (Natsume Seisakusho Co., Ltd.) was attached to a HAMILTON microsyringe. 64.6 μL of 6 mM NaOH was added to the Aβ tube prepared in the previous section, and the mixture was ultrasonically stirred in a room temperature water bath for 10 minutes to dissolve the Aβ. The dissolved Aβ was dispensed into 1.5 mL Eppendorf tubes in 32.3 μL aliquots. 34.5 μL of 10x PBS was transferred to a new Eppendorf tube, 279.4 μL of Milli-Q was added, and the mixture was vortexed. 142.7 μL was then dispensed and added to the Eppendorf tube containing 32.3 μL of Aβ. This prepared a microsyringe containing Aβ dissolved in 1x PBS. (iv) Preparation of Administration Samples. Gum arabic adjusted to 2% gum arabic was added to various plant extracts (allspice extract, clove extract) and ground in a mortar. The sample was then dissolved by adding saline, and the resulting solution was adjusted to 500 mg sample / kg body weight / 2% gum arabic / 10 mL saline. The resulting administration samples were dispensed into 4.5 mL tubes at daily dosages and stored in a deep freezer. A control (saline) administration sample of 0 mg / kg body weight / 2% gum arabic / 10 mL saline was also prepared at the same time. For administration, the sample was removed from the deep freezer, placed in a water bath at 30°C, and dissolved while shaking. (v) Preparation of Mice Orally Administered with Allspice Extract and Clove Extract The allspice extract and clove extract administration samples prepared in (iv) were orally administered to mice from day 4 to day 26 after the mice's arrival, so that the dose was 500 mg / kg per mouse.(vi) Preparation of Control Orally Administered Mice: The same control (saline) administration sample as the above-mentioned dose per mouse was orally administered from day 4 to day 26 after the mice's arrival. Both mice (v) and (vi) were orally administered from day 4 to day 26 after the mice's arrival, and the sample was administered before the model mice were created. (vii) Preparation of Model Mice by Intracerebroventricular (ICV) Administration of Aβ (hereafter referred to as ICV-Aβ) (Mouse Rearing: Day 7): On day 7 after the mice's arrival, mice orally administered allspice extract, clove extract, and saline were anesthetized by inhalation using sevoflurane. A needle tip was aligned vertically at a position 0.3 mm posterior, 1.1 mm lateral, and 4 mm repositioned from the bregma of each mouse. 3 μL of Aβ solution was slowly injected, held for 5–10 seconds, and gently withdrawn (the Aβ dose was 409.8 pmol / 3 μL / mouse). This resulted in the preparation of the following three mouse groups. A group of mice in which Aβ was administered ICV to mice that had previously been orally administered a control (saline) administration sample; a group of mice in which the control administration sample was administered orally to Aβ mice (hereinafter referred to as control-administered mice); a group of mice in which Aβ was administered ICV to mice that had previously been orally administered a clove extract administration sample; a group of mice in which the clove extract administration sample was administered orally to Aβ mice (hereinafter referred to as clove extract-administered mice); a group of mice in which Aβ was administered ICV to mice that had previously been orally administered an allspice extract administration sample; a group of mice in which the allspice extract administration sample was administered orally to Aβ mice (hereinafter referred to as allspice extract-administered mice). (viii) Evaluation of Cognitive Function in Mice by Y-maze Test The cognitive functions of the control-administered mice, clove extract-administered mice, and allspice extract-administered mice were evaluated on the 19th or 20th day after arrival using the Y-maze test, which can evaluate spatial working memory in mice, to evaluate the preventive effect of sample administration on cognitive decline. In the Y-maze test, mice were allowed to explore a Y-maze for 5 minutes, and the total number of times they entered each arm and the number of times they entered three consecutive different arms were recorded. The entry criterion was defined as the tip of the nose passing halfway through the arm. The alternation rate, which is an index of spatial working memory, was calculated using the following formula.Mice with high spatial working memory exhibit a high alternation rate. Alternation rate (%) = number of three consecutive entries into different arms / total number of entries - 2 x 100...Equation (1). The alternation rate for each mouse is shown in Table 4. Mice that were orally administered a Myrtaceae extract (allspice extract or clove extract) before intracerebral administration of Aβ (allspice extract-administered mice, clove extract-administered mice) had a higher alternation rate than mice that were orally administered a control (saline) before intracerebral administration of Aβ (control-administered mice). This indicates that allspice extract and clove extract prevent / improve spatial working memory loss. These results demonstrate that Myrtaceae extract has the effect of preventing / improving spatial working memory loss, preventing cognitive decline, and improving cognitive function.

[0009] The Aβ aggregation / deposition inhibitor of the present invention can exhibit a good Aβ aggregation / deposition inhibitory effect in biological tissues (e.g., on the surface and / or inside of brain tissue cells in a living organism). Furthermore, the Aβ aggregation / deposition inhibitor of the present invention exhibits an effect of inhibiting / improving the decline in cognitive function of a subject, which is the subject's ability to recognize things via the brain, and can serve as an agent for inhibiting / improving the decline in cognitive function. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An agent for inhibiting the aggregation and / or deposition of amyloid beta consisting of an extract of a plant belonging to the Myrtaceae family.

2. The aggregation and / or deposition inhibitor described in claim 1, which exhibits an inhibitory effect on amyloid beta aggregation and / or deposition in an evaluation system comprising a step of coexisting and culturing labeled amyloid beta, a test substance and a cerebral organoid, and a step of quantifying amyloid beta aggregated on the surface and / or inside the cerebral organoid using the label as an indicator.

3. The aggregation and / or deposition inhibitor according to claim 2, which exhibits an inhibition rate of 50% or more in the evaluation system compared to a control group to which no test substance is added.

4. The coagulation and / or deposition inhibitor according to any one of claims 1 to 3, wherein the plant belonging to the Myrtaceae family is guava, Eucalyptus robusta, clove, or allspice.

5. The flocculation and / or deposition inhibitor according to claim 4, wherein the extract is obtained by crushing cloves and subjecting the resulting crushed material to solvent extraction.

6. The coagulation and / or deposition inhibitor according to claim 5, wherein the extract is obtained by subjecting the crushed material, which is obtained by crushing cloves and has a particle diameter of 5 mm or less, to solvent extraction.

7. The coagulation and / or deposition inhibitor according to claim 5 or 6, wherein the extract is obtained by subjecting the crushed product obtained by crushing cloves to solvent extraction, the crushed product being composed of particles having a particle diameter of 0.5 mm or more.

8. The coagulation and / or deposition inhibitor according to claim 5, wherein the extract is obtained by subjecting the crushed material obtained by crushing cloves to solvent extraction, the crushed material being composed of particles having a particle size range of 0.5 mm or more and 3 mm or less.

9. An inhibitor of aggregation and / or deposition according to any one of claims 1 to 3, which inhibits aggregation and / or deposition of amyloid β on the surface and / or inside brain tissue cells of a living body.

10. An agent for inhibiting and / or improving the decline in cognitive function of a subject, comprising an extract of a plant belonging to the Myrtaceae family.

11. The agent for preventing and / or improving cognitive decline according to claim 10, wherein the plant is clove or allspice.

12. A method for producing an inhibitor of amyloid β aggregation and / or deposition, comprising the step of subjecting a plant belonging to the Myrtaceae family to solvent extraction to obtain an extract.

13. The method for producing an aggregation and / or deposition inhibitor according to claim 12, wherein the plant belonging to the Myrtaceae family is guava, Eucalyptus robusta, clove, or allspice.

14. A method for producing an aggregation and / or deposition inhibitor as described in claim 13, wherein the step of obtaining the extract is a step of obtaining the extract by subjecting the pulverized material obtained by pulverizing cloves to solvent extraction.

15. A method for producing an aggregation and / or deposition inhibitor as described in claim 14, wherein the maximum particle size of the ground product obtained by grinding the cloves is 5 mm or less.

16. A method for producing an aggregation and / or deposition inhibitor as described in claim 14 or 15, wherein the maximum particle size of the ground product obtained by grinding the cloves is 0.5 mm or more.

17. A method for producing an aggregation and / or deposition inhibitor as described in claim 14, wherein the maximum particle size of the ground product obtained by grinding the cloves is in the range of 0.5 mm or more and 3 mm or less.

18. A method for producing an aggregation and / or deposition inhibitor according to any one of claims 12 to 17, wherein the solvent used in the solvent extraction is an organic solvent, water, or a mixture thereof.

19. A method for producing an aggregation and / or deposition inhibitor according to any one of claims 12 to 18, wherein the solvent used in the solvent extraction is an aqueous ethanol solution.

20. A method for producing an agent for preventing and / or improving cognitive function decline in a subject, the method comprising a step of subjecting a plant belonging to the Myrtaceae family to solvent extraction to obtain an extract.

21. The method of claim 20, wherein the plant is clove or allspice.

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