Aggregation inhibitor

Abstract

The present invention addresses the problem of developing and providing a novel aggregation inhibitor composed of a plant-derived extract and having an aggregation inhibiting effect on aggregation-prone proteins.　Provided is an aggregation inhibitor composed of an extract of a plant selected from the group consisting of plants belonging to Asteraceae, Rosaceae, Saxifragaceae, Apiaceae, Liliaceae, Campanulaceae, Ericaceae, Lycopus, Geranium, Plantago, Hypericum, Stellaria, Chelidonium, Pachysandra, and Matteuccia.

Description

Coagulation inhibitor

[0001] The present invention relates to an agent for inhibiting aggregation of an aggregating protein.

[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. As of 2019, the number of dementia patients worldwide is estimated to be over 50 million, of which approximately 70% are thought to be AD patients, and the incidence rate is on the rise. The increase in AD patients has led to rising medical costs and nursing care issues, which have become major social issues in recent years, placing financial and emotional burdens on countries and patients.

[0003] AD begins with the aggregation and accumulation of amyloid β protein (often referred to as "Aβ" herein), a hydrophobic peptide, in the patient's brain. Subsequently, tau protein, a microtubule-associated protein, becomes hyperphosphorylated and fibrillated, which then leads to nerve cell destruction and brain atrophy (Non-Patent Documents 1 to 3).

[0004] Based on this mechanism of AD pathogenesis, technologies have been developed to evaluate the Aβ aggregation inhibitory effect of test substances added in vitro and screen candidate compounds for AD treatment. For example, the microliter-scale high-throughput screening (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 the effect of inhibiting amyloid β protein aggregation in PBS solvent (Non-Patent Document 4).

[0005] 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 water or an appropriate buffer solution such as PBS, in the presence or absence of the 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.

[0006] 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, the document discloses a quantum dot nanoprobe in which a quantum dot is bound to the N-terminus or C-terminus of an amyloid-forming peptide via cysteine.

[0007] However, candidate compounds obtained using cell-free assays often do not exhibit inhibitory effects in cell-based assays (tests using cells), which is thought to be because cell-free assays do not necessarily reflect the biological environment.

[0008] International Publication No. 2020 / 138265 Japanese Patent Application Laid-Open No. 2017-007990

[0009] Hardy J. and Selkoe DJ, 2002, Science, 297(5580): 353-. 356Jack CRJr., et al., 2010, Lancet Neurol. 9(1): 119-128Akira Tamaoka, 2017, Journal of Geriatric Dementia Research, Vol.22, No.3, p.19-23Ishigaki et al., 2013, PLOS ONE,8(8): e72992

[0010] In view of the above-mentioned circumstances, the present invention aims to develop a screening method capable of searching for candidate compounds having an aggregation-inhibiting effect or an aggregation-promoting effect on aggregating proteins such as Aβ in a cell-based assay system, and to isolate and provide a novel aggregation inhibitor for aggregating proteins based on the method.

[0011] In order to solve the above problems, the present inventors have developed a cell-based assay system (test using cells) in which cells are cultured in the presence of a labeled aggregating protein and a test substance, and the aggregating protein aggregated and / or deposited on the cell surface or within the cells in the cell culture is quantified using the label as an indicator, thereby evaluating whether or not a test substance has aggregation-inhibiting activity or aggregation-promoting activity against aggregating proteins.

[0012] Using this evaluation method, the aggregation-inhibiting or aggregation-promoting activity of various test substances derived from natural products was examined, and as a result, it was found that specific types of plant extracts have a strong aggregation-inhibiting effect on aggregating proteins. The present invention is based on the novel findings obtained as a result of the above research, and provides the following.

[0013] (1) An agent for inhibiting aggregation of a coagulable protein, comprising an extract of a plant selected from the group consisting of plants belonging to the family Asteraceae, the subfamily Rosoideae, the family Saxifragaceae, the family Apiaceae, the family Liliaceae, the family Campanulaceae, the family Ericaceae, the genus Lycopus, the genus Geranium, the genus Plantago, the genus Hypericum, the genus Stellaria, the genus Chelidonium, the genus Pachysandra, and the genus Matteuccia. (2) The flocculation inhibitor according to (1), wherein the plant of the Asteraceae family is a plant of the genus Parasenecio, the genus Cirsium, the genus Artemisia, or the genus Adenocaulon. (3) The flocculation inhibitor according to (1), wherein the plant of the subfamily Rosaceae is a plant of the genus Geum, the genus Rosa, the genus Sanguisorba, or the genus Argentina. (4) The flocculation inhibitor according to (1), wherein the plant of the Apiaceae family is a plant of the genus Sanicula or the genus Cryptotaenia. (5) The flocculation inhibitor according to (1), wherein the plant of the Liliaceae family is a plant of the genus Maianthemum or the genus Allium. (6) The flocculation inhibitor according to (1), wherein the plant belonging to the Campanulaceae family is a plant belonging to the genus Lobelia or Adenophora. (7) The flocculation inhibitor according to (1), wherein the plant belonging to the Ericaceae family is a plant belonging to the genus Ledum or Vaccinium. (8) The flocculation inhibitor according to any of (1) to (7), wherein the plant is a plant belonging to the family Asteraceae, Saxifragaceae, Geranium, Hypericum, Actinidia, Sanguisorba, Dioscorea, and Stellaria. (9) The flocculation inhibitor according to any of (1) to (8), wherein the extract is derived from the whole plant.(10) The aggregation inhibitor according to any one of (1) to (9), wherein the aggregating protein is amyloid beta. (11) A composition for inhibiting aggregation of aggregating proteins, comprising the aggregation inhibitor according to any one of (1) to (10) as an active ingredient. (12) The aggregation-inhibiting composition according to (11), comprising two or more different aggregation inhibitors according to any one of (1) to (10) as active ingredients. (13) The aggregation-inhibiting composition according to (11) or (12), which is for treating or preventing a disease caused by the aggregation of aggregating proteins. (14) The aggregation-inhibiting composition according to (13), wherein the disease is Alzheimer's disease. This specification includes the disclosures of Japanese Patent Application No. 2022-153231, from which the present application claims priority.

[0014] According to the aggregation inhibitor of the present invention, a novel aggregation inhibitor can be provided which is composed of a plant-derived extract and has an aggregation-inhibiting effect on aggregating proteins.

[0015] Figure 1 shows plots of the results of evaluating the Aβ aggregation / deposition effects of eight plant extracts in a cell system. Figure 2 shows images comparing the state of cells in a butterbur-added system and a DMSO-added system in a cell system evaluation.

[0016] 1. Aggregation Inhibitor 1-1. Overview A first aspect of the present invention is an aggregation inhibitor for aggregating proteins. The aggregation inhibitor of the present invention comprises a specific type of plant extract discovered by a method for evaluating the aggregation-inhibiting activity or aggregation-promoting activity of a test substance against aggregating proteins, developed by the applicants of the present invention. The aggregation inhibitor of the present invention can inhibit the formation of aggregates by aggregating proteins by mixing with the aggregating protein.

[0017] 1-2. Definitions of Terms The following terms used in this specification are defined below. Unless otherwise specified, the following definitions in this section are also applicable to other aspects of the present invention.

[0018] (1) Aggregating Protein As used herein, the term "aggregating protein" refers to a protein that has the property of assembling to form aggregates, or a peptide fragment thereof that contains a region involved in aggregation. As used herein, the type of aggregating protein is not limited. Examples include disease-related proteins, polyglutamic acid, and autophagy-related proteins.

[0019] Examples of disease-related aggregating proteins include amyloid beta protein and tau protein (including phosphorylated tau protein), which are causative proteins of Alzheimer's disease, alpha-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, beta2 microglobulin, which is a causative protein of dialysis-related amyloidosis, and TDP-43 protein, which is considered to be a causative protein of amyotrophic lateral sclerosis. Amyloid β proteins such as, but not limited to, amyloid β40 protein, amyloid β42 protein, amyloid β43 protein, and amyloid β38 protein are particularly suitable as aggregating proteins herein.

[0020] Autophagy-related proteins include, for example, ubiquitin-like proteins Atg-8 and Atg-12.

[0021] The aggregating protein may be a natural protein present in nature, a modified protein obtained by artificially introducing mutations or modifications into a natural protein, or an artificial protein based on an artificially designed amino acid sequence.

[0022] (2) Aggregates As used herein, the term "aggregates" refers to an assembly of two or more aggregating proteins. In this specification, so-called protein complexes are also included in aggregates. Aggregates may be homoaggregates consisting of the same type of protein, or heteroaggregates consisting of different types of proteins.

[0023] (3) Deposition As used herein, the term "deposition" refers to the adhesion and / or accumulation of aggregating proteins on the cell surface and / or within the cell.

[0024] (4) 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.

[0025] (5) Plant Extract As used herein, the term "plant extract" refers to a mixture of multiple components obtained from a plant. This typically refers to plant components such as low molecular weight compounds extracted from a plant into a solvent, or plant components including proteins, nucleic acids, lipids, sugars, and low molecular weight compounds contained in the juice of a plant. All or part of the components contained in the plant extract may be unidentified. The state of the plant extract is not important. It may be in a liquid state or a solid state (including a powdered state or a granular state). As used herein, "squeezed juice" refers to a liquid component obtained by squeezing a plant. Small pieces of the plant may be included as solid components, or the liquid component may be obtained by filtering.

[0026] (6) Plant Body In this specification, the term "plant body" refers to all (whole plant) or a part of the plant body constituting an individual plant. The part 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, and fruits. The plant body may be in a raw, fermented, or dried state. The state may be selected appropriately depending on the form of the plant body. For example, if the plant body is in the form of powder, it is preferably in the dried state. Furthermore, if the plant body is in the form of juice, it may be in the raw or fermented state.

[0027] 1-3. Composition (1) Plant Extract The aggregation inhibitor of a coagulating protein of the present invention comprises a plant extract. The plant extract of the present invention can be obtained from a plant body by any extraction method or in any state, as long as it is an extract obtained from the plant body. Examples include a liquid component (so-called extract) obtained by immersing a plant body in a solvent such as water or an organic solvent (e.g., ethanol) at a predetermined temperature for a predetermined time and then removing the plant body by centrifugation or filtration; a solid (including granules and powder) remaining after removing the solvent from the extract by reducing pressure, evaporation, drying, or the like; or a paste in an intermediate state thereof. Other examples include a squeezed juice obtained by squeezing a plant body, a juice obtained by filtering the squeezed juice to remove solid components, or a solid obtained by evaporating or drying the water content of these.

[0028] The extraction method using a solvent may be any known extraction method. When the solvent is water, the solvent temperature during extraction should be in the range of 10 to 100°C, preferably 40 to 99°C, and more preferably 70 to 99°C, at 1 atmosphere. Also, when using a heat-resistant, pressure-resistant sealed vessel such as an autoclave at 1 to 2 atmospheres, the temperature should be in the range of 100 to 121°C. When the solvent is ethanol, the temperature should be in the range of 0 to 78.3°C, preferably 15 to 77°C, and more preferably 20 to 77°C, at 1 atmosphere. Furthermore, when the solvent is a mixture of water and ethanol, the conditions described above for water or ethanol can be applied appropriately depending on the ethanol concentration. The extraction time varies depending on the solvent temperature. Generally, when the solvent temperature is low (below 40°C), a long period of time, for example, several days to a year or more, is required. However, when the solvent temperature is high (between 40°C and below the boiling point at 1 atmosphere), a short period of time, for example, 2 minutes to several hours, is sufficient. Furthermore, when the solvent is water, the reaction time may be 2 to 30 minutes, preferably 10 to 20 minutes, at a temperature of 100 to 121° C. under 1 to 2 atmospheric pressure.

[0029] When preparing a plant extract by extraction or juicing, the plant may be used in its intact state, or in its fragmented or powdered form. "Intact plant" refers to the entire plant when the whole plant is used, or to a portion of the plant, such as the flower, leaf, or root, in its intact state when a portion is used. "Fragmented" refers to the plant being cut into small pieces by cutting, grating, or other methods. The size of the fragments is not particularly limited, but small fragments are preferred to improve the efficiency of extraction into a solvent and the efficiency of juicing. For example, fragments in the range of 1 to 5 mm are acceptable. "Powdered" refers to the plant being ground into a powder, either in its intact state or after freezing, drying, or lyophilization. The size of the particles constituting the powder is also not particularly limited, as long as they are 1 mm or less. For example, the fragments may be granular, finely powdered, or a mixture thereof.

[0030] Extraction methods using solvents are particularly preferred because, unlike squeezed juice, they have a low rate of contamination with impurities such as cell fragments from the plant body and the concentration of the actual active ingredients in the extract is high, so medicinal effects can be obtained with a small amount.

[0031] (2) Plant Origin The plant from which the plant extract constituting the aggregation inhibitor of the present invention for a coagulating protein is derived is any plant selected from the group consisting of species belonging to the family Asteraceae, the family Saxifragaceae, the subfamily Rosoideae, the family Apiaceae, the family Liliaceae, the family Campanulaceae, the family Ericaceae, the genus Lycopus, the genus Geranium, the genus Plantago, the genus Hypericum, the genus Stellaria, the genus Chelidonium, the genus Pachysandra, and the genus Matteuccia. Each plant will be specifically described below.

[0032] (Asteraceae Plants) As shown in the Examples below, the aggregation inhibitory effect of extracts from various species of Asteraceae plants in different genera belonging to the same family has been observed, and therefore any plant species belonging to the same family may be used. For example, Asteraceae plants include species belonging to the genus Parasenecio, thistle (Cirsium), artemisia, or Adenocaulon.

[0033] Specific examples of P. kamtschaticus plants include P. peltifolius, P. yatabei, P. adenostyloides, P. amagiensis, P. maximowiczianus, P. delphiniifolius, and P. farfarifolius.

[0034] Specific examples of thistle plants include C. japonicum, C. purpratum, C. nipponicum, C. kamtschaticum, C. martitimum, C. dipsacolepis, and C. borealinipponense.

[0035] Specific examples of Artemisia plants include Artemisia indica, Artemisia montana, Artemisia inslaris, Artemisia capillaris, Artemisia japonica, Artemisia montana, Artemisia stelleriana, Artemisia stelleriana, and the like.

[0036] Specific examples of plants of the genus Aconitum include Aconitum himalaicum, Aconitum bicolor, Aconitum lyratum, Aconitum chilense, and the like.

[0037] (Saxifragaceae Plants) The Saxifragaceae plant may be any species that belongs to the family, such as species belonging to the genus Saxifraga, species belonging to the genus Micranthes, and species belonging to the genus Rodgersia.

[0038] Specific examples of plants of the genus Saxifragaceae include Saxifragaceae (S. stolonifera) and S. fortunei.

[0039] Specific examples of plants of the genus M. include M. punctata, M. fusca, and M. japonica.

[0040] A specific example of a Centaurea plant is R. podophylla.

[0041] (Rosaceae Plants) As shown in the Examples, the aggregation inhibitory effect of extracts from various species belonging to different genera in the Rosaceae subfamily has been observed, and therefore any plant species belonging to the subfamily may be used, such as species belonging to the genus Geum, Rosa, Sanguisorba, or Argentina.

[0042] Specific examples of plants of the genus Geum include G. japonicum, G. aleppicum, G. pentapetalum, G. coccineum, and G. ternatum.

[0043] Specific examples of plants of the genus Rosa include R. multiflora, R. sambucina, R. onoei, R. rugosa, R. amblyotis, and R. nipponensis.

[0044] Specific examples of plants of the genus Sanguisorba include S. officinalis, S. longifolia, S. tenuifolia, S. hakusanensis, S. stipulata, and S. albiflora.

[0045] A specific example of a plant of the genus A. anserina is A. anserina.

[0046] (Apiaceae Plants) As shown in the Examples, the aggregation inhibitory effect of extracts from different species of Apiaceae family has been confirmed in the same family, so any plant species belonging to the same family may be used. For example, species belonging to the Sanicula genus or the Cryptotaenia genus can be used. Specific examples of Sanicula plants include Sanicula chinensis and Cryptotaenia rubriflora. Specific examples of Cryptotaenia plants include Cryptotaenia japonica.

[0047] (Plants of the Liliaceae family) As shown in the Examples, the aggregation inhibitory effect of extracts from different species of liliaceae genera was observed, and therefore any plant species belonging to the same family may be used, such as species belonging to the genus Maianthemum or species belonging to the genus Allium.

[0048] Specific examples of plants of the genus M. include M. dilatatum, M. bifolium, M. bicolor, M. dahuricum, M. canadense, M. japonicum, M. trifolium, M. stellatum, and M. racemosum.

[0049] Specific examples of Allium plants include leeks (A. porrum), Chinese chives (A. tuberosum), garlic (A. sativum), and wild onion (A. victtorialis).

[0050] (Campanulaceae Plants) As shown in the examples, the aggregation inhibitory effect of extracts from different species of Campanulaceae genera belonging to the same family was observed, so any plant species belonging to the same family may be used. For example, species belonging to the Lobelia genus or species belonging to the Adenophora genus can be used. Specific examples of Lobelia plants include L. sessilifolia and L. chinensis. Specific examples of Adenophora plants include A. triphylla and A. remotiflora.

[0051] (Ericaceae Plants) As shown in the Examples, the aggregation inhibitory effect of each extract was observed in different species of Ericaceae genera belonging to the same family, so any plant species belonging to the same family may be used. For example, species belonging to the Ledum genus or Vaccinium genus may be used. Specific examples of Ledum plants include L. palustre. Specific examples of Vaccinium plants include V. vitis and Vaccinium smallii.

[0052] (Plants of the Genus Lycopus) Specific examples of plants of the genus Lycopus include Lycopus lucidus, Lycopus cavaleriei, Lycopus maackianus, and Lycopus uniflorus.

[0053] (Geranium plants) Specific examples of Geranium plants include G. thunbergii, G. yezoense, G. wilfordii, G. yesoense, G. erianthum, G. carolinianum, and G. molle.

[0054] (Plantago plants) Specific examples of Plantago plants include P. asiatica, P. camtschatica, P. japonica, P. aristata, P. depressa, P. major, and P. lanceolata.

[0055] (Hypericum plants) Specific examples of plants of the genus Hypericum include H. erectum, H. hakonense, H. japonicum, H. kamtschaticum, H. ascyron, H. perforatum, H. patulum, and H. pseudopetiolatum.

[0056] (Stellaria plants) Specific examples of Stellaria plants include S. media, S. neglecta, S. filicaulis, S. aquatica, S. uliginosa, S. sessiflora, S. diversiflora, S. radians, S. humifusa, and S. longifolia.

[0057] (Plants of the Genus Chelidonium) A specific example of a plant of the genus Chelidonium is C. majus.

[0058] (Plants of the Genus Pachysandra) Specific examples of plants of the genus Pachysandra include Pachysandra terminalis and Pachysandra axillaris.

[0059] (Plants of the Genus Matteuccia) Specific examples of plants of the genus Matteuccia include Matteuccia struthiopteris.

[0060] 2. Aggregation-Inhibitory Composition 2-1. Overview A second aspect of the present invention is a composition for inhibiting aggregation of an aggregating protein. The composition of the present invention includes one or more aggregation inhibitors described in the first aspect as an active ingredient. The aggregation-inhibitory composition of the present invention makes it possible to apply the aggregation inhibitor in an easy-to-handle and stable state. As a result, for example, as a pharmaceutical composition, the burden and invasiveness upon administration to a living body can be reduced, and the composition can be provided in a form that is easier to administer.

[0061] 2-2. Composition 2-2-1. Constituent Factors The aggregation-inhibiting composition of this embodiment (hereinafter often abbreviated as "the composition") contains an active ingredient as an essential constituent factor, and a solvent and / or a carrier as optional constituent factors. Each of the constituent factors will be specifically described below.

[0062] (1) Active ingredient The present composition contains the aggregation inhibitor described in the first aspect as an active ingredient. The present composition may contain one type, or two or more different types of aggregation inhibitors. Furthermore, the composition may selectively contain, in addition to the aggregation inhibitor described in the first aspect, a known substance (such as a nucleic acid, a polypeptide, or a low-molecular-weight compound) that has a similar aggregation-inhibiting effect on aggregating proteins.

[0063] The content of the aggregation inhibitor contained in the present composition varies depending on the type and / or effective amount of the aggregation inhibitor included, the dosage form of the present composition, the type of carrier or additive described below, and the type of disease, and therefore may be determined appropriately taking into account each condition.

[0064] As used herein, the term "effective amount" refers to the amount of the aggregation inhibitor in the composition that is necessary for the agent 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.

[0065] As used herein, the term "subject" refers to a living organism to which the coagulation 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 specifically referred to as "test subjects"). Furthermore, "subject information" refers to various individual information about the living organism to which the composition is applied. For example, in the case of a test subject, this includes the overall health status, the progression and severity of any disease or injury, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant medications, and resistance to treatment. The effective amount of the coagulation inhibitor in the present composition and the dosage calculated based thereon are ultimately determined by the judgment of a physician or veterinarian, etc., depending on the information about the individual subject.

[0066] (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 has low toxicity to living organisms, is generally usable in the field of formulation technology, and is preferably usable in pharmaceutical compositions. Examples of the solvent include water or an aqueous solution, a buffer solution, an emulsifier, and an organic solvent.

[0067] Pharmaceutically acceptable aqueous solutions include, for example, 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-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Pharmaceutically acceptable organic solvents include, for example, ethanol and butanol.

[0068] (3) Carrier The composition may contain a pharmaceutically acceptable carrier, which is an optional component of the composition and may be added as needed.

[0069] Carriers include, for example, suspending agents, diluents, solubilizing agents, dispersing agents, surfactants, emulsifying agents, soothing agents, stabilizers, preservatives, antiseptics, antioxidants, buffers, isotonic agents, and the like.

[0070] In addition to the above, if necessary, the composition may contain appropriate excipients, fillers, binders, disintegrants, absorption enhancers, bulking agents, moisturizing agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, and the like that are commonly used in pharmaceuticals.

[0071] Such carriers are primarily used to facilitate the formation of dosage forms, maintain the dosage form and drug efficacy, and also to make the active ingredient, the aggregation inhibitor, less susceptible to degradation in the body, and may be used appropriately as needed.

[0072] 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 aggregation inhibitor and other known aggregation inhibitors for aggregating proteins, and is capable of exerting the pharmacological effects of the active ingredients in vivo after administration. Furthermore, the specific dosage form of the present composition may be appropriately selected depending on the administration method and / or prescription conditions. Generally, administration methods can be broadly divided into oral administration and parenteral administration, and the present composition may be prepared in a dosage form suitable for each administration method.

[0073] For oral administration, dosage forms include solids (including tablets, pills, sublinguals, capsules, and drops), granules, powders, and liquids (including oral solutions, suspensions, emulsions, and syrups). Solids can be coated as needed as known in the art, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double-layered tablets, and multi-layered tablets. Oral administration is a preferred method of administration because it is minimally invasive and easy to administer.

[0074] 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 into 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 solutions (including liniments, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), powders (including nasal drops and inhalants), pastes, gels, ointments, plasters, etc. Dosage forms suitable for rectal administration include suppositories, etc.

[0075] 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 aggregation 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, i.e., directly into the central nervous system, or systemically via the circulatory system. Specific examples include intratissue administration via 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.

[0076] Injectable solutions may be formulated by appropriately combining the above-mentioned emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dose form required by generally accepted pharmaceutical practice, and are provided in the form of unit dose ampoules or multi-dose containers.

[0077] 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.

[0078] 2-2-3. Target Diseases The present composition can be used for the treatment or prevention of various diseases caused by the aggregation of aggregating proteins. As described above, the types of target diseases are not limited, as long as they are caused by the aggregation of aggregating proteins that cause the disease and the resulting aggregates being deposited intracellularly or extracellularly. Examples of the types of diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, arteriosclerosis, systemic AL amyloidosis, and amyotrophic lateral sclerosis. The type of aggregating protein is also not particularly limited, as long as it is an aggregating protein that causes the respective disease. The relationship between the target diseases of the present composition and the aggregating proteins that cause them will be explained below using several examples.

[0079] Alzheimer's disease begins with the aggregation and accumulation of amyloid β protein in neurons in the brain, followed by hyperphosphorylation and fibrillation of tau protein, a microtubule protein, which then leads to the destruction of neurons and brain atrophy. Therefore, the aggregating proteins that cause Alzheimer's disease may include β-amyloid, tau protein, and α-synuclein. The aggregation inhibitor contained in the present composition can suppress the aggregation of these proteins, thereby suppressing or preventing the progression of Alzheimer's disease.

[0080] Parkinson's disease (PD) is thought to be caused by neuronal cell death due to the multimerization and accumulation of misfolded α-synuclein, which forms protein aggregates called Lewy bodies. Therefore, misfolded mutant α-synuclein may be an aggregating protein that causes Parkinson's disease. The aggregation inhibitor contained in the present composition can inhibit the aggregation of this protein, thereby suppressing or preventing the progression of Parkinson's disease.

[0081] It is known that Huntington's disease (HD) is caused by the expression of a mutant huntingtin (mHTT) gene with an expanded CAG repeat number, which induces neuronal dysfunction and cell death, thereby contributing to the onset of HD. Therefore, mutant huntingtin may be the aggregating protein that causes Huntington's disease. The aggregation inhibitor contained in the present composition can suppress the aggregation of mutant huntingtin, thereby suppressing or preventing the progression of Huntington's disease.

[0082] Transmissible spongiform encephalopathies (including Creutzfeldt-Jakob disease, mad cow disease, and prion diseases) are neurodegenerative diseases caused by the destruction of normal nervous tissue due to the increase in abnormal prion proteins in the central nervous system and their extracellular aggregation. Therefore, the aggregating protein that causes transmissible spongiform encephalopathies may be abnormal prion proteins. The aggregation inhibitor contained in the present composition can suppress the aggregation of abnormal prion proteins, thereby suppressing or preventing the progression of transmissible spongiform encephalopathies.

[0083] Example 1 Preparation of Plant Extracts (Purpose) Various plant extracts that are aggregation inhibitors of the present invention are prepared.

[0084] (Method) (1) Preparation of plant extract of St. John's Wort (a plant of the genus St. John's Wort) The whole plant (including leaves, stems, and roots) of St. John's Wort (L. lucidus) collected in Shiranuka, Hokkaido, was immersed in 95% ethanol as a solvent for one week. The solvent was then filtered, and the filtrate was subjected to a rotary evaporator (Tokyo Rikakikai Co., Ltd.) to distill off the solvent under reduced pressure. The resulting residue was further dried using an oil rotary pump (ULVAC, Inc.) to obtain an ethanol extract.

[0085] The resulting ethanol extract was then dispersed in water and extracted with t-butyl methyl ether using a separatory funnel. The combined t-butyl methyl ether extract layers were then placed on a rotary evaporator to remove the solvent under reduced pressure. The resulting residue was further dried using an oil-sealed rotary pump to obtain a t-butyl methyl ether fraction.

[0086] The aqueous layer was then extracted with n-butanol saturated with water using a separatory funnel. The combined t-butyl methyl ether extract was again placed on a rotary evaporator to remove the solvent under reduced pressure. The resulting residue was further dried using an oil-sealed rotary pump to obtain an n-butanol fraction.

[0087] (2) Preparation of Plant Extract of Geranium thunbergii (Genus Geranium) Plant (No. 1) The whole plant (including leaves, stems, and roots) of Geranium thunbergii (G. thunbergii) collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing the Geranium thunbergii extract were obtained in the same manner as in (1) above.

[0088] (3) Preparation of Plant Extract of Geranium yesoense (Geranium genus plant) (No. 2) The whole plant (including leaves, stems, and roots) of Geranium yesoense collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing the Geranium yesoense extract were obtained in the same manner as in (1) above.

[0089] (4) Preparation of plant extract of Parasenecio adenostyloides (Asteraceae, Parasenecio genus) plant The whole plant (including leaves, stems, and roots) of Parasenecio adenostyloides collected or cultivated in Shiranuka Town, Hokkaido was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing Parasenecio adenostyloides extract were obtained using the same procedure as in (1) above.

[0090] (5) Preparation of plant extract of Artemisia montana (Asteraceae family) Leaves and stems of Artemisia montana collected or cultivated in Shiranuka Town, Hokkaido were immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction containing Artemisia montana extract was obtained by the same procedure as in (1) above.

[0091] (6) Preparation of a plant extract of kumquat thistle (a plant of the genus Cirsium in the family Asteraceae) The whole plant (including leaves, stems, roots, and flowers) of kumquat thistle (Cirsium kamtschaticum) collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction containing a kumquat thistle extract was obtained using the same procedure as in (1) above.

[0092] (7) Preparation of Plant Extract from Plantago camtschatica (a plant of the genus Plantago) The whole plant (including leaves, stems, and roots) of Plantago camtschatica collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing Plantago camtschatica extract were obtained using the same procedures as in (1) above.

[0093] (8) Preparation of Plant Extract of Hypericum ascyron Plant The whole plant (including leaves, stems, and roots) of Hypericum ascyron collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing the Hypericum ascyron extract were obtained in the same manner as in (1) above.

[0094] (9) Preparation of plant extract of Sanicula chinensis (Apiaceae, Sanicula genus) The whole plant (including leaves, stems, and roots) of Sanicula chinensis collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing Sanicula chinensis extract were obtained using the same procedures as in (1) above.

[0095] (10) Preparation of Plant Extract of Butterbur (Saxifragaceae Plant) The whole plant (including leaves, stems, and roots) of Butterbur (Micranthes japonica) collected or cultivated in Shiranuka Town, Hokkaido, was immersed in 95% ethanol at room temperature for 1 week, and then an ethanol fraction containing a Butterbur extract was obtained by the same procedure as in (1) above.

[0096] (11) Preparation of plant extract of Geum japonicum (Rosaceae, Genus Geum) Leaves and stems of Geum japonicum (Geum japonicum) collected or cultivated in Shiranuka Town, Hokkaido, were immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction containing Geum japonicum extract was obtained by the same procedure as in (1) above.

[0097] (12) Preparation of a plant extract of Rugosa rugosa (a plant of the genus Rosa and the subfamily Rosaceae) Leaves, stems, fruits, and flowers of Rugosa rugosa collected or cultivated in Shiranuka Town, Hokkaido, were immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction containing the Rugosa rugosa extract was obtained by the same procedure as in (1) above.

[0098] (13) Preparation of Plant Extract of Potentilla egedei (Rosaceae, Genus Potentilla) The above-ground parts (including leaves, stems, and flowers) of Potentilla egedei var. grandis collected or cultivated in Shiranuka Town, Hokkaido, were immersed in 95% ethanol at room temperature for one week, and then an ethanol fraction, a t-butyl methyl ether fraction, and an n-butanol fraction containing the Potentilla egedei extract were obtained by the same procedure as in (1) above.

[0099] (14) Preparation of plant extract of Sanguisorba tenuifolia (a plant of the genus Sanguisorba and the subfamily Rosaceae) Roots of Sanguisorba tenuifolia collected or cultivated in Shiranuka Town, Hokkaido, were immersed in 95% ethanol at room temperature for 1 week, and then an ethanol fraction containing a Sanguisorba tenuifolia extract was obtained by the same procedure as in (1) above.

[0100] (15) Preparation of a plant extract of Hydrangea macrophylla (a plant of the Hydrangeaceae family, genus Hydrangea) (for control) Dried leaves of Hydrangea macrophylla (OP Bio Co., Ltd.) were ground in a mill, and then 90% ethanol was added to the ground material as a solvent and extracted by shaking for 20 hours at 28° C. The solvent was centrifuged at 3,000 rpm for 20 minutes, and the supernatant was filtered through a cotton plug, centrifuged again, and freeze-dried to obtain a Hydrangea macrophylla extract.

[0101] (16) Preparation of Plant Extract of Lobelia sessilifolia (Campanulaceae, Lobelia genus) The above-ground parts (including leaves, stems, and flowers) of Lobelia sessilifolia collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing Lobelia sessilifolia extract was obtained by the same procedure as in (1) above.

[0102] (17) Preparation of a plant extract of Nobuki (Asteraceae, Genus Nobuki) The above-ground parts (including leaves, stems, and flowers) of Nobuki (Adenocaulon himalaicum) collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing Nobuki extract was obtained by the same procedure as in (1) above.

[0103] (18) Preparation of plant extract of Maianthemum dilatatum (a plant of the genus Maianthemum in the family Liliaceae) The above-ground parts (including leaves, stems, and flowers) of Maianthemum dilatatum collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing Maianthemum dilatatum extract was obtained by the same procedure as in (1) above.

[0104] (19) Preparation of a plant extract of Stellaria radians (a plant of the genus Stellaria) The above-ground parts (including leaves, stems, and flowers) of Stellaria radians collected or cultivated in Shiranuka Town, Hokkaido, were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a chickweed extract was obtained using the same procedure as in (1) above.

[0105] (20) Preparation of a plant extract of Japanese honeywort (Cryptotaenia japonica, family Apiaceae) Leaves and stems of Japanese honeywort (Cryptotaenia japonica) collected or cultivated in Shiranuka Town, Hokkaido, were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a Japanese honeywort extract was obtained by the same procedure as in (1) above.

[0106] (21) Preparation of plant extract of Allium victorialis (Allium genus, Liliaceae family) Leaves and stems of Allium victorialis subsp. platyphyllum harvested or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing Allium victorialis extract was obtained by the same procedure as in (1) above.

[0107] (22) Preparation of Plant Extract of Adenophora triphylla (Campanulaceae, Adenophora genus) Leaves and stems of Adenophora triphylla var. japonica collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing an Adenophora triphylla extract was obtained by the same procedure as in (1) above.

[0108] (23) Preparation of a plant extract of Ledum palustre (a plant of the genus Ledum, family Ericaceae) The above-ground parts (including leaves, stems, and flowers) of Ledum palustre ssp. diversipilosum collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a Ledum palustre extract was obtained by the same procedure as in (1) above.

[0109] (24) Preparation of a plant extract of cowberry (a plant of the genus Vaccinium, Ericaceae family) The above-ground parts (including leaves, stems, and flowers) of cowberry (Vaccinium vitis-idaea L.) collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a cowberry extract was obtained using the same procedure as in (1) above.

[0110] (25) Preparation of a plant extract of chickweed (Stellaria genus plant) The above-ground parts (including leaves, stems, and flowers) of chickweed (Stellaria media) collected or cultivated in Shiranuka Town, Hokkaido, were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a chickweed extract was obtained by the same procedure as in (1) above.

[0111] (26) Preparation of Plant Extract of Matteuccia struthiopteris Plant The above-ground parts (including leaves, stems, and flowers) of Matteuccia struthiopteris collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing Matteuccia struthiopteris extract was obtained by the same procedure as in (1) above.

[0112] (27) Preparation of plant extract of celandine (Papaveraceae, Chelidonium genus) The above-ground parts (including leaves, stems, and flowers) of celandine (Chelidonium majus) collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a celandine extract was obtained by the same procedure as in (1) above.

[0113] (28) Preparation of Plant Extract of Pachysandra (Plant of the Genus Pachysandra) The above-ground parts (including leaves, stems, and flowers) of Pachysandra terminalis collected or cultivated in Shiranuka Town, Hokkaido were dried and immersed in 90% ethanol at room temperature for 24 hours, and then an ethanol fraction containing a Pachysandra extract was obtained by the same procedure as in (1) above.

[0114] Example 2: Verification of aggregation inhibitory activity of aggregation inhibitor in a cell-free system (Objective) The aggregation inhibitory activity of the aggregation inhibitor of the present invention in a cell-free system is verified.

[0115] (Method) (1) Preparation of Aggregated Protein Solution. Amyloid beta protein (Aβ) was used as the aggregated protein. 5 mg of Aβ (Human, 1-42: Peptide Institute) was added to 5 mL of HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol: Fujifilm Wako Pure Chemical Industries, Ltd.) and suspended. The suspension was then left at room temperature for 1 hour. The monomer was then sonicated at 25°C and 43 kHz for 10 minutes, and the HFIP was then allowed to evaporate in a clean bench for 24 hours. After evaporation, the HFIP was dissolved in 1071 μL of DMSO to prepare a 1 mM Aβ solution. The resulting Aβ solution is hereafter referred to as "Aβ42." Aβ42 was dispensed in 256 μL aliquots into 1.5 mL tubes and stored at -80°C until use.

[0116] (2) Preparation of Quantum Dot-Modified Amyloid β Protein (QDAβ) Aβ was modified with quantum dots (QDs) to prepare quantum dot-modified amyloid β protein (QDAβ). 125 μL of 8 μM Qdot™605 ITK™ amino(PEG) Quantum Dots was placed in two 1.5 mL tubes and centrifuged at 10,000 × g at 4°C for 1 minute. The supernatant was transferred to a centrifugal concentration tube and 4500 μL of PBS was added. The tubes were centrifuged at 3800 × g at 4°C until the combined volume of the two tubes was 50 μL or less, and the filtrate was discarded. After refilling with PBS, the tubes were centrifuged again until the combined 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 tube was left to stand at room temperature for 1 hour to prepare QD-EMCS.

[0117] After preparing the QD-EMCS, 20 μL of 100 mM K-glutamic acid was added to the solution to inactivate any unreacted N-hydroxysuccinimide groups present in the solution, and the solution was allowed to stand at room temperature for 10 minutes.

[0118] Two desalting columns were prepared, each containing approximately 800 μL of resin, and centrifuged at 1,000 × g at 4°C for 1 minute. Each column was loaded with 300 μL of PBSE, and centrifuged at 1,000 × g at 4°C for 1 minute. This process was repeated twice to prepare the desalting columns.

[0119] After 110 μL of the QD-EMCS was applied to the center of two desalting columns, 15 μL of PBSE was added as a stacker. After centrifugation at 1,000 × g for 2 minutes at 4°C, 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. To deactivate any unreacted maleimide groups, 20 μL of 100 mM 2-mercaptoethanol was added and allowed to stand at room temperature for 10 minutes. The filtrate was transferred (145 μL each) to two centrifugal concentration tubes, and 4500 μL of water was added. The mixture was then centrifuged at 3,800 × g for 17 minutes at 4°C. The filtrate was discarded, and the resulting solution was combined into one tube, bringing the total volume to approximately 140 μL.

[0120] 300 μL of water was added to the desalting column and centrifuged at 1,000 × g at 4 °C for 1 minute. This process was repeated twice. 70 μL of the filtrate was applied to each desalting column, and 15 μL of water from the stacker was added. Centrifuged at 1,000 × g at 4 °C for 2 minutes to obtain the target QDAβ.

[0121] (3) Evaluation of Aβ aggregation inhibitory activity of each plant extract. A filtration device (Amicon Ultra-0.5 Ultracel-50 membrane) was attached to a 1.5 mL tube, and MilliQ water was added dropwise to the filtration device and centrifuged at 14,000 × g for 10 minutes at room temperature (15-25°C). The filtrate was removed, and 400 μl of MilliQ water was added dropwise to the filter, followed by centrifugation at 14,000 × g for 10 minutes at room temperature (15-25°C). The filtrate was discarded, and 0.1% Tween-20 aqueous solution was added dropwise to the filtration filter and allowed to stand at room temperature (15-25°C) for 2 hours.

[0122] After the blocking step, the filter was centrifuged, the filtrate was discarded, and the mixture was centrifuged at 14,000 × g for 10 minutes at room temperature (15-25°C). The filtrate was removed, MilliQ water was added dropwise to the filter, and the mixture was centrifuged at 14,000 × g for 10 minutes at room temperature (15-25°C). The filtrate was removed again, and MilliQ water was added dropwise to the filter. The mixture was centrifuged at 14,000 × g for 10 minutes at room temperature (15-25°C). The filtrate was then removed, and the mixture was centrifuged empty at 14,000 × g for 2 minutes at room temperature (15-25°C).

[0123] The filter device was attached to a new 1.5 mL tube, and AD-iPS-derived neural cells were cultured in NbM medium (see Example 3). The culture supernatant was then dripped onto the filter and centrifuged at 14,000 × g for 30 minutes at room temperature (15-25°C), and the filtrate was collected. The Aβ42 concentration in the culture supernatant was measured in advance using a human β-amyloid ELISA kit (Fujifilm Wako Pure Chemical Industries). The filtrate (PBS or culture supernatant) was added to QD-Aβ. 40 solution (final concentration 50 nM), Aβ 42A reaction solution was prepared by adding a solution (final concentration 10 μM) of the plant extract (aggregation inhibitor) prepared in Example 1 (1) to (15). For the positive control, a rosmarinic acid (RA) solution was used as a plant extract substitute. For the negative control, a reaction solution using DMSO as a plant extract substitute was used. Multiple reaction solutions were prepared with the plant extract at final concentrations of 1500 μM, 300 μM, 60 μM, 12 μM, and 2.4 μM. These reaction solutions were incubated at 37°C for 24 hours, and the amount of Aβ aggregation at each concentration of plant extract was quantified.

[0124] The amount of Aβ aggregation was measured using the SD brightness value calculated from the fluorescence image of the aggregates, which positively correlates with the amount of Aβ aggregation. Fluorescence images were acquired by placing the microwell plate containing the reaction mixture under a Nikon ECLIPSE TE2000-S equipped with an OLYMPUS DP72 (CCD) or a Nikon ECLIPSE Ti equipped with a Nikon DS-Ri2 (CMOS) camera, irradiating it with excitation light to induce fluorescence from the reaction product, and capturing the image with the CCD camera. The excitation light was appropriately adjusted depending on the quantum dot used. For example, when using Qdot605, the excitation light wavelength was set to shorter than 580 nm, e.g., 532-552 nm. Fluorescence imaging was performed using a bandpass filter appropriate for the emitted fluorescence. For example, when using Qdot605, a bandpass filter that selectively transmits only light in the 594-646 nm wavelength range was used.

[0125] (Results) Aβ aggregation was observed in the positive control rosmarinic acid (RA) solution, but not in the negative control DMSO solution. Based on the SD of the resulting brightness values, the concentration at which 50% of the Aβ aggregated (EC50) was calculated for each plant extract. Quantification of the aggregated Aβ in the positive control confirmed that approximately 50% of the Aβ aggregated was abolished when the RA concentration was 46.0 ± 7.4 μM or less. Furthermore, the reciprocal of the EC50 indicates that the value is greater than 0 if the extract has aggregation inhibitory activity. Furthermore, the higher the value, the stronger the inhibitory activity. Based on this information, the 1 / EC50 of each plant extract is shown in Table 1.

[0126]

[0127] As shown in Table 1, all ethanol extracts of the plants of the present invention were confirmed to have Aβ aggregation inhibitory activity (1 / EC50). Furthermore, when the ethanol extracts were extracted with t-butyl methyl ether or n-butanol, the 1 / EC50 tended to show high values. Furthermore, the results in Table 1 suggest that hydrophobic extracts such as t-butyl methyl ether are particularly effective in inhibiting Aβ aggregation. On the other hand, no Aβ aggregation inhibitory activity was observed at all with the ethanol extract of the control plant, sweet tea.

[0128] Example 3: Verification of amyloid β aggregation-inhibitory activity in a cell system (Objective) The aggregation-inhibitory activity of the aggregation inhibitor of the present invention in a cell system is verified.

[0129] (Method) 1. Preparation of Neural Cells for Evaluation To evaluate the inhibitory activity against Aβ deposition or aggregation on the cell surface or inside, neural cells for evaluation were prepared by the following procedure.

[0130] (1) Preparation of medium and reagents (a) NbM medium: NbM medium was prepared by adding 1 mL of B-27 supplement (Thermo Fisher Scientific) and Penicillin-Streptomycin Mixed Solution (Nacalai Tesque) to 50 mL of Neurobasal Medium minus phenol red (Thermo Fisher Scientific).

[0131] (b) Y-27632-supplemented NbM medium: 5 mg of Y-27632 (Nacalai Tesque) was dissolved in 1561 μL of PBS. The Y-27632 was added to the NbM medium at a 2000-fold dilution to prepare Y-27632-supplemented NbM medium.

[0132] (c) Matrigel-PBS: Matrigel-PBS was prepared by adding and suspending 500 μL of hESC-Qualified Matrigel (Corning) in 25 mL of PBS.

[0133] (d) SHH-supplemented NbM medium: 100 μL of DMSO was added to 10 μg of human recombinant sonic hedgehog (Veritas) to prepare an SHH solution. 10 μL of the SHH solution was added to 10 mL of the NbM medium to prepare SHH-supplemented NbM medium.

[0134] (2) Preparation of neural cells for evaluation. Pre-cultured AD-iPS-derived neural progenitor cells were detached, centrifuged, washed, and suspended in NbM medium supplemented with Y-27632 to prepare a neural progenitor suspension. This suspension was dispensed at 100 μL / well into a 96-well flat-bottom plate pre-coated with Matrigel-PBS, and the cells were seeded. The plate was then left to stand and incubated at 37°C for 1 day.

[0135] (Day 0) The following day, the medium was completely replaced with SHH-supplemented NbM medium, and incubation continued. The day this procedure was performed was designated Day 0, and subsequent days of incubation were recorded as time. (Day 2) Fresh NbM medium was added at 50 μL / well, and incubation continued. (Day 6) Half of the medium was removed, taking care not to detach the cells from the plate, and fresh NbM medium was added, and incubation continued. (Day 9) Fresh NbM medium was added at 50 μL / well, and incubation continued. (Day 12) Preparation of neural cells for evaluation was completed.

[0136] 2. Evaluation of Aβ aggregation inhibitory activity of each plant extract Among the plant extracts confirmed to have Aβ aggregation inhibitory activity in Example 2, the Aβ aggregation inhibitory activity of each of the following plant extracts was confirmed in a cell system: Tomoe-sou, Siberian geranium, Japanese honeysuckle, Japanese butterbur, and Nagareboshiwaremoko, as well as the newly prepared plant extracts (Lobelia sessilifolia, Lobelia sessilifolia), Adenocaulon himalaicum, Maianthemum dilatatum, and Stellaria radians (Stellaria radians) prepared in (16) to (19) of Example 1. Cell system evaluation was performed using the following method.

[0137] The n-butanol fraction was used for Tomoe-sou and the t-butyl methyl ether fraction was used for Siberian geranium, but the ethanol fraction was used for all the others.

[0138] Aβ prepared in the above Example 2 (1) and (2) 42 The prepared Aβ solution and QD-Aβ solution were diluted with fresh NbM medium. 42A solution containing DMSO and a prepared QD-Aβ solution were prepared. The plant extract prepared in Example 1 was diluted with fresh NbM medium to prepare a plant extract-containing solution (300 ng / μL or 150 ng / μL). Furthermore, a solution containing DMSO alone diluted at the same dilution as the plant extract was used as a negative control. After removing the culture supernatant of the prepared neural cells for evaluation with a pipette, the prepared QD-Aβ solution was dispensed at 70 μL / well, taking care not to detach the cells. Next, 10 μL / well of the plant extract-containing solution and the prepared Aβ solution were added. 42 70 μL of the solution was injected per well (final concentration: 30 nM QD-Aβ solution, Aβ 42 (10 μM solution). A gas-permeable seal was attached to the plate, and it was set in an Incucyte® Live Cell Analysis System (Sartorius) placed in a 37°C, 5% CO2 incubator. After setting, measurement began 2 hours after setting, when the measurement conditions had stabilized, and the amount of Aβ aggregation was measured over 22 hours. The Incusyte settings were as follows:

[0139] Objective: 20x Scan Type: Standard Image Channels: Phase, Red Red Acquisition times(ms): 400 Scan time : 1 shot / hrs Picture number: 3 picture / well

[0140] (Results) The results are shown in Figures 1 and 2. Figure 1 is a plot showing the Aβ aggregation / deposition effects in a cell line for nine plant extracts. When the Y-axis plot is lower than that of DMSO, the negative control, it indicates that the plant extract has Aβ aggregation / deposition inhibitory activity in nervous system cells.

[0141] As a result, it was confirmed that all of the nine plant extracts tested had Aβ aggregation / deposition inhibitory activity not only in the cell-free system but also in the cell-based system.

[0142] From the above results, it was demonstrated that the plant extract obtained in the present invention has aggregation-inhibiting activity that inhibits the aggregation of aggregating proteins, and can serve as a novel aggregation inhibitor.

[0143] Table 2 summarizes the families and genera to which the nine plants having Aβ aggregation / deposition inhibitory activity belong.

[0144] Figure 2 shows cell observation images at 22 hours for the systems with and without the addition of Butterbur extract. This figure also confirms that Aβ aggregation / deposition in cells is suppressed in the system with the addition of Butterbur extract.

[0145] Example 4: Evaluation of Aβ aggregation inhibitory activity of each material extract Nine plant extracts newly prepared in Example 1 (20) to (28), namely, Japanese honeysuckle, allium sativum, monkshood, azalea, bilberry, chickweed, celandine, celandine, and palustrine, were examined for their effects on Aβ aggregation / deposition in cells in the same manner as in Example 2. As a result, it was confirmed that all nine plant extracts used inhibited Aβ aggregation / deposition in cells more effectively than DMSO.

[0146] Table 3 summarizes the families and genera to which the nine plants having Aβ aggregation / deposition inhibitory activity used in Example 4 belong.

[0147] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An aggregate inhibitor for aggregated proteins, comprising plant extracts selected from the group consisting of plants belonging to the following genera: Rosoideae, Saxifragaceae, Apiaceae, Liliaceae, Campanulaceae, Ericaceae, Lycopus, Geranium, Plantago, Hypericum, Stellaria, Chelidon, Pachysandra, and Matteuccia.

2. The flocculation inhibitor according to claim 1, wherein the plant belonging to the Rosaceae subfamily is a plant belonging to the genus Sanguisorba, Geum, Rosa, or Argentina.

3. The flocculation inhibitor according to claim 1, wherein the plant belonging to the Apiaceae family is a plant belonging to the genus Sanicula or Cryptotaenia.

4. The flocculation inhibitor according to claim 1, wherein the plant belonging to the Liliaceae family is a plant belonging to the genus Maianthemum or the genus Allium.

5. The flocculation inhibitor according to claim 1, wherein the plant belonging to the Campanulaceae family is a plant belonging to the genus Lobelia or Adenophora.

6. The flocculation inhibitor according to claim 1, wherein the plant belonging to the Ericaceae family is a plant belonging to the genus Ledum or Vaccinium.

7. The flocculation inhibitor according to any one of claims 1 to 6, wherein the plant is a plant belonging to the genus Sanguisorba, the family Saxifragaceae, the genus Geranium, the genus Hypericum, the genus Malus genus, and the genus Stellaria.

8. The flocculation inhibitor according to claim 1, wherein the extract is derived from the whole plant.

9. The aggregation inhibitor according to claim 1, wherein the aggregated protein is amyloid-beta.

10. A composition for inhibiting the aggregation of aggregated proteins, comprising the aggregation inhibitor described in claim 1 as an active ingredient.

11. The aggregation inhibitory composition according to claim 10, comprising two or more different aggregation inhibitors as described in claim 1 as active ingredients.

12. An aggregation-inhibiting composition according to any one of claims 10 or 11, for the treatment or prevention of diseases caused by aggregation of aggregated proteins.

13. The aggregation inhibitory composition according to claim 12, wherein the disease is Alzheimer's disease.