JP2010202606A - Aggregation inhibitor of amyloid-forming protein and decomposition agent of aggregated amyloid protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an aggregation inhibitor of an amyloid-forming protein and a decomposition agent of an aggregated amyloid protein. <P>SOLUTION: The aggregation inhibitor of the amyloid-forming protein and/or the decomposition agent of the aggregated amyloid protein include(s) one or more plant(s) or treated product of the same or an extract of the same selected from a group of red kidney beans, guava, a fermented guava, Alpinia speciosa, fermented Alpinia speciosa, Origanum vulgare, basil, Melissa officinalis, Rosa rugosa, sea lettuce, Origanum majorana, Leucaena leucocephala, a fermented Leucaena leucocephala, Citrus hystrix and Gymnema sylvestre. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an amyloidogenic protein aggregation inhibitor and an aggregated amyloid protein degradation agent.

  Amyloidosis is a group of diseases that cause dysfunction by depositing amyloid (fibrous insoluble aggregates), which are specific protein aggregates, in various organs throughout the body. Specific examples include Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes, prion disease and the like.

  AD is one of the two major diseases that cause vascular dementia and dementia, and is a neurodegenerative disease.

  The major pathological changes that the AD brain exhibits are the formation of senile plaques and neurofibrillary tangles, which cause area-selective cranial nerve cell death. Among these, cerebral deposition of amyloid β protein (Aβ), which is a component of senile plaques, is the earliest change observed pathologically.

  After the appearance of senile plaques, tau protein, a type of microtubule-associated protein, is abnormally phosphorylated, becomes insoluble, and shows neurofibrillary tangles that accumulate and accumulate outside neurons, and finally neuronal cell death Brain atrophy is observed.

  Regarding the formation process of AD, the hypothesis (amyloid cascade hypothesis) that Aβ polymerization, aggregation, accumulation and the accompanying neuronal cell damage form the core is widely accepted.

  Proteins that form amyloid such as Aβ are known to form soluble aggregates (oligomers) during the aggregation process, but both amyloid and oligomers have been reported to have cytotoxic effects. Therefore, a drug that inhibits aggregation of amyloidogenic proteins such as Aβ is expected to have a therapeutic effect on amyloidosis such as AD.

  PD is the second most common neurodegenerative disease after AD, and its pathological features include degeneration of neuromelanin-containing neurons (substantia nigra specific neurons, SNpc) found in the ventral part of the midbrain and residual neurons. Eosinophilic inclusion bodies (Lewy bodies) and neurite degeneration (Lewy neurites) are observed.

  Lewy bodies are aggregates composed mainly of α-synuclein and other cytoskeletal proteins such as ubiquitin, synaptophysin, and tau protein.

  A disease group in which abnormal accumulation of amyloidated α-synuclein is the main finding is called α-synuclein abnormal disease (α-synucleinopathy), and includes Lewy body dementia, multiple system atrophy, and the like.

  As a pathological feature of type II diabetes, deposition of amyloid aggregates containing amylin as a main component is observed in the splenic islets of Langerhans, and it is pointed out that this amyloid aggregate may be involved in the development of diabetes.

  Prion disease is a general term for central nervous system diseases caused by an increase in abnormal prion protein. Prion protein is also present in the normal body, and is particularly abundant in nerve cells, and is thought to maintain the structure of nerve cells and to transmit information between nerves.

  When prion protein becomes abnormal for some reason and becomes a pathogen, it causes prion disease.

  In lesions of prion disease, vacuolar degeneration of central nervous tissue, astroglial proliferation and accumulation of prion protein aggregates are observed.

  Examples of prion diseases include sheep scrapie, bovine bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD).

  It is known that plant bodies of certain plants and extracts thereof have an Aβ aggregation inhibitory action and / or an aggregate Aβ degradation action. For example, it has been reported that peony skin, cinnamon bark, and extracts thereof have an Aβ aggregation-inhibiting action and an aggregated Aβ-degrading action (Patent Document 1).

  Further, it has been reported that butterfly mushrooms and these extract extracts have an Aβ aggregation inhibitory action and an aggregate Aβ decomposition action (Non-patent Document 1).

  Furthermore, it has been reported that curcumin contained in turmeric has an Aβ aggregation inhibitory effect, an Aβ oligomer and a neurofibril formation inhibitory effect (Non-patent Document 2).

JP 2006-206584 A

J. Neurosci. Res. 84, 427-433 (2006) J. Biol. Chem. 280, 5922-5901 (2005)

  An object of the present invention is to provide a novel amyloidogenic protein aggregation inhibitor and a degrading agent for aggregated amyloid protein.

  The present inventors have examined natural products having an aggregation inhibitory activity of amyloidogenic protein and an activity of degrading aggregated amyloid protein. As a result, they have found several kinds of plants exhibiting the aggregation inhibitory activity of amyloidogenic protein and the degradation activity of aggregated amyloid protein. It was.

That is, the present invention is as follows.
[1] One or more plants selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Ginnemu, Fermented Ginnemu, Kobumikan and Gymnema Sylvestre An aggregation inhibitor of amyloidogenic protein comprising a plant body or a processed product thereof or an extract thereof as an active ingredient.
[2] One or more plants selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Ginnemu, Fermented Ginnemu, Kobumikan and Gymnema Sylvestre An agent for degrading aggregated amyloid protein comprising a plant body or a processed product thereof or an extract thereof as an active ingredient.

  By ingesting the agent of the present invention into a mammal, aggregation of the amyloid-forming protein in the animal can be suppressed and the aggregated amyloid protein can be degraded.

  The present invention relates to one or more plants selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Ginnemu, Fermented Ginnemu, Kobumikan and Gymnema Sylvestre The present invention relates to an aggregation inhibitor of amyloid-forming protein and / or a degrading agent of aggregated amyloid protein, which contains a plant body or a processed product thereof or an extract thereof as an active ingredient.

  In the present invention, the amyloid-forming protein means a protein that forms amyloid, which is an unbranched fibrous protein aggregate that is linear in electron microscopy. Aggregated amyloid protein means a protein aggregate that polymerizes amyloid-forming protein to form amyloid and is pathologically deposited in various organs. Specific examples of the amyloid-forming protein include Aβ, α-synuclein, amylin, prion protein, and the like, but are not limited thereto, and may include any amyloid-forming protein involved in various amyloidosis.

  In the present invention, Red Kidney Beans refers to Phaseolus vulgaris of the leguminous kidney bean, Guava refers to Psidium guajava L. belonging to the genus Vangilow, and Ghetto refers to Alpinia speciosa (Wendl.) K of the Ginger family. .Schum., Oregano, Liganaceae Origanum vulgare L., basil, Lamiaceae Ocimum basilicum, lemon verbena, Lippia triphylla, Rabbitaceae, rose Rosa rugosa of the genus Rose family, Aosa is the Monostroma nitidum wittrock, Monostroma latissimum wittrock of the Cryptaceae family, Ulva pertusa of the Cryaceae family, Marjoram is Origanum majorana L. of the Labiatae family, Ginnemu Leucaena leucocephala de wit belonging to the genus Ginkgoceae, Citrus hystrix DC. Belonging to the citrus citrus, Gymnema sylvestre (Retz.) Each means schult.

  Examples of the plant body of the present invention include, for example, a wild plant body, a plant body obtained by cultivation, or all or part of a plant body obtained by culture such as tissue culture (for example, leaves, flowers, branches, stems, fruits, roots). , Seeds, cultured cells, tissues or organs, or dedifferentiated cells or cell populations (eg, callus)), but are not limited thereto.

  Preferably, as a part of the plant body, when the plant of the present invention is red kidney beans, seeds, etc., when guava, leaves, fruits, roots, etc., when it is ghetto, leaves etc., when it is oregano Leaves, seeds, etc. for basil, leaves, etc. for lemon verbena, leaves, petals, etc. for Hermanus, alga bodies, etc. for Aosa, leaves, petals, etc. for marjoram However, in the case of Ginnemu, leaves and the like, in the case of Komikan, leaves and the like in the case of Gymnema sylvestre, respectively.

  The treated product of the plant of the present invention is not particularly limited as long as it is obtained by physically, chemically or biologically treating the plant. For example, physical treatment methods include, for example, drying treatment such as sun drying, air drying, freeze drying, etc., pulverization treatment with a blender, a homogenizer, a ball mill, etc. Products, freeze-dried products, pulverized products, and the like. Examples of the chemical treatment method include enzyme treatment such as protease and saccharifying enzyme, and examples of the chemical treatment product include an enzyme treatment product. Examples of the biological treatment method include a fermentation method, and examples of the biological treatment product include a fermentation treatment product.

  In a preferred embodiment, a fermented processed product of guava, ghetto, and ginnemu (referred to herein as “fermented guava”, “fermented ghetto”, and “fermented ginnem”) is used as the processed product of the plant of the present invention. Can do. Regarding the method for producing fermented guava, for example, the methods described in JP-A-2004-24004 and JP-A-2002-330725, and the method for producing fermented ghetto are described in JP-A-2002-330725, for example. Examples of the method for producing a fermented ginnem, such as the method described in JP-A-4-234951, include, but are not limited to, fermented guava used for various health foods, Any known method can be used as a method for producing the fermented ghetto and fermented ginnemu.

  Examples of the extract of the plant body or the processed product thereof according to the present invention include extracts obtained from the aforementioned plant body or the processed product thereof by various extraction methods. Examples of the extraction method include various solvent extractions and supercritical fluid extractions.

  In the extraction and processing of the extractant, for example, an antioxidant or a preservative can be added. As a solvent used for solvent extraction, any solvent can be used as long as it can extract a substance exhibiting an action of inhibiting aggregation of amyloidogenic protein and / or a function of degrading aggregated amyloid protein. For example, water, distilled water, deionized water, inorganic Aqueous media such as saline solution, buffer solution, monohydric alcohol such as methanol, ethanol, propanol, butanol, polyhydric alcohol such as propylene glycol, glycerol, hexane, heptane, toluene, petroleum ether, benzene, ethyl acetate, chloroform, dichloromethane , Organic solvents such as 1,1,2-trichloroethene, dimethyl sulfoxide, acetone and the like. The particularly preferred solvent varies depending on the type of plant body / plant processed product to be used, but those skilled in the art can use, for example, the aggregation inhibitory activity of amyloid-forming protein and / or the degradation activity of aggregated amyloid protein described in the Examples below as indicators. A preferred solvent can be easily selected.

  Examples of the buffer solution include a phosphate buffer solution and a citrate buffer solution. Examples of the inorganic salt of the inorganic salt aqueous solution include sodium chloride, potassium chloride, calcium chloride, sodium hydrogen carbonate and the like. The alcohol is preferably a monohydric alcohol, and the monohydric alcohol is preferably methanol or ethanol.

  These solvents can be used alone or in combination. The mixed solvent is preferably a hydrous alcohol, more preferably a hydrous monohydric alcohol, and particularly preferably hydrous methanol or ethanol. The water content is preferably 80% or less, and more preferably 60% or less.

  Further, carbon dioxide converted into a supercritical fluid can also be used as a solvent.

  Extraction is performed, for example, using 0.1 to 10000 parts by weight, preferably 1 to 100 parts by weight of the solvent with respect to 1 part by weight of the plant body. Although extraction temperature does not have a restriction | limiting in particular, 0-100 degreeC is preferable and 20-90 degreeC is more preferable. The extraction time is not particularly limited, but is preferably 1 minute to 1 week, more preferably 30 minutes to 1 day. In particular, a method of extracting the aforementioned plant body or a processed product thereof with an aqueous medium such as water, pure water, deionized water, a method of extracting a residue after extraction with an aqueous medium with alcohol or hydrous alcohol, or acetone or chloroform. A method of extracting with an organic solvent such as the above, and a method of further fractionating the extract with an aqueous medium, hydrous alcohol, alcohol, organic solvent or the like are preferred.

  Although there is no restriction | limiting in particular as an apparatus used for extraction, The container devised in order to extract efficiently, a stirrer, a reflux condenser, a Soxhlet extractor, a homogenizer, a shaker, an ultrasonic generator, etc. are mentioned.

  The extract obtained as described above includes sedimentation separation, cake filtration, clarification filtration, centrifugal filtration, centrifugal sedimentation, press separation, filter press, membrane separation, and other various solid-liquid separation methods, various concentration methods, various drying methods, You may further process by the formulation methods, such as granulation or powdering, and various purification methods.

  Examples of the purification method include solvent fractionation, column chromatography, and recrystallization. In particular, a column chromatography method using various carriers such as Diaion HP-20, SP207 (Mitsubishi Chemical Corporation), Sephadex LH-20 (Pharmacia Co.) is preferable. Concentration and drying methods include freeze drying, natural drying, hot air drying, ventilation drying, spray drying, vacuum drying, sun drying, vacuum drying, fluidized bed drying, foam layer drying, drum dryer film drying, ultrasonic drying, etc. , Drying methods such as electromagnetic wave drying, and freeze-drying methods.

  The plant of the present invention or a processed product thereof or an extract thereof has an action of inhibiting aggregation of amyloid-forming protein and degrading aggregated amyloid protein, and thus is useful as a prophylactic / therapeutic agent for diseases involving aggregated amyloid protein. is there. Examples of such diseases include diseases involving Aβ (AD, Down syndrome, etc.) diseases involving α-synuclein (PD, Lewy body dementia, multiple system atrophy, etc.), diseases involving amylin (type II) Diabetes, etc.), diseases involving prion protein (CJD, BSE, scrapie, etc.), tauopathy, polyglutamine disease, amyotrophic lateral sclerosis (ALS), peripheral amyloidosis (AL amyloidosis, AA amyloidosis, AF amyloidosis, AHCA amyloidosis, AH amyloidosis, ASc amyloidosis, ASb amyloidosis, AD amyloidosis, etc.).

  The plant of the present invention or a processed product thereof or an extract thereof has low toxicity, and a human or other animal such as a non-human mammal (eg, mouse, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.), birds, reptiles, amphibians, fish and the like. The pharmaceutical composition used for administration may contain the plant of the present invention or a processed product thereof or an extract thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

  The dosage form of the preparation should preferably be the most effective for inhibiting the aggregation of amyloidogenic protein and degrading the aggregated amyloid protein. Oral administration, for example, intravenous, intraperitoneal or subcutaneous administration, etc. Although parenteral administration can be mentioned, oral administration is preferred.

  Examples of dosage forms to be administered include tablets, powders, granules, pills, suspensions, emulsions, soaking agents, decoction, capsules, syrups, solutions, elixirs, extracts, tinctures, fluid extracts, etc. Any of parenteral preparations such as oral preparations, injection preparations, instillation preparations, cream preparations, and suppositories may be used, but they are preferably used as oral preparations.

  Liquid preparations such as syrups suitable for oral administration include saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p -Preservatives such as -hydroxybenzoic acid esters, paraoxybenzoic acid derivatives such as methyl paraoxybenzoate, preservatives such as sodium benzoate, and flavors such as strawberry flavor and peppermint can be added to prepare a preparation.

  Also suitable for oral administration, such as tablets, powders and granules are lactose, sucrose, glucose, sucrose, mannitol, sorbitol and other sugars, potato, wheat, corn and other starches, calcium carbonate, calcium sulfate, hydrogen carbonate Inorganic substances such as sodium and sodium chloride, excipients such as crystalline cellulose, licorice powder and gentian powder, etc., starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, sodium bicarbonate , Disintegrants such as sodium alginate, magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and other lubricants, polyvinyl alcohol, hydroxypropylcellulose, methylcellulose, ethylcellulose, carmellose, gelatin, starch Binding agents such as liquid, surfactants such as fatty acid esters can be formulated by adding a plasticizer such as glycerin.

  In addition, preparations suitable for oral administration include additives generally used in foods and drinks such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, color formers, bleaching agents, fungicides, A gum base, a bittering agent, an enzyme, a brightener, a sour agent, a seasoning, an emulsifier, a toughening agent, a manufacturing agent, a fragrance, a spice extract and the like may be added.

  In parenteral administration, one or more auxiliary ingredients selected from preservatives, preservatives, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified in oral preparations are used. Can be added.

  The concentration of the amyloid-forming protein aggregation inhibitor, aggregated amyloid protein degradation agent of the present invention in the plant body or a processed product thereof, or an extract thereof is appropriately determined according to the type of formulation, the effect expected by administration of the formulation, etc. The selected plant body or its treated product or its extract is usually 0.1 to 100% by weight, preferably 0.5 to 70% by weight, particularly preferably 1 to 50% by weight. The aggregation inhibitor of amyloid-forming protein and the agent for degrading aggregated amyloid protein of the present invention can contain two or more of the above-mentioned plant body or a processed product thereof or an extract thereof. It is desirable that the total amount of the plant body or the processed product or the extract thereof contained in is within the above range.

  The dosage and frequency of administration of the aggregation inhibitor of amyloidogenic protein and the degradation agent of aggregated amyloid protein of the present invention to humans vary depending on the type of active ingredient, dosage form, age of the recipient, body weight, etc. The daily dose of the plant body or the processed product or the extract thereof is usually once or several times a day so that the dose is usually 50 mg to 30 g, preferably 100 mg to 10 g, particularly preferably 200 mg to 3 g. . The administration period is not particularly limited, but is usually 1 month to 30 years, preferably 6 months to 20 years.

  The dose for administration to a non-human animal varies depending on the age, type, nature of the symptom, or severity of the animal, but usually 1 to 1 kg body weight per day as the plant or treated product or extract thereof. It is administered once to several times a day so as to be 600 mg, preferably 2 to 200 mg, particularly preferably 4 to 60 mg. The administration period is not particularly limited, but is usually 1 day to 3 years, preferably 2 weeks to 1 year.

Since the plant of the present invention or a processed product thereof or an extract thereof can be used as food or feed or an additive thereof, it is used as it is, or together with other food or feed, its raw material or an additive therefor, a food or drink or feed or It can be processed into an additive for that purpose. A food or drink or feed containing the plant of the present invention, a processed product thereof or an extract thereof, has an action of inhibiting aggregation of amyloid-forming protein and / or a function of degrading aggregated amyloid protein in humans or other animals ingesting it. As shown, foods and drinks such as health foods, functional foods, dietary supplements, foods for specified health use (or non-humans such as livestock and poultry, etc.) for the prevention and / or suppression of the above-mentioned diseases involving aggregated amyloid protein Equivalent to the food in animal feed).
Accordingly, the present invention is also 1 selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Guinem, Fermented Guinem, Kobumikan and Gymnema Sylvestre. Food / beverage products or feed for inhibiting aggregation of amyloid-forming protein and / or degrading aggregated amyloid protein, or an additive for production thereof, comprising the plant body of the above or a processed product thereof or an extract thereof To do.

  The amount of the plant of the present invention or processed product thereof or extract thereof contained in the food or drink or feed of the present invention is not particularly limited as long as the food or drink or feed exhibits the above action (effective amount). However, for example, the dry weight of the plant or its treated product or its extract is 0.001 to 100% by weight, preferably 0.01 to 100% by weight, more preferably 0.1 to 100% by weight.

  Specific foods and beverages to which the plant of the present invention or a processed product thereof or an extract thereof are added include, for example, juices, soft drinks, soups, teas, lactic acid bacteria beverages, fermented milk, frozen desserts, butter, cheese, Dairy products such as yogurt, processed milk, skimmed milk powder, livestock meat products such as ham, sausage, hamburger, fish smelted products, egg products such as sushi rolls, egg tofu, cookies, jelly, snacks, chewing gum and other confectionery, bread , Noodles, pickles, salmon products, dried fish, boiled potatoes, seasonings and the like.

  Examples of the form of food and drink include powdered foods, sheet-like foods, bottled foods, canned foods, retort foods, capsule foods, tablet-like foods, liquid foods, and drinks. The food / beverage products of the present invention are used as a health food / beverage product or a functional food / beverage product for inhibiting aggregation of amyloid-forming protein or decomposing aggregated amyloid protein.

  When the food or drink of the present invention containing the plant of the present invention or a processed product thereof or an extract thereof is ingested, the intake is not particularly limited, but usually the plant or processed product thereof per day for an adult or The dry weight of the extract is 50 mg to 30 g, preferably 100 mg to 10 g, particularly preferably 200 mg to 3 g. This intake is continued for 1 month to 30 years, preferably 6 months to 20 years. However, this intake amount is only a guideline, and can be adjusted to a suitable range according to the symptom level, age, weight, etc. of the intaker.

  The feed of the present invention can include any feed used for non-human animals such as mammals, birds, reptiles, amphibians, fish, etc. For example, feed for pets such as dogs, cats, rats, etc. And feed for livestock such as cattle and pigs, feed for poultry such as chicken and turkey, and feed for cultured fish such as Thailand and Hamachi.

  The feed of the present invention can be prepared by appropriately blending the plant material of the present invention or a processed product thereof or an extract thereof with a feed raw material. Examples of feed materials include cereals, potatoes, vegetable oil meals, animal feed materials, other feed materials, and refined products. Examples of cereals include milo, wheat, barley, oats, rye, brown rice, buckwheat, whey, acne, mushrooms, corn, and soybeans.

  Examples of rice bran include rice bran, defatted rice bran, bran, powder, wheat germ, wheat bran, pellets, corn bran, and corn germ. Examples of vegetable oil cakes include soybean oil cake, kinako powder, linseed oil cake, cottonseed oil cake, peanut oil cake, safflower oil cake, palm oil cake, palm oil cake, sesame oil cake, sunflower oil cake, rapeseed oil Examples include dregs, kapok dregs and mustard dregs.

  Examples of animal feed ingredients include fish meal (North Sea meal, imported meal, whole meal, coastal meal), fish solubil, meat meal, meat and bone meal, blood meal, decomposed hair, bone meal, livestock processing by-products, feather meal, rice cake, Non-fat dry milk, casein, dry whey and the like can be mentioned. Other feed ingredients include plant foliage (alfalfa, hay cube, alfalfa leaf meal, false acacia powder, etc.), corn processing industry by-products (corn gluten, meal, corn gluten feed, corn stapler, etc.), processed starch products (starch) Etc.), sugar, fermentation industrial products (yeast, beer grounds, malt root, alcohol grounds, soy sauce grounds, etc.), agricultural production by-products (citrus processed grounds, tofu grounds, coffee grounds, cocoa grounds, etc.), others (cassava, broad beans, Guamir, seaweed, krill, spirulina, chlorella, mineral, etc.).

  Examples of purified products include proteins (casein, albumin, etc.), amino acids, carbohydrates (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins, and the like.

  When the feed of the present invention containing the plant of the present invention or a processed product thereof or an extract thereof is ingested, the intake is not particularly limited, but usually the plant body per day per kg of the animal body weight. Alternatively, the dry weight of the processed product or the extract thereof is 1 to 600 mg, preferably 2 to 200 mg, particularly preferably 4 to 60 mg. This intake is usually continued for 1 day to 3 years, preferably 2 weeks to 1 year. However, this intake is only a guideline and can be adjusted to a suitable range according to the type, age, weight, etc. of the ingested animal.

  The additive for food or drink or feed of the present invention containing the plant of the present invention or a processed product thereof or an extract thereof is generally added to the plant or processed product or extract thereof as needed. Or additives used in feeds, such as sweeteners, colorants, preservatives, thickening stabilizers, oxidation agents described in the Food Additives Display Handbook (Japan Food Additives Association, issued on January 6, 1997) Add additives such as inhibitors, color formers, bleaches, fungicides, gum bases, bitters, enzymes, brighteners, sours, seasonings, emulsifiers, tougheners, manufacturing agents, fragrances, spice extracts May be. Moreover, you may add the carrier illustrated to the above-mentioned pharmaceutical formulation.

  Examples of sweeteners include aspartame, licorice, stevia, xylose and lacanca. Examples of the colorant include carotenoids, turmeric pigments, flavonoids, caramel pigments, chicone pigments, spirulina pigments, chlorophylls, purple potato pigments, purple potato pigments, perilla pigments, blueberry pigments and the like.

  Examples of the preservative include sodium sulfite, benzoic acids, udo extract, scallop extract, Chinese mugwort extract, sorbic acids, propionic acids and the like. Examples of thickening stabilizers include gums such as gum arabic and xanthan gum, alginic acids, chitin, chitosan, kidachi aloe extract, guar gum, hydroxypropylcellulose, sodium caseinate, corn starch, carboxymethylcelluloses, gelatin, agar, dextrin, methylcellulose, Examples include polyvinyl alcohol, microfibrous cellulose, microcrystalline cellulose, seaweed cellulose, sodium polyacrylate, sodium polyphosphate, carrageenan, yeast cell wall, konjac potato extract, nata de coco, mannan and the like.

  Antioxidants include vitamin C, sodium ethylenediaminetetraacetate, calcium ethylenediaminetetraacetate, erythorbic acid, oryzanol, catechin, quercetin, clove extract, enzyme-treated rutin, apple extract, sesame oil extract, dibutylhydroxytoluene, fennel extract , Horseradish extract, seri extract, tea extract, tempeh extract, dokudami extract, tocotrienol, tocopherols, rapeseed oil extract, raw coffee bean extract, sunflower seeds, ferulic acid, butylhydroxyanisole, blueberry Examples include leaf extract, propolis extract, hego-ginkgo extract, hesperetin, pepper extract, spinach extract, gallic acid, bayberry extract, eucalyptus extract, rosemary extract and the like.

  Examples of the color former include sodium nitrite, and examples of the bleaching agent include sodium sulfite. Examples of fungicides include orthophenylphenol. Gum base includes methyl acetylricinoleate, urushi wax, ester gum, elemi resin, cucumber lily wax, ozokerite, opopanax resin, kauri gum, carnauba wax, guaiac resin, gutta kachu, gutta hankan, gutta percha, glycerin fatty acid ester, gay wax, copaoba balsam, copal Resin, rubber, rice bran wax, sugar cane wax, shellac, gelton, sucrose fatty acid ester, solva, sorbitan fatty acid ester, talc, calcium carbonate, dammar resin, chicle, tilte, tunou, low molecular rubber, paraffin wax, far balsam, propylene glycol fatty acid Esters, powdered pulp, powdered chaff, jojoba wax, polyisobutylene, polybutene, microcrystal wax, mastic, Saran Dubai chocolate, beeswax, such as calcium phosphate, and the like.

  Bitterings include isoalpha bitter acid, caffeine, Kawaratake extract, kina extract, yellowfin extract, gentian extract, spice extract, enzyme-treated naringin, Jamaica cassia extract, theobromine, naringin, nigaki extract, Examples include wormwood extract, chickworm extract, himematsutake extract, borapets, methylthioadenosine, litchi extract, olive tea, daidai extract, hop extract, mugwort extract and the like.

  Enzymes or enzyme sources include amylase, trypsin, rennet, lactic acid bacteria and the like. Examples of brighteners include urushiro and mole. Examples of the acidulant include adipic acid, itaconic acid, citric acid, succinic acid, sodium acetate, tartaric acid, carbon dioxide, lactic acid, phytic acid, fumaric acid, malic acid, phosphoric acid and the like.

  Seasonings include asparagine, aspartic acid, glutamic acid, glutamine, alanine, isoleucine, glycine, serine, cystine, tyrosine, leucine, proline and other amino acids, sodium inosinate, sodium uridylate, sodium guanylate, sodium cytidylate, ribonucleotide Nucleic acids such as calcium and sodium ribonucleotide, organic acids such as citric acid and succinic acid, potassium chloride, low salt water sodium salt solution, crude seawater chloride, whey salt, tripotassium phosphate, dipotassium hydrogen phosphate, phosphate Examples include potassium dihydrogen, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, and chlorella extract.

  Zinc salts, vitamin C, various amino acids, 5-adenylic acid, iron chloride, hesperidin, various calcined calcium, various calcined calcium, dibenzoylthiamine, calcium hydroxide, calcium carbonate, thiamine hydrochloride, Dunaliella carotene , Tocopherol, nicotinic acid, carrot carotene, palm oil carotene, calcium pantothenate, vitamin A, hydroxyproline, calcium dihydrogen pyrophosphate, ferrous pyrophosphate, ferric pyrophosphate, ferritin, heme iron, menaquinone, folic acid, Riboflavin etc. are mentioned.

  Manufacturing agents include processing aids such as acetone and ion exchange resin, fig leaf extract, Inawara ash extract, kaolin, glycerin fatty acid ester, mulberry extract, bone ash, perilla extract, ginger extract, various tannins, phafia Examples include pigments, grape seed extracts, and ethanol. In addition, these various additives can also be added and used for the above-mentioned food / beverage products or feed of this invention.

Example 1 Extraction from Plant and Preparation of Purified Fraction Red kidney beans (seed), guava (leaves), ghetto (leaves), oregano (leaves), basil (leaves), lemon verbena (leaves) by the following method ), Hermanus (fruit), Aosa (algae), Marjoram (leaves), Ginnemu (leaves) and Kobumikan (leaves) were subjected to fractionation and purification operations to prepare purified plant bodies fractions. The same extraction operation was performed for turmeric, butterfly, and peony skin, which have been reported for Aβ aggregation inhibition.
(1) Preparation for extraction A dried plant body (30 g) or a fresh plant body (140 g) was pulverized with a small mill or a food processor and immersed in 70% acetone (for 12 hours or more).
(2) Extraction The immersed product was irradiated with ultrasonic waves for 1 hour or longer and then filtered to obtain a filtrate (i). Further, the residue was immersed in 100% acetone and subjected to the same treatment to obtain a filtrate (ii). The filtrate (i) and the filtrate (ii) were mixed, concentrated under reduced pressure to remove acetone, and an acetone extraction concentrate was obtained. A portion of this extract concentrate was concentrated to dryness to obtain a crude acetone extract. Further, the residue after extraction with 100% acetone was immersed in chloroform, subjected to ultrasonic treatment for 1 hour or longer, filtered and concentrated to dryness to obtain a crude chloroform extract.
(3) Fractionation After the above acetone extract concentrate was charged into a column packed with hydrophobic adsorbent resin (Diaion HP-20, manufactured by Mitsubishi Chemical), deionized water (2RV amount), 33% methanol ( 2RV amount), 66% methanol (2RV amount), 100% methanol (2RV amount), and acetone (2RV amount) were sequentially eluted. The obtained eluate was dried using a centrifugal concentrator and a freeze dryer to obtain each fraction. Here, RV represents the amount of resin (Resin Volume), and the amount of liquid flowing when a solution of the same amount as the amount of resin is passed is 1 RV amount.

Example 2 Preparation of Extract from Plant According to the following method, an extraction operation was performed from red kidney beans, guava, fermented guava, fermented ghetto, oregano, basil, lemon verbena, hermanus, aosa, marjoram, fermented ginnemu and kobumikan. A plant body extract was prepared. The fermented guava, fermented ghetto, and fermented ginnemu were prepared by adding and fermenting several types of bacteria including sugar sources and lactic acid bacteria to each leaf.
(1) Preparation for extraction The dried plant (5 g) or fresh plant (10 g) was pulverized with a small mill or a food processor.
(2) Extraction Add 10 times volume (V / W) deionized water or 100% ethanol to the pulverized plant, stir for 3 hours, filter after extraction, extract water, 100% ethanol An extract was obtained. The extraction residue with deionized water was further added with 10 times amount (V / W) of 100% ethanol, stirred for 3 hours, and filtered after extraction to obtain a hydrous ethanol extract. Each extract was concentrated to dryness to obtain a water extract, a 100% ethanol extract, and a water-containing ethanol extract (the ratio of ethanol was converted from the amount of the water extract).

Test Example 1 Aβ aggregation inhibition evaluation (thioflavin T method evaluation)
(1) Materials and methods (i) Reagent preparation 4 × PBS solution: 250 mL of ultrapure water was added to 9.6 g of PBS powder (Nissui Pharmaceutical, Cat. No. 05913) and dissolved.
-100 μM thioflavin T / PBS solution: Thioflavin T (Sigma, Cat. No. T3516) was dissolved in PBS solution to a concentration of 100 μM.
Aβ stock solution: Aβ1-40 (Peptide Institute, Cat. No. 4379-v) powder was dissolved to a concentration of 1 mM using ultrapure water and stored at −80 ° C. It melted at the time of use, and it was confirmed that it was completely dissolved by sonication.
・ Sample solution: Dissolve the above crude extract (dried product) and fraction (dried product) in 2% DMSO ultrapure aqueous solution to prepare sample solutions of various concentrations (0 to 10μg / mL). did.
For Gymnema sylvestre, a commercially available aqueous ethanol extract powder was used.
(Ii) Measurement procedure by thioflavin T method (a) 20 μL / well of the sample solution was added to a 96-well plate. Ultra pure water containing 2% DMSO having the same concentration as the sample solution was added to the vehicle well, and only 20 μL / well of ultra pure water was added to the blank well.
(B) The Aβ stock solution was diluted with ultrapure water to prepare 60 μM. This was added at 10 μL / well in addition to the blank well. To the blank well, 10 μL / well of ultrapure water was added. The plate was mixed well.
(C) 10 μL / well of 4 × PBS solution was added to all wells. The plate was mixed well. The plate was sealed and incubated at room temperature for 18 hours.
(D) In order to measure the autofluorescence of the specimen itself, fluorescence was measured with a fluorescence reader (ARVO SX, Wallac) under the conditions of ex.485 nm and em.585 nm.
(E) Thioflavin T stock solution was added to all wells at a volume of 20 μL / well, mixed well, and left at room temperature for 1 hour.
(F) The fluorescence was measured with a fluorescence reader under the conditions of ex.485 nm and em.585 nm.
(G) For each well, the measurement value of (d) was subtracted from the measurement value of (f). Furthermore, the average value of Blank well was subtracted from the value of each well. The amount of Aβ aggregation in each well was calculated as a relative value (%) to the average of the measured values of Vehicle well calculated above. In addition, an IC50 value (analyte concentration that inhibits Aβ aggregation by 50%) was calculated from the analyte concentration and the aggregation rate.

(2) Aβ protein aggregation inhibitory activity results (IC50 values) are shown in Tables 1-1 and 1-2 below.

  Extracts or purification of Red Kidney Beans, Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Guinnemu, Fermented Guinneum, Kobumikan, Fermented Guava and Gymnema Sylvestre from Tables 1-1 and 1-2 It was revealed that the product has an Aβ aggregation inhibitory effect. By appropriately selecting a solvent for extraction and fractionation, an effect equal to or higher than that of a conventionally known plant extract having an Aβ aggregation inhibitory action was obtained.

Test Example 2 Evaluation of redissolving action of aggregated Aβ (Thioflavin T method evaluation)
(1) Materials and methods (i) Reagent preparation / PBS solution: 1 L of ultrapure water was added to 9.6 g of PBS powder (Nissui Pharmaceutical, Cat. No. 05913) and dissolved.
-100 μM thioflavin T / PBS solution: Thioflavin T (Sigma, Cat. No. T3516) was dissolved in PBS solution to a concentration of 100 μM.
Aβ stock solution: Aβ1-40 (Peptide Institute, Cat. No. 4379-v) powder was dissolved to a concentration of 1 mM using ultrapure water and stored at −80 ° C. It melt | dissolved at the time of use, and it confirmed that it melt | dissolved completely through sonication.
・ Sample solution: Sample solutions of various concentrations (0 to 10 μg / mL) were prepared by dissolving the above crude extract (dried product) and fraction (dried product) in 2% DMSO ultrapure aqueous solution. .
For Gymnema sylvestre, a commercially available aqueous ethanol extract powder was used.
Aβ amyloid suspension: Aβ stock solution was added to a PBS solution to a final concentration of 40 mM and incubated at room temperature for 24 hours. At the time of use, Aβ amyloid was precipitated by centrifugation at 5000 rpm for 10 minutes, and the supernatant was removed, and an equal amount of fresh PBS solution to the removed PBS solution was added and resuspended.
(Ii) Measurement procedure by thioflavin T method (a) 20 μL / well of the sample solution was added to a 96-well plate. A PBS solution containing the same amount of DMSO as contained in the sample solution was added to the vehicle well, and only 20 μL / well of the PBS solution was added to the blank well.
(B) Aβ amyloid suspension was added at 10 μL / well in addition to Blank well. PBS solution was added at 10 μL / well to the blank well. The plate was mixed well.
(C) PBS solution was added at 10 μL / well to all wells. The plate was mixed well. The plate was sealed and incubated at room temperature for 2 days.
(D) In order to measure the autofluorescence of the specimen itself, fluorescence was measured with a fluorescence reader (ARVO SX, Wallac) under conditions of ex. 485 nm and em. 585 nm.
(E) Thioflavin T stock solution was added to all wells at a volume of 20 μL / well, mixed well, and left at room temperature for 1 hour.
(F) Fluorescence was measured with a fluorescence reader under the conditions of ex.485 nm and em.585 nm.
(G) For each well, the measurement value of (d) was subtracted from the measurement value of (f). Furthermore, the average value of Blank well was subtracted from the value of each well. The amount of Aβ aggregation in each well was calculated as a relative value (%) to the average of the measured values of Vehicle well calculated above. Further, from the sample concentration and the aggregation rate, the ED50 value (the sample concentration for redissolving 50% of the aggregated Aβ amyloid protein) was calculated.

(2) The results of redissolving activity (ED50 value) of aggregated Aβ are shown in Tables 2-1 and 2-2 below.

  From Tables 2-1 and 2-2, extracts of Red Kidney Beans, Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Guinnemu, Fermented Guinem, Kobumikan, Fermented Guava and Gymnema Sylvestre, It was revealed that the purified product had an effect of redissolving aggregated Aβ. By appropriately selecting a solvent for extraction and fractionation, an effect equivalent to or higher than that of a conventionally known plant extract having an aggregation Aβ re-dissolution action was obtained.

Test Example 3 Evaluation of aggregation inhibitory action of amylin (Thioflavin T method evaluation)
(1) Materials and methods (i) Reagent preparation 4 × PBS solution: 250 mL of ultrapure water was added to 9.6 g of PBS powder (Nissui Pharmaceutical, Cat. No. 05913) and dissolved.
-100 μM thioflavin T / PBS solution: Thioflavin T (Sigma, Cat. No. T3516) was dissolved in PBS solution to a concentration of 100 μM.
-Amylin stock solution: Human amylin (Peptide Institute, Cat. No. 4219-v) powder was dissolved in ultrapure water to a concentration of 1 mM and stored at -80 ° C. It melt | dissolved at the time of use, and it confirmed that it melt | dissolved completely through sonication.
・ Sample solution: Sample solutions of various concentrations (0 to 10 ug / mL) were prepared by dissolving the above crude extract (dried product) and fraction (dried product) in 2% DMSO ultrapure aqueous solution. .
(Ii) Measurement procedure by thioflavin T method (a) 20 μL / well of the sample solution was added to a 96-well plate. Ultra pure water with the same amount of DMSO as contained in the sample solution was added to the vehicle well, and only 20 μL / well of ultra pure water was added to the blank well.
(B) The amylin stock solution was diluted with ultrapure water to prepare 40 μM. This was added at 10 μL / well in addition to the blank well. To the blank well, 10 μL / well of ultrapure water was added. The plate was mixed well.
(C) 10 μL / well of 4 × PBS solution was added to all wells. The plate was mixed well. The plate was sealed and incubated at room temperature for 18 hours.
(D) In order to measure the autofluorescence of the specimen itself, fluorescence was measured with a fluorescence reader (ARVO SX, Wallac) under the conditions of ex.485 nm and em.585 nm.
(E) Thioflavin T stock solution was added to all wells at a volume of 20 μL / well, mixed well, and left at room temperature for 1 hour.
(F) Fluorescence was measured with a fluorescence reader under the conditions of ex.485 nm and em.585 nm.
(G) For each well, the measurement value of (d) was subtracted from the measurement value of (f). Furthermore, the average value of Blank well was subtracted from the value of each well. The amount of amylin aggregation in each well was calculated as a relative value (%) to the average of the measured values of Vehicle well calculated above. In addition, an IC50 value (analyte concentration that inhibits amylin aggregation by 50%) was calculated from the analyte concentration and the aggregation rate.

(2) The amylin aggregation inhibitory activity results (IC50 values) are shown in the following 3-1 and 3-2.

  From Tables 3-1 and 3-2, extracts of Red Kidney Beans, Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Guinnemu, Fermented Guinem, Kobumikan, Fermented Guava and Gymnema Sylvestre, It was revealed that the purified product has an amylin aggregation inhibitory effect. By appropriately selecting a solvent for extraction and fractionation, an effect equal to or higher than that of a conventionally known plant extract having an inhibitory action on amyloid aggregation was obtained.

Example 3 Production of Pharmaceutical and Feed Containing Plant Extract (1) Using the water extract of guava leaf prepared in Example 2, tablets having the following composition were produced according to a conventional method.
Composition Blending amount (% by weight)
Guava (leaf) water extract (as dry weight) 24
Lactose 63
Cornstarch 12
Guar gum 1

(2) Using the 74% ethanol extract of guava leaf prepared in Example 2, a feed having the following composition was produced according to a conventional method.
Composition Blending amount (% by weight)
Guava (leaf) EtOH extract (as dry weight) 3
Sucrose (Kishida Chemical Co., Ltd.) 20
Corn oil (manufactured by Nacalai Tesque) 5
Choline bitartrate (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.4
Corn starch (manufactured by Nissho Chemical Co., Ltd.) 37.1
AIN-76 vitamins (Oriental Yeast) 1
AIN-76 mineral (made by Oriental Yeast Co., Ltd.) 3.5
Cellulose (Oriental Yeast Co., Ltd.) 5
Casein (Wako Pure Chemical Industries, Ltd.) 25

  The aggregation inhibitor of amyloidogenic protein and / or the degradation agent of aggregated amyloid protein of the present invention is useful for the prevention and / or treatment of various diseases in which aggregated amyloid protein is involved. Moreover, the food / beverage products or feed of this invention, or the additive for it is useful at the point which can aim at prevention and / or progress suppression of the said disease from the surface of dietary life.

Claims (2)

  Plant body of one or more plants selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Ginnemu, Fermented Ginnemu, Kobumikan, and Gimnema Sylvestre An aggregation inhibitor of amyloidogenic protein containing the treated product or the extract as an active ingredient. Plant body of one or more plants selected from the group consisting of Red Kidney Beans, Guava, Fermented Guava, Ghetto, Fermented Ghetto, Oregano, Basil, Lemon Verbena, Hermanus, Aosa, Marjoram, Ginnemu, Fermented Ginnemu, Kobumikan, and Gimnema Sylvestre An agent for degrading aggregated amyloid protein containing the treated product or the extract as an active ingredient.