WO2016072392A1 - タウ蛋白産生促進剤、タウ蛋白の欠乏に起因する疾患の治療薬・予防薬および治療用・予防用食品組成物 - Google Patents

タウ蛋白産生促進剤、タウ蛋白の欠乏に起因する疾患の治療薬・予防薬および治療用・予防用食品組成物 Download PDF

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WO2016072392A1
WO2016072392A1 PCT/JP2015/080934 JP2015080934W WO2016072392A1 WO 2016072392 A1 WO2016072392 A1 WO 2016072392A1 JP 2015080934 W JP2015080934 W JP 2015080934W WO 2016072392 A1 WO2016072392 A1 WO 2016072392A1
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Prior art keywords
tau protein
earthworm
therapeutic
deficiency
disease caused
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PCT/JP2015/080934
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English (en)
French (fr)
Japanese (ja)
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WO2016072392A8 (ja
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陽一 石井
隆行 根本
猛 岡本
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Well Stone Co
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Well Stone Co
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Priority to AU2015344361A priority Critical patent/AU2015344361B2/en
Priority to RU2017118158A priority patent/RU2742658C2/ru
Priority to KR1020177015237A priority patent/KR101948913B1/ko
Priority to CN201580045874.1A priority patent/CN106794199B/zh
Priority to EP15856572.1A priority patent/EP3216456B1/en
Priority to PH1/2017/500743A priority patent/PH12017500743B1/en
Priority to CA2964316A priority patent/CA2964316C/en
Priority to US15/523,767 priority patent/US10398740B2/en
Application filed by Well Stone Co filed Critical Well Stone Co
Priority to BR112017008734-0A priority patent/BR112017008734B1/pt
Publication of WO2016072392A1 publication Critical patent/WO2016072392A1/ja
Publication of WO2016072392A8 publication Critical patent/WO2016072392A8/ja
Anticipated expiration legal-status Critical
Priority to US16/513,927 priority patent/US10576108B2/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/204—Animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding

Definitions

  • the present invention relates to a tau protein production promoter, a therapeutic / prophylactic agent for diseases caused by deficiency of tau protein, and a food composition for treatment / prevention of diseases caused by deficiency of tau protein.
  • Tau protein is a kind of microtubule-associated protein. Tau protein is particularly abundant in nerve cells of the central nervous system, and binds to a protein called tubulin, which is the main component of microtubules, one of the cytoskeleton. It has a function to promote meeting with It is also known that tau protein associates with other signal molecule groups (Src family, PI3K, Fyn) in addition to tubulin, and promotes neurite outgrowth and nerve growth cone elongation (eg, non-patented). References 1-3).
  • Src family, PI3K, Fyn signal molecule groups
  • tau protein In vivo, excessive phosphorylation of tau protein can occur. Excessive phosphorylation of tau protein inhibits binding to tubulin, leading to a decrease in microtubules, or destabilization of microtubules and suppression of intracellular mass transport (for example, non- Patent Documents 4 and 5). Overphosphorylated tau protein aggregates to form aggregates called neurofibrillary tangles. Alzheimer's disease and progressive supranuclear palsy in which neurofibrillary tangles occur in the brain are classified as a neurodegenerative disease called tauopathy.
  • Patent Document 1 describes an effective therapy for tauopathy treatment using epothilone D, which is a microtubule stabilizer.
  • earthworm extract and earthworm dry powder have been used as preventive and therapeutic agents for various diseases mainly in Eastern countries since ancient times, and so far, calculus reducing agent and elimination accelerator for bladder, treatment for jaundice, delivery Use as an accelerator, a tonic, a hair restorer, a tonic, an antipyretic, a curative treatment, a blood circulation promoter, a half-body involuntary treatment, an indirect analgesic, a micturition, a bronchial asthma, and a hypertension treatment is known.
  • an accelerator a tonic, a hair restorer, a tonic, an antipyretic, a curative treatment, a blood circulation promoter, a half-body involuntary treatment, an indirect analgesic, a micturition, a bronchial asthma, and a hypertension treatment.
  • tauopathy such as Alzheimer's disease.
  • tauopathy such as Alzheimer's disease
  • drugs safe and low-side-effect drugs are particularly necessary, supplementing the function of tau protein, which did not work normally due to phosphorylation
  • tau protein which did not work normally due to phosphorylation
  • an object of the present invention is to provide a tau protein production promoter containing a natural product as an active ingredient, a prophylactic / therapeutic agent for a disease caused by a deficiency in tau protein, and a food composition for improving a symptom of a disease caused by a deficiency in tau protein And providing an improved pharmaceutical composition.
  • the tau protein production promoter of the present invention is characterized by containing a dry earthworm powder, a ground product and / or an extract as an active ingredient.
  • the therapeutic or prophylactic agent for a disease caused by a deficiency of tau protein according to the present invention is characterized by containing the tau protein production promoter.
  • the food composition for treating or preventing a disease caused by a deficiency of tau protein of the present invention is characterized by containing the above-mentioned tau protein production promoter.
  • the disease caused by deficiency of tau protein is preferably Alzheimer's disease.
  • the disease caused by deficiency of tau protein is preferably Alzheimer's disease.
  • the method for producing a tau protein production promoter of the present invention is characterized by using a dry earthworm powder, a ground product or an extract.
  • the method for producing a therapeutic or prophylactic agent for a disease caused by a deficiency of tau protein according to the present invention is characterized by using a dry earthworm powder, a ground product or an extract.
  • the method for producing a food composition for treating or preventing a disease caused by a deficiency of tau protein according to the present invention is characterized by using a dry earthworm powder, a ground product or an extract.
  • the method for promoting the production of tau protein according to the present invention is characterized by using earthworm dry powder, ground product and / or extract.
  • the dry earthworm powder, ground product or extract of the present invention is for use in the treatment of diseases caused by deficiency of tau protein.
  • the method for treating or preventing a disease caused by a deficiency of tau protein according to the present invention is characterized by administering an effective amount of earthworm dry powder, ground product and / or extract to a subject.
  • a tau protein production promoter containing a natural product as an active ingredient, a prophylactic / therapeutic agent for a disease caused by a deficiency in tau protein, a food composition for improving a symptom of a disease caused by a deficiency in tau protein, and an improvement
  • a pharmaceutical composition for medical use can be provided.
  • FIG. 4 is a photographic diagram showing the results of Western blotting examining changes and changes in phosphorylation amounts of tau protein (Tau), Akt and GSK-3 ⁇ (pTau, pAkt and pGSK-3 ⁇ , respectively). “ ⁇ ” And “+” indicate the results of culturing rat hippocampal neurons in culture medium NM5 and culture medium (100 ng / ml) in which earthworm dry powder is dissolved in culture medium NM5, respectively.
  • the tau protein was evaluated using Ser396, the Akt using Ser473, and the GSK-3 ⁇ using Ser9 as an index of phosphorylation.
  • 1 shows the concentrations of tau protein (Tau), Akt and GSK-3 ⁇ quantified using the image analysis software ImageJ64, and the concentrations of pTau, pAkt and pGSK-3 ⁇ phosphorylated from these results. It is a graph.
  • A is the concentration of pTau and the concentration of tau protein based on ⁇ -actin and the ratio of pTau to tau protein
  • B is the concentration of Akt and pAkt based on ⁇ -actin and pAkt relative to Akt from the left
  • C represents the concentration of GSK-3 ⁇ and pGSK-3 ⁇ based on ⁇ -actin from the left, and the ratio of pGSK-3 ⁇ to GSK-3 ⁇ .
  • FIG. 6A is a photograph (A) showing a change in the concentration of tau protein based on ⁇ -actin quantified using image analysis software ImageJ64.
  • FIG. 2 is a photograph (A) showing the result of blotting, and a graph (B) showing a change in tau protein concentration based on ⁇ -actin quantified using image analysis software ImageJ64.
  • the earthworm used as a raw material is not particularly limited, and for example, red earthworm (Lumbricus rubellus), LT earthworm (Lumbricus terrestris), earthworm (Eisenia foetida), brown earthworm (Allophophora cricket), Dendrobena octaedra), Sakuramimizu (Allolobophora japonica Michaelsen), Hatamizumi (Drawida hatamimizu Haemi), Semimizumi-Puretims (Petetima mizumifu).
  • a dry earthworm powder means a powder obtained by drying an untreated or pretreated earthworm ground product or extract.
  • the earthworm ground product is a liquid or paste-like material obtained by grinding an untreated or pretreated earthworm.
  • the earthworm extract means an extract obtained by dissolving or removing an untreated or pretreated earthworm or a ground product thereof in water or an organic solvent, and removing or separating the insoluble fraction.
  • the said pre-processing is not specifically limited, The removal process etc. of the filth etc. which are mentioned later are mentioned.
  • the earthworm dry powder, ground product and extract may be post-treated, and examples of the post-treatment include granulation, filtration, purification, concentration, dilution and pH adjustment.
  • the grinding method for obtaining a ground product of earthworms is not particularly limited, and for example, grinding can be performed using a homogenizer, a blender, a homomixer, a grinder, a pressurized cell disrupter, or the like.
  • the extraction method for obtaining the earthworm extract is not particularly limited.
  • the earthworm dry powder or the ground product can be dissolved in the extraction solvent, and the insoluble fraction can be removed or separated for extraction.
  • the extraction solvent include water, aqueous solutions, and organic solvents such as ethanol, acetone, and ethyl acetate. One kind may be used alone, or two or more kinds may be combined. Among these, water, ethanol or an aqueous ethanol solution is preferable.
  • the drying method for obtaining a dried product of earthworms is not particularly limited, and drying can be performed by a drying method such as freeze drying, heat drying, and spray drying. Among these, lyophilization is preferable for the reasons described later.
  • an effective amount of the earthworm dry powder, ground product or extract may be blended depending on the purpose.
  • the appropriate blending amount depends on various factors such as the purpose, administration route and form, and the production method of earthworm dry powder, etc., but digested matter remaining in the digestive tract of earthworms described later, soil adhering to the body skin, etc.
  • it is converted to the weight of dry earthworm powder obtained by grinding earthworms and freeze-drying the ground product.
  • Is preferably 1 to 15,000 mg per day, more preferably 12 to 1,800 mg per day, and still more preferably 120 to 180 mg per day.
  • For the purpose of treating severe diseases caused by deficiency of tau protein it is preferably 1 to 15,000 mg per day, more preferably 18 to 3,600 mg per day, more preferably 180 to 180 per day. 360 mg.
  • the shape of the tau protein production promoter, therapeutic agent, preventive agent and food composition of the present invention is not particularly limited, and may be any of solid, powder, semi-solid and liquid.
  • the dry powder, ground product or extract of earthworm can be used as it is.
  • the tau protein production promoter, therapeutic agent and preventive agent of the present invention may contain a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, tablets, granules, powders, capsules, soft capsules, liquids, injections
  • it can be administered orally or parenterally (for example, intravenous administration, direct administration to the affected area, etc.) as a suppository, sustained release agent and the like.
  • an excipient As a pharmaceutically acceptable carrier, an excipient, a binder, a disintegrant, a fluidizing agent, a lubricant, a coating agent, a suspending agent, a coloring agent, a sweetening agent, a surfactant, or the like may be used. And can be in the form of a general pharmaceutical preparation according to known methods. In addition, other therapeutic / prophylactic ingredients and pharmaceutically acceptable additives may be included.
  • the present invention may contain additives usually used in foods.
  • an excipient a binder, a disintegrant, a fluidizing agent, a lubricant, a coating agent, a suspending agent, a coloring agent, a sweetening agent, or a surfactant can be used. It can be in the form of a simple food composition. Moreover, you may contain the component derived from another foodstuff or foodstuff.
  • earthworm dry powder among the earthworm dry powder, ground product and extract, it is preferable to use earthworm dry powder from the viewpoint of storage stability in the production process.
  • the earthworm dry powder may be dissolved and / or dispersed in a liquid such as water in advance, and then mixed with other components such as conventional carriers and additives used pharmaceutically and / or in foods.
  • the disease caused by deficiency of tau protein is not particularly limited, but is preferably tauopathy, Alzheimer's disease, Pick's disease, cortical degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy, No.
  • Frontotemporal dementia linked to chromosome and associated with parkinsonism Guam-Parkinson dementia complex, Alzheimer's disease-like neurofibrillary tangle with amyloid plaques, senile dementia, ganglioglioma More preferably, it is selected from the group consisting of: gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, Hallelfolden-Spatz disease, frontotemporal dementia, frontotemporal lobar degeneration. Particularly preferred is Alzheimer's disease.
  • the removal method is not particularly limited, and can be removed using a known method.
  • a method in which an earthworm organism is immersed in an aqueous solution of an alkali salt such as sodium salt or potassium salt to excrete the loess in the digestive tract JP-A-1-47718, JP-A-1-47719, 1-47720 and Japanese Patent Application Laid-Open No. 1-268639
  • a method in which the earthworm organism is left in an acid aqueous solution maintained at 6 to 26 ° C. for 0.1 to 5 hours to remove feces in the digestive tract (Method described in JP-A-3-72427) can be used.
  • the metal chloride is at least one metal chloride selected from the group consisting of potassium, sodium, magnesium and calcium. That is, it is at least one selected from the group consisting of potassium chloride, sodium chloride, magnesium chloride and calcium chloride. Moreover, these mixtures may be sufficient and the mixture of these and the other harmless component which can be added to a foodstuff may be sufficient. Examples of such a mixture include salt, rock salt, and sun salt.
  • the metal chloride can be used by sprinkling a powdered product on a raw earthworm, which causes contact between the earthworm and the metal chloride.
  • hydroxycarboxylic acid it is preferable to contact the raw earthworm with hydroxycarboxylic acid as described below after contacting the earthworm with the metal chloride. Moreover, without making contact with the metal chloride, the contact between hydroxycarboxylic acid and earthworm can be performed as described below.
  • the contact with the hydroxycarboxylic acid can also be carried out by sprinkling powdered hydroxycarboxylic acid over raw earthworms. Further, it may be immersed in an aqueous hydroxycarboxylic acid solution having a pH of 2 to 5.
  • the contact with the hydroxycarboxylic acid is preferably carried out immediately after the contact with the metal chloride.
  • the earthworm and hydroxycarboxylic acid are contacted after removing the metal chloride by washing with water, a dry earthworm powder with high enzyme activity is obtained.
  • washing with water before contact with the hydroxycarboxylic acid washing with water is preferably performed within 30 minutes, more preferably within 20 minutes after the start of contact with the metal chloride.
  • the washing method is not particularly limited, and a known method can be employed.
  • hydroxycarboxylic acid forms an uncomfortable living environment for earthworms, raw earthworms try to improve their living environment by releasing bodily fluids and excreta by self-preserving instinct. Moreover, since hydroxycarboxylic acid has bactericidal properties, it plays the role of promoting excretion of digests remaining in the digestive organ as described above, and can be expected to have an effect of sterilizing miscellaneous bacteria attached to earthworms.
  • the crystalline hydroxycarboxylic acid used in the above method can be used regardless of the number of hydroxy groups or carboxyl groups as long as it shows a crystalline form under the conditions of use. That is, any of monohydroxymonocarboxylic acid, monohydroxypolycarboxylic acid, polyhydroxymonocarboxylic acid, and polyhydroxypolycarboxylic acid may be used.
  • hydroxycarboxylic acid used in the present invention examples include glycolic acid, lactic acid, acetic acid, ⁇ -hydroxypropionic acid, ⁇ -hydroxy-n-butyric acid, ⁇ -hydroxy-n-butyric acid, ⁇ -hydroxy-n-valeric acid, Examples include ⁇ -hydroxy-n-valeric acid, malic acid, ⁇ -methylmalic acid, ⁇ -hydroxyglutaric acid, ⁇ -hydroxyglutaric acid, citric acid, malonic acid, and succinic acid. Of these, lactic acid, acetic acid, malic acid, citric acid, malonic acid and succinic acid are preferred because they can be used for foods and are easily available. Hydroxycarboxylic acid may be used individually by 1 type, and 2 or more types may be mixed and used for it.
  • the earthworm living body treated with hydroxycarboxylic acid is washed with water and then ground into a liquid or pasty ground product. Washing is preferably performed with pure water.
  • the washing method is not particularly limited, and a known water washing method can be employed.
  • the total time of the treatment process before attrition that is, the time from sprinkling metal chlorides to raw earthworms until the washing with hydroxycarboxylic acid with water is preferably within 240 minutes in total.
  • the above-mentioned grinding method is not particularly limited, and for example, a homogenizer, a blender, a homomixer, a grinder, or a pressurized cell disrupter is usually used at 1 to 25 ° C. From the viewpoint of suppressing the decomposition of earthworm components, it is preferably carried out at a low temperature, and a temperature of 2 to 15 ° C. is preferred.
  • the ground product obtained by grinding the earthworms is accommodated in, for example, a stainless steel tray and freeze-dried.
  • the enzyme contained in the earthworm organism does not act on the living cells but acts on the dead cells instantaneously, so there is a risk of generating spoilage gas.
  • instantaneously It is preferable to perform freeze-drying after quenching and freezing to 18 ° C. to ⁇ 35 ° C. to suppress the action of the enzyme.
  • the freezing is preferably performed at a low temperature of ⁇ 18 ° C. to ⁇ 35 ° C. for 20 to 240 hours, more preferably 50 to 170 hours.
  • the ground product is frozen at a temperature of ⁇ 18 ° C. to ⁇ 35 ° C. for 20 to 240 hours, and then at several temperatures at a temperature of ⁇ 60 ° C. to + 90 ° C.
  • the sterilized light yellow earthworm dry powder can be obtained by freeze-drying for 10 to 60 hours while increasing the temperature and dividing the pressure in several steps at a pressure of 25 to 40 Pa and reducing the pressure in several steps.
  • a step of removing or separating the insoluble fraction by dissolving the lyophilized product in water or an aqueous ethanol solution.
  • the step of removing or separating the insoluble fraction can be performed by standing precipitation, centrifugation, filtration, or the like, as described above.
  • the step of dissolving in water or an aqueous ethanol solution is preferably performed while stirring or shaking.
  • the time required for dissolution in water is preferably 1 to 120 minutes, more preferably 5 to 80 minutes.
  • the ethanol concentration of the aqueous ethanol solution is not particularly limited, but is preferably 10 to 70% (v / v), more preferably 30 to 60%.
  • the tau protein production promoter, therapeutic agent, prophylactic agent and food composition of the present invention may be used as it is in the state of an aqueous solution as it is dissolved in water or an aqueous ethanol solution as described above, and the water is removed. It may be used as a concentrated liquid, or may be dried and used in a powder form. A supernatant obtained by drying and powdering may be used by dissolving in water. Moreover, the powder which freeze-dried earthworm paste can also be used as it is, without melt
  • the raw earthworm is placed in a bread box before the treatment for placing the raw earthworm in an uncomfortable environment, that is, prior to contacting the raw earthworm with a metal chloride or hydroxycarboxylic acid. It is preferable to move to a flat box such as the above and leave it in a light place for 10 to 50 hours to remove the dirt adhering to the body skin.
  • the standing time in the light is more preferably 12 to 24 hours.
  • the accommodation amount at this time is preferably such an amount that earthworms are stacked to a thickness of 30 to 60 mm, preferably 40 to 50 mm.
  • the filth attached to the earthworm skin can be peeled off, for example, by covering the raw earthworm with a nonwoven fabric and adsorbing the filth on it.
  • a nonwoven fabric By combining this leaving in the light place and removal of dirt adhering to the body skin and contact with the above-mentioned metal chlorides and / or hydroxycarboxylic acids, it is possible to further discharge and remove toxic substances in the earthworm body. I can expect.
  • the following method is particularly preferable from the viewpoint of storage stability of the dry powder.
  • A-1 contacting the earthworm with at least one metal chloride selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, the powdered hydroxycarboxylic acid and raw earthworm are brought into contact, diluted with water to adjust the pH to 2 to 5, held for 3 to 180 minutes, washed with water, ground, and the obtained ground product.
  • the manufacturing method of the dry powder of earthworm provided with the process of freeze-drying.
  • A-2) contacting the earthworm with at least one metal chloride selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, the raw earthworms are immersed in a hydroxycarboxylic acid aqueous solution adjusted to pH 2 to 5 and held for 3 to 180 minutes, and then the raw earthworms are washed with water and ground, and the obtained ground product is freeze-dried.
  • Manufacturing method of earthworm dry powder (A-3) In (A-1) or (A-2), the milled product is freeze-dried and dissolved in water or an aqueous ethanol solution, and the insoluble fraction is removed or separated, followed by further freeze-drying.
  • the manufacturing method of the dry powder of earthworm provided with a process.
  • the heating temperature is less than 110 ° C., the sterilization of the dried product may be insufficient, and when it is 130 ° C. or more, the enzyme contained in the dried earthworm is deactivated and the activity is lowered, which is not preferable. More preferably, it is 115 to 125 ° C.
  • the heating method is not particularly limited, and examples thereof include a method of applying hot air, a method of using a heating jacket, a method of heating on a tray or the like and a method of using a constant temperature and temperature chamber.
  • the heating time is preferably 30 seconds to 130 minutes, more preferably 30 minutes to 90 minutes, still more preferably 60 minutes to 90 minutes. If the heating time is too short, sterilization may be insufficient, and if the heating time is too long, the enzyme activity is lost. In addition, since the enzyme activity is lost when the above heat treatment is performed on the enzyme in the liquid, it is preferable to use a dry earthworm powder in the present invention.
  • the following method is preferred as a method for obtaining a ground product of earthworm.
  • B-1 contacting the earthworm with at least one metal chloride selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, the powdered hydroxycarboxylic acid and raw earthworm are brought into contact with each other, diluted with water to adjust the pH to 2 to 5, held for 3 to 180 minutes, washed with water, and ground.
  • Method for producing crushed material is preferred as a method for obtaining a ground product of earthworm.
  • (B-2) contacting the earthworm with a chloride of a metal selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, a method for producing a ground product of earthworms comprising a step of immersing the raw earthworms in a hydroxycarboxylic acid aqueous solution adjusted to pH 2 to 5 and holding for 3 to 180 minutes, and then washing and grinding the raw earthworms.
  • the following method is preferred as a method for obtaining the earthworm extract.
  • C-1 contacting the earthworm with at least one metal chloride selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, the powdered hydroxycarboxylic acid and raw earthworm are brought into contact, diluted with water to adjust the pH to 2 to 5, held for 3 to 180 minutes, washed with water, ground, and the obtained ground product.
  • a method for producing an extract of earthworm comprising a step of dissolving a freeze-dried product in water or an aqueous ethanol solution and removing or separating an insoluble fraction.
  • C-2 contacting the earthworm with a chloride of a metal selected from the group consisting of potassium, sodium, magnesium and calcium; Thereafter, the raw earthworms are immersed in a hydroxycarboxylic acid aqueous solution adjusted to pH 2 to 5 and held for 3 to 180 minutes. The raw earthworms are washed with water and ground, and the obtained ground product is freeze-dried.
  • a method for producing an extract of earthworm comprising a step of dissolving in water or an aqueous ethanol solution to remove or separate an insoluble fraction.
  • raw earthworms were taken out from the soiled aqueous citric acid solution, washed with water, and ground at 10 ° C. using a homogenizer to prepare earthworm paste.
  • this earthworm paste is sucked and degassed to remove the gas contained therein, and then transferred to a stainless steel tray, where it is rapidly cooled to ⁇ 35 ° C. and maintained at this temperature for 50 hours. And gradually frozen.
  • the frozen earthworm paste is kept at -35 ° C. and a pressure of 0 Pa for 2 hours, and then heated to a temperature of 25 ° C., then 40 Pa for 10 hours, then 40 ° C. and a pressure of 35 Pa for 14 hours, and then 65 ° C. and a pressure of 35 Pa for 12 hours. Drying was performed, and finally, the temperature was set to 80 ° C., and vacuum lyophilization was performed by maintaining the pressure at 25 Pa for 6 hours. By this treatment, a light yellow earthworm dry powder having a water content of 8% by mass was obtained.
  • the earthworm dry powder obtained above was subjected to heat treatment using an RM-50D heating device (manufactured by Okawara Seisakusho Co., Ltd.). As heating conditions, after heating to 120 ° C. over 90 minutes, heating was performed at 120 ° C. for 20 minutes, cooling to 40 ° C. over 240 minutes, and then dried earthworm powder was taken out.
  • the heat-treated earthworm dry powder was dissolved in a 50% aqueous ethanol solution so that the ethanol: freeze-dried powder was 20: 1 (v / w) and shaken at 1500 rpm for 1 hour at room temperature (25 ° C.).
  • the mixture was centrifuged at 10000 ⁇ g for 15 minutes at 4 ° C., and the supernatant was separated and concentrated under reduced pressure at 75 ° C. for 15 minutes.
  • the resulting supernatant was transferred to a stainless steel tray, instantly cooled to ⁇ 35 ° C., maintained at this temperature for 50 hours and gradually frozen.
  • the frozen earthworm paste is kept at -35 ° C. and a pressure of 0 Pa for 2 hours, and then heated to a temperature of 25 ° C., then 40 Pa for 10 hours, then 40 ° C. and a pressure of 35 Pa for 14 hours, and then 65 ° C. and a pressure of 35 Pa for 12 hours.
  • the temperature was set to 80 ° C., and the mixture was kept at a pressure of 25 Pa for 6 hours, followed by vacuum freeze-drying to obtain earthworm dry powder A-1.
  • Rat hippocampal neuron culture Neurons (neurons) isolated from the hippocampal region of rat fetuses on day 18 of gestation were cultured at 37 ° C. for 7 days.
  • the rat hippocampal neurons cultured as described above were obtained by dissolving the earthworm dry powder A-1 obtained above in the culture medium Neural Media 5 (NM5) so as to be 0, 1, 10, 100 and 1000 ng / mL, respectively. and cultured at 37 ° C. in earthworm lyophilized powder dissolved culture 1 ml (cell number 1 ⁇ 10 6 / 12well dish) .
  • the composition of the culture solution NM5 is as follows.
  • rat hippocampal neurons were collected after an arbitrary period of time, washed twice with PBS solution ( ⁇ ), adjusted with sample buffer solution, and then tau protein amount and tau protein phosphorylation amount were determined by Western blotting. evaluated. The amount of protein and the amount of phosphorylation were measured from the results of Western blotting using image analysis software ImageJ64. Similarly, the amount of protein and the amount of phosphorylation were also evaluated for the molecules Akt and GSK-3 ⁇ located upstream of the tau protein.
  • earthworm dry powder significantly increases the amount of tau protein, and in particular, reduces the amount of phosphorylation at the neurite site.

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BR112017008734-0A BR112017008734B1 (pt) 2014-11-04 2015-11-02 Acelerador de produção de proteína tau e agente preventivo ou terapêutico e composição alimentícia preventiva ou terapêutica para doenças causadas por deficiência de proteína tau
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