WO2017006407A1 - Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor - Google Patents

Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor Download PDF

Info

Publication number
WO2017006407A1
WO2017006407A1 PCT/JP2015/069363 JP2015069363W WO2017006407A1 WO 2017006407 A1 WO2017006407 A1 WO 2017006407A1 JP 2015069363 W JP2015069363 W JP 2015069363W WO 2017006407 A1 WO2017006407 A1 WO 2017006407A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell death
liquid
methanol
distributed
death inhibitor
Prior art date
Application number
PCT/JP2015/069363
Other languages
French (fr)
Japanese (ja)
Inventor
高橋 知也
一紅 潘
淑芳 ▲温▼
亦晃 呂
凱安 ▲荘▼
Original Assignee
株式会社Aob慧央グループ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社Aob慧央グループ filed Critical 株式会社Aob慧央グループ
Priority to CN201580081025.1A priority Critical patent/CN107708717B/en
Priority to PCT/JP2015/069363 priority patent/WO2017006407A1/en
Priority to JP2017526812A priority patent/JP6365914B2/en
Priority to TW105118524A priority patent/TWI610676B/en
Publication of WO2017006407A1 publication Critical patent/WO2017006407A1/en

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/21Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/222Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/19Acanthaceae (Acanthus family)

Definitions

  • the present invention relates to a nerve cell death inhibitor, an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, a drug or food having an anti-Alzheimer's disease action or an anti-brain function-lowering action, and a method for producing a nerve cell death inhibitor.
  • Alzheimer's disease Alzheimer's disease
  • Lewy body dementia Lewy body dementia
  • cerebrovascular dementia cerebrovascular dementia
  • amyloid cascade hypothesis is widely supported as the mechanism of Alzheimer's disease.
  • the amyloid cascade hypothesis is that amyloid ⁇ , an amyloid protein, accumulates in the brain, resulting in Alzheimer's neurofibrillary tangles, neuronal death or loss due to direct or indirect toxicity, etc. It is a hypothesis that various symptoms of the disease occur.
  • the hypothesis is widely supported at present, and serves as a guideline for research and development of preventive and therapeutic methods (see, for example, Non-Patent Document 1).
  • the white crane reishi (Rhinacanthus natusus (L.) Kurz. Also called white crane reishi grass) is an evergreen small shrub belonging to the genus Linacanthus vulgaris, which originates in the Deccan Plateau in southern India.
  • Hakutsuru Ganoderma is known to have an antibacterial action against anthelmintic, anti-inflammatory, and skin fungi (see, for example, Non-Patent Document 3), mainly in China, Taiwan, etc., and recently in Japan, Used as food.
  • the white crane reishi has an active oxygen scavenging ability (for example, see Patent Document 1), has an excretion promoting action (for example, see Patent Document 2), and antiallergic. It is disclosed that there is an action (for example, see Patent Document 3) and that it has an antitumor action (for example, see Patent Document 4).
  • linacantin C contained in a predetermined distribution, a predetermined fraction, or a white crane reishi obtained from a white crane reishi extract by a predetermined method has a neuronal cell death inhibitory effect on the toxicity of amyloid ⁇ . unknown.
  • the present invention provides a neuronal cell death inhibitor, an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, and an anti-Alzheimer's disease action or an anti-brain function-lowering drug or food having an excellent neuronal death inhibitory effect.
  • Another object of the present invention is to provide a method for producing a nerve cell death inhibitor capable of producing a nerve cell death inhibitor having an excellent nerve cell death inhibitory action.
  • Ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol, and the liquid distributed to the methanol is liquid-liquid distributed with methylene dichloride and water and distributed to the methylene dichloride.
  • the partition is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol, The nerve cell death inhibitor which contains only the predetermined fraction which is a fraction eluted with the said elution solvent from a silica gel column as an active ingredient.
  • the predetermined fraction is eluted in a volume of 0.1 to 4 times the volume of silica gel used in the silica gel column chromatography.
  • a neuronal cell death inhibitor which is a fraction eluted when a solvent is used.
  • a neuronal cell death inhibitor containing only linacantin C as an active ingredient [6] A neuronal cell death inhibitor containing only linacantin C as an active ingredient.
  • An anti-Alzheimer's disease agent comprising the neuronal cell death inhibitor according to any one of [1] to [6] as an active ingredient.
  • a method for producing a nerve cell death inhibitor comprising only the predetermined distribution as an active ingredient.
  • a method for producing a neuronal cell death inhibitor comprising only the predetermined fraction as an active ingredient, comprising the fourth step obtained in this order.
  • a neuronal death inhibitor an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, an anti-Alzheimer's disease action or an anti-brain function having an excellent inhibitory effect on neuronal cell death.
  • a pharmaceutical or food having a lowering action can be provided.
  • the above [1] is, for example, “use of the predetermined distribution as an active ingredient for producing a neuronal death inhibitor containing only the following predetermined distribution as an active ingredient.
  • the ethanol extract of Hakutsuru Ganoderma is liquid-liquid distributed with hexane and methanol, and the partition distributed to methanol is liquid-liquid distributed with methylene dichloride and water, the distribution is distributed to the methylene dichloride. It can also be expressed as “the predetermined distribution which is an object”.
  • the predetermined fraction as an active ingredient for producing a nerve cell death inhibitor containing only the following predetermined fraction as an active ingredient
  • the Hakutsuru Reishi lawn ethanol extract was liquid-liquid distributed with hexane and methanol, and the methanol-distributed distribution was liquid-liquid distributed with methylene dichloride and water and distributed to the methylene dichloride.
  • the predetermined fraction which is a fraction eluted from a silica gel column with the elution solvent when the partition is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol. "Can also be expressed.
  • the above [6] may be expressed as, for example, “use of linacantin C as an active ingredient for producing a neuronal cell death inhibitor containing only linacantin C as an active ingredient”. it can.
  • [7] above for example, “use of the neuronal cell death inhibitor according to any of [1] to [6] as an active ingredient for producing an anti-Alzheimer's disease agent”. It can also be expressed as follows.
  • [8] for example, “use of the nerve cell death inhibitor according to any of [1] to [6] as an active ingredient for producing an antibrain function-lowering agent”.
  • the anti-Alzheimer's disease agent according to [7] or the above [8] for producing a pharmaceutical or food having an anti-Alzheimer's disease action or an anti-brain function lowering action "Use of the described antibrain function-lowering agent as an active ingredient.”
  • the nerve cell death inhibitor the anti-Alzheimer's disease agent, the anti-brain function-lowering agent, the drug or food having the anti-Alzheimer's disease action or the anti-brain function-lowering action, and the method for producing the nerve cell death inhibitor of the present invention will be described.
  • the neuronal cell death inhibitor containing only a predetermined distribution which is a distribution as an active ingredient is a "first step of obtaining an ethanol extract by extracting white crane reishi with ethanol, Liquid-liquid partition with hexane and methanol to obtain a partition distributed to the methanol, and liquid-liquid partition of the partition distributed to methanol with methylene dichloride and water to the methylene dichloride. And a third step of obtaining a predetermined distribution which is a distribution to be distributed in this order, and a method for producing a neuronal cell death inhibitor containing only the predetermined distribution as an active ingredient ” It can be.
  • the predetermined distribution in the present invention can be obtained under various conditions as long as the conditions described are satisfied.
  • the “partition” means a purified product obtained by liquid-liquid partition.
  • “containing only a predetermined distribution as an active ingredient” refers to containing only a predetermined distribution as an active ingredient having an inhibitory action on nerve cell death, and is not an active ingredient ( It does not deny that it contains various solvents and auxiliary additives.
  • the raw Hakutsuru Reishi leaves, stems, roots, flowers, etc. are collected at an appropriate time and then used as they are or are usually subjected to a drying process such as ventilation drying to extract raw materials. Can be used.
  • the white crane reishi as an extraction raw material is preferably used after being crushed or chopped.
  • ethanol simply means a “solvent containing ethanol as a main component (containing 50% or more of ethanol)”. That is, “ethanol” includes not only so-called anhydrous ethanol but also a mixed solvent of ethanol and another solvent.
  • a mixed solvent of ethanol and water hydroous ethanol
  • the extraction temperature is usually 0 to 100 ° C., preferably 5 to 50 ° C.
  • the extraction time is about 1 hour to 10 days, and the amount of solvent is usually 1 to 30 times by weight, preferably 5 to 10 times by weight per dry raw material.
  • the extraction operation may be stirring or simple immersion.
  • the extraction operation may be repeated a plurality of times as necessary. Moreover, you may combine extraction operation from which conditions differ. Furthermore, it is preferable to perform an operation of removing insoluble residues from the crude extract obtained by the above operation by filtration or centrifugation.
  • Liquid-liquid distribution can also be carried out by known methods usually used industrially and experimentally.
  • Liquid-liquid partitioning is a method called two-phase partitioning, two-phase solvent partitioning, liquid-liquid extraction, etc., and is a method of recovering a target compound by using a difference in solubility using two kinds of solvents having different polarities. .
  • After the liquid-liquid partition it is preferable to obtain a partition in a dry state by removing the solvent. However, this is not the case when the removal of the solvent is unnecessary for the purpose of use.
  • methanol simply means “a solvent containing methanol as a main component (containing 50% or more of methanol)”. That is, “methanol” includes not only so-called anhydrous methanol but also a mixed solvent of methanol and another solvent. When performing liquid-liquid partitioning, it is preferable to use a mixed solvent of methanol and water, particularly 90% methanol.
  • anhydrous methanol refers to anhydrous methanol and does not include a mixed solvent containing methanol. It can be obtained by a “method for producing a nerve cell death inhibitor containing only a predetermined distribution as an active ingredient”.
  • the ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol
  • the liquid distributed to methanol is liquid-liquid distributed with methylene dichloride and water and distributed to methylene dichloride.
  • Neuronal cell death inhibitor contained as an active ingredient includes “first step of extracting white crane reishi with ethanol to obtain an ethanol extract, and liquid-liquid distribution of the ethanol extract with hexane and methanol to the methanol. A second step of obtaining a partition to be distributed; and a liquid-liquid partition of the partition distributed to methanol with methylene dichloride and water, The third step to obtain a partition distributed to methylene chloride and the partition distributed to methylene dichloride were subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol. And a fourth step of obtaining a predetermined fraction which is a fraction eluted from the silica gel column with the elution solvent in this order, and a neuronal cell death inhibitor containing the predetermined fraction as an active ingredient.
  • the predetermined fraction in the present invention can be obtained under various conditions as long as the described conditions are satisfied.
  • the “fraction” means a purified product obtained by fractionation by column chromatography.
  • “containing only a predetermined fraction as an active ingredient” means containing only a predetermined fraction as an active ingredient having a nerve cell death inhibitory action, and is not an active ingredient. It does not deny that it contains ingredients (various solvents, auxiliary additives, etc.). Since the extraction and the liquid-liquid distribution have already been described, the description thereof will be omitted. After silica gel column chromatography, it is preferable to obtain a fraction in a dry state by removing the elution solvent. However, this is not the case when the removal of the solvent is unnecessary for the purpose of use.
  • the predetermined fraction is more preferably a fraction that elutes when an elution solvent having a volume of 0.1 to 4 times the volume of silica gel used for silica gel column chromatography is used. It is even more preferable that it is a fraction that elutes when an elution solvent having a volume of 0.2 to 2.5 times is used (see test examples described later).
  • the predetermined distribution and the predetermined fraction may be obtained by further performing a purification method other than the above as long as the object of the present invention is not impaired.
  • the solvent and the mixed solvent used for obtaining the predetermined distribution and the predetermined fraction can be replaced with a solvent or a mixed solvent having the same degree of polarity.
  • a solvent it can be used to obtain a predetermined distribution and a predetermined fraction.
  • solvents include water, various alcohols, esters such as ethyl acetate, linear, branched and cyclic hydrocarbons, methylene dichloride (dichloromethane), chloroform, acetone, various ethers, etc.
  • those suitable for the method and purpose can be used.
  • the predetermined distribution and the predetermined fraction in the present invention contain linacantin C as one component.
  • the linacantin C in the “nerve cell death inhibitor containing only linacantin C as an active ingredient” of the present invention may be obtained by extracting and purifying white crane reishi as a raw material (see experimental examples described later). It may be obtained from other plants. Also, linacantin C may be obtained by synthesis. Linacantin C is a compound represented by the following chemical formula (1).
  • the linacantin C of the present invention may be linacantin C in the mechanism of action in the body, and may be in the form of a pharmaceutically acceptable salt before administration.
  • the predetermined distribution, the predetermined fraction, and linacantin C have the effect of suppressing neuronal cell death due to the toxicity of amyloid ⁇ , as shown in the experimental examples described later. For this reason, it is thought that the nerve cell death inhibitor of this invention which uses these as an active ingredient has the effect
  • the “anti-Alzheimer's disease agent” in the present specification can be used for at least one of prevention and treatment of Alzheimer's disease.
  • the “anti-brain function-lowering agent” can be used for at least one of prevention and treatment of a decrease in brain function.
  • the route of administration of the neuronal death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceuticals (herein, drugs for internal use or internal use) of the present invention is not particularly limited, and examples thereof include oral administration and rectal administration. And mucosal administration such as nasal administration, injection administration such as intravenous administration and subcutaneous administration, and the like.
  • Any dosage form can take the form of a formulation suitable for the administration method. For example, tablets, powders, fine granules, granules, capsules, powders, pills, lozenges and other solid agents, solutions , Suspensions, emulsions, syrups, liquids such as injections, gel preparations, and the like.
  • the neuronal cell death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceutical of the present invention may be administered as they are, or may be administered together with a pharmacologically acceptable excipient.
  • a pharmacologically acceptable excipient any monosaccharides, disaccharides, polysaccharides, inorganic salts, oils and fats, distilled water, etc. that can be generally used as preparations can be used.
  • additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used.
  • the effective dosage of the neuronal cell death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceutical of the present invention varies depending on the administration route, dosage form, disease symptom, age of the subject, etc.
  • the predetermined fraction or linacantin C is considered to be usually 0.1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg per day for an adult.
  • the content of a predetermined distribution, a predetermined fraction or linacantin C is determined based on the data on the dosage form, the effective dosage, and the dosage as the dosage, and the optimal active ingredient content in the dosage form for each dosage form. Can be set.
  • the pharmaceutical product of the present invention may be an external pharmaceutical product.
  • the form of the external medicine is not particularly limited.
  • an ointment, a cream, a foaming agent, a tape agent, an external preparation etc. can be mentioned, for example.
  • the external medicine of the present invention can contain various pharmaceutical ingredients in a predetermined distribution, a predetermined fraction or linacantin C as required.
  • additives such as a binder, a dispersant, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, and a bacteria inhibitor can also be used.
  • Examples of the food of the present invention include foods in the form of tea with the addition of active ingredients and foods in the form of processed foods containing the active ingredients.
  • tea it is preferable to use it by mixing with a dried white crane reishi leaf / stem or root or by mixing with other tea ingredients.
  • tea materials green tea, oolong tea, puer tea, black tea, roasted green tea, brown rice tea, Tochu tea, kashiwanoha tea, mulberry leaf tea, etc., as long as they are used as normal teas, any tea can be used. Can do.
  • the dried white crane reishi leaf / stem or root can be roasted and used in the same manner as other tea ingredients.
  • a form of food containing the active ingredient drinks, jelly, biscuits, tablets, pills, soft capsules, hard capsules, powders, fine granules, granules, etc. Either form can be used.
  • additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used as food auxiliary materials. .
  • the effective intake amount of the food of the present invention varies depending on the intake form, the health condition of the subject, the age of the subject, etc., but for a given distribution, a given fraction or linacantin C, it is usually 0. It is considered to be 1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg.
  • the content of the predetermined distribution, the predetermined fraction or linacantin C in the food of the present invention varies depending on the form of the food, but is usually 0.0001 to 1 wt%, preferably 0.001 to 0.5 wt%. More preferably, it is considered to be 0.01 to 0.1 wt%.
  • Example of distribution / fractionation / isolation of the distribution of the present invention, the fraction of the present invention and linacantin C The distribution / fractionation of the distribution of the present invention and the fraction of the present invention follows the flow shown in FIG. I went.
  • 2 kg of dried roots of Rhinacanthus natusus (L.) Kurz was prepared and pulverized with a general-purpose grinder.
  • extraction with 90% ethanol was performed.
  • the extraction was performed by immersing Hakutsuru Reishi in 20 L of 90% ethanol for 3 days and then refluxing the solvent for 1 hour.
  • the extract and the extraction residue were separated by filter paper.
  • the extraction was performed three times on the same raw material, and the solvent was removed from the extract under reduced pressure to obtain 77.82 g of a dry ethanol extract.
  • the remaining 73.82 g ethanol extract was subjected to liquid-liquid partition with hexane and 90% methanol.
  • 500 mL of 90% methanol was added to the ethanol extract, and then 500 mL of hexane was added and shaken for 1 minute.
  • the solvent was separated for about 10 minutes, and a 90% methanol phase was collected.
  • the same operation was further repeated twice, and the obtained 90% methanol phase was combined, and then the solvent was removed under reduced pressure to obtain 54.32 g of a dried product.
  • predetermined distribution A After removing 4 g from the above-mentioned distribution as an analytical sample, the remaining 50.32 g of the entire distribution was subjected to liquid-liquid distribution with methylene dichloride and water. First, 500 mL of water (purified water) was added to the distribution, and then 500 mL of methylene dichloride was added and shaken for 1 minute. The solvent was separated for about 10 minutes and the methylene dichloride phase was collected. Further, the same operation was repeated twice, and the obtained methylene chloride phase was combined, and then the solvent was removed under reduced pressure to obtain 27.55 g of a predetermined distribution. In the following description, the predetermined distribution obtained here is referred to as “predetermined distribution A”.
  • 20.00 g of the predetermined distribution A was subjected to silica gel column chromatography.
  • 20.00 g of a predetermined distribution A and 60 g of silica gel (a general-purpose product of 0.063 to 0.2 mm) were mixed.
  • a column (open column with a diameter of 6 cm, a length of 34 cm, and a volume of about 204 mL) packed with 500 g of silica gel (0.040 to 0.063 mm general-purpose product) was prepared, and an elution solvent (n-hexane: ethyl acetate: anhydrous)
  • a mixture of a predetermined partition A and silica gel was placed, and elution with an elution solvent was performed.
  • fraction 1-2 (solvent amount of about 120 to 480 mL), obtained in the 25th to 39th test tube, fraction 1-3 (solvent amount of about 480 to 780 mL), obtained in the 40th to 70th test tube
  • fraction 1-4 (solvent amount: about 780 to 1400 mL)
  • the product obtained with the remaining elution solvent, acetone and absolute ethanol was designated fraction 1-5.
  • the fractions 1-1 to 1-4 are predetermined fractions.
  • silica gel column chromatography method is different from the method of obtaining the predetermined fraction A, only the portions different from the above method will be described.
  • a predetermined distribution A and 10 g of silica gel (a general-purpose product of 0.063 to 0.2 mm) were mixed.
  • fractions 2-1 to 2-3 were analyzed by comparing 1 HNMR and 13 CNMR data with literature values (Journal of Natural Products, 59, 808-811, 1996). It was confirmed that the fraction 2-2 was linacantin C. The amount of obtained linacantin C was 444.2 mg.
  • a nuclear magnetic resonance spectrum apparatus As a nuclear magnetic resonance spectrum apparatus, a JEOL JNM-GSX500 type nuclear magnetic resonance spectrum apparatus (manufactured by JEOL Ltd.) was used.
  • test example on neuronal cell death inhibitory action of predetermined partition, predetermined fraction and linacantin C In this test example, cytotoxicity by amyloid ⁇ and predetermined partition, predetermined fraction and neuronal cell death of linacantin C A test for inhibitory action was performed. For the evaluation of neurotoxicity of amyloid ⁇ , amyloid ⁇ (25-35), which is known as the action center of neurotoxicity of amyloid ⁇ , was used.
  • PC12 cells rat adrenal pheochromocytoma-derived cell line
  • BCRC Bioresource Collection and Research Center, Bioresource Conservation and Research Center, Taiwan
  • the obtained PC12 cells were prepared using RPMI-1640 medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 units / ml penicillin and 100 mg / ml streptomycin (both obtained from Invitrogen-Gibco), 5% carbonic acid.
  • the culture was performed at 37 ° C. and saturated humidity in the presence of gas.
  • a 96-well microplate coated with poly-L-lysine was used to evaluate the cytotoxicity of amyloid ⁇ (25-35).
  • the microplate 2 ⁇ 10 4 / 100 ⁇ L of PC12 cells per well were seeded in the presence of 5% carbon dioxide, 37 ° C., and cultured under saturated humidity. After 24 hours of culture, 0.1% DMSO (manufactured by JT Baker) is used for the control group, each sample (see below) is used for the evaluation group, and 10 ⁇ M galantamine hydrobromide (anti-Alzheimer's disease agent) is used for the positive control group. It is known that it has a neuroprotective action, obtained through Sigma-Aldrich.
  • test sections were tested for both those with and without the addition of 15 ⁇ M amyloid ⁇ (25-35) (obtained through Sigma-Aldrich) (described later). 72 hours after the start of the culture, the cell viability for each test group was calculated by measuring the absorbance at 570 nm by a predetermined method using MTT (Molecular Probes).
  • cell viability was calculated for the control, the positive control, and each sample without adding amyloid ⁇ .
  • cell viability was calculated for each sample under the same conditions except that amyloid ⁇ was added.
  • the value of the cell viability when amyloid ⁇ is added is divided by the value of the cell viability when amyloid ⁇ is not added, and the value calculated by the calculation is multiplied by 100 to give% units.
  • the cell viability recovery rate was obtained by subtracting the cell viability in the control from there. This cell survival recovery rate is an index showing how much the cell survival rate has recovered from the control in a state where the influence of the sample on the cell survival rate is corrected.
  • Samples include a predetermined distribution A (methylene dichloride distribution), a predetermined fraction 1-1, a predetermined fraction 1-2, a predetermined fraction.
  • Fraction 1-3 which is a predetermined fraction, and linacantin C (fraction 2-2) were used.
  • concentration of each sample the screening regarding cytotoxicity etc. was performed in advance and the appropriate density
  • FIG. 3 (a) is a table showing test results for positive control
  • FIG. 3 (b) is a table showing test results for each sample of the present invention.
  • amyloid ⁇ has high toxicity to PC12 cells (see the control item in FIG. 3).
  • the predetermined distribution A the predetermined fraction (fractions 1-1 to 1-4) and linacantin C showed a high cell survival recovery rate.
  • the cell viability recovery rate of the fraction 1-1 and the fraction 1-2 was comparable to that of galantamine hydrobromide.
  • the cell viability recovery rate by linacantin C is 30.2% while the concentration is as low as 1 ⁇ M. Since the cell survival recovery rate by 10 ⁇ M galantamine hydrobromide is 17.2%, it was possible to find the fact that linacantin C shows a particularly high cell survival recovery rate.
  • the nerve cell death inhibitor of the present invention has a good effect of suppressing nerve cell death due to the toxicity of amyloid ⁇ , that is, an excellent nerve cell death inhibitory effect. For this reason, it is thought that the nerve cell death inhibitor of this invention which uses these as an active ingredient has the effect
  • Example 10 By the following examples, a method for preparing pharmaceuticals and foods containing linacantin C, which is an active ingredient of the neuronal death inhibitor of the present invention and an active ingredient of the anti-Alzheimer's disease agent or anti-brain function-lowering agent of the present invention. Describe. Of course, it is also possible to change the item of linacantin C to a predetermined distribution or a predetermined fraction.
  • Linacantin C a tablet is prepared according to the following formulation.
  • Linacantin C 0.2g Lactose 95.8g 2.0g dried corn starch Talc 1.8g Calcium stearate 0.2g
  • Linacantin C 0.2 g
  • talc 1.8 g
  • calcium stearate 0.2 g
  • a tablet is produced by a conventional method using a single-shot tablet press.
  • hard capsules (360 mg per capsule) are prepared according to the following formulation.
  • Linacantin C (5 g) is mixed with lactose (220 g) and corn starch (110 g), and an aqueous solution of hydroxypropylcellulose (25 g) is added thereto and kneaded.
  • granules are produced by an ordinary method using an extrusion granulator.
  • a hard capsule is prepared by filling the granule into a gelatin hard capsule.
  • soft capsules (170 mg per capsule) are prepared according to the following formulation.
  • Linacantin C 0.5mg Soybean oil 169.5mg (Preparation method)
  • Linacantin C (0.5 g) is added to and mixed with soybean oil (169.5 g).
  • soft capsules are prepared by filling soft capsules using a rotary soybean automatic molding machine according to a conventional method.
  • Pills Using linacantin C pills (100 mg per capsule) are prepared according to the following prescription.
  • Linacantin C 0.5mg Morohaya powder 20.0mg Starch 30.0mg Molasses 20.0mg Tea extract 15.0mg Soy fiber 14.0mg Shellac 0.5mg (Preparation method) After mixing the raw materials with the above blending, adding a suitable amount of water, producing a homogeneous kneaded product with a kneader, rolling the kneaded product, making a round shape using a round machine and drying it to produce a pill To do.
  • a powder (1000 mg per packet) is prepared by the following method using a conventional method.
  • Linacantin C 1mg Lactose 799mg Cornstarch 200mg
  • jelly (100 g) is prepared by a conventional method with the following prescription.
  • Linacantin C 0.002g Gelatin 2.0g Orange juice 20.0g 77.998 g of water (Preparation method)
  • the above ingredients are mixed and heated to 90 ° C. After confirming the dissolution of gelatin, fill the container and cool.
  • a jelly is prepared by solidifying gelatin.
  • an ointment (100 g) is prepared by a conventional method with the following formulation.
  • (Oil phase component) Linacantin C 0.1g White petrolatum 20.0g Mineral oil 20.0g Stearyl alcohol 5.0g Steareth-2 3.0g Propylparaben 0.1g Natural vitamin E 0.1g (Aqueous phase component) 1,3-butylene glycol 5.0 g 0.4 g of phenoxyethanol Polysorbate 60 4.5g Purified water Appropriate amount 100g (Preparation method)
  • the oil phase component and the water phase component are each heated to 80 ° C. to be uniform, and the water phase is added to the oil phase with stirring. After emulsification, the mixture is cooled to prepare an ointment.
  • tape agent (100 g) is prepared by a conventional method using linacantin C according to the following formulation.
  • the pressure-sensitive adhesive solvent and the medicinal component are made uniform, the medicinal component and the transdermal absorption accelerator are added to the pressure-sensitive adhesive solvent, and the composition is stirred at room temperature. This composition is spread on a silicone-treated polyester film, dried at 120 ° C. and cooled, and then the pressure-sensitive adhesive layer is transferred to a polyethylene film to produce a tape agent.

Abstract

A nerve cell death inhibitor, an anti-Alzheimer's disease agent, a brain hypofunction inhibitor and a drug or food having an anti-Alzheimer's disease effect or a brain hypofunction-inhibiting effect, each containing as an active ingredient "a preset partitioned portion that is a portion partitioned in methylene dichloride when an ethanol extract of Rhinacanthus nasuta (L.) Kurz is liquid-liquid partitioned with hexane and methanol and then the portion partitioned in methanol is liquid-liquid partitioned with methylene dichloride and water", "a preset fraction that is a fraction eluted from a silica gel column with an elution solvent, said elution solvent consisting of n-hexane, ethyl acetate and anhydrous methanol, when the preset partitioned portion is subjected to silica gel column chromatography using the elution solvent" or Rhinacanthin C. According to the present invention, a nerve cell death inhibitor having an excellent effect of inhibiting nerve cell death, an anti-Alzheimer's disease agent, a brain hypofunction inhibitor and a drug or food having an anti-Alzheimer's disease effect or a brain hypofunction-inhibiting effect can be provided.

Description

神経細胞死抑制剤、抗アルツハイマー病剤、抗脳機能低下剤、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品及び神経細胞死抑制剤の製造方法Neuronal cell death inhibitor, anti-Alzheimer's disease agent, anti-brain function-lowering agent, drug or food having anti-Alzheimer's disease action or anti-brain function-lowering action, and method for producing neuronal cell death inhibitor
 本発明は、神経細胞死抑制剤、抗アルツハイマー病剤、抗脳機能低下剤、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品及び神経細胞死抑制剤の製造方法に関する。 The present invention relates to a nerve cell death inhibitor, an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, a drug or food having an anti-Alzheimer's disease action or an anti-brain function-lowering action, and a method for producing a nerve cell death inhibitor.
 近年、我が国の急激な高齢化に伴い、認知症の患者が増加している。認知症の患者数は、認知症に対する画期的な予防法及び治療法が確立されない限り、今後も増え続けると予想されている。このため、医学的な観点だけではなく、行政の観点からも認知症の予防法及び治療法が希求されている。 In recent years, with the rapid aging of our country, the number of patients with dementia is increasing. The number of patients with dementia is expected to continue to increase unless innovative prevention and treatment methods for dementia are established. For this reason, the prevention method and the treatment method of a dementia are calculated | required not only from a medical viewpoint but from an administrative viewpoint.
 ところで、認知症の原因となる疾患には様々なものがあるが、アルツハイマー病(アルツハイマー型認知症)、レビー小体型認知症及び脳血管性認知症の三種類がその多くを占めているとされる。その中でもアルツハイマー病は患者数が多く、かつ、根本的な予防法及び治療法が確立されていないため、現在もアルツハイマー病について様々な研究が行われている。 By the way, there are various diseases that cause dementia. Alzheimer's disease (Alzheimer-type dementia), Lewy body dementia, and cerebrovascular dementia are said to account for most of them. The Among them, Alzheimer's disease has a large number of patients, and since fundamental preventive and therapeutic methods have not been established, various studies are still being conducted on Alzheimer's disease.
 アルツハイマー病のメカニズムとしては、アミロイドカスケード仮説が広く支持されている。アミロイドカスケード仮説とは、脳にアミロイドタンパク質であるアミロイドβが蓄積し、その結果、アルツハイマー神経原線維変化、直接的又は間接的な毒性による神経細胞の死滅や脱落等が発生し、それに伴って認知症の諸症状が発生するという仮説である。当該仮説は現在のところ広く支持されており、予防法及び治療法の開発研究における指針となっている(例えば、非特許文献1参照。)。 The amyloid cascade hypothesis is widely supported as the mechanism of Alzheimer's disease. The amyloid cascade hypothesis is that amyloid β, an amyloid protein, accumulates in the brain, resulting in Alzheimer's neurofibrillary tangles, neuronal death or loss due to direct or indirect toxicity, etc. It is a hypothesis that various symptoms of the disease occur. The hypothesis is widely supported at present, and serves as a guideline for research and development of preventive and therapeutic methods (see, for example, Non-Patent Document 1).
 従来、アルツハイマー病の治療には、ドネペジル、ガランタミン、リバスチグミン等が用いられている(例えば、非特許文献2参照。)。上記のような物質を有効成分として含有する抗アルツハイマー病剤によれば、認知症の症状の進行を抑制することが可能となる。 Conventionally, donepezil, galantamine, rivastigmine and the like have been used for the treatment of Alzheimer's disease (for example, see Non-Patent Document 2). According to the anti-Alzheimer's disease agent containing the above-mentioned substance as an active ingredient, it becomes possible to suppress the progression of symptoms of dementia.
 ところで、白鶴霊芝(Rhinacanthus nasutus (L.) Kurz。白鶴霊芝草ともいう。)は、インド南部デカン高原の原産とされるリナカンサス属キツネノマゴ科に属する常緑小低木である。白鶴霊芝の全草は駆虫、消炎、皮膚真菌に対する抗菌作用があることが知られ(例えば、非特許文献3参照。)、主に中国、台湾等において、また、最近では日本国において漢方薬や食品として用いられている。その他、本出願人による以前の出願で、白鶴霊芝に活性酸素消去能があること(例えば、特許文献1参照。)、排泄促進作用があること(例えば、特許文献2参照。)、抗アレルギー作用があること(例えば、特許文献3参照。)及び抗腫瘍作用があること(例えば、特許文献4参照。)等が開示されている。 By the way, the white crane reishi (Rhinacanthus natusus (L.) Kurz. Also called white crane reishi grass) is an evergreen small shrub belonging to the genus Linacanthus vulgaris, which originates in the Deccan Plateau in southern India. Hakutsuru Ganoderma is known to have an antibacterial action against anthelmintic, anti-inflammatory, and skin fungi (see, for example, Non-Patent Document 3), mainly in China, Taiwan, etc., and recently in Japan, Used as food. In addition, in the previous application by the present applicant, the white crane reishi has an active oxygen scavenging ability (for example, see Patent Document 1), has an excretion promoting action (for example, see Patent Document 2), and antiallergic. It is disclosed that there is an action (for example, see Patent Document 3) and that it has an antitumor action (for example, see Patent Document 4).
特開平9-143091号公報Japanese Patent Laid-Open No. 9-143091 特開平9-169662号公報Japanese Patent Laid-Open No. 9-169662 特開2001-10964号公報JP 2001-10964 A 特開2002-53481号公報JP 2002-53481 A
 しかしながら、白鶴霊芝の抽出物から所定の方法により得られる所定の分配物、所定の分画物又は白鶴霊芝に含有されるリナカンチンCがアミロイドβの毒性に対する神経細胞死抑制作用を有することは知られていない。 However, it is possible that linacantin C contained in a predetermined distribution, a predetermined fraction, or a white crane reishi obtained from a white crane reishi extract by a predetermined method has a neuronal cell death inhibitory effect on the toxicity of amyloid β. unknown.
 そこで、本発明は、優れた神経細胞死抑制作用を有する神経細胞死抑制剤、抗アルツハイマー病剤、抗脳機能低下剤及び抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品を提供することを目的とする。また、優れた神経細胞死抑制作用を有する神経細胞死抑制剤を製造することが可能な神経細胞死抑制剤の製造方法を提供することも目的とする。 Therefore, the present invention provides a neuronal cell death inhibitor, an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, and an anti-Alzheimer's disease action or an anti-brain function-lowering drug or food having an excellent neuronal death inhibitory effect. For the purpose. Another object of the present invention is to provide a method for producing a nerve cell death inhibitor capable of producing a nerve cell death inhibitor having an excellent nerve cell death inhibitory action.
 本発明者らは、アルツハイマー病の予防又は治療に用いることが可能であると考えられるものの1つである、アミロイドβの直接毒性に対する神経細胞死抑制作用を有するものについて鋭意研究を行った結果、白鶴霊芝の抽出物から所定の方法により得られる所定の分配物又は所定の分画物、及び、白鶴霊芝に含有されるリナカンチンCに神経細胞死抑制作用があることを見出し、本発明を完成させるに至った。本発明は、下記の事項により構成される。
 なお、以下の説明において特段の記載がなければ、溶媒の比率は体積で示している。
As a result of intensive studies on what has an action of suppressing neuronal cell death against the direct toxicity of amyloid β, which is one of those that can be used for the prevention or treatment of Alzheimer's disease, It has been found that a predetermined distribution or a predetermined fraction obtained from a white crane reishi extract by a predetermined method and linacantin C contained in the white crane reishi have a neuronal cell death inhibitory effect. It came to complete. The present invention includes the following items.
In the following description, unless otherwise specified, the solvent ratio is indicated by volume.
[1]白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配するとき、前記二塩化メチレンに分配される分配物である所定の分配物のみを有効成分として含有する神経細胞死抑制剤。 [1] When the ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol, and the liquid distributed to the methanol is liquid-liquid distributed with methylene dichloride and water, it is distributed to the methylene dichloride. A neuronal cell death inhibitor containing only a predetermined distribution, which is a distribution, as an active ingredient.
[2]白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付すとき、
 シリカゲルカラムから前記溶出溶媒により溶出される分画物である所定の分画物のみを有効成分として含有する神経細胞死抑制剤。
[2] Ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol, and the liquid distributed to the methanol is liquid-liquid distributed with methylene dichloride and water and distributed to the methylene dichloride. When the partition is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol,
The nerve cell death inhibitor which contains only the predetermined fraction which is a fraction eluted with the said elution solvent from a silica gel column as an active ingredient.
[3]上記[2]に記載の神経細胞死抑制剤において、前記溶出溶媒は、n-ヘキサン:酢酸エチル:無水メタノール=3:4:2の比率からなる溶出溶媒である神経細胞死抑制剤。 [3] The nerve cell death inhibitor according to [2], wherein the elution solvent is an elution solvent having a ratio of n-hexane: ethyl acetate: anhydrous methanol = 3: 4: 2. .
[4]上記[3]に記載の神経細胞死抑制剤において、前記所定の分画物は、前記シリカゲルカラムクロマトグラフィーに用いるシリカゲルの体積に対して0.1倍~4倍の体積の前記溶出溶媒を用いたときに溶出する分画物である神経細胞死抑制剤。 [4] In the neuronal cell death inhibitor according to [3] above, the predetermined fraction is eluted in a volume of 0.1 to 4 times the volume of silica gel used in the silica gel column chromatography. A neuronal cell death inhibitor, which is a fraction eluted when a solvent is used.
[5]上記[4]に記載の神経細胞死抑制剤において、前記所定の分画物は、前記シリカゲルカラムクロマトグラフィーに用いる前記シリカゲルの体積に対して0.2倍~2.5倍の体積の前記溶出溶媒を用いたときに溶出する分画物である神経細胞死抑制剤。 [5] The neuronal cell death inhibitor according to [4] above, wherein the predetermined fraction has a volume of 0.2 to 2.5 times the volume of the silica gel used in the silica gel column chromatography. A neuronal cell death inhibitor, which is a fraction eluted when the elution solvent is used.
[6]リナカンチンCのみを有効成分として含有する神経細胞死抑制剤。 [6] A neuronal cell death inhibitor containing only linacantin C as an active ingredient.
[7]上記[1]~[6]のいずれかに記載の神経細胞死抑制剤を有効成分として含有する抗アルツハイマー病剤。 [7] An anti-Alzheimer's disease agent comprising the neuronal cell death inhibitor according to any one of [1] to [6] as an active ingredient.
[8]上記[1]~[6]のいずれかに記載の神経細胞死抑制剤を有効成分として含有する抗脳機能低下剤。 [8] An anti-brain function-lowering agent containing the nerve cell death inhibitor according to any one of [1] to [6] as an active ingredient.
[9]上記[7]に記載の抗アルツハイマー病剤又は上記[8]に記載の抗脳機能低下剤を含有し、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品。 [9] A pharmaceutical or food containing the anti-Alzheimer's disease agent according to [7] or the anti-brain function-lowering agent according to [8], and having an anti-Alzheimer's disease action or an anti-brain function-lowering action.
[10]白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、前記エタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を得る第2工程と、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物である所定の分配物を得る第3工程とをこの順序で含む、前記所定の分配物のみを有効成分として含有する神経細胞死抑制剤の製造方法。 [10] A first step of extracting white crane reishi with ethanol to obtain an ethanol extract, and a second step of liquid-liquid partitioning the ethanol extract with hexane and methanol to obtain a partition distributed to the methanol And a third step of liquid-liquid partitioning the partition distributed to the methanol with methylene dichloride and water to obtain a predetermined partition which is a partition distributed to the methylene dichloride in this order. A method for producing a nerve cell death inhibitor comprising only the predetermined distribution as an active ingredient.
[11]白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、前記エタノール抽出物をヘキサン及びメタノールで液-液分配して、前記メタノールに分配される分配物を得る第2工程と、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配して、前記二塩化メチレンに分配される分配物を得る第3工程と、前記二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付して、シリカゲルカラムから前記溶出溶媒により溶出される分画物である所定の分画物を得る第4工程とをこの順序で含む、前記所定の分画物のみを有効成分として含有する神経細胞死抑制剤の製造方法。 [11] A first step of extracting a white crane reishi with ethanol to obtain an ethanol extract, and a liquid-liquid partition of the ethanol extract with hexane and methanol to obtain a partition distributed to the methanol. A third step of liquid-liquid partitioning the partition distributed to the methanol with methylene dichloride and water to obtain a partition distributed to the methylene dichloride; and partitioning to the methylene dichloride The partition was subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol, and a predetermined fraction which was a fraction eluted from the silica gel column with the elution solvent was obtained. A method for producing a neuronal cell death inhibitor comprising only the predetermined fraction as an active ingredient, comprising the fourth step obtained in this order.
 本発明によれば、後述する試験例からもわかるように、優れた神経細胞死抑制作用を有する神経細胞死抑制剤、抗アルツハイマー病剤、抗脳機能低下剤、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品を提供することができる。
 また、本発明によれば、優れた神経細胞死抑制作用を有する神経細胞死抑制剤の製造方法を提供することが可能となる。
According to the present invention, as can be seen from the test examples described later, a neuronal death inhibitor, an anti-Alzheimer's disease agent, an anti-brain function-lowering agent, an anti-Alzheimer's disease action or an anti-brain function having an excellent inhibitory effect on neuronal cell death. A pharmaceutical or food having a lowering action can be provided.
Moreover, according to this invention, it becomes possible to provide the manufacturing method of the nerve cell death inhibitor which has the outstanding nerve cell death inhibitory effect.
 なお、上記[1]については、例えば、「以下の所定の分配物のみを有効成分として含有する神経細胞死抑制剤を製造するための、前記所定の分配物の有効成分としての使用であって、白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配するとき、前記二塩化メチレンに分配される分配物である前記所定の分配物。」のように表現することもできる。
 また、上記[2]については、例えば、「以下の所定の分画物のみを有効成分として含有する神経細胞死抑制剤を製造するための、前記所定の分画物の有効成分としての使用であって、白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付すとき、シリカゲルカラムから前記溶出溶媒により溶出される分画物である前記所定の分画物。」のように表現することもできる。
 また、上記[6]については、例えば、「リナカンチンCのみを有効成分として含有する神経細胞死抑制剤を製造するための、前記リナカンチンCの有効成分としての使用。」のように表現することもできる。
 また、上記[7]については、例えば、「抗アルツハイマー病剤を製造するための、上記[1]~[6]のいずれかに記載の神経細胞死抑制剤の有効成分としての使用。」のように表現することもできる。
 また、上記[8]については、例えば、「抗脳機能低下剤を製造するための、上記[1]~[6]のいずれかに記載の神経細胞死抑制剤の有効成分としての使用。」のように表現することもできる。
 また、上記[9]については、例えば、「抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品を製造するための、上記[7]に記載の抗アルツハイマー病剤又は上記[8]に記載の抗脳機能低下剤の有効成分としての使用。」のように表現することもできる。
In addition, the above [1] is, for example, “use of the predetermined distribution as an active ingredient for producing a neuronal death inhibitor containing only the following predetermined distribution as an active ingredient. When the ethanol extract of Hakutsuru Ganoderma is liquid-liquid distributed with hexane and methanol, and the partition distributed to methanol is liquid-liquid distributed with methylene dichloride and water, the distribution is distributed to the methylene dichloride. It can also be expressed as “the predetermined distribution which is an object”.
As for the above [2], for example, “in the use of the predetermined fraction as an active ingredient for producing a nerve cell death inhibitor containing only the following predetermined fraction as an active ingredient” The Hakutsuru Reishi lawn ethanol extract was liquid-liquid distributed with hexane and methanol, and the methanol-distributed distribution was liquid-liquid distributed with methylene dichloride and water and distributed to the methylene dichloride. The predetermined fraction which is a fraction eluted from a silica gel column with the elution solvent when the partition is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol. "Can also be expressed.
In addition, the above [6] may be expressed as, for example, “use of linacantin C as an active ingredient for producing a neuronal cell death inhibitor containing only linacantin C as an active ingredient”. it can.
As for [7] above, for example, “use of the neuronal cell death inhibitor according to any of [1] to [6] as an active ingredient for producing an anti-Alzheimer's disease agent”. It can also be expressed as follows.
As for the above [8], for example, “use of the nerve cell death inhibitor according to any of [1] to [6] as an active ingredient for producing an antibrain function-lowering agent”. It can also be expressed as
As for the above [9], for example, “the anti-Alzheimer's disease agent according to [7] or the above [8] for producing a pharmaceutical or food having an anti-Alzheimer's disease action or an anti-brain function lowering action” "Use of the described antibrain function-lowering agent as an active ingredient."
白鶴霊芝から所定の分配物及び所定の分画物を分配・分画した手順を示すフローチャートである。It is a flowchart which shows the procedure which distributed and fractionated the predetermined distribution and the predetermined fraction from Hakutsuru Reishi. 白鶴霊芝からリナカンチンCを単離した手順を示すフローチャートである。It is a flowchart which shows the procedure which isolated the linacantine C from the white crane reishi. 試験例の結果を示す表である。It is a table | surface which shows the result of a test example.
 以下、本発明の神経細胞死抑制剤、抗アルツハイマー病剤、抗脳機能低下剤、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品及び神経細胞死抑制剤の製造方法について説明する。 Hereinafter, the nerve cell death inhibitor, the anti-Alzheimer's disease agent, the anti-brain function-lowering agent, the drug or food having the anti-Alzheimer's disease action or the anti-brain function-lowering action, and the method for producing the nerve cell death inhibitor of the present invention will be described.
 本発明の「白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、メタノールに分配される分配物を二塩化メチレン及び水で液-液分配するとき、二塩化メチレンに分配される分配物である所定の分配物のみを有効成分として含有する神経細胞死抑制剤」は、本発明の「白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、エタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を得る第2工程と、メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物である所定の分配物を得る第3工程とをこの順序で含む、所定の分配物のみを有効成分として含有する神経細胞死抑制剤の製造方法」により得ることができる。 According to the present invention, when the ethanol extract of Hakutsuru Reishi is liquid-liquid partitioned with hexane and methanol, and the partition distributed to methanol is liquid-liquid distributed with methylene dichloride and water, it is distributed to methylene dichloride. The neuronal cell death inhibitor containing only a predetermined distribution which is a distribution as an active ingredient is a "first step of obtaining an ethanol extract by extracting white crane reishi with ethanol, Liquid-liquid partition with hexane and methanol to obtain a partition distributed to the methanol, and liquid-liquid partition of the partition distributed to methanol with methylene dichloride and water to the methylene dichloride. And a third step of obtaining a predetermined distribution which is a distribution to be distributed in this order, and a method for producing a neuronal cell death inhibitor containing only the predetermined distribution as an active ingredient ” It can be.
 本発明における所定の分配物は、記載された条件を満たす限り種々の条件で得ることができる。以下、用いることができる方法等について説明する。
 なお、本明細書において「分配物」とは、精製物であって液-液分配により得られるもののことをいう。
 また、本明細書において「所定の分配物のみを有効成分として含有する」とは、神経細胞死抑制作用を有する有効成分として所定の分配物のみを含有することをいい、有効成分ではない成分(各種溶媒や補助的な添加剤等)を含有することを否定しない。
The predetermined distribution in the present invention can be obtained under various conditions as long as the conditions described are satisfied. Hereinafter, methods that can be used will be described.
In the present specification, the “partition” means a purified product obtained by liquid-liquid partition.
Further, in the present specification, “containing only a predetermined distribution as an active ingredient” refers to containing only a predetermined distribution as an active ingredient having an inhibitory action on nerve cell death, and is not an active ingredient ( It does not deny that it contains various solvents and auxiliary additives.
 白鶴霊芝については、原料である白鶴霊芝の葉、茎、根、花等を、適当な時期に採取した後、そのままのもの、又は、通常通風乾燥等の乾燥工程に付し、抽出原料としたものを用いることができる。抽出原料としての白鶴霊芝は、粉砕若しくは細切して用いることが好ましい。 For Hakutsuru Reishi, the raw Hakutsuru Reishi leaves, stems, roots, flowers, etc. are collected at an appropriate time and then used as they are or are usually subjected to a drying process such as ventilation drying to extract raw materials. Can be used. The white crane reishi as an extraction raw material is preferably used after being crushed or chopped.
 エタノールによる抽出は、白鶴霊芝及びエタノールを用いる限り、通常工業的・実験的に用いられる公知の抽出方法(例えば、「ファイトメディシン、第16巻、929~934ページ、2009年」を参照。)により行うことができる。当該抽出により、白鶴霊芝のエタノール抽出物を得ることができる。 As long as Hakutsuru Ganoderma and ethanol are used for extraction with ethanol, a known extraction method that is usually used industrially and experimentally (see, for example, “Phytomedicine, Vol. 16, pages 929-934, 2009”). Can be performed. By this extraction, an ethanol extract of white crane reishi can be obtained.
 なお、本明細書において単に「エタノール」という場合には、「エタノールを主成分とする(エタノールを50%以上含有する)溶媒」のことをいう。つまり、「エタノール」には、いわゆる無水エタノールだけではなく、エタノールと他の溶媒との混合溶媒も含む。白鶴霊芝から抽出を行うときには、エタノールと水との混合溶媒(含水エタノール)を用いることが好ましく、特に90%エタノールを用いることが一層好ましい。
 抽出温度は、通常0~100℃、好ましくは5~50℃である。溶媒が沸騰するような条件で抽出を行う場合には、溶媒を還流させながら抽出を行うことが好ましい。抽出時間は、1時間~10日間程度であり、溶媒量は、乾燥原料あたり1回ごとに通常1~30倍重量、好ましくは5~10倍重量である。抽出操作は、攪拌であっても単なる浸漬であってもよい。抽出操作は、必要に応じて複数回繰り返してもよい。また、条件が異なる抽出操作を組み合わせてもよい。
 さらに、上記の操作で得られた粗抽出液から不溶性残渣を濾過若しくは遠心分離により取り除く操作を行うことが好ましい。
In the present specification, the term “ethanol” simply means a “solvent containing ethanol as a main component (containing 50% or more of ethanol)”. That is, “ethanol” includes not only so-called anhydrous ethanol but also a mixed solvent of ethanol and another solvent. When extracting from Hakutsuru Reishi, it is preferable to use a mixed solvent of ethanol and water (hydrous ethanol), more preferably 90% ethanol.
The extraction temperature is usually 0 to 100 ° C., preferably 5 to 50 ° C. When performing extraction under conditions where the solvent boils, it is preferable to perform extraction while refluxing the solvent. The extraction time is about 1 hour to 10 days, and the amount of solvent is usually 1 to 30 times by weight, preferably 5 to 10 times by weight per dry raw material. The extraction operation may be stirring or simple immersion. The extraction operation may be repeated a plurality of times as necessary. Moreover, you may combine extraction operation from which conditions differ.
Furthermore, it is preferable to perform an operation of removing insoluble residues from the crude extract obtained by the above operation by filtration or centrifugation.
 液-液分配についても、通常工業的・実験的に用いられる公知の方法により行うことができる。
 液-液分配とは、二相分配、二相溶媒分配法、液-液抽出等ともいわれる方法であり、極性が異なる2種の溶媒を用い、溶解度の差により目的化合物を回収する方法である。
 液-液分配の後には、溶媒を除去して乾固した状態の分配物を得ることが好ましい。ただし、溶媒の除去が使用目的上不要である場合にはこの限りではない。
Liquid-liquid distribution can also be carried out by known methods usually used industrially and experimentally.
Liquid-liquid partitioning is a method called two-phase partitioning, two-phase solvent partitioning, liquid-liquid extraction, etc., and is a method of recovering a target compound by using a difference in solubility using two kinds of solvents having different polarities. .
After the liquid-liquid partition, it is preferable to obtain a partition in a dry state by removing the solvent. However, this is not the case when the removal of the solvent is unnecessary for the purpose of use.
 なお、本明細書において単に「メタノール」という場合には、「メタノールを主成分とする(メタノールを50%以上含有する)溶媒」のことをいう。つまり、「メタノール」には、いわゆる無水メタノールだけではなく、メタノールと他の溶媒との混合溶媒も含む。液-液分配を行うときには、メタノールと水との混合溶媒、特に90%メタノールを用いることが好ましい。一方、本明細書において「無水メタノール」という場合には、無水メタノールのことをいい、メタノールを含む混合溶媒は含まない。
所定の分配物のみを有効成分として含有する神経細胞死抑制剤の製造方法」により得ることができる。
In the present specification, the term “methanol” simply means “a solvent containing methanol as a main component (containing 50% or more of methanol)”. That is, “methanol” includes not only so-called anhydrous methanol but also a mixed solvent of methanol and another solvent. When performing liquid-liquid partitioning, it is preferable to use a mixed solvent of methanol and water, particularly 90% methanol. On the other hand, in the present specification, “anhydrous methanol” refers to anhydrous methanol and does not include a mixed solvent containing methanol.
It can be obtained by a “method for producing a nerve cell death inhibitor containing only a predetermined distribution as an active ingredient”.
 また、本発明の「白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付すとき、シリカゲルカラムから溶出溶媒により溶出される分画物である所定の分画物のみを有効成分として含有する神経細胞死抑制剤」は「白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、エタノール抽出物をヘキサン及びメタノールで液-液分配して、前記メタノールに分配される分配物を得る第2工程と、メタノールに分配される分配物を二塩化メチレン及び水で液-液分配して、前記二塩化メチレンに分配される分配物を得る第3工程と、二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付して、シリカゲルカラムから前記溶出溶媒により溶出される分画物である所定の分画物を得る第4工程とをこの順序で含む、前記所定の分画物を有効成分として含有する神経細胞死抑制剤の製造方法」により得ることができる。 In addition, according to the present invention, the ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol, and the liquid distributed to methanol is liquid-liquid distributed with methylene dichloride and water and distributed to methylene dichloride. When the obtained fraction is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol, only a predetermined fraction which is a fraction eluted from the silica gel column with the elution solvent is obtained. “Neuronal cell death inhibitor contained as an active ingredient” includes “first step of extracting white crane reishi with ethanol to obtain an ethanol extract, and liquid-liquid distribution of the ethanol extract with hexane and methanol to the methanol. A second step of obtaining a partition to be distributed; and a liquid-liquid partition of the partition distributed to methanol with methylene dichloride and water, The third step to obtain a partition distributed to methylene chloride and the partition distributed to methylene dichloride were subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol. And a fourth step of obtaining a predetermined fraction which is a fraction eluted from the silica gel column with the elution solvent in this order, and a neuronal cell death inhibitor containing the predetermined fraction as an active ingredient The production method of
 本発明における所定の分画物は、記載された条件を満たす限り種々の条件で得ることができる。以下、用いることができる方法等について説明する。
 なお、本明細書において「分画物」とは、精製物であってカラムクロマトグラフィーでの分画により得られるもののことをいう。
 また、本明細書において「所定の分画物のみを有効成分として含有する」とは、神経細胞死抑制作用を有する有効成分として所定の分画物のみを含有することをいい、有効成分ではない成分(各種溶媒や補助的な添加剤等)を含有することを否定しない。
 抽出及び液-液分配については既に説明したので、再度の説明は省略する。
 シリカゲルカラムクロマトグラフィーの後には、溶出溶媒を除去して乾固した状態の分画物を得ることが好ましい。ただし、溶媒の除去が使用目的上不要である場合にはこの限りではない。
The predetermined fraction in the present invention can be obtained under various conditions as long as the described conditions are satisfied. Hereinafter, methods that can be used will be described.
In the present specification, the “fraction” means a purified product obtained by fractionation by column chromatography.
Further, in the present specification, “containing only a predetermined fraction as an active ingredient” means containing only a predetermined fraction as an active ingredient having a nerve cell death inhibitory action, and is not an active ingredient. It does not deny that it contains ingredients (various solvents, auxiliary additives, etc.).
Since the extraction and the liquid-liquid distribution have already been described, the description thereof will be omitted.
After silica gel column chromatography, it is preferable to obtain a fraction in a dry state by removing the elution solvent. However, this is not the case when the removal of the solvent is unnecessary for the purpose of use.
 シリカゲルカラムクロマトグラフィーについても、通常工業的・実験的に用いられる公知の方法により行うことができる。
 溶出溶媒は、n-ヘキサン:酢酸エチル:無水メタノール=3:4:2の比率からなる溶出溶媒であることが好ましい(後述の実験例参照。)。この場合、所定の分画物は、シリカゲルカラムクロマトグラフィーに用いるシリカゲルの体積に対して0.1倍~4倍の体積の溶出溶媒を用いたときに溶出する分画物であることが一層好ましく、0.2倍~2.5倍の体積の溶出溶媒を用いたときに溶出する分画物であることがより一層好ましい(後述の試験例参照。)。
Silica gel column chromatography can also be carried out by known methods usually used industrially and experimentally.
The elution solvent is preferably an elution solvent having a ratio of n-hexane: ethyl acetate: anhydrous methanol = 3: 4: 2 (see the experimental examples described later). In this case, the predetermined fraction is more preferably a fraction that elutes when an elution solvent having a volume of 0.1 to 4 times the volume of silica gel used for silica gel column chromatography is used. It is even more preferable that it is a fraction that elutes when an elution solvent having a volume of 0.2 to 2.5 times is used (see test examples described later).
 所定の分配物及び所定の分画物は、本発明の目的を損なわない限り、上記以外の精製方法をさらに行って得たものであってもよい。 The predetermined distribution and the predetermined fraction may be obtained by further performing a purification method other than the above as long as the object of the present invention is not impaired.
 所定の分配物及び所定の分画物を得るために用いる溶媒及び混合溶媒は、同程度の極性を有する溶媒又は混合溶媒により代替可能であると考えられる。この場合、溶媒として使用可能なものであれば所定の分配物及び所定の分画物を得るために用いることができる。溶媒の例として、水、各種アルコール類、酢酸エチルのようなエステル類、直鎖状・分枝状・環状の各種炭化水素類、二塩化メチレン(ジクロロメタン)、クロロホルム、アセトン、各種エーテル類等を挙げることができ、使用可能な溶媒の中から、方法及び目的に合致するものを用いることができる。 It is considered that the solvent and the mixed solvent used for obtaining the predetermined distribution and the predetermined fraction can be replaced with a solvent or a mixed solvent having the same degree of polarity. In this case, as long as it can be used as a solvent, it can be used to obtain a predetermined distribution and a predetermined fraction. Examples of solvents include water, various alcohols, esters such as ethyl acetate, linear, branched and cyclic hydrocarbons, methylene dichloride (dichloromethane), chloroform, acetone, various ethers, etc. Among the solvents that can be used, those suitable for the method and purpose can be used.
 なお、後述する実験例の結果から考えると、本発明における所定の分配物及び所定の分画物には、1成分としてリナカンチンCが含有されていると考えられる。 In view of the results of the experimental examples described later, it is considered that the predetermined distribution and the predetermined fraction in the present invention contain linacantin C as one component.
 本発明の「リナカンチンCのみを有効成分として含有する神経細胞死抑制剤」におけるリナカンチンCは、白鶴霊芝を原料に抽出・精製することにより得てもよい(後述する実験例を参照。)し、その他の植物体から得てもよい。また、リナカンチンCは、合成によって得てもよい。
 リナカンチンCとは、以下の化学式(1)により表される化合物である。
Figure JPOXMLDOC01-appb-C000001
The linacantin C in the “nerve cell death inhibitor containing only linacantin C as an active ingredient” of the present invention may be obtained by extracting and purifying white crane reishi as a raw material (see experimental examples described later). It may be obtained from other plants. Also, linacantin C may be obtained by synthesis.
Linacantin C is a compound represented by the following chemical formula (1).
Figure JPOXMLDOC01-appb-C000001
 本発明のリナカンチンCは、体内の作用機序においてリナカンチンCであればよく、投与前においては、薬学上許容される塩の形態をとっていてもよい。 The linacantin C of the present invention may be linacantin C in the mechanism of action in the body, and may be in the form of a pharmaceutically acceptable salt before administration.
 所定の分配物、所定の分画物及びリナカンチンCは、後述する実験例に示すように、アミロイドβの毒性による神経細胞死を抑制する効果がある。このため、これらを有効成分とする本発明の神経細胞死抑制剤は、抗アルツハイマー病剤及び抗脳機能低下剤としての作用を有すると考えられる。
 なお、本明細書における「抗アルツハイマー病剤」とは、アルツハイマー病の予防及び治療のうち少なくとも一方に用いることができるものである。また、「抗脳機能低下剤」とは、脳機能の低下の予防及び治療のうち少なくとも一方に用いることができるものである。
The predetermined distribution, the predetermined fraction, and linacantin C have the effect of suppressing neuronal cell death due to the toxicity of amyloid β, as shown in the experimental examples described later. For this reason, it is thought that the nerve cell death inhibitor of this invention which uses these as an active ingredient has the effect | action as an anti- Alzheimer's disease agent and an anti-brain function reducing agent.
In addition, the “anti-Alzheimer's disease agent” in the present specification can be used for at least one of prevention and treatment of Alzheimer's disease. Further, the “anti-brain function-lowering agent” can be used for at least one of prevention and treatment of a decrease in brain function.
 本発明の神経細胞死抑制剤、抗脳機能低下剤、抗アルツハイマー病剤及び医薬品(ここでは、内用又は内服の医薬品)の投与経路は特に限定されないが、例えば、経口投与・直腸内投与等の経腸投与、経鼻投与などの粘膜投与、静脈内投与・皮下投与などの注射投与等を挙げることができる。剤型としては、いずれも投与方法に適した製剤の形態をとることができ、例えば、錠剤、散剤、細粒剤、顆粒剤、カプセル剤、粉末、丸剤、トローチ剤等の固形剤、溶液、懸濁剤、乳剤、シロップ剤、注射剤などの液剤、ゲル状の製剤等を挙げることができる。本発明の神経細胞死抑制剤、抗脳機能低下剤、抗アルツハイマー病剤及び医薬品は、そのまま投与してもよいが、薬理的に許容される賦形剤とともに投与しても良い。賦形剤としては、単糖類、二糖類、多糖類、無機塩類、油脂、蒸留水など、製剤として一般に使用可能なものであればいずれも用いることができる。製剤化する際には、結合剤、滑沢剤、分散剤、懸濁剤、乳化剤、希釈剤、緩衝剤、抗酸化剤、細菌抑制剤等の添加剤を用いることもできる。 The route of administration of the neuronal death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceuticals (herein, drugs for internal use or internal use) of the present invention is not particularly limited, and examples thereof include oral administration and rectal administration. And mucosal administration such as nasal administration, injection administration such as intravenous administration and subcutaneous administration, and the like. Any dosage form can take the form of a formulation suitable for the administration method. For example, tablets, powders, fine granules, granules, capsules, powders, pills, lozenges and other solid agents, solutions , Suspensions, emulsions, syrups, liquids such as injections, gel preparations, and the like. The neuronal cell death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceutical of the present invention may be administered as they are, or may be administered together with a pharmacologically acceptable excipient. As the excipient, any monosaccharides, disaccharides, polysaccharides, inorganic salts, oils and fats, distilled water, etc. that can be generally used as preparations can be used. In formulating, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used.
 本発明の神経細胞死抑制剤、抗脳機能低下剤、抗アルツハイマー病剤及び医薬品の有効投与量は、投与経路、剤形、疾患の症状、対象者の年齢等により異なるが、所定の分配物、所定の分画物又はリナカンチンCについて、通常成人一日あたり0.1~1000mg、好ましくは0.5~300mg、さらに好ましくは1~100mgであると考えられる。所定の分配物、所定の分画物又はリナカンチンCの含有量は、製剤の形態・有効投与量・製剤としての投与量のデータに基づき、各投与形態に最適な製剤中の有効成分含有量を設定することができる。 The effective dosage of the neuronal cell death inhibitor, anti-brain function-lowering agent, anti-Alzheimer's disease agent and pharmaceutical of the present invention varies depending on the administration route, dosage form, disease symptom, age of the subject, etc. The predetermined fraction or linacantin C is considered to be usually 0.1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg per day for an adult. The content of a predetermined distribution, a predetermined fraction or linacantin C is determined based on the data on the dosage form, the effective dosage, and the dosage as the dosage, and the optimal active ingredient content in the dosage form for each dosage form. Can be set.
 本発明の医薬品は、外用医薬品であってもよい。外用医薬品の形態は、特に限定されない。
 外用医薬品の形態としては、例えば、軟膏剤、クリーム剤、発布剤、テープ剤、外用剤等を挙げることができる。本発明の外用医薬品は、所定の分配物、所定の分画物又はリナカンチンCに、必要に応じて種々の医薬成分を含有することができる。また、結合剤、分散剤、懸濁剤、乳化剤、希釈剤、緩衝剤、抗酸化剤、細菌抑制剤等の添加剤を用いることもできる。
The pharmaceutical product of the present invention may be an external pharmaceutical product. The form of the external medicine is not particularly limited.
As a form of external medicine, an ointment, a cream, a foaming agent, a tape agent, an external preparation etc. can be mentioned, for example. The external medicine of the present invention can contain various pharmaceutical ingredients in a predetermined distribution, a predetermined fraction or linacantin C as required. In addition, additives such as a binder, a dispersant, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, and a bacteria inhibitor can also be used.
 本発明の食品としては、有効成分を加えたお茶としての形態の食品や、有効成分を配合した加工食品としての形態の食品を例示することができる。 Examples of the food of the present invention include foods in the form of tea with the addition of active ingredients and foods in the form of processed foods containing the active ingredients.
 お茶としては、白鶴霊芝の葉・茎若しくは根の乾燥物と混合して、又は、他の茶原料と混合して用いることが好ましい。茶原料としては、緑茶、ウーロン茶、プーアル茶、紅茶、ほうじ茶、玄米茶、杜仲茶、柿の葉茶、桑の葉茶等、通常お茶として用いられるものであれば、どのようなものでも用いることができる。なお、白鶴霊芝の葉・茎または根の乾燥物は、他の茶原料と同様に焙煎して用いることもできる。 As tea, it is preferable to use it by mixing with a dried white crane reishi leaf / stem or root or by mixing with other tea ingredients. As tea materials, green tea, oolong tea, puer tea, black tea, roasted green tea, brown rice tea, Tochu tea, kashiwanoha tea, mulberry leaf tea, etc., as long as they are used as normal teas, any tea can be used. Can do. The dried white crane reishi leaf / stem or root can be roasted and used in the same manner as other tea ingredients.
 有効成分を配合した食品の形態としては、ドリンク剤、ゼリー、ビスケット、錠剤、丸剤、ソフトカプセル剤、ハードカプセル剤、散剤、細粒剤、顆粒剤等、通常食品として提供可能な形態であれば、いずれの形態も用いることができる。また、食品の副原料として、賦形剤、結合剤、滑沢剤、分散剤、懸濁剤、乳化剤、希釈剤、緩衝剤、抗酸化剤、細菌抑制剤等の添加剤を用いることもできる。 As a form of food containing the active ingredient, drinks, jelly, biscuits, tablets, pills, soft capsules, hard capsules, powders, fine granules, granules, etc. Either form can be used. In addition, additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used as food auxiliary materials. .
 本発明の食品の有効摂取量は、摂取形態、対象者の健康状態、対象者の年齢等により異なるが、所定の分配物、所定の分画物又はリナカンチンCについて、通常成人一日あたり0.1~1000mg、好ましくは0.5~300mg、さらに好ましくは1~100mgであると考えられる。 The effective intake amount of the food of the present invention varies depending on the intake form, the health condition of the subject, the age of the subject, etc., but for a given distribution, a given fraction or linacantin C, it is usually 0. It is considered to be 1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg.
 本発明の食品中の所定の分配物、所定の分画物又はリナカンチンCの含有量は、食品の形態によっても異なるが、通常0.0001~1wt%、好ましくは0.001~0.5wt%、より好ましくは、0.01~0.1wt%であると考えられる。 The content of the predetermined distribution, the predetermined fraction or linacantin C in the food of the present invention varies depending on the form of the food, but is usually 0.0001 to 1 wt%, preferably 0.001 to 0.5 wt%. More preferably, it is considered to be 0.01 to 0.1 wt%.
[実験例]
 以下、所定の分配物、所定の分画物及びリナカンチンCの分配・分画・単離例や神経細胞死抑制作用に関する試験例により本発明をさらに詳しく説明する。なお、以下の実験例はいわば具体例であり、本発明は以下の実験例に制約されるものではない。
[Experimental example]
Hereinafter, the present invention will be described in more detail with reference to predetermined distributions, predetermined fractions, and examples of partitioning, fractionation and isolation of linacantin C, and test examples relating to neuronal cell death inhibitory action. The following experimental examples are specific examples, and the present invention is not limited to the following experimental examples.
1.本発明の分配物、本発明の分画物及びリナカンチンCの分配・分画・単離例
 本発明の分配物及び本発明の分画物の分配・分画は、図1に示すフローに沿って行った。
 まず、原料として白鶴霊芝(Rhinacanthus nasutus(L.)Kurz)の根の乾燥物2kgを準備し、汎用のグラインダーで粉砕した。
 続いて、90%エタノールによる抽出を行った。抽出は、白鶴霊芝を20Lの90%エタノールに3日間浸漬し、その後、溶媒を1時間還流させることで行った。抽出液と抽出残渣とは、濾紙により分離した。当該抽出を同じ原料に対して計3回行い、減圧により抽出液から溶媒を取り除き、乾固したエタノール抽出物77.82gを得た。
1. Example of distribution / fractionation / isolation of the distribution of the present invention, the fraction of the present invention and linacantin C The distribution / fractionation of the distribution of the present invention and the fraction of the present invention follows the flow shown in FIG. I went.
First, as a raw material, 2 kg of dried roots of Rhinacanthus natusus (L.) Kurz was prepared and pulverized with a general-purpose grinder.
Subsequently, extraction with 90% ethanol was performed. The extraction was performed by immersing Hakutsuru Reishi in 20 L of 90% ethanol for 3 days and then refluxing the solvent for 1 hour. The extract and the extraction residue were separated by filter paper. The extraction was performed three times on the same raw material, and the solvent was removed from the extract under reduced pressure to obtain 77.82 g of a dry ethanol extract.
 上記エタノール抽出物から4gを分析用サンプルとして取り除いた後、残りのエタノール抽出物73.82g全量について、ヘキサン及び90%メタノールで液-液分配を行った。まず、エタノール抽出物に90%メタノール500mLを加え、その後、ヘキサン500mLを加えて1分間振とうした。溶媒が分離するまで10分程度待ち、90%メタノール相を採取した。さらに2回同様の操作を繰り返し、得られた90%メタノール相を併せた後、減圧により溶媒を取り除き、乾固した分配物54.32gを得た。 After removing 4 g from the ethanol extract as a sample for analysis, the remaining 73.82 g ethanol extract was subjected to liquid-liquid partition with hexane and 90% methanol. First, 500 mL of 90% methanol was added to the ethanol extract, and then 500 mL of hexane was added and shaken for 1 minute. The solvent was separated for about 10 minutes, and a 90% methanol phase was collected. The same operation was further repeated twice, and the obtained 90% methanol phase was combined, and then the solvent was removed under reduced pressure to obtain 54.32 g of a dried product.
 上記分配物から4gを分析用サンプルとして取り除いた後、残りの分配物50.32g全量について、二塩化メチレン及び水で液-液分配を行った。まず、分配物に水(精製水)500mLを加え、その後、二塩化メチレン500mLを加えて1分間振とうした。溶媒が分離するまで10分程度待ち、二塩化メチレン相を採取した。さらに2回同様の操作を繰り返し、得られた塩化メチレン相を併せた後、減圧により溶媒を取り除き、所定の分配物27.55gを得た。なお、以下の記載においては、ここで得られた所定の分配物を「所定の分配物A」と記載する。 After removing 4 g from the above-mentioned distribution as an analytical sample, the remaining 50.32 g of the entire distribution was subjected to liquid-liquid distribution with methylene dichloride and water. First, 500 mL of water (purified water) was added to the distribution, and then 500 mL of methylene dichloride was added and shaken for 1 minute. The solvent was separated for about 10 minutes and the methylene dichloride phase was collected. Further, the same operation was repeated twice, and the obtained methylene chloride phase was combined, and then the solvent was removed under reduced pressure to obtain 27.55 g of a predetermined distribution. In the following description, the predetermined distribution obtained here is referred to as “predetermined distribution A”.
 次に、上記所定の分配物Aのうち20.00gをシリカゲルカラムクロマトグラフィーに付した。
 まず、所定の分配物A20.00gとシリカゲル(0.063~0.2mmの汎用品)60gとを混合した。その後、シリカゲル(0.040~0.063mmの汎用品)500gを詰めたカラム(直径6cm、長さ34cm、体積約204mLのオープンカラム)を用意し、溶出溶媒(n-ヘキサン:酢酸エチル:無水メタノール=3:4:2)で平衡化させた後、所定の分配物Aとシリカゲルとの混合物を乗せ、溶出溶媒による溶出を行った。なお、溶出溶媒1500mLを用いた後、アセトン500mL及び無水エタノール500mLをこの順序でカラムに通し、シリカゲル吸着物を溶出した。
 シリカゲルカラムクロマトグラフィーにおいては、約20mLずつに分けて試験管で採取し、TLCにより溶出のパターンを観察した。おおよその溶出のパターンにより、1~6本目の試験管で得られたものを分画物1-1(溶媒量約0~120mL)、7~24本目の試験管で得られたものを分画物1-2(溶媒量約120~480mL)、25~39本目の試験管で得られたものを分画物1-3(溶媒量約480~780mL)、40~70本目の試験管で得られたものを分画物1-4(溶媒量約780~1400mL)、残りの溶出溶媒、アセトン及び無水エタノールにより得られたものを分画物1-5とした。
 なお、上記分画物1-1~1-4が所定の分画物である。
Next, 20.00 g of the predetermined distribution A was subjected to silica gel column chromatography.
First, 20.00 g of a predetermined distribution A and 60 g of silica gel (a general-purpose product of 0.063 to 0.2 mm) were mixed. Thereafter, a column (open column with a diameter of 6 cm, a length of 34 cm, and a volume of about 204 mL) packed with 500 g of silica gel (0.040 to 0.063 mm general-purpose product) was prepared, and an elution solvent (n-hexane: ethyl acetate: anhydrous) After equilibration with methanol = 3: 4: 2), a mixture of a predetermined partition A and silica gel was placed, and elution with an elution solvent was performed. After using 1500 mL of the elution solvent, 500 mL of acetone and 500 mL of absolute ethanol were passed through the column in this order to elute the silica gel adsorbate.
In silica gel column chromatography, about 20 mL each was collected in a test tube and the elution pattern was observed by TLC. Fractions 1-1 (solvent amount: about 0 to 120 mL) obtained from the 1st to 6th test tubes and those obtained from the 7th to 24th test tubes were fractionated according to the approximate elution pattern. Product 1-2 (solvent amount of about 120 to 480 mL), obtained in the 25th to 39th test tube, fraction 1-3 (solvent amount of about 480 to 780 mL), obtained in the 40th to 70th test tube The obtained product was fraction 1-4 (solvent amount: about 780 to 1400 mL), and the product obtained with the remaining elution solvent, acetone and absolute ethanol was designated fraction 1-5.
The fractions 1-1 to 1-4 are predetermined fractions.
 リナカンチンCの単離は、図2示すフローに沿って行った。所定の分画物Aを得た方法とはシリカゲルカラムクロマトグラフィーの方法が異なるため、上記の方法とは異なる部分についてのみ説明する。
 まず、所定の分配物A2.50gとシリカゲル(0.063~0.2mmの汎用品)10gとを混合した。その後、シリカゲル(0.040~0.063mmの汎用品)200gを詰めたカラム(直径2cm、長さ30cm、体積約60mLのオープンカラム)を用意し、溶出溶媒(n-ヘキサン:酢酸エチル=4:1)で平衡化させた後、所定の分配物Aとシリカゲルとの混合物を乗せ、溶出溶媒による溶出を行った。なお、溶出溶媒の量は500mLとした。
 シリカゲルカラムクロマトグラフィーにおいては、約10mLずつに分けて試験管で採取し、TLCにより溶出のパターンを観察した。おおよその溶出のパターンにより、1本目の試験管で得られたものを分画物2-1(溶媒量約0~10mL)、2~3本目の試験管で得られたものを分画物2-2(溶媒量約10~30mL)、4~10本目の試験管で得られたものを分画物2-3(溶媒量約30~100mL)とした。
Isolation of linacantin C was performed according to the flow shown in FIG. Since the silica gel column chromatography method is different from the method of obtaining the predetermined fraction A, only the portions different from the above method will be described.
First, 2.50 g of a predetermined distribution A and 10 g of silica gel (a general-purpose product of 0.063 to 0.2 mm) were mixed. Thereafter, a column (open column with a diameter of 2 cm, a length of 30 cm, and a volume of about 60 mL) packed with 200 g of silica gel (0.040 to 0.063 mm general-purpose product) was prepared, and an elution solvent (n-hexane: ethyl acetate = 4). After equilibrating in 1), a mixture of a predetermined partition A and silica gel was placed, and elution with an elution solvent was performed. The amount of elution solvent was 500 mL.
In silica gel column chromatography, about 10 mL each was collected in a test tube, and the elution pattern was observed by TLC. According to the approximate elution pattern, the fraction obtained in the first test tube was fraction 2-1 (solvent amount of about 0 to 10 mL), and the fraction obtained in the second and third test tubes was fraction 2. -2 (amount of solvent: about 10 to 30 mL), what was obtained in the fourth to tenth test tubes was designated as fraction 2-3 (amount of solvent: about 30 to 100 mL).
 その後、分画物2-1~2-3についてHNMR及び13CNMRのデータを文献値(ジャーナル オブナチュラル プロダクツ、59巻、808~811ページ、1996年)と比較することで構造の解析を行い、分画物2-2がリナカンチンCであることを確認した。得られたリナカンチンCの量は、444.2mgであった。 Thereafter, the structure of the fractions 2-1 to 2-3 was analyzed by comparing 1 HNMR and 13 CNMR data with literature values (Journal of Natural Products, 59, 808-811, 1996). It was confirmed that the fraction 2-2 was linacantin C. The amount of obtained linacantin C was 444.2 mg.
 なお、核磁気共鳴スペクトル装置としては、JEOL JNM-GSX500型核磁気共鳴スペクトル装置(日本電子株式会社製)を用いた。 As a nuclear magnetic resonance spectrum apparatus, a JEOL JNM-GSX500 type nuclear magnetic resonance spectrum apparatus (manufactured by JEOL Ltd.) was used.
2.所定の分配物、所定の分画物及びリナカンチンCの神経細胞死抑制作用に関する試験例
 当該試験例では、アミロイドβによる細胞毒性及び所定の分配物、所定の分画物及びリナカンチンCの神経細胞死抑制作用についての試験を行った。アミロイドβの神経細胞毒性評価には、アミロイドβの神経毒性の作用中心として知られるアミロイドβ(25-35)を用いた。
2. Test example on neuronal cell death inhibitory action of predetermined partition, predetermined fraction and linacantin C In this test example, cytotoxicity by amyloid β and predetermined partition, predetermined fraction and neuronal cell death of linacantin C A test for inhibitory action was performed. For the evaluation of neurotoxicity of amyloid β, amyloid β (25-35), which is known as the action center of neurotoxicity of amyloid β, was used.
 本試験例では、神経細胞に対する作用(毒性や薬効)を評価する試験に汎く用いられているPC12細胞(ラット副腎褐色細胞腫由来細胞株)を用いた。PC12細胞は、BCRC(Bioresource Collection and Research Center、生物資源保存及研究中心、台湾)から入手したものを用いた。
 入手したPC12細胞は、5%牛胎児血清、10%馬血清、100units/mlペニシリン及び100mg/mlストレプトマイシンを添加したRPMI-1640培地(いずれも、インビトロジェン-ギブコ社から入手)を用い、5%炭酸ガス存在下、37℃、飽和湿度下で培養した。
In this test example, PC12 cells (rat adrenal pheochromocytoma-derived cell line) commonly used in tests for evaluating actions (toxicity and drug efficacy) on nerve cells were used. PC12 cells were obtained from BCRC (Bioresource Collection and Research Center, Bioresource Conservation and Research Center, Taiwan).
The obtained PC12 cells were prepared using RPMI-1640 medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 units / ml penicillin and 100 mg / ml streptomycin (both obtained from Invitrogen-Gibco), 5% carbonic acid. The culture was performed at 37 ° C. and saturated humidity in the presence of gas.
 アミロイドβ(25-35)の細胞毒性の評価には、ポリ-L-リジンをコートした96穴マイクロプレートを用いた。当該マイクロプレートに1穴あたり2×10/100μLのPC12細胞を播種し、5%炭酸ガス存在下、37℃、飽和湿度下で培養した。
 培養24時間後に、コントロール区には0.1%DMSO(J.T.Baker社製)、評価区には各サンプル(下記参照。)、ポジティブコントロール区には10μM ガランタミンハイドロブロマイド(抗アルツハイマー病剤であり、神経保護作用を有することが知られている。シグマ-アルドリッチ社を通じて入手。)を添加した。
A 96-well microplate coated with poly-L-lysine was used to evaluate the cytotoxicity of amyloid β (25-35). The microplate 2 × 10 4 / 100μL of PC12 cells per well were seeded in the presence of 5% carbon dioxide, 37 ° C., and cultured under saturated humidity.
After 24 hours of culture, 0.1% DMSO (manufactured by JT Baker) is used for the control group, each sample (see below) is used for the evaluation group, and 10 μM galantamine hydrobromide (anti-Alzheimer's disease agent) is used for the positive control group. It is known that it has a neuroprotective action, obtained through Sigma-Aldrich.
 全ての試験区について、アミロイドβ(25-35)(シグマ-アルドリッチ社を通じて入手)15μMを添加したものと、添加しないものとの両方について試験を行った(後述)。培養開始72時間後に、MTT(モレキュラー プローブス社製)による所定の方法により、吸光度570nmでの吸光度を測定することで試験区ごとの細胞生存率を算出した。 All the test sections were tested for both those with and without the addition of 15 μM amyloid β (25-35) (obtained through Sigma-Aldrich) (described later). 72 hours after the start of the culture, the cell viability for each test group was calculated by measuring the absorbance at 570 nm by a predetermined method using MTT (Molecular Probes).
 本試験例においては、まず、アミロイドβを添加しない状態でコントロール、ポジティブコントロール及び各サンプルについて細胞生存率を算出した。次に、アミロイドβを添加すること以外は同じ条件で各サンプルについて細胞生存率を算出した。そして、アミロイドβを添加したときの細胞生存率の数値を、アミロイドβを添加していないときの細胞生存率の数値で割る計算を行い、当該計算で算出した数値を100倍して%単位とし、そこからからコントロールにおける細胞生存率を差し引いたものを細胞生存回復率とした。
 この細胞生存回復率は、サンプルによる細胞生存率への影響を補正した状態において、コントロールよりも何%細胞生存率が回復したのかを示す指標である。
In this test example, first, cell viability was calculated for the control, the positive control, and each sample without adding amyloid β. Next, cell viability was calculated for each sample under the same conditions except that amyloid β was added. Then, the value of the cell viability when amyloid β is added is divided by the value of the cell viability when amyloid β is not added, and the value calculated by the calculation is multiplied by 100 to give% units. The cell viability recovery rate was obtained by subtracting the cell viability in the control from there.
This cell survival recovery rate is an index showing how much the cell survival rate has recovered from the control in a state where the influence of the sample on the cell survival rate is corrected.
 サンプルとしては、所定の分配物A(二塩化メチレン分配物)、所定の分画物である分画物1-1、所定の分画物である分画物1-2、所定の分画物である分画物1-3、所定の分画物である分画物1-4、及び、リナカンチンC(分画物2-2)を用いた。なお、各サンプルの濃度については、事前に細胞毒性に関するスクリーニング等を行い、適切な濃度を決定した。
 なお、ポジティブコントロールに関する実験と本発明の各サンプルに関する実験とは分けて行ったので、図3に示すように、それぞれでコントロールにおける細胞生存率の数値が異なっている。
Samples include a predetermined distribution A (methylene dichloride distribution), a predetermined fraction 1-1, a predetermined fraction 1-2, a predetermined fraction. Fraction 1-3, which is a predetermined fraction, and linacantin C (fraction 2-2) were used. In addition, about the density | concentration of each sample, the screening regarding cytotoxicity etc. was performed in advance and the appropriate density | concentration was determined.
Since the experiment relating to the positive control and the experiment relating to each sample of the present invention were conducted separately, as shown in FIG. 3, the numerical values of the cell viability in the control are different.
 試験例の結果を、図3に示す。図3(a)はポジティブコントロールに関する試験結果を示す表であり、図3(b)は本発明の各サンプルに関する試験結果を示す表である。
 図3に示すように、まず、アミロイドβはPC12細胞に対して高い毒性を有することが明らかになった(図3のコントロールの項目参照。)。また、所定の分配物A、所定の分画物(分画物1-1~1-4)及びリナカンチンCは、高い細胞生存回復率を示すことが明らかになった。
 分画物1-1及び分画物1-2による細胞生存回復率はガランタミンハイドロブロマイドに匹敵するものであった。
 また、濃度は1μMと低い値でありながら、リナカンチンCによる細胞生存回復率は30.2%である。10μM ガランタミンハイドロブロマイドによる細胞生存回復率が17.2%であるため、リナカンチンCは特に高い細胞生存回復率を示すという事実を見出すことができた。
The result of the test example is shown in FIG. FIG. 3 (a) is a table showing test results for positive control, and FIG. 3 (b) is a table showing test results for each sample of the present invention.
As shown in FIG. 3, first, it was revealed that amyloid β has high toxicity to PC12 cells (see the control item in FIG. 3). Further, it was revealed that the predetermined distribution A, the predetermined fraction (fractions 1-1 to 1-4) and linacantin C showed a high cell survival recovery rate.
The cell viability recovery rate of the fraction 1-1 and the fraction 1-2 was comparable to that of galantamine hydrobromide.
Further, the cell viability recovery rate by linacantin C is 30.2% while the concentration is as low as 1 μM. Since the cell survival recovery rate by 10 μM galantamine hydrobromide is 17.2%, it was possible to find the fact that linacantin C shows a particularly high cell survival recovery rate.
 上記試験例により、本発明の神経細胞死抑制剤は、アミロイドβの毒性による神経細胞死を抑制する良好な効果、つまり、優れた神経細胞死抑制効果を有することが確認できた。このため、これらを有効成分とする本発明の神経細胞死抑制剤は、抗アルツハイマー病剤及び抗脳機能低下剤としての作用を有すると考えられる。 From the above test examples, it was confirmed that the nerve cell death inhibitor of the present invention has a good effect of suppressing nerve cell death due to the toxicity of amyloid β, that is, an excellent nerve cell death inhibitory effect. For this reason, it is thought that the nerve cell death inhibitor of this invention which uses these as an active ingredient has the effect | action as an anti- Alzheimer's disease agent and an anti-brain function reducing agent.
[実施例]
 以下の実施例により、本発明の神経細胞死抑制剤の有効成分であり、本発明の抗アルツハイマー病剤又は抗脳機能低下剤の有効成分でもあるリナカンチンCを含有する医薬品及び食品の調製法について記載する。なお、リナカンチンCの項目を所定の分配物や所定の分画物に変更することも当然可能である。
[Example]
By the following examples, a method for preparing pharmaceuticals and foods containing linacantin C, which is an active ingredient of the neuronal death inhibitor of the present invention and an active ingredient of the anti-Alzheimer's disease agent or anti-brain function-lowering agent of the present invention. Describe. Of course, it is also possible to change the item of linacantin C to a predetermined distribution or a predetermined fraction.
(1)錠剤
 リナカンチンCを用いて、次の処方で錠剤を作製する。

   リナカンチンC                 0.2g
   乳糖                     95.8g
   乾燥コーンスターチ               2.0g
   タルク                     1.8g
   ステアリン酸カルシウム             0.2g

(調製法)
 乳糖(95.8g)に、リナカンチンC(0.2g)、乾燥コーンスターチ(2g)、タルク(1.8g)、ステアリン酸カルシウム(0.2g)を添加して混合する。次いで、単発式打錠機を用いて常法により錠剤を作製する。
(1) Tablet Using Linacantin C, a tablet is prepared according to the following formulation.

Linacantin C 0.2g
Lactose 95.8g
2.0g dried corn starch
Talc 1.8g
Calcium stearate 0.2g

(Preparation method)
Linacantin C (0.2 g), dried corn starch (2 g), talc (1.8 g), and calcium stearate (0.2 g) are added to lactose (95.8 g) and mixed. Subsequently, a tablet is produced by a conventional method using a single-shot tablet press.
(2)ハードカプセル剤
 リナカンチンCを用いて、次の処方でハードカプセル剤(1カプセルあたり360mg)を作製する。

   リナカンチンC                   5mg
   乳糖                      220mg
   コーンスターチ                 110mg
   ヒドロキシプロピルセルロース           25mg

(調製法)
 リナカンチンC(5g)に、乳糖(220g)及びコーンスターチ(110g)を添加して混合し、これにヒドロキシプロピルセルロース(25g)の水溶液を添加して練合する。次いで、押し出し造粒機を用いて、常法により顆粒を製造する。この顆粒をゼラチンハードカプセルに充填することにより、ハードカプセル剤を作製する。
(2) Hard capsules Using Linacantin C, hard capsules (360 mg per capsule) are prepared according to the following formulation.

Linacantin C 5mg
Lactose 220mg
Corn starch 110mg
Hydroxypropylcellulose 25mg

(Preparation method)
Linacantin C (5 g) is mixed with lactose (220 g) and corn starch (110 g), and an aqueous solution of hydroxypropylcellulose (25 g) is added thereto and kneaded. Next, granules are produced by an ordinary method using an extrusion granulator. A hard capsule is prepared by filling the granule into a gelatin hard capsule.
(3)ソフトカプセル剤
 リナカンチンCを用いて、次の処方でソフトカプセル剤(1カプセルあたり170mg)を作製する。

   リナカンチンC                 0.5mg
   大豆油                   169.5mg

(調製法)
 大豆油(169.5g)に、リナカンチンC(0.5g)を添加して混合する。次いで、ロータリー・ダイズ式自動成型機を用いて、常法に従い、ソフトカプセルに充填することにより、ソフトカプセル剤を作製する。
(3) Soft capsules Using linacantin C, soft capsules (170 mg per capsule) are prepared according to the following formulation.

Linacantin C 0.5mg
Soybean oil 169.5mg

(Preparation method)
Linacantin C (0.5 g) is added to and mixed with soybean oil (169.5 g). Next, soft capsules are prepared by filling soft capsules using a rotary soybean automatic molding machine according to a conventional method.
(4)丸剤
 リナカンチンCを用いて、次の処方で丸剤(1粒あたり100mg)を作製する。

   リナカンチンC                 0.5mg
   モロヘイヤ末                 20.0mg
   デンプン                   30.0mg
   糖蜜                     20.0mg
   茶抽出物                   15.0mg
   大豆ファイバー                14.0mg
   セラック                    0.5mg

(調製法)
 上記配合で原料を混合し、適量加水後、練合機で均質な練合物を製造し、得られた練合物を圧延し製丸機を用いて製丸後乾燥して丸剤を作製する。
(4) Pills Using linacantin C, pills (100 mg per capsule) are prepared according to the following prescription.

Linacantin C 0.5mg
Morohaya powder 20.0mg
Starch 30.0mg
Molasses 20.0mg
Tea extract 15.0mg
Soy fiber 14.0mg
Shellac 0.5mg

(Preparation method)
After mixing the raw materials with the above blending, adding a suitable amount of water, producing a homogeneous kneaded product with a kneader, rolling the kneaded product, making a round shape using a round machine and drying it to produce a pill To do.
(5)散剤
 リナカンチンCを用いて、次の処方で常法により散剤(1包あたり1000mg)を作製する。

   リナカンチンC                   1mg
   乳糖                      799mg
   コーンスターチ                 200mg
(5) Powder Using Linacantin C, a powder (1000 mg per packet) is prepared by the following method using a conventional method.

Linacantin C 1mg
Lactose 799mg
Cornstarch 200mg
(6)ゼリー
 リナカンチンCを用いて、次の処方で、常法によりゼリー(100g)を作製する。

   リナカンチンC               0.002g
   ゼラチン                  2.0g
   オレンジ果汁               20.0g
   水                    77.998g

(調製法)
 上記成分を混合し、90℃へ加熱する。ゼラチンの溶解を確認してから容器に充填し、冷却する。ゼラチンを固化することでゼリーを作製する。
(6) Jelly Using linacantin C, jelly (100 g) is prepared by a conventional method with the following prescription.

Linacantin C 0.002g
Gelatin 2.0g
Orange juice 20.0g
77.998 g of water

(Preparation method)
The above ingredients are mixed and heated to 90 ° C. After confirming the dissolution of gelatin, fill the container and cool. A jelly is prepared by solidifying gelatin.
(7)軟膏
 リナカンチンCを用いて、次の処方で、常法により軟膏(100g)を作製する。

 (油相成分)
   リナカンチンC                 0.1g
   白色ワセリン                 20.0g
   ミネラルオイル                20.0g
   ステアリルアルコール              5.0g
   ステアレス-2                 3.0g
   プロピルパラベン                0.1g
   天然ビタミンE                 0.1g
 (水相成分)
   1,3-ブチレングリコール           5.0g
   フェノキシエタノール              0.4g
   ポリソルベート 60              4.5g
   精製水                    適量
                      全量 100g

(調製法)
 油相成分及び水相成分をそれぞれ80℃に熱して均一にし、水相を油相に攪拌しながら加え、乳化後冷却し軟膏を作製する。
(7) Ointment Using linacantin C, an ointment (100 g) is prepared by a conventional method with the following formulation.

(Oil phase component)
Linacantin C 0.1g
White petrolatum 20.0g
Mineral oil 20.0g
Stearyl alcohol 5.0g
Steareth-2 3.0g
Propylparaben 0.1g
Natural vitamin E 0.1g
(Aqueous phase component)
1,3-butylene glycol 5.0 g
0.4 g of phenoxyethanol
Polysorbate 60 4.5g
Purified water Appropriate amount 100g

(Preparation method)
The oil phase component and the water phase component are each heated to 80 ° C. to be uniform, and the water phase is added to the oil phase with stirring. After emulsification, the mixture is cooled to prepare an ointment.
(8)テープ剤
 リナカンチンCを用いて、次の処方で、常法によりテープ剤(100g)を作製する。

 (粘着剤溶剤)
   スチレン-イソプロピレン-スチレンブロック共重合体  7.0g
   ピコライト                     25.0g
   イソプロピレンゴム                  5.0g
   トルエン                      15.0g
   酢酸エチル                     14.2g
   ヘキサン                      25.0g
 (薬効成分)
   リナカンチンC                    0.1g
   エタノール                      5.0g
 (経皮吸収促進剤)
   オレイルアルコール                  0.8g
                         全量 100g

(調製法)
 粘着剤溶剤及び薬効成分をそれぞれ均一にし、薬効成分及び経皮吸収促進剤を粘着剤溶剤に加え、室温で攪拌し組成物を作製する。この組成物をシリコーン処理したポリエステルフィルム上に延展し、120℃で乾燥させ冷却後、ポリエチレンフィルムへ粘着剤層を転写させ、テープ剤を作製する。
(8) Tape agent A tape agent (100 g) is prepared by a conventional method using linacantin C according to the following formulation.

(Adhesive solvent)
Styrene-Isopropylene-Styrene Block Copolymer 7.0g
Picolite 25.0g
Isopropylene rubber 5.0g
Toluene 15.0g
14.2 g of ethyl acetate
Hexane 25.0g
(Medicinal ingredients)
Linacantin C 0.1g
Ethanol 5.0g
(Transdermal absorption enhancer)
Oleyl alcohol 0.8g
Total amount 100g

(Preparation method)
The pressure-sensitive adhesive solvent and the medicinal component are made uniform, the medicinal component and the transdermal absorption accelerator are added to the pressure-sensitive adhesive solvent, and the composition is stirred at room temperature. This composition is spread on a silicone-treated polyester film, dried at 120 ° C. and cooled, and then the pressure-sensitive adhesive layer is transferred to a polyethylene film to produce a tape agent.

Claims (11)

  1.  白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配するとき、
     前記二塩化メチレンに分配される分配物である所定の分配物のみを有効成分として含有する神経細胞死抑制剤。
    When the ethanol extract of Hakutsuru Reishi is liquid-liquid distributed with hexane and methanol, and the distribution distributed to the methanol is liquid-liquid distributed with methylene dichloride and water,
    The nerve cell death inhibitor which contains only the predetermined | prescribed distribution which is a distribution distributed to the said methylene dichloride as an active ingredient.
  2.  白鶴霊芝のエタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付すとき、
     シリカゲルカラムから前記溶出溶媒により溶出される分画物である所定の分画物のみを有効成分として含有する神経細胞死抑制剤。
    The Hakutsuru Ganodermae ethanol extract is liquid-liquid distributed with hexane and methanol, and the partition distributed to methanol is liquid-liquid distributed with methylene dichloride and water, and the partition distributed to the methylene dichloride is divided into When subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol,
    The nerve cell death inhibitor which contains only the predetermined fraction which is a fraction eluted with the said elution solvent from a silica gel column as an active ingredient.
  3.  請求項2に記載の神経細胞死抑制剤において、
     前記溶出溶媒は、n-ヘキサン:酢酸エチル:無水メタノール=3:4:2の比率からなる溶出溶媒である神経細胞死抑制剤。
    In the nerve cell death inhibitor according to claim 2,
    The elution solvent is a neuronal cell death inhibitor which is an elution solvent having a ratio of n-hexane: ethyl acetate: anhydrous methanol = 3: 4: 2.
  4.  請求項3に記載の神経細胞死抑制剤において、
     前記所定の分画物は、前記シリカゲルカラムクロマトグラフィーに用いるシリカゲルの体積に対して0.1倍~4倍の体積の前記溶出溶媒を用いたときに溶出する分画物である神経細胞死抑制剤。
    In the neuronal cell death inhibitor according to claim 3,
    The predetermined fraction is a neuronal cell death inhibitor that is a fraction eluted when the elution solvent is used in a volume of 0.1 to 4 times the volume of silica gel used in the silica gel column chromatography. Agent.
  5.  請求項4に記載の神経細胞死抑制剤において、
     前記所定の分画物は、前記シリカゲルカラムクロマトグラフィーに用いる前記シリカゲルの体積に対して0.2倍~2.5倍の体積の前記溶出溶媒を用いたときに溶出する分画物である神経細胞死抑制剤。
    In the neuronal cell death inhibitor according to claim 4,
    The predetermined fraction is a nerve fraction that elutes when the elution solvent having a volume of 0.2 to 2.5 times the volume of the silica gel used in the silica gel column chromatography is used. Cell death inhibitor.
  6.  リナカンチンCのみを有効成分として含有する神経細胞死抑制剤。 Neuronal cell death inhibitor containing only linacantin C as an active ingredient.
  7.  請求項1~6のいずれかに記載の神経細胞死抑制剤を有効成分として含有する抗アルツハイマー病剤。 An anti-Alzheimer's disease agent comprising the nerve cell death inhibitor according to any one of claims 1 to 6 as an active ingredient.
  8.  請求項1~6のいずれかに記載の神経細胞死抑制剤を有効成分として含有する抗脳機能低下剤。 An anti-brain function-lowering agent comprising the nerve cell death inhibitor according to any one of claims 1 to 6 as an active ingredient.
  9.  請求項7に記載の抗アルツハイマー病剤又は請求項8に記載の抗脳機能低下剤を含有し、抗アルツハイマー病作用又は抗脳機能低下作用を有する医薬品又は食品。 A pharmaceutical or food comprising the anti-Alzheimer's disease agent according to claim 7 or the anti-brain function-lowering agent according to claim 8, and having an anti-Alzheimer's disease action or an anti-brain function-lowering action.
  10.  白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、
     前記エタノール抽出物をヘキサン及びメタノールで液-液分配し、前記メタノールに分配される分配物を得る第2工程と、
     前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配し、前記二塩化メチレンに分配される分配物である所定の分配物を得る第3工程とをこの順序で含む、前記所定の分配物のみを有効成分として含有する神経細胞死抑制剤の製造方法。
    A first step of extracting white crane reishi with ethanol to obtain an ethanol extract;
    A second step of liquid-liquid partitioning the ethanol extract with hexane and methanol to obtain a partition distributed to the methanol;
    A third step of liquid-liquid partitioning of the partition distributed to the methanol with methylene dichloride and water to obtain a predetermined partition which is a partition distributed to the methylene dichloride, in this order, A method for producing a nerve cell death inhibitor comprising only a predetermined distribution as an active ingredient.
  11.  白鶴霊芝をエタノールで抽出してエタノール抽出物を得る第1工程と、
     前記エタノール抽出物をヘキサン及びメタノールで液-液分配して、前記メタノールに分配される分配物を得る第2工程と、
     前記メタノールに分配される分配物を二塩化メチレン及び水で液-液分配して、前記二塩化メチレンに分配される分配物を得る第3工程と、
     前記二塩化メチレンに分配される分配物をn-ヘキサン、酢酸エチル及び無水メタノールから構成される溶出溶媒を用いるシリカゲルカラムクロマトグラフィーに付して、シリカゲルカラムから前記溶出溶媒により溶出される分画物である所定の分画物を得る第4工程とをこの順序で含む、前記所定の分画物のみを有効成分として含有する神経細胞死抑制剤の製造方法。
    A first step of extracting white crane reishi with ethanol to obtain an ethanol extract;
    A second step of liquid-liquid partitioning the ethanol extract with hexane and methanol to obtain a partition distributed to the methanol;
    A third step of liquid-liquid partitioning the partition distributed to methanol with methylene dichloride and water to obtain a partition distributed to the methylene dichloride;
    The fraction distributed to the methylene dichloride is subjected to silica gel column chromatography using an elution solvent composed of n-hexane, ethyl acetate and anhydrous methanol, and the fraction eluted from the silica gel column with the elution solvent. A method for producing a neuronal cell death inhibitor containing only the predetermined fraction as an active ingredient, in this order.
PCT/JP2015/069363 2015-07-04 2015-07-04 Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor WO2017006407A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN201580081025.1A CN107708717B (en) 2015-07-04 2015-07-04 Application of rhinacanthin quinone C as nerve cell apoptosis inhibitor
PCT/JP2015/069363 WO2017006407A1 (en) 2015-07-04 2015-07-04 Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor
JP2017526812A JP6365914B2 (en) 2015-07-04 2015-07-04 Neuronal cell death inhibitor, anti-Alzheimer's disease agent, anti-brain function-lowering agent, drug or food having anti-Alzheimer's disease action or anti-brain function-lowering effect, method for producing neuronal cell death inhibitor, method for producing anti-Alzheimer's disease agent, Method for producing anti-brain function-lowering agent, and method for producing drug or food having anti-Alzheimer's disease action or anti-brain function-lowering action
TW105118524A TWI610676B (en) 2015-07-04 2016-06-14 Method for producing nerve cell death inhibitor, anti-Alzheimer's disease agent, anti-brain hypofunction agent, medicine having anti-Alzheimer's disease effect or anti-brain function, and nerve cell death inhibitor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2015/069363 WO2017006407A1 (en) 2015-07-04 2015-07-04 Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor

Publications (1)

Publication Number Publication Date
WO2017006407A1 true WO2017006407A1 (en) 2017-01-12

Family

ID=57685054

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/069363 WO2017006407A1 (en) 2015-07-04 2015-07-04 Nerve cell death inhibitor, anti-alzheimer's disease agent, brain hypofunction inhibitor, drug or food having anti-alzheimer's disease effect or brain hypofunction-inhibiting effect, and method for producing nerve cell death inhibitor

Country Status (4)

Country Link
JP (1) JP6365914B2 (en)
CN (1) CN107708717B (en)
TW (1) TWI610676B (en)
WO (1) WO2017006407A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018029793A1 (en) * 2016-08-09 2018-02-15 株式会社Aob慧央グループ Il-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, anti-neuropsychiatric disease agent, medicine and food
CN111671089A (en) * 2020-06-24 2020-09-18 天津市泉又今生物科技有限公司 Application of biological enhanced cell nutrient in preparation of anti-beta-amyloid protein cytotoxicity functional food
CN115385795B (en) * 2022-10-11 2023-11-17 广西中医药大学赛恩斯新医药学院 Preparation method and application of Rhinacanthus nasuta (L.) kurz naphthoquinone monomer Rhinacanthin-C

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011190250A (en) * 2010-02-19 2011-09-29 Arsoa Honsya Corp New compound, antitumor agent and pharmaceutical, food and cosmetic having antitumor action
JP2011190251A (en) * 2010-02-19 2011-09-29 Arsoa Honsya Corp Drug, food, or cosmetic which has anti-obesity agent and fat accumulation inhibiting action
CN102391277A (en) * 2011-10-10 2012-03-28 南京泽朗医药科技有限公司 Method for preparing high-purity rhinacanthin C

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011190250A (en) * 2010-02-19 2011-09-29 Arsoa Honsya Corp New compound, antitumor agent and pharmaceutical, food and cosmetic having antitumor action
JP2011190251A (en) * 2010-02-19 2011-09-29 Arsoa Honsya Corp Drug, food, or cosmetic which has anti-obesity agent and fat accumulation inhibiting action
CN102391277A (en) * 2011-10-10 2012-03-28 南京泽朗医药科技有限公司 Method for preparing high-purity rhinacanthin C

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRIMSON, J.M. ET AL.: "Rhinacanthus nasutus Extracts Prevent Glutamate and Amyloid-beta Neurotoxicity in HT-22 Mouse Hippocampal Cells: Possible Active Compounds Include Lupeol, Stigmasterol and beta-Sitosterol", INT. J. MOL. SCI., vol. 13, 2012, pages 5074 - 5097, XP055342327 *
BRIMSON, J.M. ET AL.: "Rhinacanthus nasutus Protects Cultured Neuronal Cells against Hypoxia Induced Cell Death", MOLECULES, vol. 16, no. 8, 2011, pages 6322 - 6338, XP055342328 *
SIRIPONG, P. ET AL.: "Induction of apoptosis in tumor cells by three naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ", BIOL. PHARM. BULL., vol. 29, no. 10, 2006, pages 2070 - 2076, XP055342335 *

Also Published As

Publication number Publication date
TW201705970A (en) 2017-02-16
JP6365914B2 (en) 2018-08-01
CN107708717B (en) 2021-05-14
JPWO2017006407A1 (en) 2017-09-21
TWI610676B (en) 2018-01-11
CN107708717A (en) 2018-02-16

Similar Documents

Publication Publication Date Title
US9770479B2 (en) Extract of Rehmannia glutinasa Libosch for reducing blood sugar, reducing blood fat, treating leukemia, and preparation method and uses thereof
CN102212093B (en) Flavonoid glycoside compounds, method for preparing same and application
JP5410683B2 (en) Hepatoprotective agent and anti-TNF-α agonist obtained from Kankaniku Juyo
CN1813900B (en) Kadsura longipedunculata lignin extract and its preparing method and use
US20110160152A1 (en) Extracts of aquilaria hulls and use thereof in the treatment of cancer
JP6365914B2 (en) Neuronal cell death inhibitor, anti-Alzheimer's disease agent, anti-brain function-lowering agent, drug or food having anti-Alzheimer's disease action or anti-brain function-lowering effect, method for producing neuronal cell death inhibitor, method for producing anti-Alzheimer's disease agent, Method for producing anti-brain function-lowering agent, and method for producing drug or food having anti-Alzheimer's disease action or anti-brain function-lowering action
CN108472325B (en) Composition for improving memory comprising petasites japonicus leaf extract
TWI673059B (en) IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, anti-psychiatric agent, pharmaceutical
TWI453016B (en) Anti-obesity agents and pharmaceuticals, foods or cosmetics that have a fat accumulation inhibitory effect
CN102875615B (en) Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf
JP5620847B2 (en) Novel compounds, antitumor agents and pharmaceuticals, foods or cosmetics having antitumor activity
CN105796764B (en) Preparation method and application of negundo chastetree fruit total lignans
TWI389701B (en) Extracts of aquilaria hulls and use thereof in the treatment of cancer
KR102175269B1 (en) A pharmaceutical composition comprising compounds isolated from Phlomoides umbrosa(Turcz.) Kamelin and Makhm for preventing or treating cancer
CN104840451B (en) It is a kind of for treating coronary heart disease, the effective ingredient in Chinese of hyperlipidemia, preparation method and the therefrom method of separating effective ingredient
KR101529279B1 (en) Liver cytoprotective composition comprising an extract of brassica juncea coss. and compounds isolated therefrom
KR101349113B1 (en) Pharmacological composition for dementia prevention or treatment comprising specific substance extracted from Hericium erinacium and preparation method thereof
EP3733169A1 (en) Cognitive function improvement agent
CN104523819B (en) A kind of false indigo extract and preparation method thereof
KR102021453B1 (en) Pharmaceutical Composition for Prevention or Treatment of Alzheimer's Disease Including Dendrobium nobile Extract
KR100965304B1 (en) Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27
JP2024008233A (en) Phloroglucinol derivatives
JP5441433B2 (en) Liver function improving agent containing fruit juice
KR20090087684A (en) Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27
KR101057483B1 (en) Composition for the prevention or treatment of nerve cell damage, including 9-hydroxy-alpha-tocopherone

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15897670

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017526812

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15897670

Country of ref document: EP

Kind code of ref document: A1