WO2023166796A1 - Essential oil composition - Google Patents

Essential oil composition Download PDF

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Publication number
WO2023166796A1
WO2023166796A1 PCT/JP2022/043319 JP2022043319W WO2023166796A1 WO 2023166796 A1 WO2023166796 A1 WO 2023166796A1 JP 2022043319 W JP2022043319 W JP 2022043319W WO 2023166796 A1 WO2023166796 A1 WO 2023166796A1
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Prior art keywords
oil
mass
composition
medium
essential
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PCT/JP2022/043319
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French (fr)
Japanese (ja)
Inventor
利彦 岡野
研之 服部
Original Assignee
学校法人 明治薬科大学
カラーズ株式会社
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Priority to JP2023540778A priority Critical patent/JP7436975B2/en
Publication of WO2023166796A1 publication Critical patent/WO2023166796A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13—Coniferophyta (gymnosperms)
    • A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/534—Mentha (mint)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to a composition and the like for suppressing activation of microglial cells.
  • Microglial cells are small, non-neuronal stromal cells of mesodermal origin that form part of the supporting tissue of the central nervous system. Normally, microglial cells have multiple elongated branching processes called laminid type. When stimuli such as nerve cell injury are applied, microglial cells are activated and morphologically change to amoeba-like ameboids. In recent years, activation of microglial cells in the brain can cause depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, and Huntington's disease. It is known that activation of microglial cells in the spinal cord can cause chronic fatigue syndrome.
  • Essential oils refer to oils containing volatile aromatic components that are mainly extracted from plants, and are used as aromatherapy, fragrances, and skin emollients, as well as anti-inflammatory and antibacterial effects. reported to have It has also been reported that some essential oil components migrate into the brain when volatilized essential oils are inhaled. However, the relationship between essential oils and microglial cells, particularly the effect of essential oils on the activation of microglial cells, has not been investigated.
  • One of the objects of the present invention is to provide compositions such as foods, drinks, cosmetics, etc. for suppressing the activation of microglial cells.
  • One of the objects of the present invention is to provide compositions for inhibiting the release of IL-1 ⁇ .
  • One of the objects of the present invention is to provide a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells.
  • One of the objects of the present invention is to provide a pharmaceutical composition for treating or preventing neurological diseases caused by activation of microglial cells.
  • the present inventors have discovered that a specific essential oil has an effect of suppressing the activation of microglial cells and/or an effect of suppressing the release of IL-1 ⁇ , leading to the present invention. More specifically, the present invention can have the following aspects.
  • 1 essential oil selected from the group consisting of citrus medica vulgaris pericarp oil, cypress oil, lemon pericarp oil, lime oil, red pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil
  • a composition for inhibiting activation of microglial cells comprising more than one species.
  • the essential oil is one or more essential oils selected from the group consisting of cypress oil and Roman chamomile flower oil.
  • composition according to [1] or [2] above wherein the content of the essential oil is 0.001 to 30% by mass when the composition is 100% by mass.
  • composition according to any one of [1] to [3] above wherein the composition is a pharmaceutical composition, food or drink, cosmetics, animal feed, or miscellaneous goods.
  • composition according to any one of [1] to [3] above which is a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells.
  • the suppression of inflammation is accompanied by suppression of the release of pro-inflammatory cytokines selected from IL-1 ⁇ , IL-1 ⁇ , IL-6, IFN- ⁇ , MCP-1 and TNF- ⁇ ;
  • pro-inflammatory cytokines selected from IL-1 ⁇ , IL-1 ⁇ , IL-6, IFN- ⁇ , MCP-1 and TNF- ⁇ ; The composition according to [5] above.
  • the neurological disease is selected from depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's chorea, and chronic fatigue syndrome, [8 ] The pharmaceutical composition as described in ].
  • the essential oil is one or more essential oils selected from the group consisting of Nelumbo japonicum oil and Roman chamomile flower oil.
  • the present invention it is possible to provide an effect of suppressing activation of microglial cells and/or an effect of suppressing release of IL-1 ⁇ . Moreover, according to the present invention, it is possible to provide an effect of suppressing inflammation caused by activation of microglial cells and an effect of treating or preventing neurological diseases caused by activation of microglial cells.
  • compositions comprising a particular essential oil.
  • the composition is not limited to forms such as solution, gel, solid, and mist as long as it is the essential oil itself or a mixture containing the essential oil.
  • Compositions may be not only pharmaceutical compositions and formulations, but also food and drink, cosmetics, animal feeds, miscellaneous goods, etc., and existing pharmaceutical compositions, food and drink, and cosmetics using the composition as a raw material. , animal feed, miscellaneous goods, and the like.
  • An essential oil means an oil obtained mainly by extraction from plants, preferably an oil containing volatile aromatic components.
  • Essential oils include animal-derived essential oils and plant-derived essential oils, and plant-derived essential oils are preferred in the present invention.
  • the essential oil is preferably liquid at room temperature (25°C ⁇ 5°C).
  • essential oils with high volatility are preferable because the active ingredients in the essential oils can be taken into the body by inhalation as in the case of using an aroma diffuser, and they are a simple administration form.
  • the part of the plant from which the essential oil is extracted is not particularly limited, but examples include leaves, roots, stems, bark, seeds, flowers, fruits, fruit peels, and combinations thereof. The plant can be used as it is or after drying and crushing the plant.
  • the essential oils that can be used as active ingredients in the present invention include Citrus medica vulgaris pericarp oil, Pyrrhiza medicinalis oil, Lemon pericarp oil, Lime oil, Scots pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. one or more essential oils selected from the group;
  • Citrus medica vulgaris pericarp oil refers to oil containing aromatic components obtained by extraction from the pericarp of Citrus medica vulgaris.
  • the oil of Nyukoju is an oil containing aromatic components obtained by extracting a part or the whole of Nyukoju, including the wood, bark or sap of Nyukoju.
  • Lemon peel oil refers to oil containing aromatic components obtained by extraction from lemon peel.
  • Lime oil refers to oil containing aromatic components obtained by extracting a part or the whole of lime, including lime fruit, peel or pulp.
  • Scots pine leaf oil refers to oil containing aromatic components obtained by extracting from the leaves of Scots pine.
  • Roman chamomile flower oil refers to oil containing aromatic components obtained by extraction from Roman chamomile flowers.
  • Spearmint oil refers to oil containing aromatic components obtained by extracting a part or the whole of spearmint including leaves, stems, roots, etc. of spearmint.
  • Ylang-ylang flower oil refers to oil containing aromatic components obtained by extracting from ylang-ylang flowers. A preferred extraction method will be described later.
  • These essential oils were specifically found to have remarkable effects such as suppression of activation of microglial cells, and are preferred. This effect is surprising given that bergamot fruit oil, mint leaf oil and eucalyptus leaf oil, which are similar to these essential oils, do not exhibit this effect.
  • These essential oils that can be used as active ingredients in the present invention may be used alone or in combination of two or more. When two types of essential oils are used in combination, the mass ratio is, for example, 99:1 to 1:99, preferably 9:1 to 1:9, more preferably 5:1 to 1:5, still more preferably 2:1. ⁇ 1:2 is suitable.
  • the content of the essential oil in the composition of the present invention is, when the composition is 100% by mass, for example, 0.001 to 30% by mass, preferably 0.01 to 20% by mass, more preferably 0.01 to 10% by mass, more preferably 0.1 to 10% by mass.
  • the content of the essential oil in the composition of the present invention is, for example, 0.001 to 30% by mass, preferably 0.01 to 20% by mass, more preferably It is suitably 0.01 to 10% by mass, more preferably 0.1 to 10% by mass.
  • the content of the essential oil varies depending on the form of the composition. may be 30 to 100% by mass, 70 to 100% by mass, or 95 to 98% by mass.
  • the composition of the present invention may contain other essential oils and other active ingredients in addition to the above essential oils as active ingredients. Although other essential oils cannot be expected to have the action and effect of the present invention, other relaxing effects, moisturizing effects, and the like can be obtained.
  • Other essential oils include yuzu peel oil, bergamot leaf oil, bergamot fruit oil, damask rose flower oil, shouzuku seed oil, coccineum oil, chamomile flower oil, salvia oil, eucalyptus leaf oil, sycamore oil, ginger root oil.
  • active ingredients are the effect of the present invention such as the effect of suppressing the activation of microglial cells and the effect of suppressing the release of IL-1 ⁇ , and other effects such as antibacterial, insecticidal, anti-inflammatory.
  • active ingredients include, for example, ⁇ -lactam antibacterial agents, fosfomycin, vancomycin, minocycline, ciclesonide, and the like.
  • the content of other active ingredients in the composition can be added in any amount as long as it is an amount that does not impair the effect of the original active ingredient (essential oil). It is suitable to contain 90 mass %, preferably 0.01 mass % to 70 mass %, more preferably 0.1 mass % to 50 mass %.
  • composition of the present invention may optionally contain excipients, binders, emulsifiers, solvents, adhesives, disintegrants, thickeners, lubricants, colorants, Additives such as flow agents and moisturizing agents may be added.
  • excipients include alcohol, glycerin, dextrin, starch and the like.
  • Binders can include, for example, cellulose, methylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose.
  • emulsifiers include lecithin, polyethylene glycol (PG), glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester and the like.
  • Solvents include, for example, monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, propylene glycol, dipropylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, tetrahydrofuran (THF ); organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane, and dimethylformamide; avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil , persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, linagiri oil, Japanese paul
  • Preferred solvents include ethanol, olive oil, squalane, myristyl myristate and water. These additives can be used singly or in combination of two or more. Those that act as both excipients and solvents, such as alcohols, act as both excipient and solvent additives even when one is included.
  • the content of the additive in the composition can be added in any amount as long as it is an amount that does not impair the effect of the active ingredient. It is suitable to contain from 0.1% to 30% by mass, more preferably from 0.1% by mass to 50% by mass.
  • the organic solvent is, for example, 1% by mass to 99.9% by mass, preferably 1% by mass to 99% by mass, more preferably about 20% by mass to 90% by mass. It is appropriate to contain
  • the oil content is, for example, 0.01 to 95% by mass, preferably 1 to 90% by mass, more preferably 5 to 80% by mass.
  • a method for producing an essential oil a method generally used for preparing an essential oil can be used, and a method generally known as a method of extracting a plant to obtain an essential oil can be used.
  • the extraction method for example, pressing, squeezing, solvent extraction, steam distillation, supercritical extraction, carbon dioxide gas extraction, cold immersion method, digestion method, etc. can be used alone or in combination.
  • the essential oil can be obtained by optionally removing the solvent and the like from the liquid extract obtained by these extraction methods.
  • extraction solvents include monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, propylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, tetrahydrofuran (THF) and the like. ethers; organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane and dimethylformamide; and water.
  • the extraction conditions for extraction vary depending on the plant and solvent used, but the temperature, pressure, extraction time, etc. generally used in extraction methods can be used.
  • the steam distillation method using steam at a temperature of 40 to 120 ° C., preferably 50 to 100 ° C. below the boiling point of general essential oils (150 to 350 ° C.), for example, 5 to 200 kPa, preferably 10 to 100 kPa may be carried out under a reduced pressure of
  • the extraction method using an alcohol such as ethanol may be carried out at room temperature (25° C. ⁇ 5° C.) under high pressure of 300 to 1000 kPa, preferably about 500 to 800 kPa.
  • the extraction solvent may be removed after the extraction operation.
  • compositions of the present invention can be used not only in pharmaceutical compositions, but also in foods and beverages, cosmetics, animal feeds, and miscellaneous goods.
  • Pharmaceutical compositions include, for example, oral administration agents such as tablets, pills, capsules, powders, granules, syrups, and sublingual agents; injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, Parenteral administration agents such as drops, external agents (ointments, creams, poultices, patches, inhalants), and suppositories can be mentioned.
  • Food and drink products include beverages and foods, and examples of beverages include tea, coffee, soft drinks, fruit juice drinks, carbonated drinks, sports drinks, non-alcoholic drinks, and alcoholic drinks.
  • Foods in general given to humans such as plant foods, animal foods, synthetic foods, cereals, bread, noodles, cakes, noodles, freeze-dried foods, dairy products, seasonings, frozen foods, nutritional supplements , and medical foods.
  • cosmetics include lotions, milky lotions, moisturizers, serums, skin oils, perfumes, sunscreens, shaving lotions, packs, hair tonics, hair balms, hair waxes, hair greases, hair oils, hair treatments, hair colors, and face washes.
  • cosmetics body shampoos, shampoos, rinses, conditioners, cleansers, creams, body creams, hand creams, makeup bases, foundations, mascara, eyeliners, concealers, lipsticks, lip balms, glosses and nail colors.
  • Animal feeds include, for example, feeds for animals such as dogs, cats, horses, sheep, pigs, monkeys, rabbits, guinea pigs, mice, birds, chickens, ducks, and goats, especially mammals.
  • miscellaneous goods include aromatics, aroma diffusers, aroma candles, aroma stones, aroma mists, clothing mists, deodorant mists, pillow mists, insect repellents, clothing detergents, softeners, and bath agents.
  • the content of the composition depends on the type of pharmaceutical composition, etc., but is an amount that does not impair the effects of the present invention.
  • 0.001% to 70% by mass preferably 0.01% to 50% by mass, more preferably 0.1% to 30% by mass with respect to the total amount
  • animal feeds it is suitable to contain, for example, 0.001% to 50% by mass, preferably 0.01% to 30% by mass, more preferably 0.1% to 10% by mass.
  • miscellaneous goods it is suitable to contain, for example, 0.01% by mass to 100% by mass, preferably 0.01% by mass to 80% by mass, more preferably 0.1% by mass to 50% by mass.
  • the composition of the present invention is used to suppress activation of microglial cells (Method for Suppressing Microglial Cell Activation).
  • Microglial cells are small, non-neuronal stromal cells of mesodermal origin that form part of the supporting tissue of the central nervous system.
  • Essential oils are generally known to have anti-inflammatory effects, but they are not known to have the effect of suppressing the activation of microglial cells. was found to be effective. It is surprising to see such an effect, and it is expected that diseases and conditions caused by activation of microglial cells can be alleviated and cured.
  • the composition of the present invention can also be used to treat or prevent neurological diseases caused by activation of microglial cells (method for treating or preventing neurological diseases caused by activation of microglial cells).
  • neurological diseases include depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's disease, chronic fatigue syndrome, and the like.
  • the composition of the present invention is also used to inhibit the release of IL-1 ⁇ (IL-1 ⁇ release inhibition method). In particular, it was found that by using the specific essential oil of the present invention, an IL-1 ⁇ production inhibitory effect can be obtained without showing substantial or significant cytotoxicity.
  • the composition of the present invention is also used to suppress inflammation caused by activation of microglial cells (method for suppressing inflammation caused by activation of microglial cells). Inflammation here mainly includes inflammation in the brain and spinal cord. Examples of intracerebral sites where inflammation occurs include hippocampus, cerebral cortex, cerebellum, basal ganglia, and nigrostriatum. The spinal cord site where inflammation occurs includes the dorsal horn of the spinal cord and the like.
  • the inflammation is, for example, an inflammation selected from IL-1 ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-12, IL-17, IL-18, IFN- ⁇ , MCP-1 and TNF- ⁇ Expression of an inducible cytokine, preferably with release (expression) of a pro-inflammatory cytokine selected from IL-1 ⁇ , IL-1 ⁇ , IFN- ⁇ , MCP-1 and TNF- ⁇ .
  • a pro-inflammatory cytokine selected from IL-1 ⁇ , IL-1 ⁇ , IFN- ⁇ , MCP-1 and TNF- ⁇ .
  • the composition of the present invention particularly the specific essential oil as an active ingredient, suppresses the release (expression) of pro-inflammatory cytokines such as IL-1 ⁇ , thereby suppressing the activation of microglial cells.
  • microglial cells include MG6 cells, iCell (R) microglia, and BV2 cells.
  • composition of the present invention is in the form of a pharmaceutical composition or food (especially functional food or dietary supplement), it varies depending on the dosage form, administration subject, administration route, target disease, symptom, active ingredient concentration, etc.
  • the dosage of the active ingredient per day is, for example, 0.1 to 20 mg/kg body weight, preferably 0.2 to 10 mg/kg body weight, more preferably 0.5 to 10 mg/kg body weight. , it is desirable to administer this amount once to several times a day (eg, 2, 3, 4 or 8 times).
  • the amount is, for example, 0.1 to 20 mg/day, preferably 0.2 to 10 mg/day, more preferably 0.5 to 10 mg/day. is appropriate.
  • the administration method varies depending on the dosage form and form of the pharmaceutical composition or food. For example, when a liquid agent is administered to a subject by inhalation, it is preferably administered by an aroma diffuser, a spray, a mist, or the like.
  • Example 1 [Preparation of composition] As each essential oil, the essential oils listed in Table 2 were used. A composition of each essential oil was prepared as a 10% (w/w) ethanol solution by weighing 2-3 mg of each essential oil and adding 9 volumes (w/w) of ethanol.
  • Comparative Example 1 [Preparation of composition] The essential oils listed in Table 3 were used as comparative essential oils. As in Example 1, 2-3 mg of the essential oil was weighed and 9 times the amount (w/w) of ethanol was added to prepare each composition as a 10% (w/w) ethanol solution.
  • MG6 cells a mouse-derived microglial cell line, were purchased from the RIKEN Cell Bank (cell number RCB2403).
  • the medium used for culture was D-MEM (manufactured by Fujifilm Wako, product number; 049-32645) 500mL, fetal bovine serum (manufactured by Biofill) 50mL was added, and insulin (Fujifilm Wako Pure Chemical Co., Ltd. 099-06473) and 2-mercaptoethanol at final concentrations of 10 ⁇ g/mL and 0.1 mmol/L, respectively (hereinafter referred to as medium A).
  • MG6 cells were cultured according to the method specified by Riken's cell bank.
  • MG6 cells were seeded in a 24-well plate at 0.5 ⁇ 10 5 cells/well, and the next day, the adherence of the cells was confirmed. , performed the following: First, medium 1 was prepared by adding LPS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181) to medium A at a final concentration of 1 ⁇ g/mL.
  • LPS manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181
  • medium 2 was prepared by adding ATP (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511) to a new medium A in such an amount that the final concentration was 1 mmol/L. After removing the medium from each well, 0.5 mL/well of medium 1 was added to the MG6 cells cultured in the 24-well plate prepared as described above, and cultured for 4 hours. Next, the medium in each well was removed, 0.5 mL/well of medium 2 was added to each well of MG6 cells, and the cells were cultured for 30 minutes.
  • ATP manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511
  • the medium was collected from each well as a medium for measuring activation, and a solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the new medium A was prepared. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.
  • medium 3 or medium 3 prepared in the same manner as medium 1 or medium 2, except that LPS or ATP was added at a final concentration twice that of medium 1 and medium 2, respectively.
  • Medium 4 was prepared.
  • 10 ⁇ L of each composition (10% (w/w) ethanol solution of essential oil) prepared in Example 1 and Comparative Example 1 was aliquoted and added to 5 mL of Medium A. After that, it was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 5.
  • an ultrasonic homogenizer manufactured by Branson, product number: SFX150
  • MG6 cells were seeded in a 24-well plate at 0.5 ⁇ 10 5 cells/well in the same manner as in the previous [Method for activating microglia with LPS and ATP using MG6 cells].
  • MG6 cells cultured on a 24-well plate were prepared. After removing the medium from each well of the MG6 cells cultured on the prepared 24-well plate, 0.5 mL/well of medium 6 was added to each well, and the MG6 cells were cultured for 4 hours. Next, after removing the medium from each well, 0.5 mL/well of medium 7 was added to each well, and the MG6 cells were further cultured for 30 minutes.
  • the medium was collected from each well as a medium for measuring activation, and a solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the new medium A was prepared. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.
  • MCC950 is an inhibitor of NLRP3 inflammasome, MCC950 (Funakoshi Co., Ltd., product number: AG-CR1-3615-M001), added to medium 1 and medium 2 to a final concentration of 10 ⁇ mol / L. It was used as a positive control to confirm that IL-1 ⁇ release was actually suppressed.
  • the IL-1 ⁇ concentrations in Tables 4-1 to 4-5 are average values measured three times.
  • the cytotoxicity rate was measured by LDH assay.
  • LDH assay kit product number: 341-91754 manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.
  • the total LDH activity in the medium for activation measurement in each well and the cell lysate was defined as the total LDH activity, and the ratio of the LDH activity in the cell lysate to the total LDH activity was expressed as a cytotoxicity rate in %.
  • control and MCC950 used are the same as in Tables 4-1 to 4-5.
  • essential oils that do not show a significant increase in the cytotoxicity rate compared to the control, those that suppress the release of IL-1 ⁇ into the medium are particularly effective in suppressing the activation of MG6 cells. I assumed.
  • the cytotoxicity rates in Tables 5-1 to 5-5 are the average values measured three times.
  • iCell (R) microglia [IL-1 ⁇ release inhibition test for iCell (R) microglia] ⁇ Preparation of cell lysate containing human iPS cell-derived microglial cells and no essential oil (for control) As microglial cells, iCell (R) microglia (manufactured by FUJIFILM Cellular Dynamics, Inc.), which are human iPS cell-derived microglial cells, were used. Note that iCell is a registered trademark of Fujifilm Cellular Dynamics Inc. iCell (R) microglia were cultured according to the culture method recommended by Fujifilm Wako Pure Chemical Industries, Ltd.
  • iCell (R) microglial basal medium manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 556-36861
  • iCell (R) microglial supplement A product number: 550-36881
  • Microglial supplement B product number: 557-36891
  • iCell (R) nerve supplement C product number: 553-36871
  • medium B medium
  • the adhesiveness of iCell (R) microglia is weak, the culture vessel was coated with Cell-Tak cell and tissue adhesive (Cell-Tak, manufactured by Corning, product number: CLS354240) during the culture.
  • the iCell (R) microglia were suspended in the medium B at 1 ⁇ 10 5 cells/mL, and seeded onto a 48-well plate at 0.25 mL/well. After seeding, centrifugation was performed at 20°C and 1,000 rpm for 5 minutes using a Tomy centrifuge (part number: CAX-571) and a rotor (part number: TS-4C) to spread the cells to the bottom of the plate. Collected to promote adhesion. Two days after seeding, the medium was removed, and medium 8 obtained by adding LPS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181) at a final concentration of 1 ⁇ g/mL to the new medium B was added to each well.
  • LPS manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181
  • iCell (R) microglia were cultured for 4 hours.
  • the medium 8 was removed from each well, and ATP (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511) was added to the new culture medium B so that the final concentration was 1 mmol/L.
  • 0.25 mL/well of medium 9 was added and the iCell (R) microglia were further cultured for 30 minutes.
  • the medium After culturing for 30 minutes, the medium is collected from each well as a medium for measuring activation, and the remaining iCell (R) microglia is added to 10 mL of medium B as a medium for MTT assay (manufactured by Dojindo Laboratories, product number: 0.25 mL/well of a mixed solution containing 1 mL of CK04) was added to each well and cultured for 4 hours. The culture medium after culturing for 4 hours was used as a cell viability measurement medium.
  • MTT assay manufactured by Dojindo Laboratories, product number: 0.25 mL/well of a mixed solution containing 1 mL of CK04
  • medium 10 or medium 10 prepared in the same manner as medium 8 or medium 9, except that LPS or ATP was added at a final concentration twice that of medium 8 or medium 9, respectively.
  • a medium 11 was prepared.
  • 10 ⁇ L of each composition (10% (w/w) ethanol solution of essential oil) prepared in Example 1 and Comparative Example 1 above was aliquoted and added to 5 mL of Medium B above. After that, it was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 12. 3 mL each of this medium 12 and medium 10 or medium 11 were mixed to adjust the final concentration of each essential oil in the culture medium containing LPS or ATP to 100 ppm, which was designated as medium 13 or 14, respectively.
  • iCell (R) microglia were suspended in the above medium B to a concentration of 1 x 10 5 cells/mL, and 0.0. Seeded at 25 mL/well. After removing the medium from each well of iCell (R) microglial cells cultured on the 48-well plate, 0.25 mL/well of medium 13 was added to each well, and MG6 cells were cultured for 4 hours.
  • MTT reagent manufactured by Dojindo Laboratories, product number: 0.25 mL/well of a mixed solution containing 1 mL of CK04 was added to each well and cultured for 4 hours. The culture medium after culturing for 4 hours is used as the cell viability measurement medium.
  • MTT assay ⁇ Measurement of absorbance
  • the relative absorbance value (%) of each addition group was defined as the survival rate.
  • the effect of suppressing the activation of microglial cells derived from human iPS cells was excellent.
  • the cytotoxicity rate in Table 6 is the average value measured three times.
  • Formulation Example 1 [Aroma Diffuser Oil 1] Nyukoju oil (FRANKINCENSE OIL, Boswellia Carterii Oil, Cas RN (R) : 89957-98-2): 30% by mass ethanol: residue

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Abstract

The purpose of the present invention is to provide, for example, a composition for suppressing the activation of microglial cells. Specifically, the present invention provides, for example, a composition that is for suppressing the activation of microglial cells and that comprises one or more essential oils selected from the group consisting of Citrus medica vulgaris peel oil, Boswellia carterii oil, lemon peel oil, lime oil, Pinus sylvestris leaf oil, Anthemis nobilis flower oil, spearmint oil, and ylang-ylang flower oil.

Description

精油組成物essential oil composition
この発明は、ミクログリア細胞の活性化を抑制するための組成物等に関する。 The present invention relates to a composition and the like for suppressing activation of microglial cells.
ミクログリア細胞は、中枢神経系の支持組織の一部を構成する中胚葉起源の小型、非ニューロン性間質細胞である。通常、ミクログリア細胞は、ラミファイド型と呼ばれる複数の細長い分岐突起を有する。神経細胞の傷害などの刺激が加わると、ミクログリア細胞が活性化されてアメーバ状のアメボイド型に形態が変化する。近年、脳内のミクログリア細胞の活性化が、うつ病、統合失調症、認知症、アルツハイマー病、パーキンソン病、多発硬化症、側索硬化症、及びハンチントン舞踏病などの原因となり得ること、また、脊髄内のミクログリア細胞の活性化が慢性疲労症候群の原因となり得ることが知られている。一方、精油は、主に植物から抽出して得られる揮発性の芳香成分を含む油分を意味し、アロマテラピーや芳香剤、皮膚への塗布剤として使用されるほか、抗炎症作用や抗菌作用も有することが報告されている。一部の精油成分には、揮発した精油を吸入すると、精油の成分が脳内に移行することも報告されている。
しかし、精油とミクログリア細胞との関係、特に、精油におけるミクログリア細胞の活性化を抑制する効果について、検討されてこなかった。 Microglial cells are small, non-neuronal stromal cells of mesodermal origin that form part of the supporting tissue of the central nervous system. Normally, microglial cells have multiple elongated branching processes called laminid type. When stimuli such as nerve cell injury are applied, microglial cells are activated and morphologically change to amoeba-like ameboids. In recent years, activation of microglial cells in the brain can cause depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, and Huntington's disease. It is known that activation of microglial cells in the spinal cord can cause chronic fatigue syndrome. Essential oils, on the other hand, refer to oils containing volatile aromatic components that are mainly extracted from plants, and are used as aromatherapy, fragrances, and skin emollients, as well as anti-inflammatory and antibacterial effects. reported to have It has also been reported that some essential oil components migrate into the brain when volatilized essential oils are inhaled.
However, the relationship between essential oils and microglial cells, particularly the effect of essential oils on the activation of microglial cells, has not been investigated.
特開２０２１－６３０１８号公報Japanese Patent Application Laid-Open No. 2021-63018 国際公開第２０１８／０２９７９３号WO2018/029793
本発明の目的の一つは、ミクログリア細胞の活性化を抑制するための組成物、例えば、飲食品や化粧品等を提供することである。本発明の目的の一つは、ＩＬ-１βの放出を抑制するための組成物を提供することである。本発明の目的の一つは、ミクログリア細胞の活性化に起因する炎症を抑制するための医薬組成物を提供することである。本発明の目的の一つは、ミクログリア細胞の活性化に起因する神経性疾患を治療又は予防するための医薬組成物を提供することである。 One of the objects of the present invention is to provide compositions such as foods, drinks, cosmetics, etc. for suppressing the activation of microglial cells. One of the objects of the present invention is to provide compositions for inhibiting the release of IL-1β. One of the objects of the present invention is to provide a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells. One of the objects of the present invention is to provide a pharmaceutical composition for treating or preventing neurological diseases caused by activation of microglial cells.
本発明者らは、特定の精油にはミクログリア細胞の活性化を抑制する効果があることを見出し、及び／又はＩＬ-１βの放出を抑制する効果があることを見出し、本発明に至った。より具体的には、本発明は以下の態様であり得る。
〔１〕シトルスメジカブルガリス果皮油、ニュウコウジュ油、レモン果皮油、ライム油、セイヨウアカマツ葉油、ローマカミツレ花油、スペアミント油、及びイランイラン花油からなる群から選択される精油を１種以上含む、ミクログリア細胞の活性化を抑制するための組成物。
〔２〕前記精油が、ニュウコウジュ油及びローマカミツレ花油からなる群から選択される１種以上の精油である、前記〔１〕に記載の組成物。
〔３〕前記精油の含有量が、前記組成物を１００質量％とした場合、０．００１～３０質量％である、前記〔１〕又は〔２〕に記載の組成物。
〔４〕前記組成物が、医薬組成物、飲食品、化粧品、動物用飼料、又は雑貨品である、前記〔１〕～〔３〕のいずれかに記載の組成物。
〔５〕前記組成物が、ミクログリア細胞の活性化に起因する炎症を抑制するための医薬組成物である、前記〔１〕～〔３〕のいずれかに記載の組成物。
〔６〕前記炎症の抑制が、ＩＬ-１α、ＩＬ-１β、ＩＬ-６、ＩＦＮ－γ、ＭＣＰ－１及びＴＮＦ－αから選択される炎症誘導性サイトカインの放出の抑制を伴うものである、前記〔５〕に記載の組成物。
〔７〕前記炎症が、脳内及び／又は脊髄内の炎症である、前記〔５〕又は〔６〕に記載の医薬組成物。
〔８〕前記組成物が、ミクログリア細胞の活性化に起因する神経性疾患を治療又は予防するための医薬組成物である、前記〔１〕～〔３〕のいずれかに記載の組成物。
〔９〕前記神経性疾患が、うつ病、統合失調症、認知症、アルツハイマー病、パーキンソン病、多発硬化症、側索硬化症、ハンチントン舞踏病、及び慢性疲労症候群から選択される、前記〔８〕に記載の医薬組成物。
〔１０〕シトルスメジカブルガリス果皮油、ニュウコウジュ油、レモン果皮油、ライム油、セイヨウアカマツ葉油、ローマカミツレ花油、スペアミント油、及びイランイラン花油からなる群から選択される精油を１種以上含む、ＩＬ-１βの放出を抑制するための組成物。
〔１１〕前記精油が、ニュウコウジュ油及びローマカミツレ花油からなる群から選択される１種以上の精油である、前記〔１０〕に記載の組成物。
〔１２〕前記精油の含有量が、組成物を１００質量％とした場合、０．００１～３０質量％である、前記〔１０〕又は〔１１〕に記載の組成物。 The present inventors have discovered that a specific essential oil has an effect of suppressing the activation of microglial cells and/or an effect of suppressing the release of IL-1β, leading to the present invention. More specifically, the present invention can have the following aspects.
[1] 1 essential oil selected from the group consisting of citrus medica vulgaris pericarp oil, cypress oil, lemon pericarp oil, lime oil, red pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil A composition for inhibiting activation of microglial cells, comprising more than one species.
[2] The composition according to [1] above, wherein the essential oil is one or more essential oils selected from the group consisting of cypress oil and Roman chamomile flower oil.
[3] The composition according to [1] or [2] above, wherein the content of the essential oil is 0.001 to 30% by mass when the composition is 100% by mass.
[4] The composition according to any one of [1] to [3] above, wherein the composition is a pharmaceutical composition, food or drink, cosmetics, animal feed, or miscellaneous goods.
[5] The composition according to any one of [1] to [3] above, which is a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells.
[6] the suppression of inflammation is accompanied by suppression of the release of pro-inflammatory cytokines selected from IL-1α, IL-1β, IL-6, IFN-γ, MCP-1 and TNF-α; The composition according to [5] above.
[7] The pharmaceutical composition of [5] or [6] above, wherein the inflammation is intracerebral and/or spinal cord inflammation.
[8] The composition according to any one of [1] to [3] above, which is a pharmaceutical composition for treating or preventing a neurological disease caused by activation of microglial cells.
[9] The neurological disease is selected from depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's chorea, and chronic fatigue syndrome, [8 ] The pharmaceutical composition as described in ].
[10] 1 essential oil selected from the group consisting of citrus medica vulgaris pericarp oil, citrus medica vulgaris pericarp oil, nephrolithus oil, lemon pericarp oil, lime oil, red pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil A composition for inhibiting the release of IL-1β, comprising more than one species.
[11] The composition according to [10] above, wherein the essential oil is one or more essential oils selected from the group consisting of Nelumbo japonicum oil and Roman chamomile flower oil.
[12] The composition according to [10] or [11] above, wherein the content of the essential oil is 0.001 to 30% by mass based on 100% by mass of the composition.
本発明によれば、ミクログリア細胞の活性化を抑制する効果、及び／又はＩＬ-１βの放出を抑制する効果を提供することができる。また、本発明によれば、ミクログリア細胞の活性化に起因する炎症を抑制する効果、及びミクログリア細胞の活性化に起因する神経性疾患を治療又は予防する効果を提供することができる。 According to the present invention, it is possible to provide an effect of suppressing activation of microglial cells and/or an effect of suppressing release of IL-1β. Moreover, according to the present invention, it is possible to provide an effect of suppressing inflammation caused by activation of microglial cells and an effect of treating or preventing neurological diseases caused by activation of microglial cells.
ここで、発明を実施するための形態を詳説するが、以下で例示する好ましい態様やより好ましい態様等は、「例えば」、「好ましい」、及び「より好ましい」等の表現にかかわらず適宜相互に組み合わせて使用することができる。また、数値範囲の記載は例示であって、各範囲の上限と下限並びに実施例の数値とを適宜組み合わせた範囲も好ましく使用することができる。さらに、「含有する」又は「含む」等の用語は、「本質的になる」や「のみからなる」と読み替えてもよい。 Here, the mode for carrying out the invention will be described in detail. Can be used in combination. Moreover, the description of the numerical range is an example, and a range obtained by appropriately combining the upper limit and the lower limit of each range and the numerical values of the examples can also be preferably used. Further, terms such as "contain" or "include" may be read as "consisting essentially of" or "consisting only of".
＜組成物＞
本発明の一態様は、特定の精油を含む組成物である。ここで、組成物は、精油それ自体や精油を含む混合物であれば、溶液、ゲル、固形物、及びミストなどの形態にとらわれることはない。組成物は、医薬組成物や製剤のみならず、飲食品、化粧品、動物用飼料、及び雑貨品等であってもよいし、当該組成物を原料とし、既存の医薬組成物、飲食品、化粧品、動物用飼料、及び雑貨品等と組み合わせてもよい。
＜精油＞
精油は、主に植物から抽出して得られる油分、好ましくは揮発性の芳香成分を含む油分を意味する。精油は動物由来精油と植物由来精油とが存在するが、本発明では植物由来精油が好ましい。精油は常温（２５℃±５℃）にて液体であることが好ましい。本発明では、揮発性が高い精油である方が、アロマディフューザーを使用する場合のように吸引により精油中の有効成分を体内に取り入れることができ、簡易な投与形態であるから好ましい。精油を抽出する植物の部位については、特に限定はないが、例えば、葉、根、茎、樹皮、種、花、果実、果実の果皮、及びこれらの組み合わせを挙げることができる。植物は、そのまま、あるいは、植物を乾燥し、破砕して使用することもできる。 <Composition>
One aspect of the invention is a composition comprising a particular essential oil. Here, the composition is not limited to forms such as solution, gel, solid, and mist as long as it is the essential oil itself or a mixture containing the essential oil. Compositions may be not only pharmaceutical compositions and formulations, but also food and drink, cosmetics, animal feeds, miscellaneous goods, etc., and existing pharmaceutical compositions, food and drink, and cosmetics using the composition as a raw material. , animal feed, miscellaneous goods, and the like.
<Essential oil>
An essential oil means an oil obtained mainly by extraction from plants, preferably an oil containing volatile aromatic components. Essential oils include animal-derived essential oils and plant-derived essential oils, and plant-derived essential oils are preferred in the present invention. The essential oil is preferably liquid at room temperature (25°C ± 5°C). In the present invention, essential oils with high volatility are preferable because the active ingredients in the essential oils can be taken into the body by inhalation as in the case of using an aroma diffuser, and they are a simple administration form. The part of the plant from which the essential oil is extracted is not particularly limited, but examples include leaves, roots, stems, bark, seeds, flowers, fruits, fruit peels, and combinations thereof. The plant can be used as it is or after drying and crushing the plant.
本発明の有効成分として使用できる精油は、シトルスメジカブルガリス果皮油、ニュウコウジュ油、レモン果皮油、ライム油、セイヨウアカマツ葉油、ローマカミツレ花油、スペアミント油、及びイランイラン花油からなる群から選択される１種以上の精油である。ここで、シトルスメジカブルガリス果皮油は、シトルスメジカブルガリスの果皮から抽出して得られる芳香成分を含む油分を言う。ニュウコウジュ油は、ニュウコウジュの木部、樹皮または樹液を含むニュウコウジュの一部または全体から抽出して得られる芳香成分を含む油分を言う。レモン果皮油は、レモンの果皮から抽出して得られる芳香成分を含む油分を言う。ライム油は、ライムの果実、果皮または果肉等を含むライムの一部または全体から抽出して得られる芳香成分を含む油分を言う。セイヨウアカマツ葉油は、セイヨウアカマツの葉から抽出して得られる芳香成分を含む油分を言う。ローマカミツレ花油は、ローマカミツレの花から抽出して得られる芳香成分を含む油分を言う。スペアミント油は、スペアミントの葉、茎、根等を含むスペアミントの一部または全体から抽出して得られる芳香成分を含む油分を言う。イランイラン花油は、イランイランの花から抽出して得られる芳香成分を含む油分を言う。好ましい抽出方法については、後述する。 The essential oils that can be used as active ingredients in the present invention include Citrus medica vulgaris pericarp oil, Pyrrhiza medicinalis oil, Lemon pericarp oil, Lime oil, Scots pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. one or more essential oils selected from the group; Here, Citrus medica vulgaris pericarp oil refers to oil containing aromatic components obtained by extraction from the pericarp of Citrus medica vulgaris. The oil of Nyukoju is an oil containing aromatic components obtained by extracting a part or the whole of Nyukoju, including the wood, bark or sap of Nyukoju. Lemon peel oil refers to oil containing aromatic components obtained by extraction from lemon peel. Lime oil refers to oil containing aromatic components obtained by extracting a part or the whole of lime, including lime fruit, peel or pulp. Scots pine leaf oil refers to oil containing aromatic components obtained by extracting from the leaves of Scots pine. Roman chamomile flower oil refers to oil containing aromatic components obtained by extraction from Roman chamomile flowers. Spearmint oil refers to oil containing aromatic components obtained by extracting a part or the whole of spearmint including leaves, stems, roots, etc. of spearmint. Ylang-ylang flower oil refers to oil containing aromatic components obtained by extracting from ylang-ylang flowers. A preferred extraction method will be described later.
より具体的には以下の表１のような精油であることが好ましい。
　表１
Figure JPOXMLDOC01-appb-I000001
More specifically, the essential oils shown in Table 1 below are preferred.
Table 1
Figure JPOXMLDOC01-appb-I000001
好ましくは、シトルスメジカブルガリス果皮油、ニュウコウジュ油、ライム油、及びローマカミツレ花油からなる群から選択される１種以上の精油、又は、ニュウコウジュ油、レモン果皮油、ライム油、及びイランイラン花油からなる群から選択される１種以上の精油である。より好ましくは、ニュウコウジュ油及びローマカミツレ花油からなる群から選択される１種以上の精油である。これらの精油は、ミクログリア細胞の活性化を抑制する等の顕著な効果を有することが具体的に見いだされたものであり、好ましい。この効果は、これらの精油に類似するベルガモット果実油、ハッカ葉油、及びユーカリ葉油では発揮されないことからすると、驚くべきである。これらの本発明の有効成分として使用できる精油は、単独で使用する場合のほか、２種以上の精油を組み合わせて使用してもよい。２種の精油を組み合わせて使用する場合、質量比で例えば９９：１～１：９９、好ましくは９：１～１：９、より好ましくは５：１～１：５、更に好ましくは２：１～１：２であることが適当である。 Preferably, one or more essential oils selected from the group consisting of Citrus medica vulgaris pericarp oil, medica vulgaris oil, lime oil, and Roman chamomile flower oil; One or more essential oils selected from the group consisting of ylang-ylang flower oil. More preferably, it is one or more essential oils selected from the group consisting of cypress oil and Roman chamomile flower oil. These essential oils were specifically found to have remarkable effects such as suppression of activation of microglial cells, and are preferred. This effect is surprising given that bergamot fruit oil, mint leaf oil and eucalyptus leaf oil, which are similar to these essential oils, do not exhibit this effect. These essential oils that can be used as active ingredients in the present invention may be used alone or in combination of two or more. When two types of essential oils are used in combination, the mass ratio is, for example, 99:1 to 1:99, preferably 9:1 to 1:9, more preferably 5:1 to 1:5, still more preferably 2:1. ~1:2 is suitable.
本願発明の組成物中の精油の含有量は、組成物を１００質量％とした場合、例えば、０．００１～３０質量％、好ましくは０．０１～２０質量％、より好ましくは０．０１～１０質量％、更に好ましくは０．１～１０質量％であることが適当である。当該組成物がエタノールを含有する組成物である場合、本願発明の組成物中の精油の含有量は、例えば、０．００１～３０質量％、好ましくは０．０１～２０質量％、より好ましくは０．０１～１０質量％、更に好ましくは０．１～１０質量％であることが適当である。また、精油の含有量は、組成物の形態によっても異なり、例えば当該組成物が医薬品等の原料として利用されたり、アロマディフューザーのような形態で使用される場合、本願発明の組成物中、精油が３０～１００質量％、７０～１００質量％又は９５～９８質量％であってもよい。 The content of the essential oil in the composition of the present invention is, when the composition is 100% by mass, for example, 0.001 to 30% by mass, preferably 0.01 to 20% by mass, more preferably 0.01 to 10% by mass, more preferably 0.1 to 10% by mass. When the composition is a composition containing ethanol, the content of the essential oil in the composition of the present invention is, for example, 0.001 to 30% by mass, preferably 0.01 to 20% by mass, more preferably It is suitably 0.01 to 10% by mass, more preferably 0.1 to 10% by mass. In addition, the content of the essential oil varies depending on the form of the composition. may be 30 to 100% by mass, 70 to 100% by mass, or 95 to 98% by mass.
＜任意成分＞
本発明の組成物には、上記有効成分である精油のほか、その他の精油やその他の有効成分を含んでもよい。その他の精油は本発明の作用効果は期待できないものの、それ以外のリラックス効果や保湿効果などを得ることができる。その他の精油としては、ユズ果皮油、ベルガモット葉油、ベルガモット果実油、ダマスクバラ花油、ショウズク種子油、エンピツビャクシン油、カミツレ花油、オニサルビア油、ユーカリ葉油、ニオイテンジクアオイ油、ショウガ根油、オレンジ油、ビターオレンジ葉／枝油、ビターオレンジ花油、ビターオレンジ果皮油、マンダリンオレンジ果皮油、ローズマリー葉油、ヒノキ油、クスノキ葉油、ラバンデュラハイブリダ油、ラベンダー油、ギンコウボク葉油、ギンコウボク花油、マヨラナ葉油、メリッサ葉油、パルマローザ油、ヒロハラベンダー花油、ビャクダン油、オオベニミカン果皮油、セイヨウハッカ油、タチジャコウソウ花／葉油、ハッカ葉油、コリアンダー果実油、イタリアイトスギ葉／実／茎油、セイヨウネズ果実油、レモングラス油、アオモジ果実油、ティーツリー葉油、ベチベル根油、メボウキ油、セイヨウカノコソウ根油等を挙げることができる（ここで「／」は「または」を意味する）。これらその他の精油は、本発明と同様に、精油において通常使用される抽出方法により得られたものであり、例えば、圧搾、搾汁、溶媒抽出、水蒸気蒸留、超臨界抽出、炭酸ガス抽出、冷浸法、温浸法などを単独あるいは組み合わせた方法で抽出された精油を使用することができる。
組成物中のその他の精油の含有量は、本来の有効成分（精油）の効果を損なわない量であれば、任意の量を加えることができるが、例えば、例えば、０．００１質量％～７０質量％、好ましくは０．０１質量％～５０質量％、より好ましくは０．１質量％～３０質量％含有することが適当である。 <Optional component>
The composition of the present invention may contain other essential oils and other active ingredients in addition to the above essential oils as active ingredients. Although other essential oils cannot be expected to have the action and effect of the present invention, other relaxing effects, moisturizing effects, and the like can be obtained. Other essential oils include yuzu peel oil, bergamot leaf oil, bergamot fruit oil, damask rose flower oil, shouzuku seed oil, coccineum oil, chamomile flower oil, salvia oil, eucalyptus leaf oil, sycamore oil, ginger root oil. , orange oil, bitter orange leaf/branch oil, bitter orange flower oil, bitter orange peel oil, mandarin orange peel oil, rosemary leaf oil, cypress oil, camphor leaf oil, lavandula hybrida oil, lavender oil, ginkgo leaf oil , ginkgo flower oil, marjoram leaf oil, melissa leaf oil, palmarosa oil, lavender flower oil, sandalwood oil, mandarin orange peel oil, mint oil, thyme flower/leaf oil, mint leaf oil, coriander fruit oil, Italian cypress leaf / fruit / stem oil, juniper fruit oil, lemongrass oil, aomoji fruit oil, tea tree leaf oil, vetiver root oil, basil oil, valerian root oil, etc. (where "/" is "or" means). These other essential oils are obtained by extraction methods commonly used for essential oils, such as pressing, squeezing, solvent extraction, steam distillation, supercritical extraction, carbon dioxide extraction, cold An essential oil extracted by a method such as an immersion method or a digestion method alone or in combination can be used.
The content of other essential oils in the composition can be added in any amount as long as it is an amount that does not impair the effect of the original active ingredient (essential oil). % by mass, preferably 0.01% to 50% by mass, more preferably 0.1% to 30% by mass.
その他の有効成分は、ミクログリア細胞の活性化を抑制する効果や、ＩＬ-１βの放出を抑制する効果のような本願発明が奏する効果及びそれ以外の効果、例えば、抗菌、除虫、抗炎症、鎮痛、鎮静、リラックス効果等の効果が得られる公知の有効成分を挙げることができる。その他の有効成分としては、例えば、βラクタム系抗菌剤、ホスホマイシン、バンコマイシン、ミノサイクリン、シクレソニド等を挙げることができる。
組成物中のその他の有効成分の含有量は、本来の有効成分（精油）の効果を損なわない量であれば、任意の量を加えることができるが、例えば、例えば、０．００１質量％～９０質量％、好ましくは０．０１質量％～７０質量％、より好ましくは０．１質量％～５０質量％含有することが適当である。 Other active ingredients are the effect of the present invention such as the effect of suppressing the activation of microglial cells and the effect of suppressing the release of IL-1β, and other effects such as antibacterial, insecticidal, anti-inflammatory, Known active ingredients that provide analgesic, sedative, relaxing effects, and the like can be used. Other active ingredients include, for example, β-lactam antibacterial agents, fosfomycin, vancomycin, minocycline, ciclesonide, and the like.
The content of other active ingredients in the composition can be added in any amount as long as it is an amount that does not impair the effect of the original active ingredient (essential oil). It is suitable to contain 90 mass %, preferably 0.01 mass % to 70 mass %, more preferably 0.1 mass % to 50 mass %.
さらに、本発明の組成物には、組成物の用途や目的に応じて、任意に賦形剤、結合剤、乳化剤、溶剤、粘着剤、崩壊剤、粘稠剤、滑沢剤、着色剤、流動剤、保湿剤等の添加剤を添加してもよい。賦形剤としては、例えば、アルコール、グリセリン、デキストリン、澱粉等を挙げることができる。結合剤としては、例えば、セルロース、メチルセルロース、ヒドロキシエチルセルロース、及びカルボキシメチルセルロースナトリウムを挙げることができる。乳化剤としては、例えば、レシチン、ポリエチレングリコール（PG）、グリセリン脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステル、ショ糖脂肪酸エステル等を挙げることができる。溶剤としては、例えば、メタノール、エタノール、プロパノール、ｎ－ブタノール、エチレングリコール、プロピレングリコール、ジプロピレングリコールなどの一価又は多価のアルコール類；アセトン、メチルエチルケトンなどのケトン類；ジエチルエーテル、テトラヒドロフラン（ＴＨＦ）などのエーテル類；ヘキサン、ベンゼン、トルエン、クロロホルム、ジクロロメタン、ジメチルホルムアミドなどの有機溶媒；アボガド油、ツバキ油、タートル油、マカデミアナッツ油、トウモロコシ油、ミンク油、オリーブ油、ナタネ油、卵黄油、ゴマ油、パーシック油、小麦胚芽油、サザンカ油、ヒマシ油、アマニ油、サフラワー油、綿実油、エノ油、大豆油、落花生油、茶実油、カヤ油、コメヌカ油、シナギリ油、日本キリ油、ホホバ油、胚芽油、トリグリセリン等の液体油脂；流動パラフィン、オゾケライト、スクワラン、プリスタン、パラフィン、セレシン、スクワレン、ワセリン、マイクロクリスタリンワックス等の炭化水素油；オクタン酸セチル、ミリスチン酸ミリスチル、トリ２－エチルヘキサン酸グリセリル、テトラ２－エチルヘキサン酸ペンタエリトリット、コハク酸ジオクチル、ジネオペンタン酸トリプロピレングリコール等の合成エステル油などの化粧品や医薬部外品に通常用いられる油分及び水などを挙げることができる。好ましい溶剤としては、エタノール、オリーブ油、スクワラン、ミリスチン酸ミリスチル及び水を挙げることができる。これらの添加剤は１種又は２種以上を組み合わせて使用することができる。例えばアルコール類など、賦形剤としても溶剤としても作用するものは、１種類を含む場合でも賦形剤及び溶剤の双方の添加剤として作用する。組成物中の添加剤の含有量は、有効成分の効果を損なわない量であれば、任意の量を加えることができるが、例えば、０．００１質量％～７０質量％、好ましくは０．０１質量％～５０質量％、より好ましくは０．１質量％～３０質量％含有することが適当である。なかでも有機溶媒については、組成物を１００質量％とした場合、例えば、１質量％～９９．９質量％、好ましくは１質量％～９９質量％、より好ましくは２０質量％～９０質量％程度含有することが適当である。油分については、例えば、０．０１～９５質量％、好ましくは１～９０質量％、より好ましくは５～８０質量％程度含有することが適当である。 Furthermore, the composition of the present invention may optionally contain excipients, binders, emulsifiers, solvents, adhesives, disintegrants, thickeners, lubricants, colorants, Additives such as flow agents and moisturizing agents may be added. Examples of excipients include alcohol, glycerin, dextrin, starch and the like. Binders can include, for example, cellulose, methylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose. Examples of emulsifiers include lecithin, polyethylene glycol (PG), glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester and the like. Solvents include, for example, monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, propylene glycol, dipropylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, tetrahydrofuran (THF ); organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane, and dimethylformamide; avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil , persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, linagiri oil, Japanese paulownia oil, jojoba Liquid fats and oils such as oil, germ oil, and triglycerin; Hydrocarbon oils such as liquid paraffin, ozokerite, squalane, pristane, paraffin, ceresin, squalene, vaseline, and microcrystalline wax; cetyl octanoate, myristyl myristate, tri-2-ethyl Examples include oils and water commonly used in cosmetics and quasi-drugs, such as synthetic ester oils such as glyceryl hexanoate, pentaerythritol tetra-2-ethylhexanoate, dioctyl succinate, and tripropylene glycol dineopentanoate. Preferred solvents include ethanol, olive oil, squalane, myristyl myristate and water. These additives can be used singly or in combination of two or more. Those that act as both excipients and solvents, such as alcohols, act as both excipient and solvent additives even when one is included. The content of the additive in the composition can be added in any amount as long as it is an amount that does not impair the effect of the active ingredient. It is suitable to contain from 0.1% to 30% by mass, more preferably from 0.1% by mass to 50% by mass. Among them, when the composition is 100% by mass, the organic solvent is, for example, 1% by mass to 99.9% by mass, preferably 1% by mass to 99% by mass, more preferably about 20% by mass to 90% by mass. It is appropriate to contain The oil content is, for example, 0.01 to 95% by mass, preferably 1 to 90% by mass, more preferably 5 to 80% by mass.
＜製造方法・抽出方法＞
精油の製造方法としては、一般的に精油の調製に使用される方法を用いることができるが、一般に植物を抽出して精油を得る方法として公知の方法を用いることができる。抽出方法としては、例えば、圧搾、搾汁、溶媒抽出、水蒸気蒸留、超臨界抽出、炭酸ガス抽出、冷浸法、温浸法などを単独あるいは組み合わせて用いることができる。これらの抽出方法によって得られた抽出液から、さらに任意に溶媒等を除去することにより、精油を得ることができる。抽出溶媒としては、例えば、メタノール、エタノール、プロパノール、ｎ－ブタノール、エチレングリコール、プロピレングリコールなどの一価又は多価のアルコール類；アセトン、メチルエチルケトンなどのケトン類；ジエチルエーテル、テトラヒドロフラン（ＴＨＦ）などのエーテル類；ヘキサン、ベンゼン、トルエン、クロロホルム、ジクロロメタン、ジメチルホルムアミドなどの有機溶媒；及び水などを挙げることができる。
抽出における抽出条件は、使用する植物や溶媒によっても異なるが、一般的に抽出方法で使用される温度、圧力、抽出時間等を使用することができる。例えば、水蒸気を使用する水蒸気蒸留法では、一般的な精油の沸点（１５０～３５０℃）以下の４０～１２０℃、好ましくは５０～１００℃の温度で、例えば５～２００ｋＰａ、好ましくは１０～１００ｋＰａの減圧下で行われてもよい。エタノール等のアルコールを用いた抽出法では、例えば、常温（２５℃±５℃）で３００～１０００ｋＰａ、好ましくは５００～８００ｋＰａ程度の高圧下で行われてもよい。抽出溶媒は、抽出操作の後で除去してもよい。 <Manufacturing method / extraction method>
As a method for producing an essential oil, a method generally used for preparing an essential oil can be used, and a method generally known as a method of extracting a plant to obtain an essential oil can be used. As the extraction method, for example, pressing, squeezing, solvent extraction, steam distillation, supercritical extraction, carbon dioxide gas extraction, cold immersion method, digestion method, etc. can be used alone or in combination. The essential oil can be obtained by optionally removing the solvent and the like from the liquid extract obtained by these extraction methods. Examples of extraction solvents include monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, propylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, tetrahydrofuran (THF) and the like. ethers; organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane and dimethylformamide; and water.
The extraction conditions for extraction vary depending on the plant and solvent used, but the temperature, pressure, extraction time, etc. generally used in extraction methods can be used. For example, in the steam distillation method using steam, at a temperature of 40 to 120 ° C., preferably 50 to 100 ° C. below the boiling point of general essential oils (150 to 350 ° C.), for example, 5 to 200 kPa, preferably 10 to 100 kPa may be carried out under a reduced pressure of The extraction method using an alcohol such as ethanol may be carried out at room temperature (25° C.±5° C.) under high pressure of 300 to 1000 kPa, preferably about 500 to 800 kPa. The extraction solvent may be removed after the extraction operation.
＜用途＞
本発明の組成物は、医薬組成物のみならず、飲食料品、化粧品、動物用飼料、雑貨品に含めて使用することができる。医薬組成物としては、例えば、錠剤、丸剤、カプセル剤、散剤、顆粒剤、シロップ剤、及び舌下剤等の経口投与剤；注射剤（静脈注射、筋肉注射、局所注射等）、含嗽剤、点滴剤、外用剤（軟膏、クリーム、パップ剤、貼付薬、吸入薬）、及び座剤等の非経口投与剤等を挙げることができる。飲食料品としては、飲料と食品があり、飲料としては、例えば、茶、コーヒー、清涼飲料水、果汁飲料、炭酸飲料、スポーツ飲料、ノンアルコール飲料、及びアルコール飲料等を挙げることができる。食品としては、ヒトに与えられる食糧全般、例えば、植物性食品、動物性食品、合成食品、穀類、パン、麺、ケーキ、麺、フリーズドライ食品、乳製品、調味料、冷凍食品、栄養補助食品、及び医療食品等を挙げることができる。化粧品としては、例えば、化粧水、乳液、保湿剤、美容液、スキンオイル、香水、サンスクリーン、シェービングローション、パック、ヘアトニック、ヘアバーム、ヘアワックス、ヘアグリース、ヘアオイル、ヘアトリートメント、ヘアカラー、洗顔料、ボディシャンプー、シャンプー、リンス、コンディショナー、クレンジング、クリーム、ボディクリーム、ハンドクリーム、化粧下地、ファンデーション、マスカラ、アイライナー、コンシーラー、口紅、リップクリーム、グロス及びネイルカラー等を挙げることができる。動物用飼料としては、例えば、イヌ、ネコ、ウマ、ヒツジ、ブタ、サル、ウサギ、モルモット、マウス、トリ、ニワトリ、アヒル、及びヤギ等の動物用、特に哺乳動物用のエサを挙げることができる。雑貨品としては、例えば、芳香剤、アロマディフューザー、アロマキャンドル、アロマストーン、アロマミスト、衣料用ミスト、消臭ミスト、ピローミスト、防虫剤、衣料用洗剤、柔軟剤及び入浴剤を挙げることができる。
医薬組成物、飲食料品、化粧品、動物用飼料、及び雑貨品等に含める場合の組成物の含有量は、含められる医薬組成物等の種類にもよるが、本発明の効果を損なわない量で、全体量に対して、医薬組成物では、例えば、０．００１質量％～７０質量％、好ましくは０．０１質量％～５０質量％、より好ましくは０．１質量％～３０質量％含有することが適当である。飲食料品では、例えば、０．００１質量％～５０質量％、好ましくは０．０１質量％～３０質量％、より好ましくは０．１質量％～１０質量％含有することが適当である。化粧品では、例えば、０．００１質量％～７０質量％、好ましくは０．０１質量％～５０質量％、より好ましくは０．１質量％～３０質量％含有することが適当である。動物用飼料では、例えば、０．００１質量％～５０質量％、好ましくは０．０１質量％～３０質量％、より好ましくは０．１質量％～１０質量％含有することが適当である。雑貨品では、例えば、０．０１質量％～１００質量％、好ましくは０．０１質量％～８０質量％、より好ましくは０．１質量％～５０質量％含有することが適当である。 <Application>
The composition of the present invention can be used not only in pharmaceutical compositions, but also in foods and beverages, cosmetics, animal feeds, and miscellaneous goods. Pharmaceutical compositions include, for example, oral administration agents such as tablets, pills, capsules, powders, granules, syrups, and sublingual agents; injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, Parenteral administration agents such as drops, external agents (ointments, creams, poultices, patches, inhalants), and suppositories can be mentioned. Food and drink products include beverages and foods, and examples of beverages include tea, coffee, soft drinks, fruit juice drinks, carbonated drinks, sports drinks, non-alcoholic drinks, and alcoholic drinks. Foods in general given to humans, such as plant foods, animal foods, synthetic foods, cereals, bread, noodles, cakes, noodles, freeze-dried foods, dairy products, seasonings, frozen foods, nutritional supplements , and medical foods. Examples of cosmetics include lotions, milky lotions, moisturizers, serums, skin oils, perfumes, sunscreens, shaving lotions, packs, hair tonics, hair balms, hair waxes, hair greases, hair oils, hair treatments, hair colors, and face washes. cosmetics, body shampoos, shampoos, rinses, conditioners, cleansers, creams, body creams, hand creams, makeup bases, foundations, mascara, eyeliners, concealers, lipsticks, lip balms, glosses and nail colors. Animal feeds include, for example, feeds for animals such as dogs, cats, horses, sheep, pigs, monkeys, rabbits, guinea pigs, mice, birds, chickens, ducks, and goats, especially mammals. . Examples of miscellaneous goods include aromatics, aroma diffusers, aroma candles, aroma stones, aroma mists, clothing mists, deodorant mists, pillow mists, insect repellents, clothing detergents, softeners, and bath agents. .
When included in pharmaceutical compositions, food and drink, cosmetics, animal feeds, miscellaneous goods, etc., the content of the composition depends on the type of pharmaceutical composition, etc., but is an amount that does not impair the effects of the present invention. In the pharmaceutical composition, for example, 0.001% to 70% by mass, preferably 0.01% to 50% by mass, more preferably 0.1% to 30% by mass with respect to the total amount It is appropriate to For food and drink, it is suitable to contain, for example, 0.001% to 50% by mass, preferably 0.01% to 30% by mass, more preferably 0.1% to 10% by mass. In cosmetics, it is suitable to contain, for example, 0.001% to 70% by mass, preferably 0.01% to 50% by mass, more preferably 0.1% to 30% by mass. For animal feeds, it is suitable to contain, for example, 0.001% to 50% by mass, preferably 0.01% to 30% by mass, more preferably 0.1% to 10% by mass. For miscellaneous goods, it is suitable to contain, for example, 0.01% by mass to 100% by mass, preferably 0.01% by mass to 80% by mass, more preferably 0.1% by mass to 50% by mass.
＜使用方法＞
本発明の組成物は、ミクログリア細胞の活性化を抑制するために使用される（ミクログリア細胞活性化の抑制方法）。ミクログリア細胞とは、中枢神経系の支持組織の一部を構成する中胚葉起源の小型、非ニューロン性間質細胞である。精油は一般的に抗炎症作用を有することが知られているが、ミクログリア細胞の活性化を抑制する効果は知られておらず、この度、ごく一部の精油にのみミクログリア細胞の活性化を抑制する効果が認められた。このような効果が見られることは驚くべきことであり、ひいてはミクログリア細胞の活性化に起因する疾病や症状を軽減及び治癒できることが期待される。従って、本発明の組成物は、ミクログリア細胞の活性化に起因する神経性疾患を治療又は予防にも使用され得る（ミクログリア細胞の活性化に起因する神経性疾患の治療又は予防方法）。ここで神経性疾患としては、例えば、うつ病、統合失調症、認知症、アルツハイマー病、パーキンソン病、多発硬化症、側索硬化症、ハンチントン舞踏病、慢性疲労症候群等を挙げることができる。 <How to use>
The composition of the present invention is used to suppress activation of microglial cells (Method for Suppressing Microglial Cell Activation). Microglial cells are small, non-neuronal stromal cells of mesodermal origin that form part of the supporting tissue of the central nervous system. Essential oils are generally known to have anti-inflammatory effects, but they are not known to have the effect of suppressing the activation of microglial cells. was found to be effective. It is surprising to see such an effect, and it is expected that diseases and conditions caused by activation of microglial cells can be alleviated and cured. Therefore, the composition of the present invention can also be used to treat or prevent neurological diseases caused by activation of microglial cells (method for treating or preventing neurological diseases caused by activation of microglial cells). Examples of neurological diseases include depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's disease, chronic fatigue syndrome, and the like.
本発明の組成物は、また、ＩＬ-１βの放出を抑制するためにも使用される（ＩＬ-１β放出抑制方法）。特に本発明の特定の精油を使用することにより、実質的な、又は、大きな細胞毒性を示さずに、ＩＬ-１βの産生抑制効果を得られることがわかった。
本発明の組成物は、さらに、ミクログリア細胞の活性化に起因する炎症を抑制するためにも使用される（ミクログリア細胞の活性化に起因する炎症抑制方法）。ここで炎症としては、脳内や脊髄内の炎症を主に挙げることができる。炎症が起きる脳内部位としては、例えば、海馬、大脳皮質、小脳、基底核、黒質線条体等を挙げることができる。炎症が起きる脊髄部位としては脊髄後角等を挙げることができる。当該炎症は、例えば、ＩＬ－１α、ＩＬ-１β、ＩＬ－２、ＩＬ－６、ＩＬ－１２、ＩＬ－１７、ＩＬ－１８、ＩＦＮ－γ、ＭＣＰ－１及びＴＮＦ－αから選択される炎症誘導性サイトカインの発現、好ましくは、ＩＬ－１α、ＩＬ-１β、ＩＦＮ－γ、ＭＣＰ－１、及びＴＮＦ－αから選択される炎症誘導性サイトカインの放出（発現）を伴う。本発明の組成物、特に有効成分である特定の精油は、これらＩＬ-１βをはじめとする炎症誘導性サイトカインの放出（発現）を抑制し、もってミクログリア細胞の活性化を抑制するものと推察される。ここで、ミクログリア細胞としては、ＭＧ６細胞、ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａ、及びＢＶ２細胞を挙げることができる。 The composition of the present invention is also used to inhibit the release of IL-1β (IL-1β release inhibition method). In particular, it was found that by using the specific essential oil of the present invention, an IL-1β production inhibitory effect can be obtained without showing substantial or significant cytotoxicity.
The composition of the present invention is also used to suppress inflammation caused by activation of microglial cells (method for suppressing inflammation caused by activation of microglial cells). Inflammation here mainly includes inflammation in the brain and spinal cord. Examples of intracerebral sites where inflammation occurs include hippocampus, cerebral cortex, cerebellum, basal ganglia, and nigrostriatum. The spinal cord site where inflammation occurs includes the dorsal horn of the spinal cord and the like. The inflammation is, for example, an inflammation selected from IL-1α, IL-1β, IL-2, IL-6, IL-12, IL-17, IL-18, IFN-γ, MCP-1 and TNF-α Expression of an inducible cytokine, preferably with release (expression) of a pro-inflammatory cytokine selected from IL-1α, IL-1β, IFN-γ, MCP-1 and TNF-α. It is presumed that the composition of the present invention, particularly the specific essential oil as an active ingredient, suppresses the release (expression) of pro-inflammatory cytokines such as IL-1β, thereby suppressing the activation of microglial cells. be. Here, microglial cells include MG6 cells, iCell (R) microglia, and BV2 cells.
＜投与方法＞
本発明の組成物は、医薬組成物や食品（特に機能性食品や栄養補助食品）の形態である場合、その剤型、投与対象、投与ルート、対象疾患、症状、有効成分濃度等によっても異なるが、例えば、一日あたりの有効成分の投与量として、例えば、０．１～２０ｍｇ／ｋｇ体重、好ましくは０．２～１０ｍｇ／ｋｇ体重、さらに好ましくは０．５～１０ｍｇ／ｋｇ体重であり、この量を１日１回～数回（例、２回、３回、４回又は８回）投与するのが望ましい。本発明の組成物を成人（体重７０ｋｇ）に投与する場合、例えば、０．１～２０ｍｇ／日、好ましくは０．２～１０ｍｇ／日、さらに好ましくは０．５～１０ｍｇ／日の量であることが適当である。
投与方法としては、医薬組成物や食品の剤型や形態によって異なるが、例えば液剤を投与対象に吸引によって投与する場合、アロマディフューザー、スプレー、及びミスト等によって投与することが好ましい。 <Administration method>
When the composition of the present invention is in the form of a pharmaceutical composition or food (especially functional food or dietary supplement), it varies depending on the dosage form, administration subject, administration route, target disease, symptom, active ingredient concentration, etc. However, for example, the dosage of the active ingredient per day is, for example, 0.1 to 20 mg/kg body weight, preferably 0.2 to 10 mg/kg body weight, more preferably 0.5 to 10 mg/kg body weight. , it is desirable to administer this amount once to several times a day (eg, 2, 3, 4 or 8 times). When the composition of the present invention is administered to an adult (body weight 70 kg), the amount is, for example, 0.1 to 20 mg/day, preferably 0.2 to 10 mg/day, more preferably 0.5 to 10 mg/day. is appropriate.
The administration method varies depending on the dosage form and form of the pharmaceutical composition or food. For example, when a liquid agent is administered to a subject by inhalation, it is preferably administered by an aroma diffuser, a spray, a mist, or the like.
　以下、本発明について、実施例及び比較例に基づき具体的に説明するが、本発明はこれら実施例に限定されるものではない。 The present invention will be specifically described below based on examples and comparative examples, but the present invention is not limited to these examples.
実施例１［組成物の調製］
各精油として、表２のリストに記載された精油を使用した。各精油の２～３ｍｇを秤量し、９倍量（ｗ／ｗ）のエタノールを加えて、１０％（ｗ／ｗ）のエタノール溶液としての各精油の組成物を調製した。 Example 1 [Preparation of composition]
As each essential oil, the essential oils listed in Table 2 were used. A composition of each essential oil was prepared as a 10% (w/w) ethanol solution by weighing 2-3 mg of each essential oil and adding 9 volumes (w/w) of ethanol.
表２
Figure JPOXMLDOC01-appb-I000002
Table 2
Figure JPOXMLDOC01-appb-I000002
比較例１［組成物の調製］
比較例の精油として、表３のリストに記載された精油を使用した。実施例１と同様に、精油の２～３ｍｇを秤量し、９倍量（ｗ／ｗ）のエタノールを加えて、１０％（ｗ／ｗ）のエタノール溶液としての各組成物を調製した。 Comparative Example 1 [Preparation of composition]
The essential oils listed in Table 3 were used as comparative essential oils. As in Example 1, 2-3 mg of the essential oil was weighed and 9 times the amount (w/w) of ethanol was added to prepare each composition as a 10% (w/w) ethanol solution.
表３
Figure JPOXMLDOC01-appb-I000003
Table 3
Figure JPOXMLDOC01-appb-I000003
［ＭＧ６細胞の入手方法と培地の調製法］
マウス由来ミクログリア細胞株のＭＧ６細胞は、理化学研究所の細胞バンク（細胞番号ＲＣＢ２４０３）から購入した。培養に用いた培地は、Ｄ－ＭＥＭ（富士フィルム和光製、製品番号；０４９－３２６４５）５００ｍＬに対して、牛胎児血清（Ｂｉｏｆｉｌｌ社製）を５０ｍＬ添加し、インスリン（富士フィルム和光純薬株式会社製、製品番号：０９９－０６４７３）と２－メルカプトエタノールとを、それぞれ終濃度で１０μｇ／ｍＬと０．１ｍｍｏｌ／Ｌとになるように添加して調製した（以下、培地Ａとする）。ＭＧ６細胞の培養は、理化学研究所の細胞バンクが指定する方法に従った。 [How to obtain MG6 cells and how to prepare the medium]
MG6 cells, a mouse-derived microglial cell line, were purchased from the RIKEN Cell Bank (cell number RCB2403). The medium used for culture was D-MEM (manufactured by Fujifilm Wako, product number; 049-32645) 500mL, fetal bovine serum (manufactured by Biofill) 50mL was added, and insulin (Fujifilm Wako Pure Chemical Co., Ltd. 099-06473) and 2-mercaptoethanol at final concentrations of 10 μg/mL and 0.1 mmol/L, respectively (hereinafter referred to as medium A). MG6 cells were cultured according to the method specified by Riken's cell bank.
［ＭＧ６細胞を用いたミクログリアのＬＰＳおよびＡＴＰによる活性化の方法］
ＭＧ６細胞からのＩＬ－１β放出量を測定する前に、０．５×１０5　ｃｅｌｌｓ／ｗｅｌｌとなるように２４ｗｅｌｌプレートにＭＧ６細胞を播種し、翌日、細胞が接着していることを確認して、以下の操作を行った。まず、培地Ａに終濃度で１μｇ／ｍＬとなるような量のＬＰＳ（富士フィルム和光純薬株式会社製、製品番号：１２５－０５１８１）を添加した培地１を準備した。また、新たな培地Ａに終濃度で１ｍｍｏｌ／Ｌとなるような量のＡＴＰ（富士フィルム和光純薬株式会社製、製品番号：３０３－５０５１１）を添加した培地２を準備した。上記の通り準備した２４ｗｅｌｌプレートに培養したＭＧ６細胞に対して、各ｗｅｌｌの培地を除去した後、培地１を０．５ｍＬ／ｗｅｌｌずつ添加し、４時間の培養を行った。次に各ｗｅｌｌの培地を除去して、各ｗｅｌｌのＭＧ６細胞に対して培地２を０．５ｍＬ／ｗｅｌｌずつ添加し、３０分間の培養を行った。当該３０分の培養後に、各ｗｅｌｌから培地を活性化測定用培地として回収するとともに、新たな上記培地Ａに０．１％のＴｒｉｔｏｎ　Ｘ－１００（ナカライテスク社製）を加えて調製した溶液を、各Ｗｅｌｌに０．５　ｍＬ／ｗｅｌｌずつ加えて、ＭＧ６細胞を含有する細胞溶解液を調製した。 [Method for activation of microglia with LPS and ATP using MG6 cells]
Before measuring the amount of IL-1β released from MG6 cells, MG6 cells were seeded in a 24-well plate at 0.5×10 5 cells/well, and the next day, the adherence of the cells was confirmed. , performed the following: First, medium 1 was prepared by adding LPS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181) to medium A at a final concentration of 1 μg/mL. In addition, medium 2 was prepared by adding ATP (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511) to a new medium A in such an amount that the final concentration was 1 mmol/L. After removing the medium from each well, 0.5 mL/well of medium 1 was added to the MG6 cells cultured in the 24-well plate prepared as described above, and cultured for 4 hours. Next, the medium in each well was removed, 0.5 mL/well of medium 2 was added to each well of MG6 cells, and the cells were cultured for 30 minutes. After culturing for 30 minutes, the medium was collected from each well as a medium for measuring activation, and a solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the new medium A was prepared. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.
［精油及びＬＰＳまたはＡＴＰを含有する培地の調製方法］
精油等の効果を検討するために、まず、終濃度で、上記培地１及び培地２のそれぞれ２倍量のＬＰＳまたはＡＴＰを添加した以外は、培地１または培地２と同様に調製した培地３または培地４を用意した。また、上記実施例１及び比較例１で調製した各組成物（精油の１０％（ｗ／ｗ）のエタノール溶液）の１０μＬを分取し、５ｍＬの上記培地Ａに加えた。その後、超音波ホモジナイザー（Ｂｒａｎｓｏｎ社製、製品番号：ＳＦＸ１５０）を用いて懸濁させ、培地５とした。この培地５と培地３または培地４を３ｍＬずつ混和し、ＬＰＳまたはＡＴＰを含有する培養培地中の各精油の終濃度が１００ｐｐｍとなるように調製し、これをそれぞれ培地６または７とした。 [Method for preparing medium containing essential oil and LPS or ATP]
In order to examine the effects of essential oils, first, medium 3 or medium 3 prepared in the same manner as medium 1 or medium 2, except that LPS or ATP was added at a final concentration twice that of medium 1 and medium 2, respectively. Medium 4 was prepared. In addition, 10 μL of each composition (10% (w/w) ethanol solution of essential oil) prepared in Example 1 and Comparative Example 1 was aliquoted and added to 5 mL of Medium A. After that, it was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 5. 3 mL each of this medium 5 and medium 3 or medium 4 were mixed to adjust the final concentration of each essential oil in the culture medium containing LPS or ATP to 100 ppm, which was designated as medium 6 or 7, respectively.
［精油を添加した条件におけるＭＧ６細胞の活性化の方法］
前項の［ＭＧ６細胞を用いたミクログリアのＬＰＳおよびＡＴＰによる活性化の方法］と同様に、０．５×１０5　ｃｅｌｌｓ／ｗｅｌｌとなるように２４ｗｅｌｌプレートにＭＧ６細胞を播種し、翌日、細胞が接着していることを確認して２４ｗｅｌｌプレート上で培養したＭＧ６細胞を準備した。当該準備した２４ｗｅｌｌプレート上で培養したＭＧ６細胞に対して、各ｗｅｌｌから培地を除去した後、培地６を各ｗｅｌｌに０．５　ｍＬ／ｗｅｌｌずつ添加し、ＭＧ６細胞を４時間培養した。次に、各ｗｅｌｌから培地を除去した後、培地７を各ｗｅｌｌに０．５　ｍＬ／ｗｅｌｌずつ添加し、ＭＧ６細胞をさらに３０分間培養した。当該３０分の培養後、各ｗｅｌｌから培地を活性化測定用培地として回収するとともに、新たな上記培地Ａに０．１％のＴｒｉｔｏｎ　Ｘ－１００（ナカライテスク社製）を加えて調製した溶液を、各Ｗｅｌｌに０．５　ｍＬ／ｗｅｌｌずつ加えて、ＭＧ６細胞を含有する細胞溶解液を調製した。 [Method for activation of MG6 cells under the condition of adding essential oil]
MG6 cells were seeded in a 24-well plate at 0.5 × 10 5 cells/well in the same manner as in the previous [Method for activating microglia with LPS and ATP using MG6 cells]. MG6 cells cultured on a 24-well plate were prepared. After removing the medium from each well of the MG6 cells cultured on the prepared 24-well plate, 0.5 mL/well of medium 6 was added to each well, and the MG6 cells were cultured for 4 hours. Next, after removing the medium from each well, 0.5 mL/well of medium 7 was added to each well, and the MG6 cells were further cultured for 30 minutes. After culturing for 30 minutes, the medium was collected from each well as a medium for measuring activation, and a solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the new medium A was prepared. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.
［ＭＧ６細胞によるＩＬ－１β放出量の測定］
培養後の上記の活性化測定用培地および細胞溶解液のＩＬ－１β濃度は、ＢｉｏＬｅｇｅｎｄ社のＥＬＩＳＡキット（ＥＬＩＳＡ　ＭＡＸTM　Ｄｅｌｕｘｅ　Ｓｅｔ　Ｍｏｕｓｅ　ＩＬ－１β、製品番号：４３２６０４）を用いて測定した。以下、各試験において「コントロール」は、ＭＧ６細胞を含有し、［ＭＧ６細胞を用いたミクログリアのＬＰＳおよびＡＴＰによる活性化の方法］で調製した精油を含有しない細胞溶解液である。「ＭＣＣ９５０」は、ＮＬＲＰ３インフラマソームの阻害剤であるＭＣＣ９５０（フナコシ株式会社、製品番号：AG-CR1-3615-M001）を、さらに終濃度１０μｍｏｌ／Ｌとなるように培地１および培地２に添加したものであり、実際にＩＬ－１β放出が抑制されることを確認するための陽性対照として用いた。なお、表４－１～表４－５中のＩＬ－１β濃度は３回測定した平均の値である。 [Measurement of IL-1β release by MG6 cells]
The concentrations of IL-1β in the culture medium for activation measurement and cell lysate after culture were measured using an ELISA kit (ELISA MAX ™ Deluxe Mouse Set Mouse IL-1β, product number: 432604) from BioLegend. Hereinafter, the “control” in each test is a cell lysate containing MG6 cells and not containing essential oils prepared in [Method for activation of microglia with LPS and ATP using MG6 cells]. "MCC950" is an inhibitor of NLRP3 inflammasome, MCC950 (Funakoshi Co., Ltd., product number: AG-CR1-3615-M001), added to medium 1 and medium 2 to a final concentration of 10 μmol / L. It was used as a positive control to confirm that IL-1β release was actually suppressed. The IL-1β concentrations in Tables 4-1 to 4-5 are average values measured three times.
表４－１
Figure JPOXMLDOC01-appb-I000004
Table 4-1
Figure JPOXMLDOC01-appb-I000004
表４－２
Figure JPOXMLDOC01-appb-I000005
Table 4-2
Figure JPOXMLDOC01-appb-I000005
表４－３
Figure JPOXMLDOC01-appb-I000006
Table 4-3
Figure JPOXMLDOC01-appb-I000006
表４－４
Figure JPOXMLDOC01-appb-I000007
Table 4-4
Figure JPOXMLDOC01-appb-I000007
表４－５
Figure JPOXMLDOC01-appb-I000008
Table 4-5
Figure JPOXMLDOC01-appb-I000008
［ＭＧ６細胞に対する細胞障害率試験］
精油のＭＧ６活性化抑制効果が細胞障害による非特異的な効果ではないことを確認するために、ＬＤＨアッセイによる細胞障害率の測定を行った。富士フィルム和光純薬社製のＬＤＨアッセイキット（製品番号：３４１－９１７５４）を用いて、培養後の活性化測定用培地及び細胞溶解液のＬＤＨ活性を測定した。各ｗｅｌｌの活性化測定用培地と細胞溶解液のＬＤＨ活性の合計を総ＬＤＨ活性とし、細胞溶解液のＬＤＨ活性の総ＬＤＨ活性に対する比を細胞障害率として％で表した。使用した「コントロール」及び「ＭＣＣ９５０」は表４－１～表４－５と同じである。コントロールと比較して、細胞障害率の大きな上昇を認めない精油の中で、ＩＬ－１βの培地中への放出量を抑制したものを、特にＭＧ６細胞の活性化を抑制する効果が顕著に優れたものとした。なお、表５－１～表５－５中の細胞障害率は３回測定した平均の値である。 [Cytotoxicity test against MG6 cells]
In order to confirm that the MG6 activation inhibitory effect of the essential oil is not a non-specific effect due to cytotoxicity, the cytotoxicity rate was measured by LDH assay. Using an LDH assay kit (product number: 341-91754) manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., the LDH activity of the culture medium for activation measurement and the cell lysate was measured. The total LDH activity in the medium for activation measurement in each well and the cell lysate was defined as the total LDH activity, and the ratio of the LDH activity in the cell lysate to the total LDH activity was expressed as a cytotoxicity rate in %. The "control" and "MCC950" used are the same as in Tables 4-1 to 4-5. Among the essential oils that do not show a significant increase in the cytotoxicity rate compared to the control, those that suppress the release of IL-1β into the medium are particularly effective in suppressing the activation of MG6 cells. I assumed. The cytotoxicity rates in Tables 5-1 to 5-5 are the average values measured three times.
表５－１
Figure JPOXMLDOC01-appb-I000009
Table 5-1
Figure JPOXMLDOC01-appb-I000009
表５－２
Figure JPOXMLDOC01-appb-I000010
Table 5-2
Figure JPOXMLDOC01-appb-I000010
表５－３
Figure JPOXMLDOC01-appb-I000011
Table 5-3
Figure JPOXMLDOC01-appb-I000011
表５－４
Figure JPOXMLDOC01-appb-I000012
Table 5-4
Figure JPOXMLDOC01-appb-I000012
表５－５
Figure JPOXMLDOC01-appb-I000013
Table 5-5
Figure JPOXMLDOC01-appb-I000013
［ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａに対するＩＬ－１β放出抑制試験］
・ヒトｉＰＳ細胞由来のミクログリア細胞を含有し、精油を含有しない細胞溶解液の調製（コントロール用）
ミクログリア細胞として、ヒトｉＰＳ細胞由来のミクログリア細胞であるｉＣｅｌｌ(R)　ミクログリア（ＦＵＪＩＦＩＬＭ　Ｃｅｌｌｕｌａｒ　Ｄｙｎａｍｉｃｓ，　Ｉｎｃ．製）を使用した。なお、ｉＣｅｌｌはフジフイルム　セルラー　ダイナミクス　インコーポレイテッドの登録商標である。ｉＣｅｌｌ(R)ミクログリアの培養は、富士フィルム和光純薬社の推奨している培養方法に従って行った。具体的には、まず、ｉＣｅｌｌ(R)ミクログリア用基礎培地（富士フィルム和光純薬株式会社製、品番：５５６－３６８６１）にｉＣｅｌｌ(R)ミクログリア用サプリメントＡ（品番：５５０－３６８８１）とｉＣｅｌｌ(R)ミクログリア用サプリメントＢ（品番：５５７－３６８９１）とｉＣｅｌｌ(R)神経用サプリメントＣ（品番：５５３－３６８７１）を添加し、培地を調製した（以下、培地Ｂとする）。ｉＣｅｌｌ(R)ミクログリアの接着性が弱いため、培養の際に培養容器をセルタック細胞及び組織接着剤（Ｃｅｌｌ－Ｔａｋ、コーニング社製、製品番号：ＣＬＳ３５４２４０）にてコーティングした。
上記ｉＣｅｌｌ(R)ミクログリアを、上記培地Ｂに１×１０5　ｃｅｌｌｓ／ｍＬとなるように懸濁し、４８ｗｅｌｌプレートに０．２５ｍＬ／ｗｅｌｌずつ播種した。播種の後、Ｔｏｍｙ社製の遠心機（品番：ＣＡＸ－５７１）とローター（品番：ＴＳ－４Ｃ）を用いて、２０℃、１，０００ｒｐｍで５分間の遠心操作を行い、プレート底面に細胞を集めて接着を促進した。
播種の２日後、培地を除去した後、新たな上記培地Ｂに終濃度で１μｇ／ｍＬのＬＰＳ（富士フィルム和光純薬株式会社製、製品番号：１２５－０５１８１）を添加した培地８を各ｗｅｌｌに０．２５ｍＬ／ｗｅｌｌずつ加えて、ｉＣｅｌｌ(R)ミクログリアを４時間培養した。次に、各ｗｅｌｌから培地８を除去し、新たな上記培養用培地Ｂに終濃度で１ｍｍｏｌ／ＬとなるようにＡＴＰ（富士フィルム和光純薬株式会社製、製品番号：３０３－５０５１１）を添加した培地９を０．２５ｍＬ／ｗｅｌｌずつ加えてｉＣｅｌｌ(R)ミクログリアをさらに３０分間培養した。当該３０分の培養後に、各ｗｅｌｌから培地を活性化測定用培地として回収するとともに、残ったｉＣｅｌｌ(R)ミクログリアにＭＴＴアッセイ用の培地として培地Ｂ１０ｍＬにＭＴＴ試薬（同仁化学研究所製、品番：ＣＫ０４）を１ｍＬ加えた混合液を、各ｗｅｌｌに０．２５ｍＬ／ｗｅｌｌずつ加えて、４時間の培養を行った。当該４時間後の培養後の培地を細胞生存率測定用培地とした。 [IL-1β release inhibition test for iCell (R) microglia]
・Preparation of cell lysate containing human iPS cell-derived microglial cells and no essential oil (for control)
As microglial cells, iCell (R) microglia (manufactured by FUJIFILM Cellular Dynamics, Inc.), which are human iPS cell-derived microglial cells, were used. Note that iCell is a registered trademark of Fujifilm Cellular Dynamics Inc. iCell (R) microglia were cultured according to the culture method recommended by Fujifilm Wako Pure Chemical Industries, Ltd. Specifically, first, iCell (R) microglial basal medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 556-36861), iCell (R) microglial supplement A (product number: 550-36881) and iCell ( R) Microglial supplement B (product number: 557-36891) and iCell (R) nerve supplement C (product number: 553-36871) were added to prepare a medium (hereinafter referred to as medium B). Since the adhesiveness of iCell (R) microglia is weak, the culture vessel was coated with Cell-Tak cell and tissue adhesive (Cell-Tak, manufactured by Corning, product number: CLS354240) during the culture.
The iCell (R) microglia were suspended in the medium B at 1×10 5 cells/mL, and seeded onto a 48-well plate at 0.25 mL/well. After seeding, centrifugation was performed at 20°C and 1,000 rpm for 5 minutes using a Tomy centrifuge (part number: CAX-571) and a rotor (part number: TS-4C) to spread the cells to the bottom of the plate. Collected to promote adhesion.
Two days after seeding, the medium was removed, and medium 8 obtained by adding LPS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 125-05181) at a final concentration of 1 μg/mL to the new medium B was added to each well. 0.25 mL/well, and iCell (R) microglia were cultured for 4 hours. Next, the medium 8 was removed from each well, and ATP (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511) was added to the new culture medium B so that the final concentration was 1 mmol/L. 0.25 mL/well of medium 9 was added and the iCell (R) microglia were further cultured for 30 minutes. After culturing for 30 minutes, the medium is collected from each well as a medium for measuring activation, and the remaining iCell (R) microglia is added to 10 mL of medium B as a medium for MTT assay (manufactured by Dojindo Laboratories, product number: 0.25 mL/well of a mixed solution containing 1 mL of CK04) was added to each well and cultured for 4 hours. The culture medium after culturing for 4 hours was used as a cell viability measurement medium.
［精油及びＬＰＳまたはＡＴＰを含有する培地の調製方法］
精油等の効果を検討するために、まず、終濃度で、上記培地８及び培地９のそれぞれ２倍量のＬＰＳまたはＡＴＰを添加した以外は、培地８または培地９と同様に調製した培地１０または培地１１を用意した。また、上記実施例１及び比較例１で調製した各組成物（精油の１０％（ｗ／ｗ）のエタノール溶液）の１０μＬを分取し、５ｍＬの上記培地Ｂに加えた。その後、超音波ホモジナイザー（Ｂｒａｎｓｏｎ社製、製品番号：ＳＦＸ１５０）を用いて懸濁させ、培地１２とした。この培地１２と培地１０または培地１１を３ｍＬずつ混和し、ＬＰＳまたはＡＴＰを含有する培養培地中の各精油の終濃度が１００ｐｐｍとなるように調製し、これをそれぞれ培地１３または１４とした。 [Method for preparing medium containing essential oil and LPS or ATP]
In order to examine the effects of essential oils, first, medium 10 or medium 10 prepared in the same manner as medium 8 or medium 9, except that LPS or ATP was added at a final concentration twice that of medium 8 or medium 9, respectively. A medium 11 was prepared. In addition, 10 μL of each composition (10% (w/w) ethanol solution of essential oil) prepared in Example 1 and Comparative Example 1 above was aliquoted and added to 5 mL of Medium B above. After that, it was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 12. 3 mL each of this medium 12 and medium 10 or medium 11 were mixed to adjust the final concentration of each essential oil in the culture medium containing LPS or ATP to 100 ppm, which was designated as medium 13 or 14, respectively.
［精油を添加した条件におけるヒトｉＰＳ細胞由来のミクログリア細胞の活性化の方法］
前項の［ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａに対するＩＬ－１β放出抑制試験］と同様に、ｉＣｅｌｌ(R)ミクログリアを、上記培地Ｂに１×１０5　ｃｅｌｌｓ／ｍＬとなるように懸濁し、４８ｗｅｌｌプレートに０．２５ｍＬ／ｗｅｌｌずつ播種した。当該４８ｗｅｌｌプレート上で培養したｉＣｅｌｌ(R)ミクログリア細胞の各ｗｅｌｌから培地を除去した後、培地１３を各ｗｅｌｌに０．２５ｍＬ／ｗｅｌｌずつ添加し、ＭＧ６細胞を４時間培養した。次に、各ｗｅｌｌから培地を除去した後、培地１４を各ｗｅｌｌに０．２５ｍＬ／ｗｅｌｌずつ添加し、ｉＣｅｌｌ(R)ミクログリア細胞をさらに３０分間培養した。当該３０分の培養後、各ｗｅｌｌから培地を活性化測定用培地として回収するとともに、残ったｉＣｅｌｌ(R)ミクログリアにＭＴＴアッセイ用の培地として培地Ｂ１０ｍＬにＭＴＴ試薬（同仁化学研究所製、品番：ＣＫ０４）を１ｍＬ加えた混合液を、各ｗｅｌｌに０．２５ｍＬ／ｗｅｌｌずつ加えて４時間の培養を行った。当該４時間後の培養後の培地を細胞生存率測定用培地とする。 [Method for activation of human iPS cell-derived microglial cells under the condition of adding essential oil]
In the same manner as in the previous section [IL-1β release inhibition test on iCell (R) microglia], iCell (R) microglia were suspended in the above medium B to a concentration of 1 x 10 5 cells/mL, and 0.0. Seeded at 25 mL/well. After removing the medium from each well of iCell (R) microglial cells cultured on the 48-well plate, 0.25 mL/well of medium 13 was added to each well, and MG6 cells were cultured for 4 hours. Next, after removing the medium from each well, 0.25 mL/well of medium 14 was added to each well, and the iCell (R) microglial cells were further cultured for 30 minutes. After culturing for 30 minutes, the medium is collected from each well as an activation measurement medium, and the remaining iCell (R) microglia is added to 10 mL of medium B as a medium for MTT assay. MTT reagent (manufactured by Dojindo Laboratories, product number: 0.25 mL/well of a mixed solution containing 1 mL of CK04) was added to each well and cultured for 4 hours. The culture medium after culturing for 4 hours is used as the cell viability measurement medium.
・吸光度の測定（ＭＴＴアッセイ）
精油のＭＧ６活性化抑制効果が細胞障害による非特異的な効果ではないことを確認するために、ＭＴＴアッセイによる細胞生存率の測定を行った。上記の方法で、活性化測定用培地を回収した後にｉＣｅｌｌ(R)ミクログリア細胞にＭＴＴアッセイ用の培地を添加し、４時間経過後に細胞生存率測定用培地を回収し、４５０ｎｍの吸光度（ＯＤ450）を測定することによってＭＴＴアッセイを行った。４時間の培養時に細胞を含まないｗｅｌｌにＭＴＴアッセイ用の培地を添加し、細胞に添加した培地と同様に４時間の培養後に４５０ｎｍの吸光度を測定したものをバックグラウンドとして、各試料の吸光度の値から差し引いた。コントロールの吸光度の値を１００％として、各添加群の吸光度の相対値（％）を生存率とした。吸光度の値が小さいほど、精油によって細胞が非特異的に障害されていると考えるため、コントロールと比較して、細胞生存率の低下よりもＩＬ－１βの放出量がより大きく低下したものを、ヒトｉＰＳ細胞由来のミクログリア細胞の活性化を抑制する効果が優れたものとした。なお、表６中の細胞障害率は３回測定した平均の値である。 ・Measurement of absorbance (MTT assay)
In order to confirm that the MG6 activation inhibitory effect of the essential oil is not a non-specific effect due to cytotoxicity, cell viability was measured by MTT assay. After collecting the activation measurement medium by the above method, the medium for MTT assay was added to the iCell (R) microglial cells, and after 4 hours, the medium for cell viability measurement was collected, and the absorbance at 450 nm (OD 450 ), the MTT assay was performed. MTT assay medium was added to wells containing no cells during 4 hours of culture, and absorbance at 450 nm was measured after 4 hours of culture in the same manner as the medium added to the cells. subtracted from the value. Taking the absorbance value of the control as 100%, the relative absorbance value (%) of each addition group was defined as the survival rate. The smaller the absorbance value, the more non-specifically damaged cells are considered to be caused by the essential oil. The effect of suppressing the activation of microglial cells derived from human iPS cells was excellent. In addition, the cytotoxicity rate in Table 6 is the average value measured three times.
表６
Figure JPOXMLDOC01-appb-I000014
Table 6
Figure JPOXMLDOC01-appb-I000014
［ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａによるＩＬ－１β放出量の測定］
培養後の上記［ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａに対するＩＬ－１β放出抑制試験］の活性化測定用培地中のＩＬ－１β濃度は、ＢｉｏＬｅｇｅｎｄ社のＥＬＩＳＡキット（ＥＬＩＳＡ　ＭＡＸTM　Ｄｅｌｕｘｅ　Ｓｅｔ　Ｈｕｍａｎ　ＩＬ－１β、製品番号：４３７００４）を用いて測定し、ｉＣｅｌｌ(R)ミクログリア細胞活性化の指標とした。コントロールは、ｉＣｅｌｌ(R)ミクログリア細胞を含有し、［ｉＣｅｌｌ(R)　ｍｉｃｒｏｇｌｉａに対するＩＬ－１β放出抑制試験］で調製した精油を含有しない活性化測定用培地である。なお、表７中のＩＬ－１β濃度は３回測定した平均の値である。 [Measurement of IL-1β release by iCell (R) microglia]
The concentration of IL-1β in the medium for measuring the activation of the [IL-1β release inhibition test on iCell (R) microglia] after culture was measured using an ELISA kit (ELISA MAX ™ Deluxe Set Human IL-1β, product number: BioLegend). : 437004) and used as an index of iCell (R) microglial cell activation. The control is a culture medium for activation measurement containing iCell (R) microglial cells and containing no essential oil prepared in the [IL-1β release suppression test for iCell (R) microglia]. The IL-1β concentration in Table 7 is the average value measured three times.
表７
Figure JPOXMLDOC01-appb-I000015
Table 7
Figure JPOXMLDOC01-appb-I000015
以下、本発明の組成物の配合例を示す。これらの配合例も、本発明の効果を発揮するものと理解できる。なお、質量％は全体（配合例１であればアロマディフューザー用オイル１全体）に対する割合である。
配合例１［アロマディフューザー用オイル１］
ニュウコウジュ油（FRANKINCENSE OIL、Boswellia Carterii Oil、Cas RN(R)：89957-98-2）：３０質量％
エタノール：残余 Hereinafter, formulation examples of the composition of the present invention are shown. It can be understood that these compounding examples also exhibit the effects of the present invention. It should be noted that % by mass is a ratio to the whole (in the case of Formulation Example 1, the entire aroma diffuser oil 1).
Formulation Example 1 [Aroma Diffuser Oil 1]
Nyukoju oil (FRANKINCENSE OIL, Boswellia Carterii Oil, Cas RN (R) : 89957-98-2): 30% by mass
ethanol: residue
配合例２［アロマディフューザー用オイル２］
レモン果皮油（Citrus Limon（Lemon）Peel Oil、Cas RN(R)：68916-89-2）：１０質量％
イランイラン花油（Cananga Odorata Flower Oil、Cas RN(R)：8006-81-3）：５．０質量％
エタノール：残余 Formulation Example 2 [Aroma Diffuser Oil 2]
Lemon peel oil (Citrus Limon (Lemon) Peel Oil, Cas RN (R) : 68916-89-2): 10% by mass
Ylang-ylang flower oil (Cananga Odorata Flower Oil, Cas RN (R) : 8006-81-3): 5.0% by mass
ethanol: residue
配合例３［ハンドミスト］
ローマカミツレ花油（Anthemis Nobilis Flower Oil、Cas RN(R)：8015-92-7）：０．２５質量％
シトルスメジカブルガリス果皮油（Citrus Medica Vulgaris Peel Oil、Cas RN(R)：93685-55-3）：０．２質量％
イランイラン花油（Cananga Odorata Flower Oil、Cas RN(R)：8006-81-3）：０．０５質量％
グリセリン：３．０質量％
エタノール：７０質量％
精製水：残余 Formulation Example 3 [Hand Mist]
Roman chamomile flower oil (Anthemis Nobilis Flower Oil, Cas RN (R) : 8015-92-7): 0.25% by mass
Citrus Medica Vulgaris Peel Oil (Citrus Medica Vulgaris Peel Oil, Cas RN (R) : 93685-55-3): 0.2% by mass
Ylang-ylang flower oil (Cananga Odorata Flower Oil, Cas RN (R) : 8006-81-3): 0.05% by mass
Glycerin: 3.0% by mass
Ethanol: 70% by mass
Purified water: remainder
配合例４［ロールオンフレグランス］
セイヨウアカマツ葉油（Pinus Sylvestris Leaf Oil、Cas RN(R)：8023-99-2）：５．４質量％
ライム油（Citrus Aurantifolia (Lime) Oil、Cas RN(R)：8008-26-2）：１．０質量％
スペアミント油（Mentha Viridis (Spearmint) Leaf Oil、Cas RN(R)：8008-79-5）：０．５質量％
ニュウコウジュ油（FRANKINCENSE OIL、Boswellia Carterii Oil、Cas RN(R)：89957-98-2）：０．１質量％
マカダミア種子油：２．５質量％
スクワラン：６０．５質量％
ホホバ種子油：３０質量％ Formulation Example 4 [Roll-on fragrance]
Japanese red pine leaf oil (Pinus Sylvestris Leaf Oil, Cas RN (R) : 8023-99-2): 5.4% by mass
Lime oil (Citrus Aurantifolia (Lime) Oil, Cas RN (R) : 8008-26-2): 1.0% by mass
Spearmint oil (Mentha Viridis (Spearmint) Leaf Oil, Cas RN (R) : 8008-79-5): 0.5% by mass
Nyukoju oil (FRANKINCENSE OIL, Boswellia Carterii Oil, Cas RN (R): 89957-98-2): 0.1% by mass
Macadamia seed oil: 2.5% by mass
Squalane: 60.5% by mass
Jojoba seed oil: 30% by mass
配合例５［超音波ディフューザー用オイル］
シトルスメジカブルガリス果皮油（Citrus Medica Vulgaris Peel Oil、Cas RN(R)：93685-55-3）：１０質量％
レモン果皮油（Citrus Limon（Lemon）Peel Oil、Cas RN(R)：68916-89-2）：１５質量％
イランイラン花油（Cananga Odorata Flower Oil、Cas RN(R)：8006-81-3）：３５質量％
ライム油（Citrus Aurantifolia (Lime) Oil、Cas RN(R)：8008-26-2）：２０質量％
スペアミント油（Mentha Viridis (Spearmint) Leaf Oil、Cas RN(R)：8008-79-5）：５．０質量％
ローマカミツレ花油（Anthemis Nobilis Flower Oil、Cas RN(R)：8015-92-7）：１５質量％ Formulation Example 5 [Oil for ultrasonic diffuser]
Citrus Medica Vulgaris Peel Oil (Citrus Medica Vulgaris Peel Oil, Cas RN (R) : 93685-55-3): 10% by mass
Lemon peel oil (Citrus Limon (Lemon) Peel Oil, Cas RN (R) : 68916-89-2): 15% by mass
Ylang-ylang flower oil (Cananga Odorata Flower Oil, Cas RN (R) : 8006-81-3): 35% by mass
Lime oil (Citrus Aurantifolia (Lime) Oil, Cas RN (R) : 8008-26-2): 20% by mass
Spearmint oil (Mentha Viridis (Spearmint) Leaf Oil, Cas RN (R) : 8008-79-5): 5.0% by mass
Roman chamomile flower oil (Anthemis Nobilis Flower Oil, Cas RN (R) : 8015-92-7): 15% by mass
配合例６［洗顔料］
ライム油（Citrus Aurantifolia Oil、Cas RN(R)：8008-26-2）：２．０質量％
ステアリン酸カリウム：２０質量％
ラウリン酸カリウム：５．０質量％
グリセリン：６．０質量％
１，３―ブチレングリコール：５．０質量％
水酸化ナトリウム：６．５質量％
酸化チタン：０．２質量％
クエン酸ナトリウム：０．０５質量％
精製水：残余 Formulation Example 6 [face wash]
Lime oil (Citrus Aurantifolia Oil, Cas RN (R) : 8008-26-2): 2.0% by mass
Potassium stearate: 20% by mass
Potassium laurate: 5.0% by mass
Glycerin: 6.0% by mass
1,3-butylene glycol: 5.0% by mass
Sodium hydroxide: 6.5% by mass
Titanium oxide: 0.2% by mass
Sodium citrate: 0.05% by mass
Purified water: remainder
配合例７［化粧水］
ローマカミツレ花油（Anthemis Nobilis Flower Oil、Cas RN(R)：8015-92-7）：０．５質量％
ソルビット：４．０質量％
ジプロピレングリコール：６．０質量％
グリセリン：０．５質量％
ＰＥＧ－３２：５．０質量％
ポリクオタニウム－５１：１．０質量％
アラントイン：０．０１質量％
エタノール：１０質量％
クエン酸：０．３質量％
精製水：残余 Formulation Example 7 [Lotion]
Roman chamomile flower oil (Anthemis Nobilis Flower Oil, Cas RN (R) : 8015-92-7): 0.5% by mass
Sorbit: 4.0% by mass
Dipropylene glycol: 6.0% by mass
Glycerin: 0.5% by mass
PEG-32: 5.0% by mass
Polyquaternium-51: 1.0% by mass
Allantoin: 0.01% by mass
Ethanol: 10% by mass
Citric acid: 0.3% by mass
Purified water: remainder
配合例８［クリーム］
ニュウコウジュ油（FRANKINCENSE OIL、Boswellia Carterii Oil、Cas RN(R)：89957-98-2）：０．５質量％
ライム油（Citrus Aurantifolia Oil、Cas RN(R)：8008-26-2）：０．３質量％
ステアリン酸：２．０質量％
ステアリルアルコール：６．０質量％
水添ラノリン：４．０質量％
スクワラン：９．０質量％
オクチルドデカノール：１０質量％
１，３－ブチレングリコール：６．０質量％
ポリエチレングリコール１５００：４．０質量％
ポリオキシエチレン（２５）セチルエーテル：３．０質量％
モノステアリン酸グリセリン：２．０質量％
精製水：残余  Formulation Example 8 [Cream]
Nyukoju oil (FRANKINCENSE OIL, Boswellia Carterii Oil, Cas RN (R) : 89957-98-2): 0.5% by mass
Lime oil (Citrus Aurantifolia Oil, Cas RN (R) : 8008-26-2): 0.3% by mass
Stearic acid: 2.0% by mass
Stearyl alcohol: 6.0% by mass
Hydrogenated lanolin: 4.0% by mass
Squalane: 9.0% by mass
Octyldodecanol: 10% by mass
1,3-butylene glycol: 6.0 mass%
Polyethylene glycol 1500: 4.0% by mass
Polyoxyethylene (25) cetyl ether: 3.0% by mass
Glyceryl monostearate: 2.0% by mass
Purified water: remainder
配合例９［飲料］
レモン果皮油（Citrus Limon（Lemon）Peel Oil、Cas RN(R)：68916-89-2）：０．３質量％
ライム油（Citrus Aurantifolia Oil、Cas RN(R)：8008-26-2）：０．３質量％
アスコルビン酸リン酸マグネシウム：０．１質量％
クエン酸：０．７質量％
果糖ブドウ糖液糖：６０質量％
精製水：残余 Formulation Example 9 [Beverage]
Lemon peel oil (Citrus Limon (Lemon) Peel Oil, Cas RN (R): 68916-89-2): 0.3% by mass
Lime oil (Citrus Aurantifolia Oil, Cas RN (R) : 8008-26-2): 0.3% by mass
Magnesium ascorbic acid phosphate: 0.1% by mass
Citric acid: 0.7% by mass
Fructose glucose liquid sugar: 60% by mass
Purified water: remainder
配合例１０［錠剤］
スペアミント油（Mentha Viridis (Spearmint) Leaf Oil、Cas RN(R)：8008-79-5）：５．０質量％
アスコルビン酸リン酸マグネシウム：５．０質量％
トウモロコシデンプン ：１０質量％
精製白糖：２０質量％
カルボキシメチルセルロースカルシウム：１０質量％
微結晶セルロース：３５質量％
ポリビニルピロリドン：５．０質量％
タルク：１０質量％ Formulation Example 10 [tablet]
Spearmint oil (Mentha Viridis (Spearmint) Leaf Oil, Cas RN (R) : 8008-79-5): 5.0% by mass
Magnesium ascorbic acid phosphate: 5.0% by mass
Corn starch: 10% by mass
Refined sucrose: 20% by mass
Carboxymethylcellulose calcium: 10% by mass
Microcrystalline cellulose: 35% by mass
Polyvinylpyrrolidone: 5.0% by mass
Talc: 10% by mass

Claims (12)

  1. 　シトルスメジカブルガリス果皮油、ニュウコウジュ油、レモン果皮油、ライム油、セイヨウアカマツ葉油、ローマカミツレ花油、スペアミント油、及びイランイラン花油からなる群から選択される精油を１種以上含む、ミクログリア細胞の活性化を抑制するための組成物。 Contains one or more essential oils selected from the group consisting of Citrus medica vulgaris pericarp oil, citrullus japonicum oil, lemon peel oil, lime oil, red pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. , a composition for inhibiting activation of microglial cells.
  2. 　前記精油が、ニュウコウジュ油及びローマカミツレ花油からなる群から選択される１種以上の精油である、請求項１に記載の組成物。 The composition according to claim 1, wherein the essential oil is one or more essential oils selected from the group consisting of Nelumbo japonicum oil and Roman chamomile flower oil.
  3. 　前記精油の含有量が、前記組成物を１００質量％とした場合、０．００１～３０質量％である、請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein the content of the essential oil is 0.001 to 30% by mass when the composition is 100% by mass.
  4. 　前記組成物が、医薬組成物、飲食品、化粧品、動物用飼料、又は雑貨品である、請求項１～３のいずれかに記載の組成物。 The composition according to any one of claims 1 to 3, wherein the composition is a pharmaceutical composition, food and drink, cosmetics, animal feed, or general merchandise.
  5. 　前記組成物が、ミクログリア細胞の活性化に起因する炎症を抑制するための医薬組成物である、請求項１～３のいずれかに記載の組成物。 The composition according to any one of claims 1 to 3, which is a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells.
  6. 　前記炎症の抑制が、ＩＬ-１α、ＩＬ-１β、ＩＬ-６、ＩＦＮ－γ、ＭＣＰ－１及びＴＮＦ－αから選択される炎症誘導性サイトカインの放出の抑制を伴うものである、請求項５に記載の組成物。 Claim 5, wherein the suppression of inflammation is accompanied by suppression of the release of pro-inflammatory cytokines selected from IL-1α, IL-1β, IL-6, IFN-γ, MCP-1 and TNF-α. The composition according to .
  7. 　前記炎症が、脳内及び／又は脊髄内の炎症である、請求項５又は６に記載の医薬組成物。 The pharmaceutical composition according to claim 5 or 6, wherein the inflammation is intracerebral and/or spinal cord inflammation.
  8. 　前記組成物が、ミクログリア細胞の活性化に起因する神経性疾患を治療又は予防するための医薬組成物である、請求項１～３のいずれかに記載の組成物。 The composition according to any one of claims 1 to 3, which is a pharmaceutical composition for treating or preventing neurological diseases caused by activation of microglial cells.
  9. 　前記神経性疾患が、うつ病、統合失調症、認知症、アルツハイマー病、パーキンソン病、多発硬化症、側索硬化症、ハンチントン舞踏病、及び慢性疲労症候群から選択される、請求項８に記載の医薬組成物。 9. The neurological disease of claim 8, wherein the neurological disease is selected from depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's chorea, and chronic fatigue syndrome. pharmaceutical composition.
  10. 　シトルスメジカブルガリス果皮油、ニュウコウジュ油、レモン果皮油、ライム油、セイヨウアカマツ葉油、ローマカミツレ花油、スペアミント油、及びイランイラン花油からなる群から選択される精油を１種以上含む、ＩＬ-１βの放出を抑制するための組成物。 Contains one or more essential oils selected from the group consisting of Citrus medica vulgaris pericarp oil, citrullus japonicum oil, lemon peel oil, lime oil, red pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. , a composition for inhibiting the release of IL-1β.
  11. 　前記精油が、ニュウコウジュ油及びローマカミツレ花油からなる群から選択される１種以上の精油である、請求項１０に記載の組成物。 The composition according to claim 10, wherein the essential oil is one or more essential oils selected from the group consisting of Nelumbo japonicum oil and Roman chamomile flower oil.
  12. 　前記精油の含有量が、組成物を１００質量％とした場合、０．００１～３０質量％である、請求項１０又は１１に記載の組成物。 The composition according to claim 10 or 11, wherein the content of the essential oil is 0.001 to 30% by mass when the composition is 100% by mass.
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