JP7436975B2 - essential oil composition - Google Patents

Description

The present invention relates to compositions and the like for suppressing activation of microglial cells.

Microglial cells are small, non-neuronal stromal cells of mesodermal origin that constitute part of the supporting tissue of the central nervous system. Normally, microglial cells have multiple elongated branching processes called lamified cells. When a stimulus such as injury to a nerve cell is applied, microglial cells are activated and change their shape into an ameboid shape. In recent years, it has been discovered that activation of microglial cells in the brain can be a cause of depression, schizophrenia, dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, lateral sclerosis, Huntington's chorea, etc. It is known that activation of microglial cells within the spinal cord can cause chronic fatigue syndrome. On the other hand, essential oils refer to oils containing volatile aromatic components that are mainly extracted from plants and are used for aromatherapy, as fragrances, and as liniments for the skin, as well as having anti-inflammatory and antibacterial effects. It has been reported that It has also been reported that some essential oil components move into the brain when the volatile essential oil is inhaled.
However, the relationship between essential oils and microglial cells, particularly the effect of essential oils on suppressing the activation of microglial cells, has not been investigated.

JP 2021-63018 Publication International Publication No. 2018/029793

Maria Graca, "Antioxidant and Anti-Inflammatory Activities of Essential Oils: A Short Review", Miguel, Molecules 2010, 15, pp. 9252-9287 Magdalena Valdivieso-Ugarte, et. al., "Antimicrobial, Antioxidant, and Immunomodulatory Properties of Essential Oils: A Systematic Review", Nutrients 2019, 11, 2786

One of the objects of the present invention is to provide a composition, such as a food/beverage product or a cosmetic product, for suppressing the activation of microglial cells. One of the objects of the present invention is to provide a composition 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.

The present inventors have discovered that certain essential oils have the effect of suppressing the activation of microglial cells and/or have the effect of suppressing the release of IL-1β, leading to the present invention. More specifically, the present invention may have the following aspects.
[1] 1 essential oil selected from the group consisting of Citrus medica vulgaris peel oil, pomegranate oil, lemon peel oil, lime oil, Scots pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. A composition for suppressing activation of microglial cells, comprising at least one species.
[2] The composition as described in [1] above, wherein the essential oil is one or more essential oils selected from the group consisting of white koji oil and 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, wherein the composition 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 proinflammatory cytokines selected from IL-1α, IL-1β, IL-6, IFN-γ, MCP-1 and TNF-α, The composition described in [5] above.
[7] The pharmaceutical composition according to [5] or [6] above, wherein the inflammation is inflammation within the brain and/or spinal cord.
[8] The composition according to any one of [1] to [3] above, wherein the composition is a pharmaceutical composition for treating or preventing a neurological disease caused by activation of microglial cells.
[9] The above-mentioned [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. The pharmaceutical composition described in ].
[10] 1 essential oil selected from the group consisting of Citrus medica vulgaris peel oil, pomegranate oil, lemon peel oil, lime oil, Scots pine leaf oil, Roman chamomile flower oil, spearmint oil, and ylang-ylang flower oil. A composition for inhibiting the release of IL-1β, comprising at least one species of IL-1β.
[11] The composition as described in [10] above, wherein the essential oil is one or more essential oils selected from the group consisting of pomegranate oil and chamomile flower oil.
[12] The composition according to [10] or [11], wherein the content of the essential oil is 0.001 to 30% by mass when the composition is 100% by mass.

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. Further, 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.

Here, the mode for carrying out the invention will be explained in detail, but preferred embodiments and more preferred embodiments exemplified below may be interchanged as appropriate regardless of expressions such as "for example," "preferable," and "more preferable." Can be used in combination. Moreover, the description of numerical ranges is an illustration, and ranges in which the upper and lower limits of each range and the numerical values of the examples are appropriately combined can also be preferably used. Furthermore, terms such as "contains" or "includes" may be read as "consist essentially of" or "consist only of."

<Composition>
One aspect of the invention is a composition that includes certain essential oils. Here, the composition is not limited to the form of a solution, gel, solid, mist, etc., as long as it is an essential oil itself or a mixture containing an essential oil. The composition may be not only a pharmaceutical composition or a preparation, but also a food/drink, cosmetic, animal feed, miscellaneous goods, etc., or it may be used as a raw material for existing pharmaceutical compositions, food/beverage, cosmetics, etc. , animal feed, miscellaneous goods, etc.
<Essential oil>
Essential oils refer to oils obtained mainly by extraction from plants, preferably oils containing volatile aromatic components. Although essential oils include animal-derived essential oils and plant-derived essential oils, 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, highly volatile essential oils 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 the form of administration is simple. The parts of the plant from which the essential oil is extracted are not particularly limited, but include, for example, leaves, roots, stems, bark, seeds, flowers, fruits, fruit peels, and combinations thereof. The plants can be used as they are or after being dried and crushed.

The essential oils that can be used as active ingredients in the present invention include Citrus medica vulgaris peel oil, pomegranate oil, lemon peel 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 peel oil refers to an oil containing aromatic components obtained by extraction from the peel of Citrus medica vulgaris. White koji oil refers to an oil containing aromatic components obtained by extraction from a part or the whole of a white koji tree, including the wood, bark, or sap of a white koji tree. Lemon peel oil refers to an oil containing aromatic components obtained by extracting from lemon peel. Lime oil refers to an oil containing aromatic components obtained by extracting part or all of a lime, including the fruit, peel, or pulp of the lime. Pinus leaf oil refers to an oil containing aromatic components that is extracted from the leaves of Pinus sylvestris. Roman chamomile flower oil refers to an oil containing aromatic components extracted from Roman chamomile flowers. Spearmint oil refers to an oil containing aromatic components obtained by extracting part or all of spearmint including spearmint leaves, stems, roots, etc. Ylang-ylang flower oil is an oil containing aromatic components extracted from ylang-ylang flowers. A preferred extraction method will be described later.

More specifically, essential oils as shown in Table 1 below are preferable.
Table 1

Preferably, one or more essential oils selected from the group consisting of Citrus medica vulgaris peel oil, pomegranate oil, lime oil, and Roman chamomile flower oil, or pomegranate oil, lemon peel oil, lime oil, and 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 nutmeg oil and chamomile flower oil. These essential oils have been specifically found to have remarkable effects such as suppressing the activation of microglial cells, and are therefore preferred. This effect is surprising since bergamot fruit oil, peppermint 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 essential oils. 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, and even more preferably 2:1. A ratio of ~1:2 is appropriate.

The content of 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 0.01 to 20% by mass, when the composition is 100% by mass. A suitable amount is 10% by weight, more preferably 0.1 to 10% by weight. When the composition is a composition containing ethanol, the content of 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 A suitable amount is 0.01 to 10% by weight, more preferably 0.1 to 10% by weight. In addition, the content of essential oils varies depending on the form of the composition. For example, when the composition is used as a raw material for medicines or in the form of an aroma diffuser, the content of essential oils in the composition of the present invention may vary depending on the form of the composition. may be 30 to 100% by weight, 70 to 100% by weight, or 95 to 98% by weight.

<Optional ingredients>
The composition of the present invention may contain other essential oils and other active ingredients in addition to the above-mentioned essential oil as an active ingredient. Although other essential oils cannot be expected to have the effects of the present invention, they can provide other relaxing effects, moisturizing effects, etc. Other essential oils include yuzu peel oil, bergamot leaf oil, bergamot fruit oil, damask rose flower oil, mint seed oil, juniper oil, chamomile oil, salvia oil, eucalyptus leaf oil, argentine oil, and 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 tree leaf oil, lavandula hybrida oil, lavender oil, ginkgo tree leaf oil , Ginkgo flower oil, Majorana leaf oil, Melissa leaf oil, Palmarosa oil, Hirohalabender flower oil, Sandalwood oil, Mandarin orange peel oil, Mentha oil, Thymus flower/leaf oil, Mentha leaf oil, Coriander fruit oil, Italian Cypress leaf Examples include /fruit/stem oil, juniper fruit oil, lemongrass oil, silverberry fruit oil, tea tree leaf oil, vetiver root oil, meboki oil, valerian root oil, etc. (here, "/" is "or") ). Similar to the present invention, these other essential oils are obtained by extraction methods commonly used for essential oils, such as compression, juice extraction, solvent extraction, steam distillation, supercritical extraction, carbon dioxide extraction, and cold extraction. Essential oils extracted by steeping, digestion, etc. alone or in combination can be used.
The content of other essential oils in the composition can be any amount as long as it does not impair the effect of the original active ingredient (essential oil), but for example, from 0.001% by mass to 70% by mass. It is suitable that the content is 0.01% by mass to 50% by mass, more preferably 0.1% to 30% by mass.

Other active ingredients include the effects 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, as well as other effects, such as antibacterial, insecticidal, anti-inflammatory, Known active ingredients that can provide effects such as analgesia, sedation, and relaxation effects can be mentioned. 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 any amount as long as it does not impair the effect of the original active ingredient (essential oil), but for example, from 0.001% by mass. It is appropriate to contain 90% by weight, preferably 0.01% to 70% by weight, more preferably 0.1% to 50% by weight.

Furthermore, the composition of the present invention may optionally include excipients, binders, emulsifiers, solvents, adhesives, disintegrants, thickeners, lubricants, colorants, etc., depending on the use and purpose of the composition. Additives such as flow agents and humectants may also be added. Examples of excipients include alcohol, glycerin, dextrin, starch, and the like. Binders include, for example, cellulose, methylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose. Examples of the emulsifier 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. Examples of solvents include monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, propylene glycol, and dipropylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, and tetrahydrofuran (THF). ); organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane, 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, sasanquat oil, castor oil, linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, chinensis oil, Japanese tung oil, jojoba Liquid oils such as oil, germ oil, and triglycerin; Hydrocarbon oils such as liquid paraffin, ozokerite, squalane, pristane, paraffin, ceresin, squalene, petrolatum, and microcrystalline wax; Cetyl octoate, myristyl myristate, and tri2-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 alone or in combination of two or more. For example, alcohols, which act both as excipients and solvents, act as both excipient and solvent additives even when one type is included. The content of the additive in the composition can be any amount as long as it does not impair the effect of the active ingredient, but for example, 0.001% by mass to 70% by mass, preferably 0.01% by mass. It is suitable that the content is from 50% by mass, more preferably from 0.1% to 30% by mass. Among them, regarding the organic solvent, when the composition is 100% by mass, for example, about 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 include it. It is appropriate to contain the oil in an amount of, for example, 0.01 to 95% by mass, preferably 1 to 90% by mass, and more preferably 5 to 80% by mass.

<Production method/Extraction method>
As a method for producing essential oils, methods generally used for preparing essential oils can be used, and generally known methods for obtaining essential oils by extracting plants can be used. As the extraction method, for example, pressing, squeezing, solvent extraction, steam distillation, supercritical extraction, carbon dioxide extraction, cold soaking, digestion, etc. can be used alone or in combination. Essential oils can be obtained by further optionally removing solvents and the like from the extracts obtained by these extraction methods. Extraction solvents include, for example, monohydric or polyhydric alcohols such as methanol, ethanol, propanol, n-butanol, ethylene glycol, and propylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether, tetrahydrofuran (THF), etc. Examples include ethers; organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane, and dimethylformamide; and water.
Extraction conditions for extraction vary depending on the plant and solvent used, but temperature, pressure, extraction time, etc. commonly used in extraction methods can be used. For example, in a steam distillation method using water vapor, the temperature is 40 to 120 °C, preferably 50 to 100 °C, which is below the boiling point of general essential oils (150 to 350 °C), and the pressure is 5 to 200 kPa, preferably 10 to 100 kPa. It may be carried out under reduced pressure. In the extraction method using alcohol such as ethanol, it may be carried out, for example, at room temperature (25° C.±5° C.) and under high pressure of about 300 to 1000 kPa, preferably about 500 to 800 kPa. The extraction solvent may be removed after the extraction operation.

<Application>
The composition of the present invention can be used not only in pharmaceutical compositions, but also in foods and beverages, cosmetics, animal feed, and miscellaneous goods. Pharmaceutical compositions include, for example, oral preparations such as tablets, pills, capsules, powders, granules, syrups, and sublingual preparations; injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, Examples include parenteral preparations such as drops, external preparations (ointments, creams, poultices, patches, and inhalants), and suppositories. Food and beverages 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 include all foods given to humans, such as plant foods, animal foods, synthetic foods, grains, bread, noodles, cakes, noodles, freeze-dried foods, dairy products, seasonings, frozen foods, and nutritional supplements. , and medical foods. Cosmetics include, for example, lotion, emulsion, moisturizer, serum, skin oil, perfume, sunscreen, shaving lotion, pack, hair tonic, hair balm, hair wax, hair grease, hair oil, hair treatment, hair color, and face wash. Examples include body shampoo, shampoo, conditioner, conditioner, cleansing, cream, body cream, hand cream, makeup base, foundation, mascara, eyeliner, concealer, lipstick, lip balm, gloss, and nail color. 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 for mammals. . Examples of miscellaneous goods include air fresheners, aroma diffusers, aroma candles, aroma stones, aroma mist, clothing mist, deodorant mist, pillow mist, insect repellent, laundry detergent, fabric softener, and bath salts. .
The content of the composition when included in pharmaceutical compositions, food and beverages, cosmetics, animal feed, miscellaneous goods, etc. depends on the type of pharmaceutical composition, etc. to be included, but is an amount that does not impair the effects of the present invention. The pharmaceutical composition contains, for example, 0.001% to 70% by weight, preferably 0.01% to 50% by weight, more preferably 0.1% to 30% by weight, based on the total amount. It is appropriate to do so. In foods and drinks, it is appropriate to contain, for example, 0.001% to 50% by weight, preferably 0.01% to 30% by weight, more preferably 0.1% to 10% by weight. For cosmetics, it is appropriate to contain, for example, 0.001% to 70% by weight, preferably 0.01% to 50% by weight, more preferably 0.1% to 30% by weight. In animal feed, it is appropriate to contain, for example, 0.001% to 50% by weight, preferably 0.01% to 30% by weight, more preferably 0.1% to 10% by weight. For miscellaneous goods, it is appropriate to contain, for example, 0.01% to 100% by weight, preferably 0.01% to 80% by weight, more preferably 0.1% to 50% by weight.

<How to use>
The composition of the present invention is used to suppress microglial cell activation (method for suppressing microglial cell activation). Microglial cells are small, non-neuronal stromal cells of mesodermal origin that constitute part of the supporting tissue of the central nervous system. Essential oils are generally known to have anti-inflammatory effects, but their effectiveness in suppressing the activation of microglial cells is not known, and we have recently discovered that only a few essential oils can suppress the activation of microglial cells. The effect of It is surprising that such an effect is observed, and it is expected that diseases and symptoms 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 chorea, and chronic fatigue syndrome.

The composition of the present invention is also used to inhibit the release of IL-1β (IL-1β release inhibition method). In particular, it has been found that by using the specific essential oil of the present invention, the effect of suppressing IL-1β production can be obtained without showing substantial or large cytotoxicity.
The composition of the present invention is further 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 within the brain and spinal cord. Examples of sites in the brain where inflammation occurs include the hippocampus, cerebral cortex, cerebellum, basal ganglia, and nigrostriatal body. Examples of spinal cord sites where inflammation occurs include the dorsal horn of the spinal cord. 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 inducible cytokines, preferably accompanied by the release (expression) of pro-inflammatory cytokines 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 that is the active ingredient, suppresses the release (expression) of inflammation-inducing cytokines such as IL-1β, thereby suppressing the activation of microglial cells. Ru. Here, examples of microglial cells include MG6 cells, iCell ^(R) microglia, and BV2 cells.

<Administration method>
When the composition of the present invention is in the form of a pharmaceutical composition or food (particularly a functional food or a nutritional supplement), it may vary depending on its dosage form, administration target, administration route, target disease, symptoms, active ingredient concentration, etc. However, the daily dosage of the active ingredient is, for example, 0.1 to 20 mg/kg body weight, preferably 0.2 to 10 mg/kg body weight, and more preferably 0.5 to 10 mg/kg body weight. It is desirable to administer this amount once to several times (eg, 2, 3, 4 or 8 times) per day. When the composition of the present invention is administered to adults (body weight 70 kg), the amount is, for example, 0.1 to 20 mg/day, preferably 0.2 to 10 mg/day, and more preferably 0.5 to 10 mg/day. That is appropriate.
The method of administration varies depending on the dosage form and form of the pharmaceutical composition or food, but for example, when a liquid preparation is administered to a subject by suction, it is preferable to administer it using an aroma diffuser, spray, mist, or the like.

Hereinafter, the present invention will be specifically explained based on Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 [Preparation of composition]
As each essential oil, the essential oils listed in Table 2 were used. A composition of each essential oil as a 10% (w/w) ethanol solution was prepared by weighing 2-3 mg of each essential oil and adding 9 times the amount (w/w) of ethanol.

Table 2

Comparative Example 1 [Preparation of composition]
The essential oils listed in Table 3 were used as essential oils for comparative examples. In the same manner as in Example 1, 2 to 3 mg of 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.

Table 3

[How to obtain MG6 cells and how to prepare culture 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 500 mL of D-MEM (manufactured by Fuji Film Wako, product number: 049-32645), 50 mL of fetal bovine serum (manufactured by Biofill), and insulin (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). (Product Number: 099-06473) and 2-mercaptoethanol were added 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 the RIKEN Cell Bank.

[Method of activating 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, it was confirmed that the cells were attached. , performed the following operations. First, medium 1 was prepared by adding LPS (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 125-05181) to medium A in an amount such that the final concentration was 1 μg/mL. In addition, a 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 an amount such that the final concentration was 1 mmol/L. After removing the medium from each well of the MG6 cells cultured in the 24-well plate prepared as described above, 0.5 mL/well of medium 1 was added and cultured for 4 hours. Next, the medium in each well was removed, and 0.5 mL/well of medium 2 was added to the MG6 cells in each well, followed by culturing for 30 minutes. After the 30-minute culture, the medium was collected from each well as a medium for activation measurement, and a new solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the above medium A was added. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.

[Method for preparing medium containing essential oil and LPS or ATP]
In order to examine the effects of essential oils, etc., first, medium 3 or medium 3 or ATP was prepared in the same manner as medium 1 or medium 2, except that twice the amount of LPS or ATP as in medium 1 and medium 2 was added at the final concentration. 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 taken out and added to 5 mL of medium A. Thereafter, the suspension was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 5. This medium 5 was mixed with 3 mL each of medium 3 or medium 4, and the final concentration of each essential oil in the culture medium containing LPS or ATP was adjusted to 100 ppm, and this was designated as medium 6 or 7, respectively.

[Method for activating MG6 cells under conditions in which essential oil is added]
In the same manner as in the previous section [Method of activating microglia with LPS and ATP using MG6 cells], MG6 cells were seeded on a 24-well plate at 0.5 x 10 ⁵ cells/well, and the cells were allowed to adhere the next day. 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 the 30-minute culture, the medium was collected from each well as a medium for activation measurement, and a new solution prepared by adding 0.1% Triton X-100 (manufactured by Nacalai Tesque) to the above medium A was added. , 0.5 mL/well was added to each well to prepare a cell lysate containing MG6 cells.

[Measurement of IL-1β release amount by MG6 cells]
The IL-1β concentration in the above activation measurement medium and cell lysate after culture was measured using an ELISA kit (ELISA MAX ^TM Deluxe Set Mouse IL-1β, product number: 432604) from BioLegend. Hereinafter, in each test, the "control" is a cell lysate containing MG6 cells and not containing essential oil prepared by [Method of activating 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), which is added to medium 1 and medium 2 at a final concentration of 10 μmol/L. This was used as a positive control to confirm that IL-1β release was actually suppressed. Note that the IL-1β concentrations in Tables 4-1 to 4-5 are the average values measured three times.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

[Cytotoxicity rate test for MG6 cells]
In order to confirm that the essential oil's inhibitory effect on MG6 activation was not a non-specific effect due to cell damage, the cell damage rate was measured by LDH assay. Using an LDH assay kit manufactured by Fuji Film Wako Pure Chemical Industries, Ltd. (product number: 341-91754), the LDH activity of the activation measurement medium and cell lysate after culture was measured. The total LDH activity of the activation measurement medium and the cell lysate in each well was defined as the total LDH activity, and the ratio of the LDH activity of the cell lysate to the total LDH activity was expressed as a cell damage rate in %. The "control" and "MCC950" used were 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 amount of IL-1β released into the medium are particularly effective in suppressing the activation of MG6 cells. It was assumed that Note that the cell damage rates in Tables 5-1 to 5-5 are the average values of three measurements.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

[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 the microglial cells, iCell ^(R) microglia (manufactured by FUJIFILM Cellular Dynamics, Inc.), which are microglial cells derived from human iPS cells, were used. Note that iCell is a registered trademark of Fujifilm Cellular Dynamics Incorporated. iCell ^(R) microglia was cultured according to the culture method recommended by Fuji Film Wako Pure Chemical Industries, Ltd. Specifically, first, iCell ^{( R)} microglial supplement A (product number: 550-36881) and iCell ^{( R)} Supplement B for microglia (product number: 557-36891) and iCell ^(R) supplement C for nerves (product number: 553-36871) were added to prepare a medium (hereinafter referred to as medium B). Because iCell ^(R) microglia has weak adhesive properties, the culture vessel was coated with Cell-Tak cell and tissue adhesive (Cell-Tak, manufactured by Corning, product number: CLS354240) during culture.
The iCell ^(R) microglia was suspended in the medium B at 1×10 ⁵ cells/mL, and seeded at 0.25 mL/well in a 48-well plate. After seeding, centrifuge for 5 minutes at 20°C and 1,000 rpm using a Tomy centrifuge (product number: CAX-571) and rotor (product number: TS-4C) to spread the cells onto the bottom of the plate. gathered to promote adhesion.
Two days after seeding, after removing the medium, add a new medium 8 to each well of the above medium B with a final concentration of 1 μg/mL LPS (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 125-05181). iCell ^(R) microglia was cultured for 4 hours. Next, remove medium 8 from each well, and add ATP (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 303-50511) to the new culture medium B at a final concentration of 1 mmol/L. The iCell ^(R) microglia was further cultured for 30 minutes by adding 0.25 mL/well of culture medium 9. After the 30-minute culture, the medium was collected from each well as a medium for activation measurement, and the remaining iCell ^(R) microglia was injected with MTT reagent (manufactured by Dojindo Laboratories, product number: A mixture containing 1 mL of CK04) was added to each well at a rate of 0.25 mL/well, and cultured for 4 hours. The medium after the 4-hour culture was used as a medium for measuring cell viability.

[Method for preparing medium containing essential oil and LPS or ATP]
In order to examine the effects of essential oils, etc., first, medium 10 or medium 10 or ATP was prepared in the same manner as medium 8 or medium 9, except that twice the amount of LPS or ATP as in medium 8 and medium 9 was added at the final concentration. 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 was taken out and added to 5 mL of the above medium B. Thereafter, the suspension was suspended using an ultrasonic homogenizer (manufactured by Branson, product number: SFX150) to obtain medium 12. This medium 12 was mixed with 3 mL each of medium 10 or medium 11, and the final concentration of each essential oil in the culture medium containing LPS or ATP was adjusted to 100 ppm, and this was designated as medium 13 or 14, respectively.

[Method for activating human iPS cell-derived microglial cells under conditions in which essential oil is added]
In the same manner as in the previous section [IL-1β release inhibition test for iCell ^(R) microglia], iCell ^(R) microglia were suspended in the above medium B to a concentration of 1 x 10 ⁵ cells/mL, and placed in a 48-well plate at 0.05 mA. It was 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 the 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 the 30-minute culture, the medium was collected from each well as a medium for activation measurement, and the remaining iCell ^(R) microglia was injected with MTT reagent (manufactured by Dojindo Laboratories, product number: A mixture containing 1 mL of CK04) was added to each well at a rate of 0.25 mL/well, and cultured for 4 hours. The medium after the 4-hour culture is used as a medium for measuring cell viability.

・Measurement of absorbance (MTT assay)
In order to confirm that the essential oil's inhibitory effect on MG6 activation was not a non-specific effect due to cell damage, cell viability was measured by MTT assay. After collecting the medium for measuring activation using the above method, add the medium for MTT assay to iCell ^(R) microglial cells, collect the medium for measuring cell viability after 4 hours, and measure the absorbance at 450 nm (OD ₄₅₀ The MTT assay was performed by measuring ). A medium for MTT assay was added to a well containing no cells during 4 hours of culture, and the absorbance at 450 nm was measured after 4 hours of culture in the same way as the medium added to the cells.The absorbance of each sample was measured using the background. subtracted from the value. The absorbance value of the control was set as 100%, and the relative value (%) of the absorbance of each addition group was defined as the survival rate. The smaller the absorbance value, the more non-specifically damaged the cells by the essential oil. It was determined that the effect of suppressing the activation of microglial cells derived from human iPS cells was excellent. Note that the cell damage rate in Table 6 is the average value of three measurements.

Table 6

[Measurement of IL-1β release amount using iCell ^(R) microglia]
The IL-1β concentration in the activation measurement medium of the above [IL-1β release inhibition test for iCell ^(R) microglia] after culturing was determined using the BioLegend ELISA kit (ELISA MAX ^TM Deluxe Set Human IL-1β, product number :437004) and used as an index of iCell ^(R) microglial cell activation. The control is an activation measurement medium containing iCell ^(R) microglia cells and not containing essential oil prepared in [IL-1β release inhibition test for iCell ^(R) microglia]. Note that the IL-1β concentration in Table 7 is the average value of three measurements.

Table 7

Examples of formulations of the composition of the present invention are shown below. It can be understood that these formulation examples also exhibit the effects of the present invention. In addition, mass % is a ratio with respect to the whole (in the case of formulation example 1, the whole aroma diffuser oil 1).
Formulation example 1 [Oil for aroma diffuser 1]
FRANKINCENSE OIL, Boswellia Carterii Oil, Cas RN ^(R) : 89957-98-2): 30% by mass
Ethanol: residual

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: residual

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 (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

Formulation example 4 [roll-on fragrance]
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
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

Formulation example 5 [Oil for ultrasonic diffuser]
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

Formulation example 6 [facial cleanser]
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

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

Formulation example 8 [cream]
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% by 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

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

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 white sugar: 20% by mass
Carboxymethylcellulose calcium: 10% by mass
Microcrystalline cellulose: 35% by mass
Polyvinylpyrrolidone: 5.0% by mass
Talc: 10% by mass

Claims (12)

A composition for suppressing activation of microglial cells, comprising one or more volatile essential oils selected from the group consisting of Citrus medica vulgaris peel oil, lemon peel oil, lime oil, and Pinus fulva leaf oil. The composition according to claim 1, wherein the essential oil is one or more essential oils selected from the group consisting of Citrus medica vulgaris peel oil, lemon peel oil, and lime oil. 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. The composition according to claim 1 or 2, wherein the composition is a pharmaceutical composition, food or drink, cosmetics, animal feed, or miscellaneous goods. The composition according to claim 1 or 2, wherein the composition is a pharmaceutical composition for suppressing inflammation caused by activation of microglial cells. 5. 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 described in. The pharmaceutical composition according to claim 5, wherein the inflammation is inflammation within the brain and/or spinal cord. The composition according to claim 1 or 2, wherein the composition 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. Pharmaceutical composition. A composition for suppressing the release of IL-1β, comprising one or more essential oils selected from the group consisting of Citrus medica vulgaris peel oil, lemon peel oil, lime oil, and Pinus fulva leaf oil. The composition according to claim 10, wherein the essential oil is one or more essential oils selected from the group consisting of Citrus medica vulgaris peel oil, lemon peel oil, and lime oil. 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.