JP2024067721A - Agent for activating phagocytic activity of macrophages and composition containing the same - Google Patents

Abstract

[Problem] The present invention aims to provide a technique for activating the phagocytic ability of macrophages. [Solution] An agent for activating the phagocytic ability of macrophages, which contains an essential oil of a plant belonging to the genus Citrus in the family Rutaceae as an active ingredient. [Selected Figure] Figure 2

Description

The present invention relates to an agent for activating the phagocytic activity of macrophages and a composition containing the same.

Macrophages, a type of white blood cell, play a central role in immune function. Macrophages are also called phagocytes because they have the ability to phagocytose various foreign substances that invade from the outside. Macrophages are present in various tissues and peripheral blood, and tissue macrophages include histiocytes in connective tissue, Kupffer cells in the liver, pulmonary macrophages in the lungs, Langerhans cells in the skin, and osteoclasts in the bones. Therefore, macrophages are present everywhere in the body and are important immune cells that process foreign substances, so in recent years, treatments that activate the phagocytic ability of macrophages to treat infectious diseases, cancer, etc. have been attracting attention.

Incidentally, essential oils obtained from plants of the genus Citrus in the family Rutaceae are widely used in aromatherapy, and for example, the scent of essential oil from bergamot, a plant of the genus Citrus in the family Rutaceae, is known to have antidepressant, antibacterial, sedative, sleep-inducing effects, etc. Specifically, it has been reported that the scent of bergamot essential oil has the effect of lowering the concentration of cortisol, a stress hormone, and has a psychological stress-reducing effect (Non-Patent Document 1).
However, it is not known whether essential oils from plants of the genus Citrus in the family Rutaceae have the effect of activating the phagocytic ability of macrophages.

Aromatherapy Journal Vol. 20, No. 3, 28-37 (2019)

The objective of the present invention is to provide a technology for activating the phagocytic ability of macrophages.

The present inventors have found that essential oils from plants belonging to the genus Citrus in the family Rutaceae activate the phagocytic ability of macrophages.

The present invention includes the following aspects.
[1] An agent for activating the phagocytic ability of macrophages, comprising an essential oil from a plant belonging to the genus Citrus in the family Rutaceae as an active ingredient.
[2] The phagocytic ability activator described in [1], wherein the essential oil of a plant belonging to the genus Citrus in the family Rutaceae is one or more selected from the group consisting of bergamot essential oil, orange essential oil, and yuzu essential oil.
[3] A composition for improving or preventing aging and/or damage to biological tissues, comprising the phagocytic ability activator according to [1] or [2].
[4] The composition described in [3], wherein the biological tissue is skin tissue.
[5] A composition comprising the phagocytic ability activator according to [1] or [2], the composition being a cosmetic, a quasi-drug, or a pharmaceutical.

The present invention has an effect of providing a technique for activating the phagocytic ability of macrophages.

Fluorescence micrograph of human monocyte-derived M1 macrophages with added fluorescent beads. Graph showing the percentage of human monocyte-derived M1 macrophages that phagocytosed fluorescent beads.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate without departing from the spirit of the present invention. The present invention is not limited to the embodiments, but only by the claims. Furthermore, each aspect disclosed in this specification can be combined with any other feature disclosed in this specification.

The agent for activating the phagocytic activity of macrophages of the present invention (hereinafter also referred to as the agent of the present invention) contains an essential oil of a plant belonging to the genus Citrus in the family Rutaceae as an active ingredient.
The essential oils of plants belonging to the genus Citrus in the family Rutaceae include, but are not limited to, bergamot (Citrus
Preferably, the essential oil is at least one selected from the group consisting of Citrus x bergamia essential oil, Citrus sinensis essential oil, and Citrus junos essential oil, and more preferably bergamot essential oil. Note that the term "essential oil" in the present invention refers to an oily component that is insoluble or poorly soluble in water among components extracted from plants.

The parts used for extraction are not particularly limited, and examples include flowers, stems, leaves, branches, roots, fruits, peels, buds, etc., and among these, it is preferable to use the peels. When extracting, these may be in a fresh or dried state.

The method of extracting essential oils is not particularly limited, and examples include squeezing, steam distillation, solvent extraction, oil adsorption, and supercritical fluid extraction, with squeezing being preferred. Commercially available essential oils may also be used.

In the present invention, "macrophages" refer to cells that are differentiated in each tissue from monocytes (mononuclear leukocytes), a type of white blood cell, under the influence of various environmental factors and inductive factors. In the present invention, "macrophages" include macrophages present in various tissues and peripheral blood, and are not particularly limited, but for example, macrophages present in peripheral blood are preferred. In other words, macrophages derived from peripheral blood are preferred.

Macrophages are broadly classified into M1 type macrophages (M1 macrophages) and M2 type macrophages (M2 macrophages) according to their differentiation type. M1 macrophages are also known as classically activated macrophages or inflammatory macrophages, and are macrophages that produce inflammatory cytokines such as TNFα to enhance immunity and promote inflammation. M2 macrophages are also known as wound-healing macrophages or anti-inflammatory macrophages, and are macrophages that produce anti-inflammatory cytokines such as IL-10 to suppress immunity and suppress inflammation.
In the present invention, the macrophage may be an M1 macrophage or an M2 macrophage, but is preferably an M1 macrophage.

Essential oils from plants belonging to the genus Citrus in the family Rutaceae have the effect of activating the phagocytic ability of macrophages, and can efficiently eliminate unnecessary substances from the body. The phagocytic ability of macrophages is the function of macrophages to take in unnecessary substances in the body and break them down, and is also called phagocytosis. Substances that macrophages phagocytose include pathogens such as viruses or bacteria, cancer cells, aging cells that accumulate in tissues and organs due to aging, aging stem cells with reduced differentiation and proliferation abilities, damaged cells that have been subjected to external stimuli (trauma, burns, etc.), dead cells, melanin granules that have dripped from the epidermis to the dermis, waste products (oxidized bad cholesterol, amyloid beta that causes dementia, melanin), etc.
Therefore, the essential oil of a plant belonging to the genus Citrus of the family Rutaceae can be used as an active ingredient of an agent for activating the phagocytic activity of macrophages, and can also be used as an active ingredient of a skin-whitening agent or an immune-enhancing agent. The agent of the present invention and a composition containing the agent of the present invention can also be used for improving or preventing aging and/or damage of biological tissue, and the biological tissue is preferably skin tissue.

When the biological tissue is skin tissue, the agent of the present invention can be used for improving or preventing aging and/or damage of skin tissue caused by dermal senescent cells accumulated in the dermis layer, waste products, external stimuli (trauma, burns, etc.), etc. In the present invention, "improvement" includes "treatment".
Furthermore, the agent of the present invention can be used for improving or preventing diseases, etc. (including diseases, symptoms, signs, and disorders in this specification) caused by aging and/or damage of skin tissue. Here, diseases, etc. caused by aging or damage of skin tissue include, but are not limited to, wrinkles, sagging, loss of firmness, pigmentation (spots), dullness, thickening of the skin, open pores, acne scars, skin atrophy (linear atrophy of the skin, photoelastosis, white fibrous papulosis of the neck, lichen sclerosus atrophicus, etc.), urticaria, contact dermatitis (rash), atopic dermatitis, wounds in the epidermis, dermis, and/or subcutaneous tissue (cuts, lacerations, punctures, bites, contusions, contusions, abrasions, etc.), bedsores, burns, scars, keloids, etc., and also include damage to the scalp and hair, such as thinning hair and hair loss. Among these, wounds (cuts, lacerations, puncture wounds, bite wounds, contusions, bruises, abrasions, etc.) in the epidermis, dermis, and/or subcutaneous tissue, bedsores, burns, scars, and keloids are preferred.
Furthermore, when the biological tissue is other than skin tissue, examples of diseases and the like caused by aging or damage to biological tissue include various age-related diseases (arteriosclerosis, osteoarthritis, cataracts, age-related macular degeneration, etc.) associated with destruction or functional decline of organs and tissues due to secretory substances of senescent cells that accumulate with age, infectious diseases (influenza, pneumonia, tuberculosis), allergic diseases (hay fever, allergic rhinitis, bronchial asthma, etc.), autoimmune diseases (rheumatoid arthritis, polymyositis, type 1 diabetes, systemic lupus erythematosus, glomerulonephritis, etc.), cancer, inflammatory bowel disease, dementia (Alzheimer's dementia, etc.), neuropathic pain, metabolic abnormalities and decline, and the like.

In the present invention, the activation of the phagocytic ability of macrophages includes the display of the phagocytic ability of macrophages when the macrophage phagocytic ability activator of the present invention is applied to a subject. In other words, in the present invention, the activation of the phagocytic ability of macrophages includes the induction or enhancement of the phagocytic ability of macrophages.

Whether or not the phagocytic ability of macrophages is activated can be confirmed by a known method for measuring the phagocytic ability of macrophages. Examples of methods for measuring the phagocytic ability of macrophages include a method in which the phagocytic ability activator of macrophages of the present invention is administered orally, intraperitoneally, intravenously, or the like to a subject, peritoneal cells, etc. are collected, fluorescent beads are added to the macrophages collected by adhesion method to cause phagocytosis, and the proportion of phagocytosed macrophages and the number of phagocytosed fluorescent beads are measured by observation with a fluorescent microscope. Another example is a method in which the phagocytic ability activator of macrophages of the present invention is added to known macrophage cells, cultured in a culture medium by a conventional method, the cells are collected, fluorescent beads are added to the collected cells to cause phagocytosis, and the number of phagocytosed macrophages is measured by observation with a fluorescent microscope.

The agent of the present invention may be used in its original form or may be diluted to any concentration. The agent of the present invention may be formulated in the form of an external preparation or an oral preparation by appropriately combining and blending with any component used in formulation.
In the present invention, the agent for activating the phagocytic activity of macrophages is preferably in the form of an external preparation or an oral preparation, and more preferably in the form of an external preparation.

Suitable examples of topical preparations include cosmetics, quasi-drugs, medicines, etc., and may contain any commonly used ingredient to the extent that the effects of the present invention are not impaired. That is, the composition containing the agent for activating the phagocytic activity of macrophages of the present invention can be used as a cosmetic, quasi-drug, or medicine.
Optional components include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japan wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, Ibota wax, lanolin, reduced lanolin, hard lanolin, and jojoba wax; liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, and wase hydrocarbons such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, undecylenic acid, and the like; higher fatty acids such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, cetostearyl alcohol, and the like; cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, Synthetic ester oils such as diisopropyl adipate, di-2-ethylhexyl sebacate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glycerin di-2-heptylundecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, and pentane erythritol tetra-2-ethylhexanoate; anionic surfactants such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl triethanolamine ether sulfate; cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, and laurylamine oxide; imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyltaurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic (registered trademark) types, POE.P Nonionic surfactants such as OP alkyl ethers (POE/POP 2-decyl tetradecyl ether, etc.), tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, erythritol, sorbitol, xylitol, maltitol, propylene glycol, and 2,4-hexanediol; sodium pyrrolidone carboxylate, lactic acid, Preferred examples of the ultraviolet absorbing agents include moisturizing components such as sodium lactate; para-aminobenzoic acid-based ultraviolet absorbers; anthranilic acid-based ultraviolet absorbers; salicylic acid-based ultraviolet absorbers; cinnamic acid-based ultraviolet absorbers; benzophenone-based ultraviolet absorbers; sugar-based ultraviolet absorbers; ultraviolet absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; and antibacterial agents such as phenoxyethanol.

Examples of oral preparations include general foods such as sweets, bread, and noodles, drink preparations, food groups for the purpose of promoting health (e.g., foods for specified health uses) in the form of capsules or tablets, and orally administered pharmaceuticals in the form of granules, powders, capsules, or tablets.
In the case of oral preparations, acceptable optional ingredients may be contained. In the case of foods, suitable examples of such optional ingredients include seasoning ingredients such as salt, sugar, sodium glutamate, sodium inosinate, and vinegar, coloring ingredients, odor-correcting ingredients such as flavors, thickeners, emulsifiers/dispersants, preservatives, stabilizers, and various vitamins. In the case of foods and medicines for health promotion, suitable examples include excipients such as crystalline cellulose and lactose, binders such as gum arabic and hydroxypropyl cellulose, disintegrants such as croscarmellose sodium and starch, lubricants such as magnesium stearate, flavorings, odor-correcting agents, coloring agents, and various vitamins. Oral preparations can be produced by processing these ingredients according to conventional methods.

The total content of essential oils from plants of the genus Citrus in the family Rutaceae in the topical preparation is not particularly limited, but may be, for example, 0.0001 to 5% by mass, preferably 0.001 to 1% by mass.
The total content of essential oils from plants of the genus Citrus in the family Rutaceae in the oral preparation is not particularly limited, but can be, for example, 0.01 to 100% by mass, preferably 10 to 80% by mass.
In addition, in the case of an external preparation or oral preparation, the essential oil of a plant belonging to the genus Citrus of the family Rutaceae can be administered in a dose of 10 to 1000 mg per day, either once or in divided doses.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist of the invention is not exceeded.

Example 1 Evaluation of the Effect of Activating the Phagocytic Ability of Macrophages The effect of essential oils from plants belonging to the genus Citrus in the family Rutaceae in activating the phagocytic ability of macrophages was evaluated by the following procedure.
Human monocyte-derived M1 macrophages (PromoCell, product number: C-12914) were seeded onto a fibronectin-coated 24-well plate ( ^20x104 cells/well) and cultured for 24 hours at 37°C in 5% _CO2 using M1 macrophage differentiation medium (PromoCell, product number: C-28055). After that, the medium was replaced with fresh M1 macrophage differentiation medium every 2-3 days and cultured at 37°C in 5% _CO2 .
The medium was then replaced with an M1 macrophage differentiation medium containing bergamot essential oil at a final concentration of 0.01% by mass. The bergamot essential oil was obtained by squeezing and distilling the peel of bergamot. As a negative control, the medium was replaced with an M1 macrophage differentiation medium that did not contain bergamot essential oil. After culturing for 24 hours in a 37°C, 5% _CO2 environment, the medium was removed and washed with PBS(-) 1000μL/well.

Thereafter, M1 macrophage differentiation medium containing fluorescent beads with a diameter of 1.0 μm (Invitrogen™ FluoSpheres™ Carboxylate-Modified Microspheres, ThermoFisher, product number: F8789) was added so that the final concentration (mass%) of fluorescent beads was 0.005%, and the cells were cultured at 37°C in a 5% _CO2 environment for 3 hours. After that, the medium was removed and the cells were washed with 1000 μL/well of PBS(-) to remove the fluorescent beads in the medium.
Thereafter, 500 μL/well of 4% paraformaldehyde solution was added to fix the cells, and the cells were observed using a fluorescence microscope with an objective lens at a magnification of ×10. The total number of cells and the number of cells containing fluorescent beads in a given area (355 μm horizontal × 266 μm vertical) within the field of view were counted, and the percentage of the number of cells that had phagocytosed the fluorescent beads relative to the total number of cells in that area was calculated.

As shown in the results in FIG. 1, it was observed that the phagocytic function of macrophages to which bergamot essential oil had been added was activated.
Also, as shown in Figure 2, the percentage of macrophages that phagocytosed fluorescent beads in the negative control was 25.8%, whereas the percentage of macrophages that phagocytosed fluorescent beads in the case where bergamot essential oil was added was 49.5%, indicating that the phagocytic ability was activated in macrophages to which bergamot essential oil was added.

Claims (5)

An agent that activates the phagocytic function of macrophages, containing the essential oil of a plant belonging to the genus Citrus in the family Rutaceae as an active ingredient. The phagocytic activity activator according to claim 1, wherein the essential oil of a plant belonging to the genus Citrus in the family Rutaceae is one or more selected from the group consisting of bergamot essential oil, orange essential oil, and yuzu essential oil. A composition for improving or preventing aging and/or damage of biological tissue, comprising the phagocytic activity activator according to claim 1 or 2. The composition according to claim 3, wherein the biological tissue is skin tissue. A composition comprising the phagocytic activity activator according to claim 1 or 2, the composition being a cosmetic, a quasi-drug, or a pharmaceutical.

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