JP2024092042A - Oral Compositions - Google Patents

Abstract

[Problem] The objective is to provide a composition that inhibits the activation of microglial cells and effectively improves related functionality. [Solution] An oral composition is provided that contains (A) apigenin and (B) at least one selected from vitamins. Also provided is a composition for inhibiting microglial cell activation, and a composition for protecting the retina and/or improving cognitive function, which contain the above ingredients. Also provided is a composition for inhibiting microglial cell activation, and a composition for protecting the retina and/or improving cognitive function, which contain at least one selected from the group consisting of guava, garlic, celery, parsley, and extracts thereof, which are food materials containing the above ingredients. [Selected Figure] Figure 1

Description

The present invention relates to an oral composition. The present invention also relates to a composition for inhibiting microglial cell activation, and a composition for protecting the retina and/or improving cognitive function.

In recent years, it has been reported that activation of microglial cells causes neuroinflammation and affects various disorders based on the mechanism of neurodegeneration. Two subpopulations of activated microglial cells are known (Non-Patent Document 1). Of these, M1 microglial cells produce inflammatory cytokines (TNF-α, IL-1β, CSF1, CSF2, CXCL10, CCL2, etc.) and have neurotoxic functions. On the other hand, M2 microglial cells produce anti-inflammatory cytokines (IL-4, IL-10, TGF-β, etc.) and have neuroprotective functions.

It is recognized that the production of IL-1β by microglial cells is an important factor in disorders involving inflammation of the central nervous system, including Alzheimer's disease (Non-Patent Document 2). It is known that in Alzheimer's disease model mice, an increase in IL-1β leads to neuroinflammation, which exacerbates Alzheimer's disease.

Microglial cells also affect various abnormal conditions not only in the central nervous system such as the brain and spinal cord, but also in the posterior segment of the eye. For example, it is known that microglial-mediated neuroinflammation is an important factor in major retinal degenerative diseases such as glaucoma, age-related macular degeneration, and diabetic retinopathy (Non-Patent Document 3). As mentioned above, microglial cells also have a neuroprotective effect by shifting to M2 microglial cells, so completely inhibiting microglial cells is not appropriate from the perspective of homeostasis, and it is desirable to target and appropriately suppress M1 microglial cells that are activated to produce inflammatory cytokines, etc.

It has been reported that the main action of microglial cells may be cytokines and chemokines, and control of the production of these inflammatory cytokines may be a new therapeutic target for diseases involving microglial cells (Non-Patent Document 4).

Sudhakar R et al. Targeting Microglial Activation States as a Therapeutic Avenue in Parkinson’s Disease. Frontiers in Aging Neuroscience. 2017, Vol. 9, Article 176, p.1-18. Solomon S Shaftel et al. The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective. Journal of Neuroinflammation. 2008, Vol. 5:7, p.1-12. Maria H. Madeira et al. Contribution of Microglia-Mediated Neuroinflammation to Retinal Degenerative Diseases. Mediators of Inflammation. Volume 2015, Article ID 673090, p.1-15. Yukari Mogami and Kaoru Sato. The regulatory function of microglia in the central nervous system development and its potential application in drug discovery and treatment. Folia Pharmacol. Jpn. 2017, Vol.150, p.268-274.

If the activation of microglial cells can be effectively suppressed, it is expected that there will be effects such as reducing the risk of, preventing, and treating abnormal conditions in the central nervous system, such as the brain and spinal cord, and the posterior segment of the eye, and therefore various pharmaceutical developments are ongoing.

However, because microglial cell activation needs to be continuously suppressed, pharmaceutical ingredients alone are not sufficient, and there is a demand for novel compositions that suppress microglial cell activation using ingredients that are highly safe and can be applied not only in the pharmaceutical field but also in the fields of functional foods and supplements.

The present invention therefore aims to provide a composition that effectively suppresses the activation of microglial cells, thereby effectively improving associated diseases and disorders.

In order to solve the above problems, the inventors conducted extensive research and discovered that ingredients that are safely used in the food and pharmaceutical fields suppress the activation of microglial cells, which led to the completion of the present invention.

That is, the present invention provides the compositions described below.
[1]
(A) apigenin,
(B) at least one selected from vitamins.
[2]
The oral composition according to [1], wherein the blending ratio of the component (A) to the component (B) is 0.01 to 100 parts by mass of the component (B) per 1 part by mass of the component (A).
[3]
The oral composition according to [1] or [2], wherein the component (B) is at least one selected from the group consisting of vitamin A and vitamin _B6 .
[4]
(A) apigenin,
(B) a composition for suppressing microglial cell activation, comprising at least one selected from vitamins.
[5]
A composition for inhibiting microglial cell activation, comprising at least one member selected from the group consisting of guava, garlic, celery, parsley, and extracts thereof.
[6]
(A) apigenin,
(B) A composition for protecting the retina and/or improving cognitive function, comprising at least one selected from vitamins.
[7]
A composition for protecting the retina and/or improving cognitive function, comprising at least one selected from the group consisting of guava, garlic, celery, parsley, and extracts thereof.
[8]
The composition described in [6] or [7], wherein the retinal protection and/or cognitive improvement is achieved by suppressing neural inflammation and/or degeneration.
[9]
The composition according to any one of claims [6] to [8], wherein the retinal protection is at least one selected from the group consisting of improved protection of the eye from light stimuli, inhibition of eye aging, improvement of eye contrast sensitivity, improvement of eye focusing function, improvement of eye fatigue, and maintenance of pigment amount in the macula of the eye.
[10]
The composition according to any one of [6] to [8], wherein the retinal protection is risk reduction, prevention, or treatment of a posterior ocular disease.
[11]
The composition according to [10], wherein the posterior ocular disease is at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreous retinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, Leber's disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, central serous chorioretinopathy, central exudative chorioretinopathy, polypoidal choroidal vasculopathy, multifocal choroiditis, neovascular maculopathy, retinal aneurysm, retinal angiomatous proliferation, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, and ischemic optic neuropathy.
[12]
The composition according to any one of [6] to [8], wherein the cognitive function improvement is at least one selected from the group consisting of maintaining memory, maintaining judgment, maintaining attention, maintaining spatial awareness, and maintaining efficiency in calculation work.
[13]
The composition according to any one of [6] to [8], wherein the cognitive function improvement is risk reduction, prevention or treatment of a neurodegenerative disease.
[14]
The composition according to [13], wherein the neurodegenerative disease is at least one selected from the group consisting of glaucoma, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, or vascular dementia.

According to the present invention, it is possible to suppress the activation of microglial cells using ingredients that are safely used in the food and pharmaceutical fields, and to effectively improve abnormalities and diseases caused by the activation of microglial cells.

FIG. 1 is a graph showing the results of a test in Test Example 1 on the inhibition of microglial cell activation by combined use of apigenin and vitamin A, using the inflammatory cytokine IL-1β as an indicator. FIG. 2 is a graph showing the results of a test of inhibition of microglial cell activation by combined use of apigenin and vitamin _B6 in Test Example 2, using the inflammatory cytokine IL-1β as an indicator. FIG. 3 is a graph showing the results of a test of inhibition of microglial cell activation by a guava extract derived from a food material containing apigenin, vitamin A, and vitamin _B6 , using the inflammatory cytokine IL-1β as an indicator in Test Example 3. FIG. 4 is a graph showing the results of a test of inhibition of microglial cell activation by a guava extract derived from a food material containing apigenin, vitamin A, and vitamin _B6 , using the inflammatory cytokine TNF-α as an indicator in Test Example 3. FIG. 5 is a graph showing the results of a test of inhibition of microglial cell activation by a guava extract derived from a food material containing apigenin, vitamin A, and vitamin _B6 , using the inflammatory cytokine CCL2 as an indicator in Test Example 3. FIG. 6 is a graph showing the results of evaluating the inhibitory effect of apigenin alone on IL-1β production at concentrations of 1 μM, 5 μM, and 10 μM, as a reference example. FIG. 7 is a graph showing the results of evaluating the effect of apigenin alone on inhibiting TNF-α production at concentrations of 1 μM, 5 μM, and 10 μM, as a reference example. FIG. 8 is a graph showing the results of evaluating the inhibitory effect of apigenin alone on CCL2 production at concentrations of 1 μM, 5 μM, and 10 μM, as a reference example. FIG. 9 is a graph showing the results of a test of inhibition of microglial cell activation by dried garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine IL-1β as an indicator in Test Example 4. FIG. 10 is a graph showing the results of a test of microglial cell activation inhibition by dried garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine TNF-α as an indicator in Test Example 4. FIG. 11 is a graph showing the results of a test of the inhibition of microglial cell activation by dried garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine CCL2 as an indicator in Test Example 4. FIG. 12 is a graph showing the results of a test of inhibition of microglial cell activation by black garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine IL-1β as an indicator in Test Example 4. FIG. 13 is a graph showing the results of a test of inhibiting microglial cell activation by black garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine TNF-α as an indicator in Test Example 4. FIG. 14 is a graph showing the results of a test of inhibition of microglial cell activation by black garlic powder derived from a food material containing apigenin and vitamin _B6 , using the inflammatory cytokine CCL2 as an indicator in Test Example 4.

Oral Compositions
In one embodiment, the oral composition of the present invention contains (A) apigenin and (B) at least one selected from vitamins.

((A) Apigenin)
Apigenin is a derivative of flavone and has the molecular formula _C15H10O5 . _It is known to be contained in plants such as thyme, dahlia, and _wisteria . Since apigenin is the aglycone of apigenin glycoside, sugar may be separated from apigenin glycoside, it may be extracted from a plant containing apigenin, it may be produced by synthesis, or a commercially available product may be used.

Examples of apigenin glycosides include apiin, such as apigetrin (apigenin-7-glucoside), videxin (apigenin-8-C-glucoside), isovitexin (apigenin-6-C-glucoside), and rhoifolin (apigenin-7-O-neohesperidoside).

When apigenin is extracted from a plant containing apigenin, the plant containing apigenin may be used in its raw form or may be dried, and there are no particular limitations on the state of the raw material. In order to increase the apigenin content, the plant containing apigenin may be roasted before use.

The extraction method and purification method are not particularly limited, and conventional methods can be used. The extraction solvent used in this specification can be the same as the extraction solvent used when using a plant extract.

(B) Vitamins
In the present invention, the vitamins of component (B) may be either water-soluble or fat-soluble vitamins as long as the effects of the present invention are achieved.

Examples of vitamins in the component (B) include vitamin A such as retinol, retinol derivatives (retinol acetate, retinol palmitate, etc.), retinal, retinoic acid, methyl retinoate, ethyl retinoate, retinol retinoic acid, d-δ-tocopheryl retinoate, α-tocopheryl retinoate, and β-tocopheryl retinoate;
Provitamin A such as β-carotene, α-carotene, γ-carotene, δ-carotene, lycopene, zeaxanthin, cryptoxanthin, and echinenone;
Vitamin E such as α-tocopherol, β-tocopherol, dl-α-tocopherol succinate, dl-α-tocopherol calcium succinate, δ-tocopherol, and tocopherol nicotinate;
Vitamin _B2 such as riboflavin, flavin mononucleotide, flavin adenine dinucleotide, riboflavin butyrate, riboflavin tetrabutyrate, riboflavin 5'-phosphate sodium, and riboflavin tetranicotinate;
Nicotinic acids such as methyl nicotinate, nicotinic acid, and nicotinamide;
Vitamin C such as ascorbyl stearate, L-ascorbyl dipalmitate, ascorbyl tetraisopalmitate (ascorbyl tetra-2-hexyldecanoate), ascorbic acid, sodium ascorbate, dehydroascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, and ascorbyl glucoside;
Vitamin D such as methylhesperidin, ergocalciferol, and cholecalciferol;
Vitamin K such as phylloquinone and falnoquinone;
Vitamin B1 class such as dibenzoyl thiamine, dibenzoyl thiamine hydrochloride, thiamine hydrochloride, thiamine cetyl hydrochloride, thiamine thiocyanate, thiamine lauryl hydrochloride, thiamine nitrate, thiamine monophosphate, thiamine lysine salt, thiamine triphosphate, thiamine monophosphate ester phosphate, thiamine monophosphate ester, thiamine diphosphate ester, thiamine diphosphate ester hydrochloride, thiamine triphosphate ester, and thiamine triphosphate ester _{monophosphate} ;
Vitamin _B6 such as pyridoxine hydrochloride, pyridoxine acetate, pyridoxal hydrochloride, 5'-pyridoxal phosphate, and pyridoxamine hydrochloride;
Vitamin _B12 , such as cyanocobalamin, hydroxocobalamin, and deoxyadenosylcobalamin;
Folic acids such as folic acid and pteroylglutamic acid;
Pantothenic acids such as pantothenic acid, calcium pantothenate, pantothenyl alcohol (panthenol), D-pantetheine, D-pantethine, coenzyme A, and pantothenyl ethyl ether;
Biotins such as biotin and biocytin; and vitamin-like factors such as carnitine, ferulic acid, α-lipoic acid, orotic acid, and γ-oryzanol.

From the viewpoint of more prominently exhibiting the effects of the present invention, the vitamins of the component (B) are preferably at least one selected from the group consisting of vitamins A, vitamin _B6 , vitamin _B12 , pantothenic acid, and vitamin _B2 , and more preferably at least one selected from the group consisting of vitamins A and vitamin _B6 . Also, it is more preferably at least one selected from the group consisting of retinol, pyridoxine, cyanocobalamin, panthenol, flavin adenine dinucleotide, and salts thereof, and particularly preferably at least one selected from the group consisting of retinoic acid and pyridoxine hydrochloride. Examples of vitamins A include retinol palmitate ester manufactured by DSM, in which 0.550 μg is vitamin A1 I.U. Here, I.U. means the international unit obtained by the method described in the Vitamin A Quantitative Method of the Japanese Pharmacopoeia, 16th Edition, etc. These vitamins can be used alone or in combination of two or more. As such vitamins, commercially available products can be used, or food materials containing vitamins can be used.

Specific examples of vitamin salts include those mentioned above, but are not particularly limited as long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable. Specific examples include organic acid salts [e.g., monocarboxylates (acetate, trifluoroacetate, butyrate, palmitate, stearate, etc.), polycarboxylates (fumarate, maleate, succinate, malonate, etc.), oxycarboxylates (lactate, tartrate, citrate, etc.), organic sulfonates (methanesulfonate, toluenesulfonate, tosylate, etc.)], inorganic acid salts (e.g., hydrochloride, sulfate, nitrate, hydrobromide, phosphate, etc.), etc.].

The content of apigenin is set appropriately depending on the type and content of other ingredients, the formulation form of the composition, etc., but is preferably 0.001 to 10% by mass, more preferably 0.005 to 5% by mass, even more preferably 0.01 to 3% by mass, particularly preferably 0.05 to 2% by mass, and most preferably 0.1 to 1% by mass, based on the total amount of the composition.

The total content of component (B) is set appropriately depending on the type and content of other blended ingredients, the formulation form of the composition, etc., but is preferably 0.001 to 10% by mass, more preferably 0.005 to 5% by mass, even more preferably 0.01 to 3% by mass, particularly preferably 0.05 to 2% by mass, and most preferably 0.1 to 1% by mass, based on the total amount of the composition.

When vitamin A is contained, the content of vitamin A alone is not particularly limited, but is, for example, preferably 0.001 to 100% by mass, more preferably 0.005 to 50% by mass, even more preferably 0.01 to 10% by mass, particularly preferably 0.05 to 2% by mass, and most preferably 0.1 to 1% by mass, based on the total amount of the composition.

When vitamin _B6 is contained, the content of vitamin _B6 alone is not particularly limited, but is, for example, preferably 0.1 to 95% by mass, more preferably 0.3 to 70% by mass, and even more preferably 0.5 to 50% by mass, based on the total amount of the composition.

The blending ratio of component (A) to component (B) is appropriately set depending on the type and content of other blended components, the formulation form of the composition, etc., but for example, the blending ratio of component (B) is preferably 0.01 to 100 parts by mass, more preferably 0.05 to 80 parts by mass, even more preferably 0.1 to 70 parts by mass, particularly preferably 0.5 to 60 parts by mass, and most preferably 1 to 50 parts by mass per part by mass of component (A).

(Food material containing component (A) and component (B))
In addition to the above-mentioned components (A) and (B), food materials containing these components can be used from the viewpoint of exerting the functionality of the present invention.
The food material containing the components (A) and (B) is not limited as long as it exerts the effects of the present invention. For example, at least one selected from the group consisting of guava, garlic, celery, parsley, and extracts thereof is preferable, at least one selected from the group consisting of guava, garlic, and extracts thereof is more preferable, and guava and/or an extract thereof is even more preferable.

(Guava and its extracts)
Guava (Psidium guajava L) is a plant belonging to the genus Psidium of the family Myrtaceae. Guava and its extracts refer to dried products, fine powders, and extracts thereof made from guava fruit, peel, or leaves. Among these, those made from guava leaves are preferred from the viewpoint of achieving the effects of the present invention, although they are not limited thereto.

The guava extract is not limited to a specific extraction method as long as it achieves the effects of the present invention. For example, guava extract refers to an extract obtained by immersing guava or its crushed product in water and/or an organic solvent and filtering the residue, or the extract from which the solvent has been removed, or a fine powder of these, or the extract or the product from which the solvent has been removed is dissolved, dispersed, or diluted with an appropriate solvent, and it is also possible to use a commercially available product. As an example of another extraction method, guava extract can be extracted by processing the fruit, peel, or leaves of GPsidium guajava L, such as by steaming them.

In the present specification, when using a plant extract (also referred to as plant extract), more specifically, as the extraction solvent, water (including hot water), alcohols such as methanol, ethanol, isopropanol, ethylene glycol, 1,3-butylene glycol, glycerin, etc., esters such as ethyl acetate, ketones such as acetone and methyl ethyl ketone, nitriles such as acetonitrile, ethers such as diethyl ether and tetrahydrofuran, saturated hydrocarbons such as pentane, hexane, cyclopentane, cyclohexane, aromatic hydrocarbons such as toluene, halogenated hydrocarbons such as dichloromethane and chloroform, and other organic solvents such as dimethylformamide and dimethyl sulfoxide (which may all contain water), etc. may be appropriately used, and may be any mixture of one or two of them. Of these solvents, water, ethanol, 1,3-butylene glycol, or a mixture of these is preferable. The extracts described in this specification can be obtained from various raw material companies, and are usually sold in a form including excipients, but are not limited thereto. Hereinafter, the amount of extract refers to the amount converted into dry solid content.

The extraction solvent for guava extract is not limited as long as it achieves the effects of the present invention, but is preferably water, ethanol, or aqueous ethanol, and more preferably aqueous ethanol.

Commercially available products of guava and its extracts include, but are not limited to, Guava Leaf Extract Powder-S (BHN Co., Ltd.).

(Garlic and its extracts)
Garlic (Allium sativum) is a plant belonging to the Allium genus of the Amaryllidaceae family. Garlic and its extracts refer to garlic bulbs, scales, or leaves as raw materials, and include dried products thereof, fine powders thereof (hereinafter also simply referred to as garlic powder), and extracts thereof. Any garlic can be used as long as it can achieve the effects of the present invention, but garlic powder is preferred.

Garlic may be fresh garlic, dried garlic, or black garlic that has been aged and fermented. Black garlic can be produced by conventional methods and is not particularly limited in the production method. For example, black garlic can be produced by subjecting raw garlic bulbs to self-fermentation. Specifically, black garlic can be produced by storing raw garlic bulbs at a temperature range of 30°C to 75°C and appropriate humidity for 5 to 40 days.

Garlic that has been heat-treated to reduce the garlic odor may also be used.

As long as the effect of the present invention is achieved, the garlic extract is not limited to a specific extraction method. For example, garlic extract refers to an extract obtained by immersing garlic or its crushed product in water and/or an organic solvent and filtering the residue, or the extract from which the solvent has been removed, or a fine powder of these, or the extract or the product from which the solvent has been removed is dissolved, dispersed, or diluted with an appropriate solvent, and commercially available products can also be used. As another example of an extraction method, garlic extract can be extracted after processing the bulbs, scales, or leaves of garlic, such as by steaming them.

The raw materials for the garlic extract and garlic powder are not particularly limited, but dried garlic is preferred from the viewpoint of achieving the most pronounced effects of the present invention.

The extraction solvent for garlic extract is not limited as long as it achieves the effects of the present invention, but is preferably water, ethanol, or aqueous ethanol, and more preferably aqueous ethanol.

Commercially available products of garlic and its extracts include, but are not limited to, Matured Black Garlic Crushed Powder (Maruzen Pharmaceutical Co., Ltd.) and Garlic Extract Powder SP (Bizen Chemical Industry Co., Ltd.).

There are no particular limitations on the origin of the garlic, but from the viewpoint of achieving the most significant effects of the present invention, garlic from Japan and/or the United States is preferred, and garlic from the United States is more preferred.

The total content of the food material containing components (A) and (B) is appropriately set depending on the type and content of other blended ingredients, the formulation form of the composition, etc., but is preferably 0.01 to 95% by mass, more preferably 0.03 to 80% by mass, even more preferably 0.05 to 75% by mass, and particularly preferably 0.1 to 70% by mass, based on the total amount of the composition.

When guava and/or an extract thereof is contained, the content of guava and/or an extract thereof alone is not particularly limited, but is, for example, preferably 0.01 to 95% by mass, more preferably 0.1 to 80% by mass, even more preferably 1 to 75% by mass, and particularly preferably 2 to 70% by mass, based on the total amount of the composition.

When garlic and/or an extract thereof is contained, the content of garlic and/or an extract thereof alone is not particularly limited, but is, for example, preferably 0.01 to 95% by mass, more preferably 0.1 to 80% by mass, even more preferably 1 to 75% by mass, and particularly preferably 2 to 70% by mass, based on the total amount of the composition.

[Application]
In the present invention, the activation of microglial cells can be effectively suppressed by using the component (A) in combination with the component (B). As shown in the examples described below, the activation of microglial cells can be suppressed even by the component (A) alone, but the effect is not sufficient. As shown in the examples of the present specification, the coexistence of the component (A) and the component (B) enables the activation of microglial cells to be significantly suppressed. In the present specification, the suppression of the activation of microglial cells can be confirmed by a known method as long as the abnormal state of microglial cells is prevented, prevented, delayed, improved, alleviated, or treated. Although not limited, the suppression of the activation of microglial cells is preferably suppressed by M1 microglial cells among the subpopulations of microglial cells, and can be confirmed by a reduction in the production of inflammatory cytokines, chemokines, etc. produced by M1 microglial cells.

The oral composition of the present invention is also suitable for use in improving conditions, symptoms, and diseases associated with the activation of microglial cells. In recent years, it has been reported that the activation of microglial cells causes neuroinflammation and affects various disorders based on the mechanism of neurodegeneration. For example, microglial cells exist as resident macrophages in the retina, and are activated by various stimuli. Upon activation of microglial cells, M1 microglial cells produce inflammatory cytokines (TNF-α, IL-1β, CSF1, CSF2, CXCL10, etc.), causing nerve disorders.

Retinal nerve damage worsens with nerve inflammation and degeneration, leading to significant impairment of retinal function. When retinal damage worsens due to nerve inflammation and/or degeneration, it is known to lead to conditions such as a decrease in the eye's protective function against light stimuli, eye aging, a decrease in visual function including visual acuity, visual field, and contrast sensitivity, eye fatigue, and a decrease in the amount of pigment in the macula of the eye.

Therefore, if it is possible to suppress the activation of microglial cells in the retina, and in particular to continuously reduce the production of inflammatory cytokines (TNF-α, IL-1β, CSF1, CSF2, CXCL10, etc.) produced by M1 microglial cells, this would be ideal for protecting the retina. In order to continuously exert such effects, it is desirable to use not only pharmaceuticals but also food ingredients that have been used in the diet for a long time and can be safely ingested continuously.

Among various proinflammatory cytokines released by activated microglia, IL-1β can activate innate immunity by inducing the expression of numerous inflammatory cytokines and chemokines. Release of IL-1β from microglia has been implicated as a major effector of neuroinflammation and neurodegenerative diseases. Therefore, inhibition of IL-1β in activated microglia is a potential molecular target for the treatment of neurodegenerative diseases. In addition, it is known that IL-1β produced by the activation of microglial cells exacerbates neuroinflammation in the retina, and reducing the amount of IL-1β produced is a therapeutic target for posterior ocular diseases such as age-related macular degeneration and glaucoma from the viewpoint of retinal protection (Riccardo Natoli et al. Microglia-derived IL-1 β promotes chemokine expression by Muller cells and RPE in focal retinal degeneration. Molecular Neurodegeneration. 2017, Vol. 12:31, p.1-11, Hui-Lan Zeng et al. The role of microglia in the progression of glaucomatous neurodegeneration- a review. Int J Ophthalmol. 2018, Vol.11, No.1, p.143-149, etc.).

The oral composition of the present invention, when used in combination with component (A) and component (B), or when used with a food material containing components (A) and (B), can significantly suppress the activation of microglial cells, thereby suppressing inflammation and/or degeneration of nerves and leading to the above-mentioned retinal protection.

In addition, by suppressing the activation of microglial cells, it is known that there is a therapeutic effect on retinal ganglion cells and photoreceptor cell disorders in the posterior segment of the eye, as well as a protective effect on retinal ganglion cells and photoreceptor cells. Therefore, the above ingredients are effective against posterior segment abnormalities and diseases, including glaucoma and age-related macular degeneration (AMD).

In this specification, the posterior segment of the eye refers to the area inside the back surface of the crystalline lens, including the retina, macula, sclera, choroid, vitreous body, and optic nerve. Of the areas of the posterior segment of the eye, it is preferable to target the retina in order to achieve a significant effect of the present invention.

In this specification, a posterior segment abnormality refers to a state in which some abnormality or finding occurs in at least one selected from the group consisting of the retina, macula, sclera, choroid, vitreous body, and optic nerve, although it does not result in a disease in the posterior segment. Abnormalities and (clinical) findings in the posterior segment can be found, for example, through a health check, a medical examination by a doctor, a clinical test, etc., but are not limited thereto. Examples of examinations of the posterior segment include fundus examination, intraocular pressure examination, fundus three-dimensional image analysis (OCT) examination, visual field examination, refraction examination, slit lamp microscopy, etc. Among these examinations, for example, in fundus examination, abnormal findings in the posterior segment are evaluated from the viewpoint of hypertensive changes and arteriosclerotic changes using the "Scheie classification". In addition, in addition to the "Scheie classification," fundus examination may show findings such as enlarged optic nidus, leopard-striated fundus, hard exudates, drusen, suspected glaucoma, hemorrhage, suspected hemorrhage, retinal choroidal atrophy, suspected cataract, crossing phenomenon, exudates, retinal degeneration, meandering, suspected macular degeneration, conus, and optic nerve findings. The present invention may also be used to reduce the risk of, prevent, or treat posterior segment abnormalities.

As used herein, prevention of posterior ocular disease refers to preventing or delaying the onset of a posterior ocular disease or symptom, or reducing the risk of onset of a posterior ocular disease or symptom.

As used herein, treatment of a posterior ocular disease refers to alleviating or improving a posterior ocular disease or symptom, preventing or delaying the worsening of a disease or symptom, or preventing, delaying, or reversing the progression of a posterior ocular disease or symptom.

Risk reduction, prevention, or treatment of posterior ocular diseases includes, but is not limited to, risk reduction, prevention, or treatment of at least one selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, proliferative vitreous retinopathy, retinal artery occlusion, retinal vein occlusion, uveitis, Leber's disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelium detachment, central serous chorioretinopathy, central exudative chorioretinopathy, polypoidal choroidal vasculopathy, multifocal choroiditis, neovascular maculopathy, retinal aneurysm, retinal angiomatous proliferation, optic neuropathy caused by these diseases, optic neuropathy caused by glaucoma, and ischemic optic neuropathy.

In glaucoma, it is known that the production of TNF-α leads to cell death of RGCs (retinal ganglion cells), and suppressing the production of inflammatory cytokines by M1 microglial cells is effective against glaucoma and/or optic nerve damage caused by glaucoma (Non-Patent Document 3).

AMD can be targeted for prevention or treatment regardless of its classification, as long as the activation of microglial cells is involved in the onset or exacerbation of the condition. However, it is preferable to target wet AMD (exudative AMD) and/or dry AMD (atrophic AMD). Although not limited thereto, it is known that in dry AMD (atrophic AMD), activated microglial cells accumulate in the outer layer of the retina, etc.

Neurological disorders in the central nervous system, such as the brain, are exacerbated by inflammation and degeneration of the nerves, leading to significant impairment of cognitive function. It is known that when central nervous disorders are exacerbated by inflammation and/or degeneration of the nerves, it can lead to conditions such as impaired memory, impaired judgment, impaired attention, impaired spatial awareness, and impaired efficiency in calculation tasks. It is known that aging causes microglial cells in the brain to produce more inflammatory cytokines such as IL-1β, which impairs cognitive functions including memory and contributes to brain aging (Susan L. et al. Immune dysregulation and cognitive vulnerability in the aging brain: Interactions of microglia, IL-1β, BDNF and synaptic plasticity. Neuropharmacology. 2015, Vol. 96(0 0), p.11-18, etc.).

Therefore, if it is possible to suppress the activation of microglial cells in the central nervous system, and in particular to continuously reduce the production of inflammatory cytokines (TNF-α, IL-1β, CSF1, CSF2, CXCL10, etc.) produced by M1 microglial cells, this would be ideal for maintaining and improving cognitive function. In order to continuously exert such effects, it is desirable to use not only pharmaceuticals but also food ingredients that have been used in the diet for a long time and can be safely ingested continuously.

Among the inflammatory cytokines produced by M1 microglial cells, IL-1β in particular is the starting point of inflammatory signals, so reducing the amount of IL-1β produced is highly significant.

The oral composition of the present invention, when used in combination with component (A) and component (B), or when used with a food material containing components (A) and (B), can significantly suppress the activation of microglial cells, thereby suppressing inflammation and/or degeneration of nerves and leading to the improvement of the above-mentioned cognitive functions.

In addition, by suppressing the activation of microglial cells, it is known that the above ingredients have a therapeutic effect against neurodegenerative diseases. Therefore, the above ingredients are effective against neurodegenerative diseases including glaucoma and Alzheimer's disease.

As used herein, prevention of neurodegenerative disease refers to preventing or delaying the onset of a disease or condition involving neurodegeneration, or reducing the risk of onset of a disease or condition involving neurodegeneration.

As used herein, treatment of a neurodegenerative disease refers to alleviating or improving a disease or symptom involving neurodegeneration, preventing or slowing the worsening of a disease or symptom, or preventing, slowing, or reversing the progression of a disease or symptom involving neurodegeneration.

The risk reduction, prevention, or treatment of neurodegenerative diseases includes, but is not limited to, risk reduction, prevention, or treatment of at least one selected from the group consisting of glaucoma, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, and vascular dementia.

The composition of the present invention can be added to or mixed with food, beverages, feed, pet food, etc., and used as such. Alternatively, it can be used as a beverage or food as it is. Alternatively, it can be added to or mixed with food and beverages that are labeled with the following functional properties, such as retinal protection, inhibition of ocular aging, improvement of ocular contrast sensitivity, improvement of ocular focusing function, relief of ocular fatigue, maintenance of pigment amount in the macula, and risk reduction or prevention of posterior segment diseases, and that have similar content to the above, i.e., health foods, functional foods, foods for the sick, and foods for specified health uses.

Health foods, functional foods, foods for the sick, and foods for specified health uses can be used in various formulations, such as solid preparations (tablets, granules, fine granules, powders, capsules, chewable tablets, etc.), liquid preparations (syrups, suspensions), and liquid foods. Foods in formulation form can be produced in the same manner as known pharmaceutical preparations, and can be produced by mixing the active ingredient with a carrier acceptable for use as a food, such as a suitable excipient, and then using conventional means.

For example, tablets can be prepared by mixing a powdered active ingredient with a pharma- ceutically acceptable carrier component (such as an excipient) and compressing the mixture, and confectionery tablets such as candy can be prepared by pouring the mixture into a mold. Tablets can also be sugar-coated to form sugar-coated tablets. Furthermore, tablets can be single-layer tablets or layered tablets such as double-layer tablets.

Granules and other powdered granules may be prepared by various granulation methods (extrusion granulation, grinding granulation, dry compaction granulation, fluidized bed granulation, rolling granulation, high-speed stirring granulation, etc.), and tablets can be prepared by an appropriate combination of the above granulation methods and tableting methods (wet tableting, direct tableting), etc.

Capsules can be prepared by filling a capsule (soft or hard capsule) with a powder (powder, granules, etc.) using conventional methods.

The liquid preparation can be prepared by dissolving or dispersing each component in an aqueous medium (purified water, purified water containing ethanol, etc.) which is a carrier component, filtering or sterilizing the mixture as necessary, filling the mixture into a specified container, and sterilizing the mixture. The preferred dosage form of the solid preparation of the present invention is a capsule or tablet, and a soft capsule (soft capsule) is more preferred.

Soft capsules have a smooth surface and are easy to swallow, making them popular with users. Common methods for manufacturing soft capsules include the flat plate method, the rotary method, and the seamless method.

In the rotary method (punching method) manufacturing, a sheet-like capsule shell sandwiches the flowing filling contents and forms a capsule shape along the holes of a rotating cylindrical die. On the other hand, in the seamless method (dropping method) manufacturing, the capsule shell composition and contents are simultaneously ejected from multiple concentric nozzles to form a seamless capsule shape.

The base material for the soft capsule shell is not particularly limited, but may be starch, pullulan, cellulose, polyvinyl alcohol, gelatin, succinated gelatin, etc., with starch, gelatin, and succinated gelatin being preferred, and gelatin and succinated gelatin being more preferred. These may be used alone or in combination of two or more kinds.

The composition of the present invention can also be manufactured as liquid beverages such as soups, juices, fruit juice drinks, milk, dairy drinks, whey drinks, lactic acid bacteria drinks, tea drinks, alcoholic drinks, coffee drinks, carbonated drinks, soft drinks, water drinks, cocoa drinks, jelly drinks, sports drinks, and diet drinks; semi-solid foods such as puddings and yogurt; noodles, confectioneries, spreads, and the like.

When the composition of the present invention is prepared as a food composition, various food additives may be added. Examples of food additives include antioxidants, colorants, flavorings, seasonings, sweeteners, acidulants, pH adjusters, quality stabilizers, preservatives, etc.

When the composition of the present invention is prepared as a pharmaceutical composition, it is prepared as a formulation containing a combination of the active ingredients (A) and (B), or a food material containing the active ingredients (A) and (B), and preferably a pharma- ceutically acceptable carrier. A pharma-ceutically acceptable carrier generally refers to an inactive, non-toxic, solid or liquid bulking agent, diluent, or encapsulating material that does not react with the active ingredient, such as water, ethanol, polyols, appropriate mixtures thereof, and solvents or dispersing media such as vegetable oils.

The pharmaceutical composition is administered orally or parenterally, for example, into the oral cavity, into the digestive tract, or into the nasal cavity. Oral preparations include solid preparations (tablets, granules, fine granules, powders, capsules, chewable tablets, etc.) and liquid preparations (syrups, suspensions, inhalants), etc. Parenteral preparations include eye drops, drops, nasal drops, injections, etc.

The pharmaceutical composition may further contain additives commonly used in the pharmaceutical field. Such additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents, etc., and can be used as needed. The composition may also be coated with a known retardant or the like to achieve sustained release so as to act for a long time. The pharmaceutical composition may further contain other additives or drugs, such as antacids and gastric mucosa protectors, as needed.

The pharmaceutical composition can be applied in the form of an oral composition, an oral composition, etc. The pharmaceutical composition can be used therapeutically or non-therapeutically.

The daily oral intake or dosage of apigenin for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or dosage of apigenin for an adult is, for example, preferably 0.005 mg/kg body weight/day or more, more preferably 0.01 mg/kg body weight/day or more, and even more preferably 0.05 mg/kg body weight/day or more. The daily oral intake or dosage of apigenin for an adult is, for example, preferably 100 mg/kg body weight/day or less, more preferably 10 mg/kg body weight/day or less, and even more preferably 1 mg/kg body weight/day or less. The daily oral intake or dosage of apigenin for an adult is, for example, preferably 0.005 to 100 mg/kg body weight/day, more preferably 0.01 to 10 mg/kg body weight/day, and even more preferably 0.05 to 1 mg/kg body weight/day. The amount of apigenin contained can be the amount that results in the above-mentioned intake or administration amount. Note that the oral intake or administration amount per day for adults can be divided into 1-6 capsules, 1-4 capsules, 1-3 capsules, or 1-2 capsules depending on the dosage form.

The daily oral intake or dosage of vitamins for adults can be determined appropriately depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or dosage of vitamins for adults is, for example, preferably 0.0001 mg/kg body weight/day or more, more preferably 0.001 mg/kg body weight/day or more, and even more preferably 0.005 mg/kg body weight/day or more. The daily oral intake or dosage of vitamins for adults is, for example, preferably 10 mg/kg body weight/day or less, more preferably 1 mg/kg body weight/day or less, and even more preferably 0.1 mg/kg body weight/day or less. The daily oral intake or dosage of vitamins for adults is, for example, preferably 0.0001 to 10 mg/kg body weight/day, more preferably 0.2 to 600 mg/kg body weight/day, even more preferably 0.001 to 1 mg/kg body weight/day, particularly preferably 2 to 100 mg/kg body weight/day, and most preferably 0.005 to 0.1 mg/kg body weight/day. The content of vitamins can be the amount that results in the above intake or dosage. The daily oral intake or dosage for adults may be divided into dosage forms, for example, 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules in the case of capsules.

The daily oral intake or dosage of vitamins A for adults can be determined appropriately depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or dosage of vitamins for adults is, for example, preferably 0.001 mg/kg body weight/day or more, more preferably 0.005 mg/kg body weight/day or more, and even more preferably 0.01 mg/kg body weight/day or more. The daily oral intake or dosage of vitamins for adults is, for example, preferably 10 mg/kg body weight/day or less, more preferably 1 mg/kg body weight/day or less, and even more preferably 0.1 mg/kg body weight/day or less. The daily oral intake or dosage of vitamins A for adults is, for example, preferably 0.001 to 10 mg/kg body weight/day, more preferably 0.005 to 1 mg/kg body weight/day, and even more preferably 0.01 to 0.1 mg/kg body weight/day. The vitamin content can be the amount that results in the intake or administration amount described above. The oral intake or administration amount for an adult per day can be divided into 1-6 capsules, 1-4 capsules, 1-3 capsules, or 1-2 capsules depending on the dosage form.

The daily oral intake or administration amount of vitamin _B6 and/or a salt thereof for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or administration amount of vitamin _B6 and/or a salt thereof for an adult is, for example, preferably 0.0001 mg/kg body weight/day or more, more preferably 0.001 mg/kg body weight/day or more, even more preferably 0.005 mg/kg body weight/day or more, particularly preferably 0.01 mg/kg body weight/day or more, and most preferably 0.02 mg/kg body weight/day or more. The daily oral intake or administration amount of vitamin _B6 and/or its salt for an adult is, for example, preferably 50 mg/kg body weight/day or less, more preferably 10 mg/kg body weight/day or less, even more preferably 5 mg/kg body weight/day or less, particularly preferably 1 mg/kg body weight/day or less, and most preferably 0.5 mg/kg body weight/day or less. The daily oral intake or administration amount of vitamin _B6 and/or its salt for an adult is, for example, preferably 0.0001 to 50 mg/kg body weight/day, more preferably 0.001 to 10 mg/kg body weight/day, even more preferably 0.005 to 5 mg/kg body weight/day, particularly preferably 0.01 to 1 mg/kg body weight/day, and most preferably 0.02 to 0.5 mg/kg body weight/day. The content of vitamin _B6 and/or its salt can be the amount that results in the above intake or administration amount. The daily oral intake or dosage for an adult may be divided into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules depending on the dosage form.

The daily oral intake or administration amount of guava for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or administration amount of guava and its extracts for an adult is, for example, preferably 0.001 g/kg body weight/day or more, more preferably 0.01 g/kg body weight/day or more, and even more preferably 0.1 g/kg body weight/day or more. The daily oral intake or administration amount of guava and its extracts for an adult is, for example, preferably 1000 g/kg body weight/day or less, more preferably 100 g/kg body weight/day or less, and even more preferably 10 g/kg body weight/day or less. The daily oral intake or administration amount of guava and its extract for an adult is, for example, preferably 0.001 to 1000 g/kg body weight/day, more preferably 0.01 to 100 g/kg body weight/day, and even more preferably 0.1 to 10 g/kg body weight/day. The content of guava and its extract can be the amount that results in the above intake or administration amount. The daily oral intake or administration amount for an adult may be divided according to the dosage form, for example, in the case of capsules, into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules.

When it is made into an extract, the daily oral intake or administration amount of guava extract for an adult can be appropriately determined depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or administration amount of guava and its extract for an adult is, for example, preferably 0.01 mg/kg body weight/day or more, more preferably 0.1 mg/kg body weight/day or more, and even more preferably 0.5 mg/kg body weight/day or more. The daily oral intake or administration amount of guava and its extract for an adult is, for example, preferably 500 mg/kg body weight/day or less, more preferably 100 mg/kg body weight/day or less, and even more preferably 50 mg/kg body weight/day or less. The daily oral intake or administration amount of guava and its extract for an adult is, for example, preferably 0.01 to 500 mg/kg body weight/day, more preferably 0.5 to 100 mg/kg body weight/day, and even more preferably 0.1 to 50 mg/kg body weight/day. The content of guava and its extract can be the amount that results in the above intake or administration amount. The daily oral intake or administration amount for an adult may be divided according to the dosage form, for example, in the case of capsules, into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules.

The daily oral intake or dosage of garlic and its extracts for adults can be determined appropriately depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors. The daily oral intake or dosage of garlic and its extracts for adults is, for example, preferably 0.01 mg/kg body weight/day or more, more preferably 0.1 mg/kg body weight/day or more, and even more preferably 0.5 mg/kg body weight/day or more. The daily oral intake or dosage of garlic and its extracts for adults is, for example, preferably 500 mg/kg body weight/day or less, more preferably 100 mg/kg body weight/day or less, and even more preferably 50 mg/kg body weight/day or less. The daily oral intake or dosage of garlic and its extract for an adult is, for example, preferably 0.01 to 500 mg/kg body weight/day, more preferably 0.1 to 100 mg/kg body weight/day, and even more preferably 0.5 to 50 mg/kg body weight/day. The content of garlic and its extract can be an amount that results in the above intake or dosage. The daily oral intake or dosage for an adult may be divided according to the dosage form, for example, in the case of capsules, into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules.

The composition of the present invention can be taken or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any desired period and interval.

Next, the present invention will be specifically explained using examples, but the present invention is not limited to the following examples. Furthermore, the amount of extract shown in the examples is the amount converted to dry solid content unless otherwise specified.

[Test Example 1: Microglial cell activation inhibition test 1]
Mouse microglial cell line (BV2, Banca Biologica e Cell Factory, hereinafter the same) was seeded at 1.0 x 105 cells/1 mL/well in a 12-well plate using DMEM/F12 medium containing ⁵ % v/v FBS and an appropriate amount of antibiotics. The next day, the medium was replaced with serum-free DMEM/F12 medium, 100 ng/mL LPS was added, and the medium was cultured for 2 hours, after which apigenin (referred to as Api) and vitamin A were added to the concentrations shown in the figure.

In Figure 1, the control shows a sample containing only DMEM/F12 medium. Retinoic acid (also written as Ret) was used as vitamin A. In the Api+Ret sample group, in addition to 5 μM apigenin, 2 μM, 1 μM, or 0.5 μM of retinoic acid was added. Unless otherwise specified, the unit in the figure is "μM", and when "%" is used, it means "w・v%". The same applies below.

After 24 hours of culture, RNA was extracted using RNA isolation using RNeasy Mini Kit (Qiagen Co., Ltd.). cDNA was synthesized from the extracted RNA sample using RevertraAce qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd.). Quantification was performed by real-time PCR using Taqman probes (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystem). Gene expression was standardized to 18s rRNA and calculated using the 2 ^−ΔΔCT method. The evaluation results of IL-1β are shown in Figure 1.

As shown in Figure 1, stimulation of microglial cells with LPS induced the production of IL-1β, an inflammatory cytokine characteristic of M1 microglial cells, a subpopulation of activated microglial cells. Even when apigenin or vitamin A is administered alone, the production of inflammatory cytokines may be suppressed to a certain extent, but this is not sufficient. On the other hand, when apigenin and vitamin A were administered together, surprisingly, the production of IL-1β was significantly suppressed. It was an unexpected result that the production of IL-1β, which is involved as a main effector of neuroinflammation and neurodegenerative diseases, was significantly suppressed by the coexistence of apigenin and vitamin A.

[Test Example 2: Microglial cell activation inhibition test 2]
Mouse microglial cell line (BV2) was seeded at 1.0 x ¹⁰⁵ cells/1mL/well in a 12-well plate using DMEM/F12 medium containing 5% v/v FBS and an appropriate amount of antibiotics. The next day, the medium was replaced with serum-free DMEM/F12 medium, 100 ng/mL LPS was added, and the medium was cultured for 2 hours, after which apigenin and vitamin _B6 were added to the concentrations shown in the figure. After 24 hours of culture, RNA was extracted using RNA isolation using RNeasy Mini Kit (Qiagen Co., Ltd.). cDNA was synthesized from the extracted RNA sample using RevertraAce qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd.). Quantification was performed by real-time PCR using Taqman probes (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems). Gene expression was normalized to 18s rRNA and calculated using the 2 ^−ΔΔCT method. The results of IL-1β evaluation are shown in Figure 2.

As shown in FIG. 2, administration of vitamin _B6 alone hardly suppressed IL-1β production from activated microglial cells, but administration of apigenin and vitamin _B6 together significantly suppressed IL-1β production.

[Test Example 3: Microglial cell activation inhibition test 3]
Mouse microglial cell line (BV2) was seeded at 1.0 x ¹⁰⁵ cells/1mL/well in a 12-well plate using DMEM/F12 medium containing 5% v/v FBS and an appropriate amount of antibiotics. The next day, the medium was replaced with serum-free DMEM/F12 medium, 100 ng/mL LPS was added, and the medium was cultured for 2 hours, after which guava leaf extract (Maruzen Pharmaceutical Co., Ltd.) was added to the concentration shown in the figure. After 24 hours of culture, RNA was extracted using RNA isolation using RNeasy Mini Kit (Qiagen Co., Ltd.). cDNA was synthesized from the extracted RNA sample using RevertraAce qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd.). Quantification was performed by real-time PCR using Taqman probes (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystem). Gene expression was normalized to 18s rRNA and calculated using the 2 ^-ΔΔCT method. The results are shown in Figures 3 to 5. Figure 3 shows the results of evaluation of IL-1β, Figure 4 shows the results of evaluation of TNF-α, and Figure 5 shows the results of evaluation of CCL2.

As shown in Figures 3 to 5, the production of inflammatory cytokines and chemokines characteristic of M1 microglial cells was significantly suppressed when guava extract derived from food materials containing apigenin, vitamin A, and vitamin _B6 was used. It is known that guava extract contains about 579 mg/kg of apigenin on a dry weight basis (Koo Hui Miean et al. Flavonoid (Myricetin, Quercetin, Kaempferol, Luteolin, and Apigenin) Content of Edible Tropical Plants. J Agric Food Chem. 2001, Vol 49(6), 3106-12). The guava extract used in Test Example 4 was calculated to contain about 0.043 μM apigenin at 0.002 w/v%, about 0.021 μM apigenin at 0.001 w/v%, and about 0.011 μM apigenin at 0.0005 w/v% (the equivalent amount of apigenin in the guava extract is also shown in each figure). Compared to the 5 μM apigenin used in Test Examples 1 and 2, the amount of apigenin contained in the guava extract was small, but since vitamins A and _B6 coexisted, it was found to significantly suppress the production of inflammatory cytokines characteristic of M1 microglial cells. As a reference example, the inhibitory effect of apigenin alone on the production of IL-1β, TNF-α, and CCL2 was evaluated at concentrations of 1 μM, 5 μM, and 10 μM (FIGS. 6 to 8) in the same manner as in Test Example 1. Even when compared with these reference examples, it was found that the guava extract derived from a food material containing vitamin A and vitamin _B6 had a significant inhibitory effect on the production of inflammatory cytokines, which indicates the inhibition of M1 microglial cells.

[Test Example 4: Microglial cell activation inhibition test 4]
Mouse microglial cell line (BV2) was seeded at 1.0 x ¹⁰⁵ cells/1mL/well in a 12-well plate using DMEM/F12 medium containing 5% v/v FBS and an appropriate amount of antibiotics. The next day, the medium was replaced with serum-free DMEM/F12 medium, 100 ng/mL LPS was added, and the medium was cultured for 2 hours. After that, dried garlic powder (Nihon Funa Yakuhin Co., Ltd.) and black garlic powder (Maruzen Pharmaceutical Co., Ltd.) were added to the concentrations shown in the figure. After 24 hours of culture, RNA was extracted using RNA isolation using RNeasy Mini Kit (Qiagen Co., Ltd.). cDNA was synthesized from the extracted RNA sample using RevertraAce qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd.). Quantification was performed by real-time PCR using Taqman probes (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystem). Gene expression was normalized to 18s rRNA and calculated using the 2 ^-ΔΔCT method. The results using dried garlic powder are shown in Figures 9 to 11, and the results using black garlic powder are shown in Figures 12 to 14. Figures 9 and 12 show the evaluation results of IL-1β, Figures 10 and 13 show the evaluation results of TNF-α, and Figures 11 and 14 show the evaluation results of CCL2.

As shown in Figures 9 to 14, when garlic powder derived from a food material containing apigenin and vitamin _B6 was used, the production of inflammatory cytokines and chemokines characteristic of M1 microglial cells was significantly suppressed.

Claims (1)

The invention described in the specification.