WO2018029793A1

WO2018029793A1 - Il-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, anti-neuropsychiatric disease agent, medicine and food

Info

Publication number: WO2018029793A1
Application number: PCT/JP2016/073506
Authority: WO
Inventors: 高橋　知也; 一紅潘; 淑芳 ▲温▼; 亦晃呂; 凱安 ▲荘▼; 明翰李; 明▲徳▼ 彭
Original assignee: 株式会社Ａｏｂ慧央グループ
Priority date: 2016-08-09
Filing date: 2016-08-09
Publication date: 2018-02-15
Also published as: JP6497791B2; TW201805015A; TWI673059B; JPWO2018029793A1

Abstract

An IL-6 production inhibitor which comprises rhinacanthin C alone as an active ingredient and inhibits IL-6 production in microglia. An anti-inflammatory agent which comprises rhinacanthin C alone as an active ingredient and inhibits IL-6 production in microglia to thereby prevent or suppress inflammation in the central nervous system. An anti-neurodegenerative disease agent or an anti-neuropsychiatric disease agent which comprises the IL-6 production inhibitor or anti-inflammatory agent as described above as an active ingredient. A medicine or a food which comprises the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent or anti-neuropsychiatric disease agent as described above. According to the present invention, the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, anti-neuropsychiatric disease agent, medicine and food each using rhinacanthin C, which is clarified as having an inhibitory effect on IL-6 production in microglia, can be provided.

Description

IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological agent, pharmaceutical and food

The present invention relates to an IL-6 production inhibitor, an anti-inflammatory agent, an anti-neurodegenerative disease agent, an antipsychotic neurological agent, a pharmaceutical and a food using linacantin C.

Technical background

Microglia are a type of glial cell that exists in the central nervous system (brain and spinal cord). Microglia are also called microglia cells or Hortega cells.
Microglia are thought to be immunocompetent cells such as macrophages. Microglia has various actions and roles such as antigen presenting action that is the starting point of immune response, innate immunity action against foreign bodies, phagocytosis against foreign bodies and wastes, formation of neural circuits, and production of various substances that affect surrounding cells. Have

Microglia become active due to external stimuli and stress, and produce useful substances such as antioxidants and nutrient factors. However, microglial secretes excitatory amino acids such as inflammatory cytokines, chemokines, nucleic acids, glutamic acid, reactive oxygen species, proteases, etc. due to pathological activation, and damages surrounding cells, thereby causing the origin of neuroinflammation. It becomes. For this reason, microglia can also cause neurodegeneration of the central nervous system (for example, refer nonpatent literature 1).

IL-6 (interleukin-6) is a kind of cytokine produced by microglia. IL-6 is known to be closely related to inflammation (see, for example, Non-Patent Document 2). IL-6 produced by microglia causes inflammation in the central nervous system and causes neurodegenerative diseases such as Parkinson's disease, Parkinson's syndrome, amyotrophic lateral sclerosis, multiple sclerosis, stroke, cerebral infarction and cerebral ischemia It becomes. In addition, inflammation in the central nervous system also causes neuropsychiatric disorders such as schizophrenia, depression, autism spectrum disorder, addiction and epilepsy (see, for example, Non-Patent Document 3).
For this reason, if IL-6 production by microglia can be suppressed, it is considered possible to prevent neurodegenerative diseases and neuropsychiatric diseases and to suppress or treat the progression of symptoms.

By the way, the white crane reishi (Rhinacanthus natusus (L.) Kurz. Also called white crane reishi grass) is an evergreen small shrub belonging to the genus Linacanthus vulgaris that is native to the Deccan Plateau in southern India. Hakutsuru Ganoderma is known to have antibacterial action against anthelmintic, anti-inflammatory, and skin fungi (see Non-Patent Document 4, for example) and is mainly used as a folk medicine or food in China, Taiwan, Thailand, India, etc. It has been. Hakutsuru Ganoderma is also recently used as a food in Japan. In the previous application by the applicant of the present application, Hakutsuru Ganoderma has active oxygen scavenging ability (for example, see Patent Document 1), excretion promoting action (for example, see Patent Document 2), anti-allergy. It is disclosed that there is an action (for example, see Patent Document 3) and that it has an antitumor action (for example, see Patent Document 4).

In addition, white crane reishi contains a compound called linacantin C (for example, see Non-Patent Document 5). Linacantin C is a compound having a naphthoquinone skeleton and is represented by the following chemical formula (1).

Japanese Patent Laid-Open No. 9-143091 Japanese Patent Laid-Open No. 9-169662 JP 2001-10964 A JP 2002-53481 A

However, it is not known that linacantin C suppresses IL-6 production in microglia.

Therefore, an object of the present invention is to provide an IL-6 production inhibitor that suppresses IL-6 production in microglia. Another object of the present invention is to provide an anti-inflammatory agent that prevents or suppresses inflammation of the central nervous system by suppressing IL-6 production in microglia. Another object of the present invention is to provide an anti-neurodegenerative disease agent, an antipsychotic neurological disease agent, a pharmaceutical and a food related to the above-mentioned IL-6 production inhibitor or anti-inflammatory agent.

The inventors of the present invention conducted intensive studies on the properties of linacantin C. As a result, it was found that linacantin C has an action of suppressing the production of IL-6 in microglia, thereby completing the present invention. The present invention includes the following items.

[1] An IL-6 production inhibitor that contains only linacantin C as an active ingredient and suppresses IL-6 production in microglia.

[2] An anti-inflammatory agent that contains only linacantin C as an active ingredient and prevents or suppresses inflammation of the central nervous system by suppressing IL-6 production in microglia.

[3] An anti-neurodegenerative disease agent comprising, as an active ingredient, the IL-6 production inhibitor according to [1] or the anti-inflammatory agent according to [2].

[4] In the anti-neurodegenerative disease agent according to [3] above, the neurodegenerative disease is Parkinson's disease, Parkinson's syndrome, amyotrophic lateral sclerosis, multiple sclerosis, stroke, cerebral infarction, or cerebral ischemia. An anti-neurodegenerative disease agent that is at least one of them.

[5] An antipsychotic neuropathy agent comprising the IL-6 production inhibitor according to [1] or the anti-inflammatory agent according to [2] as an active ingredient.

[6] The antipsychotic neuropathy according to [5], wherein the neuropsychiatric disorder is at least one of schizophrenia, depression, autism spectrum disorder, addiction and epilepsy. Agent.

[7] An IL-6 production inhibitor, an anti-inflammatory agent, an anti-neurodegenerative disease agent or an antipsychotic neurological disease agent according to any one of [1] to [6] above, A pharmaceutical product having at least one of an anti-inflammatory action, an anti-neurodegenerative disease action, and an anti-psychotic nerve disease action.

[8] An IL-6 production inhibitor, an anti-inflammatory agent, an anti-neurodegenerative disease agent or an antipsychotic neurological disease agent according to any one of [1] to [6] above, A food having at least one of an anti-inflammatory action, an anti-neurodegenerative disease action, and an anti-psychotic neuropathy action.

According to the present invention, as shown in Test Examples described later, IL that suppresses IL-6 production in microglia using linacantin C that has been clarified to have an action of suppressing IL-6 production in microglia. -6 production inhibitor can be provided. In addition, an anti-inflammatory agent that prevents or suppresses inflammation of the central nervous system can be provided by suppressing IL-6 production in microglia using linacantin C. Furthermore, it is possible to provide an anti-neurodegenerative disease agent, an antipsychotic neuropathy agent, a pharmaceutical and a food related to the above-mentioned IL-6 production inhibitor or anti-inflammatory agent, that is, using linacantin C.

As for the above [1], for example, “the active ingredient of linacantin C for producing an IL-6 production inhibitor containing only linacantin C as an active ingredient and suppressing the production of IL-6 in microglia. It can also be expressed as "Use as."
As for the above [2], for example, “manufacturing an anti-inflammatory agent containing only linacantin C as an active ingredient and preventing or suppressing inflammation of the central nervous system by inhibiting the production of IL-6 derived from microglia. Therefore, it can also be expressed as “Use of Linacantin C as an active ingredient”.
As for the above [3], for example, “the active ingredient of the IL-6 production inhibitor according to [1] or the anti-inflammatory agent according to [2] for producing an anti-neurodegenerative disease agent” It can also be expressed as "Use as."
As for the above [5], for example, “the active ingredient of the IL-6 production inhibitor according to [1] or the anti-inflammatory agent according to [2] above for producing an antipsychotic neuropathy agent” It can also be expressed as "Use as."
As for [7] above, for example, the above [for producing a pharmaceutical product having at least one of IL-6 production inhibitory action, anti-inflammatory action, anti-neurodegenerative disease action and antipsychotic neuropathy action] 1] to [6], use of IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, or antipsychotic neurological disease agent.
In addition, the above [8] is, for example, “for producing a food having at least one of IL-6 production inhibitory action, anti-inflammatory action, anti-neurodegenerative disease action, and antipsychotic nerve disease action” 1] to [6], use of IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, or antipsychotic neurological disease agent.

It is a graph which shows the result of the test using A (beta) in a test example. 6 is a graph showing the results of a test using IFN-γ and LPS in a test example.

Hereinafter, the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, anti-psychotic neurological disease agent, pharmaceutical and food of the present invention will be described.

The IL-6 production inhibitor of the present invention contains only linacantin C as an active ingredient, and suppresses IL-6 production in microglia.
In the present specification, for an IL-6 production inhibitor, “contains only linacantin C as an active ingredient” means that the IL-6 production inhibitor acts as an active ingredient having an action of inhibiting the production of IL-6 in microglia. It contains only C. Therefore, it cannot be denied that the IL-6 production inhibitor contains components that are not the active ingredients (for example, auxiliary additives, excipients, solvents, etc.).
In the present specification, the action of suppressing the production of IL-6 in microglia is referred to as “IL-6 production inhibitory action”.

The anti-inflammatory agent of the present invention contains only linacantin C as an active ingredient, and prevents or suppresses inflammation of the central nervous system by suppressing the production of microglia-derived IL-6.
In the present specification, the term “contains only linacantin C as an active ingredient” for an anti-inflammatory agent means that the anti-inflammatory agent prevents or suppresses inflammation of the central nervous system by suppressing IL-6 production in microglia. It means that only linacantin C is contained as an active ingredient having an anti-inflammatory agent, and it is not denied that the anti-inflammatory agent contains a component that is not the active ingredient (for example, an auxiliary additive, an excipient, a solvent, etc.).
In the present specification, the action of preventing or suppressing inflammation of the central nervous system by suppressing the production of IL-6 in microglia is referred to as “anti-inflammatory action”.

The anti-neurodegenerative disease agent and anti-psychotic neurological disease agent of the present invention contain the IL-6 production inhibitor or anti-inflammatory agent of the present invention (that is, substantially linacantin C) as an active ingredient.
Linacantin C suppresses IL-6 production in microglia as shown in the test examples described below. Therefore, the anti-neurodegenerative disease agent and antipsychotic neuropathy agent of the present invention containing the IL-6 production inhibitor or anti-inflammatory agent of the present invention as an active ingredient, that is, containing linacantin C as an active ingredient, In order to prevent or suppress inflammation in the nervous system, it has an effect as an anti-neurodegenerative disease agent and an antipsychotic nerve disease agent.

As used herein, the term “anti-neurodegenerative disease agent” refers to an agent that can be used for prevention, suppression or treatment of neurodegenerative diseases. As used herein, “neurodegenerative disease” refers to a disease caused by abnormalities in cells of the central nervous system, particularly inflammation of the central nervous system. Examples of neurodegenerative diseases targeted by the anti-neurodegenerative disease agent of the present invention include Parkinson's disease, Parkinson's syndrome, amyotrophic lateral sclerosis, multiple sclerosis, stroke, cerebral infarction and cerebral ischemia. The anti-neurodegenerative disease agent of the present invention preferably has an action on at least one of them.
In the present specification, the action of preventing or suppressing inflammation in the central nervous system to prevent a neurodegenerative disease or suppress the progression of symptoms is referred to as “anti-neurodegenerative disease action”.

Note that neurodegenerative diseases also include Alzheimer's disease. However, “an anti-Alzheimer's disease agent containing linacantin C as an active ingredient” is described in the scope of the application originally filed by the applicant of the present application, although the mechanism of action is different from that of the present invention. Therefore, “anti-Alzheimer's agent in ultimate use” can be excluded from the anti-neurodegenerative disease agent of the present application (see PCT / JP2015 / 69363 not published at the time of filing of the present application). ).

As used herein, the term “antipsychotic neuropathy agent” refers to an agent that can be used for the prevention, prevention or treatment of neuropsychiatric disorders. As used herein, “psycho-neurological disorder” refers to a disease mainly caused by an abnormality of a cell of the central nervous system, particularly inflammation of the central nervous system, which mainly affects the mind. “Psychiatric / neurological disorder” is sometimes referred to as “psychological / neurological disorder”. Examples of the neuropsychiatric disorder targeted by the antipsychotic neuropathy agent of the present invention include schizophrenia, depression, autism spectrum disorder, addiction and epilepsy. It is preferable to have an action on at least one of these.
In the present specification, the action of preventing or suppressing inflammation in the central nervous system, thereby preventing a neuropsychiatric disorder or suppressing the progression of symptoms is referred to as an “antipsychotic neuropathy effect”.

The anti-neurodegenerative disease agent and anti-psychotic neurological disease agent of the present invention are effective components other than the IL-6 production inhibitor or anti-inflammatory agent of the present invention as an active ingredient related to the anti-neurodegenerative disease action or anti-psychotic neurological disease action. May be contained.

The medicament and food of the present invention contain the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent or anti-psychotic neuropathy agent of the present invention, and inhibit the IL-6 production, anti-inflammatory, anti-nerve It has at least one of degenerative disease action and antipsychotic nerve disease action.
That is, the pharmaceutical and food of the present invention contain linacantin C as an active ingredient.

If the pharmaceutical and food of the present invention have at least one of IL-6 production inhibitory action, anti-inflammatory action, anti-neurodegenerative disease action, and antipsychotic neuropathy action, IL-6 production inhibitory action, anti-inflammation It may be one that does not have the main purpose of action, anti-neurodegenerative disease action and anti-psycho-neurological disease action.

The IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological disease agent, pharmaceutical and food of the present invention include natural products containing linacantin C (for example, Hakutsuru Reishi), the natural products Processed products (for example, dried products of Hakutsuru Reishi), extracts of the natural products (for example, ethanol extracts of Hakutsuru Reishi), purified products of the natural products or extracts thereof (for example, ethanol of Hakutsu Reishi) A purified product obtained by purifying the extract by column chromatography or the like can be used.
In addition, linacantin C isolated from natural products containing linacantin C should be used for the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neuropathy agent, pharmaceutical and food of the present invention. You can also.
In addition, linacantin C obtained by chemical synthesis (total synthesis or partial synthesis) is used for the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological disease agent, pharmaceutical and food of the present invention. You can also

The linacantin C used in the present invention may be in the form of linacantin C when acting in the body, and may be in the form of a pharmaceutically acceptable salt or precursor before administration, formulation or use. Good.

There are no particular limitations on the route of administration of the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological disease and pharmaceuticals (herein, pharmaceuticals for internal use or internal use) of the present invention. For example, oral administration -Examples include enteral administration such as rectal administration, mucosal administration such as nasal administration, injection administration such as intravenous administration and subcutaneous administration, and the like. Any dosage form can take the form of a formulation suitable for the administration method. For example, tablets, powders, fine granules, granules, capsules, powders, pills, lozenges and other solid agents, solutions , Suspensions, emulsions, syrups, liquids such as injections, gel preparations, and the like. The IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological disease agent and pharmaceutical of the present invention may be administered as they are, but administered together with a pharmacologically acceptable excipient. Also good. As the excipient, any monosaccharides, disaccharides, polysaccharides, inorganic salts, oils and fats, distilled water, etc. that can be generally used as preparations can be used. In formulating, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used.

Effective doses of the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent, antipsychotic neurological disease agent and pharmaceutical agent of the present invention vary depending on the administration route, dosage form, disease symptoms, age of the subject, etc. However, for linacantin C, it is considered that the daily dose is usually 0.1 to 1000 mg, preferably 0.5 to 300 mg, more preferably 1 to 100 mg per adult. The content of linacantin C can be set to the optimum active ingredient content in the preparation for each dosage form based on the data on the form of the preparation, the effective dose, and the dose as the preparation.

The medicine of the present invention may be an external medicine. Although the form of external medicine is not specifically limited, For example, an ointment, a cream agent, a poultice, a tape agent, an external preparation, etc. can be mentioned. The external medicine of the present invention can contain various pharmaceutical ingredients as needed in addition to linacantin C. In addition, additives such as a binder, a dispersant, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, and a bacteria inhibitor can also be used.

The food of the present invention is a tea or processed food containing the IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent or antipsychotic neuropathy agent of the present invention, essentially linacantin C. A food can be illustrated.

Examples of tea include natural products containing linacantin C, such as those using dried leaves, stems or roots of white crane reishi. The natural product containing linacantin C is preferably used by mixing with other tea ingredients. As tea materials, green tea, oolong tea, puer tea, black tea, hoji tea, brown rice tea, tochu tea, kashiwa leaf tea, mulberry leaf tea, and the like can be used. As long as linacantin C is not destroyed, natural products containing linacantin C (for example, dried leaves, stems or roots of white crane reishi) can be roasted and used in the same manner as other tea ingredients. .

As the form of processed food, any form can be provided as usual processed food such as drinks, jellies, biscuits, tablets, pills, soft capsules, hard capsules, powders, fine granules, granules, etc. Can also be used. In addition, additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacterial inhibitors can be used as auxiliary ingredients for processed foods. it can.

The effective intake of the food of the present invention varies depending on the intake form, the health condition of the subject, the age of the subject, etc., but for linacantin C, usually 0.1 to 1000 mg, preferably 0.5 to 300 mg per adult day More preferably, it is considered to be 1 to 100 mg.

The content of linacantin C in the food of the present invention varies depending on the form of the food, but is usually 0.0001 to 1 wt%, preferably 0.001 to 0.5 wt%, more preferably 0.01 to 0.00. It is considered to be 1 wt%.

[Test example]
Hereinafter, the present invention will be described in more detail by test examples relating to the action of linacantin C. The following test examples are specific examples, and the present invention is not limited to the following test examples.

In the test example, a test was conducted to verify the action of linacantin C together with the influence of a substance that is thought to promote the production of IL-6 in microglia.
As substances considered to promote IL-6 production in microglia, amyloid β, interferon-γ and lipopolysaccharide were used.

Amyloid β is a kind of amyloid protein and is considered to have direct or indirect toxicity to nerve cells. In the test examples, amyloid β (25-35), which is known as a neurotoxic action center, was used as amyloid β. As amyloid β (25-35), a product purchased from Sigma Aldrich of the United States (Amyloid β-Protein Fragment 25-35, Cat. No. A4559, hereinafter simply referred to as “Aβ”) was used.

Interferon-γ is a kind of cytokine and has antiviral action, NK cell activity enhancing action, macrophage activation action, and the like. On the other hand, interferon-γ also has an action of enhancing inflammation. In the test examples, interferon-γ purchased from R & D Systems, Inc. (Recombinant Mouse IFN-gamma Protein, Cat. 485-MI-100, hereinafter simply referred to as “IFN-γ”) is used. It was.

Lipopolysaccharide (lipopolysaccharide) is a glycolipid constituting the outer cell wall membrane in Gram-negative bacteria. Lipopolysaccharide acts on cells such as humans to promote the production of inflammatory cytokines. In the test examples, lipopolysaccharide purchased from Sigma-Aldrich, USA (Lipopolysaccharides from Escherichia coli 0111: B4, Cat.L4391. Hereinafter, simply referred to as “LPS”) was used.

Note that Linacantin C used in the test examples was isolated from Hakutsuru Ganoderma. The isolation method is as follows.

First, 2 kg of dried roots of white crane Ganoderma (Rhinacanthus nasutus (L.) Kurz) was prepared and pulverized with a general-purpose grinder.
Subsequently, extraction with 90% ethanol was performed. The extraction was performed by immersing the white crane reishi in 20 l of 90% ethanol for 3 days and then refluxing the solvent for 1 hour. The extract and the extraction residue were separated by filter paper. The extraction was performed three times on the same raw material. The solvent was removed from the extract under reduced pressure to obtain 77.82 g of a dry ethanol extract.

After removing 4 g from the ethanol extract as a sample for analysis, the remaining 73.82 g ethanol extract was subjected to liquid-liquid partition with hexane and 90% methanol. First, 500 ml of 90% methanol was added to the ethanol extract, and then 500 ml of hexane was further added and shaken for 1 minute. The solvent was separated for about 10 minutes, and a 90% methanol phase was collected. The same operation was further repeated twice, and the solvent was removed from the obtained 90% methanol phase under reduced pressure to obtain 54.32 g of a dried solid product.

After removing 4 g from the above-mentioned distribution as an analytical sample, the remaining 50.32 g of the entire distribution was subjected to liquid-liquid distribution with methylene dichloride and water. First, 500 ml of purified water was added to the distribution, and then 500 ml of methylene dichloride was further added and shaken for 1 minute. The solvent was separated for about 10 minutes and the methylene dichloride phase was collected. Further, the same operation was repeated twice, and the solvent was removed from the obtained methylene chloride phase under reduced pressure to obtain 27.55 g of a predetermined distribution.

Thereafter, 2.50 g of a predetermined distribution and 10 g of silica gel (a general-purpose product of 0.063 to 0.2 mm) were mixed. Thereafter, a column (open column with a diameter of 2 cm, a length of 30 cm, and a volume of about 60 ml) packed with 200 g of silica gel (0.040 to 0.063 mm general-purpose product) was prepared, and an elution solvent (n-hexane: ethyl acetate = 4). After equilibrating in 1), a mixture of a predetermined partition and silica gel was placed, and elution with an elution solvent was performed. The amount of elution solvent was 500 ml.

In silica gel column chromatography, about 10 ml each was collected in a test tube, and the elution pattern was observed by TLC. According to the approximate elution pattern, the fraction obtained in the first test tube was fraction 2-1 (solvent amount of about 0 to 10 ml), and the fraction obtained in the second and third test tubes was fraction 2. -2 (amount of solvent: about 10 to 30 ml), what was obtained in the 4th to 10th test tubes was designated as fraction 2-3 (amount of solvent: about 30 to 100 ml).

Thereafter, the structure of the fractions 2-1 to 2-3 was analyzed by comparing ¹ HNMR and ¹³ CNMR data with literature values (Journal of Natural Products, 59, 808-811, 1996). It was confirmed that the fraction 2-2 was linacantin C. The amount of obtained linacantin C was 444.2 mg.

Note that a JEOL JNM-GSX500 type nuclear magnetic resonance spectrum apparatus (manufactured by JEOL Ltd.) was used as the nuclear magnetic resonance spectrum apparatus.

Hereinafter, the test method will be described.
In the test examples, mouse-derived microglia BV-2 cells (hereinafter simply referred to as BV-2 cells) were used as microglia. BV-2 cells were prepared in the laboratory of Dr. Chen Jiayi, Taipei Medical University, Taiwan. The BV-2 cells were obtained by introducing the v-raf / v-myc oncogene into mouse microglia cultured cells by retrovirus and establishing a cell line.
The obtained BV-2 cells were precultured at 37 ° C. in the presence of 5% carbon dioxide gas using RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin.

BV-2 cells were obtained in 96-well plates (Coming # 96, Welling, Wrap Lid, # ³ , 99) from 96 Well Clear Flat Bottom PolyTC TC-Treated Microplates, Independently Wrapped, Wright Lid, Steroid # 35, 99, Corning, USA. The cells were seeded at a density and cultured at 37 ° C. for 16 hours in the presence of 5% carbon dioxide gas.
Thereafter, the sample to which linacantin C was added was pretreated for 4 hours by adding linacantin C having a predetermined concentration. Thereafter, each sample having a predetermined concentration was added to the sample to which Aβ was added, the sample to which IFN-γ was added, and the sample to which LPS was added, and cultured at 37 ° C. for 48 hours in the presence of 5% carbon dioxide gas.

After the culture, the supernatant was collected, and the amount of IL-6 produced was measured by a conventional ELISA method. ELISA measurement is conducted by US Affymetrix, eBIOSCIENCE Mouse IL-6 ELISA Ready-SET-Go! It was performed using.
Further, the cell viability was calculated by a conventional MTT test method. Specifically, 10 μl of a 5 mg / ml MTT solution (manufactured by Thermo Fisher Scientific, USA) was added to 100 μl of cell culture solution, and cultured at 37 ° C. for 1.5 hours in the presence of 5% carbon dioxide gas. Thereafter, the culture solution is removed, 100 μl of a lysis solution (isopropanol solution containing 0.04 N hydrochloric acid) is added to completely dissolve the product, and the absorbance at a wavelength of 570 nm (control 640 nm) is measured to determine the cell viability. Calculated.

In the test example, the test using Aβ and the test using IFN-γ and LPS were performed separately.
In the test using Aβ, the concentration of linacantin C was 0.25, 0.5 μM, and the concentration of Aβ was 20 μM.
In the test using IFN-γ and LPS, the concentration of linacantin C was 0.06, 0.13, 0.25, 0.5 μM, the concentration of IFN-γ was 0.1 ng / ml, and the concentration of LPS was 100 ng / ml.
The concentration of the additive in each sample was determined in advance by screening for cytotoxicity and the like.

Hereinafter, the results of the test will be described.

(1) Test using Aβ FIG. 1 is a graph showing the results of a test using Aβ in a test example. FIG. 1A is a bar graph showing the amount of IL-6 produced for each sample, and FIG. 1B is a bar graph showing the cell viability for each sample. The item “RC” described in the lower part of the horizontal axis in FIGS. 1 (a) and 1 (b) indicates whether or not linacantin C was added and the amount added. Linacantin C was not added, and linacantin C was added to the sample indicated by the numerical value by the numerical value (unit: μM). On the other hand, the item “Aβ” indicates the presence or absence of the addition of Aβ. Aβ was not added to the sample with “−”, and Aβ was added to the sample with “+”. The vertical axis in FIG. 1 (a) indicates the production amount of IL-6 (unit: pg / ml), and the vertical axis in FIG. 1 (b) indicates the cell viability (unit:%). In FIG. 1, the numerical values described above the bar graph are numerical values corresponding to the vertical axis.

The significant difference test for IL-6 production change was as described below. About the sample which added only linacantine C, it decreased with the significant difference of p <0.001 on the basis of the sample which added nothing. About the sample which added only A (beta), it increased with the significant difference of p <0.05 on the basis of the sample which added nothing. About the sample which added 0.5 micromol Linacantin C and also added A (beta), it decreased with the significant difference of p <0.05 on the basis of the sample which added only A (beta).

(2) Test using IFN-γ and LPS FIG. 2 is a graph showing the results of a test using IFN-γ and LPS in a test example. FIG. 2 (a) is a bar graph showing the amount of IL-6 produced for each sample, and FIG. 2 (b) is a bar graph showing the cell viability for each sample. The items of “RC” described in the lower part of the horizontal axis in FIGS. 2A and 2B indicate the presence or absence and addition amount of linacantin C. In the samples where “−” is described, Linacantin C was not added, and linacantin C was added to the sample indicated by the numerical value by the numerical value (unit: μM). On the other hand, the item “IFN-γ” indicates whether or not IFN-γ has been added. For samples in which “−” is described, IFN-γ is not added, and in samples where “+” is described. IFN-γ was added. The item “LPS” indicates whether or not LPS was added. LPS was not added to the sample with “−” and LPS was added to the sample with “+”. The vertical axis in FIG. 2 (a) indicates the production amount of IL-6 (unit: pg / ml), and the vertical axis in FIG. 2 (b) indicates the cell viability (unit:%). In FIG. 2, the numerical values described above the bar graph are numerical values corresponding to the vertical axis.

The significant difference test for IL-6 production change was as described below. About the sample which added only linacantine C 0.13 micromol, it fell with the significant difference of p <0.01 on the basis of the sample which added nothing. About the sample which added only linacantin C 0.25 micromol, and the sample which added 0.5 micromol, it decreased with the significant difference of p <0.001 on the basis of the sample which added nothing. For the sample to which only IFN-γ was added and the sample to which only LPS was added, there was a significant difference of p <0.05 with respect to the sample to which nothing was added. For samples to which linacantin C was added at 0.06 μM or 0.25 μM and IFN-γ was also added, the sample decreased with a significant difference of p <0.05 based on the sample to which only IFN-γ was added. In the sample to which 0.5 μM linacantin C was added and IFN-γ was also added, the sample decreased with a significant difference of p <0.001 based on the sample to which only IFN-γ was added. About the sample which added Linacantin C 0.06 micromol and LPS, it fell with the significant difference of p <0.05 on the basis of the sample which added LPS only. About the sample which added linacantin C 0.13 micromol and LPS was added, it decreased with the significant difference of p <0.01 on the basis of the sample which added LPS only. About the sample which added linacanthin C 0.25 micromol or 0.5 micromol and also added LPS, it fell with the significant difference of p <0.001 on the basis of the sample which added LPS only.

From the above test examples, as shown in FIGS. 1 and 2, it was confirmed that Aβ, IFN-γ and LPS have an effect of promoting IL-6 production in microglia.
It was also confirmed that linacantin C suppresses IL-6 production.
Furthermore, it was also confirmed that the action of linacantin C has concentration dependency.
In addition, about the cell survival rate, it was a numerical value of the level which does not have a problem in any sample.

For this reason, it is considered that the IL-6 production inhibitor of the present invention can inhibit the production of IL-6 in microglia. In addition, it is considered that the anti-inflammatory agent of the present invention can prevent or suppress central nervous system inflammation caused by IL-6 by suppressing IL-6 production in microglia. In addition, the anti-neurodegenerative disease agent, antipsychotic neuropathy agent, pharmaceutical and food of the present invention suppress IL-6 production in microglia and prevent or suppress inflammation of the central nervous system, thereby producing IL-6 production. It is considered to have at least one of an inhibitory action, an anti-inflammatory action, an anti-neurodegenerative disease action and an antipsychotic nerve disease action.

[Example]
By the following examples, linacantin C, which is an active ingredient of the IL-6 production inhibitor and anti-inflammatory agent of the present invention, and a substantial active ingredient of the anti-neurodegenerative disease agent and antipsychotic neuropathy agent of the present invention, Describes how to prepare pharmaceuticals and foods.

(1) Tablet Using Linacantin C, a tablet is prepared according to the following formulation.

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

(Preparation method)
Linacantin C (0.2 g), dried corn starch (2 g), talc (1.8 g), and calcium stearate (0.2 g) are added to lactose (95.8 g) and mixed. Subsequently, a tablet is produced by a conventional method using a single-shot tablet press.

(2) Hard capsules Using Linacantin C, hard capsules (360 mg per capsule) are prepared according to the following formulation.

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

(Preparation method)
Linacantin C (5 g) is mixed with lactose (220 g) and corn starch (110 g), and an aqueous solution of hydroxypropylcellulose (25 g) is added thereto and kneaded. Next, granules are produced by an ordinary method using an extrusion granulator. A hard capsule is prepared by filling the granule into a gelatin hard capsule.

(3) Soft capsules Using linacantin C, soft capsules (170 mg per capsule) are prepared according to the following formulation.

Linacantin C 0.5mg
Soybean oil 169.5mg

(Preparation method)
Linacantin C (0.5 g) is added to and mixed with soybean oil (169.5 g). Next, soft capsules are prepared by filling soft capsules using a rotary soybean automatic molding machine according to a conventional method.

(4) Pills Using linacantin C, pills (100 mg per capsule) are prepared according to the following prescription.

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

(Preparation method)
After mixing the raw materials with the above blending, adding a suitable amount of water, producing a homogeneous kneaded product with a kneader, rolling the kneaded product, making a round shape using a round machine and drying it to produce a pill To do.

(5) Powder Using Linacantin C, a powder (1000 mg per packet) is prepared by the following method using a conventional method.

Linacantin C 1mg
Lactose 799mg
Cornstarch 200mg

(6) Jelly Using linacantin C, jelly (100 g) is prepared by a conventional method with the following prescription.

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

(Preparation method)
The above ingredients are mixed and heated to 90 ° C. After confirming the dissolution of gelatin, fill the container and cool. A jelly is prepared by solidifying gelatin.

(7) Ointment Using linacantin C, an ointment (100 g) is prepared by a conventional method with the following formulation.

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

(Preparation method)
The oil phase component and the water phase component are each heated to 80 ° C. to be uniform, and the water phase is added to the oil phase while stirring, emulsified and cooled to prepare an ointment.

(8) Tape agent A tape agent (100 g) is prepared by a conventional method using linacantin C according to the following formulation.

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

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

Claims

An IL-6 production inhibitor containing only linacantin C as an active ingredient and suppressing IL-6 production in microglia.
An anti-inflammatory agent that contains only linacantin C as an active ingredient and prevents or suppresses inflammation of the central nervous system by suppressing IL-6 production in microglia.
An anti-neurodegenerative disease agent comprising the IL-6 production inhibitor according to claim 1 or the anti-inflammatory agent according to claim 2 as an active ingredient.
In the anti-neurodegenerative disease agent according to claim 3,
An anti-neurodegenerative disease agent, wherein the neurodegenerative disease is at least one of Parkinson's disease, Parkinson's syndrome, amyotrophic lateral sclerosis, multiple sclerosis, stroke, cerebral infarction and cerebral ischemia.
An antipsychotic neuropathy agent comprising the IL-6 production inhibitor according to claim 1 or the anti-inflammatory agent according to claim 2 as an active ingredient.
In the antipsychotic neuropathy agent according to claim 5,
An agent for neuropsychiatric disorders, wherein the neuropsychiatric disorder is at least one of schizophrenia, depression, autism spectrum disorder, addiction and epilepsy.
An IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent or anti-psychotic neurological disease agent according to any one of claims 1 to 6, comprising an IL-6 production inhibitory action, anti-inflammatory action, anti-nerve A pharmaceutical product having at least one of a degenerative disease action and an antipsychotic nerve disease action.
An IL-6 production inhibitor, anti-inflammatory agent, anti-neurodegenerative disease agent or anti-psychotic neurological disease agent according to any one of claims 1 to 6, comprising an IL-6 production inhibitory action, anti-inflammatory action, anti-nerve A food having at least one of a degenerative disease action and an antipsychotic nerve disease action.