JP2020050593A - Inhibitors of interleukin-33 production, pharmaceuticals, quasi drugs, cosmetics and food and drink compositions for preventing, treating or inhibiting allergic diseases related to increase of interleukin-33 - Google Patents

Abstract

To provide pharmaceuticals, quasi drugs, cosmetics and food and drink compositions exerting excellent prevention, treatment or inhibitory effects on allergic diseases such as allergic rhinitis and atopic dermatitis based on the interleukin-33 (IL-33) production inhibitory action.SOLUTION: The invention provides an IL-33 production inhibitor comprising 4'-demthylnobiletin as an effective ingredient, and a pharmaceutical, a quasi drug, a cosmetic and a food and drink composition for preventing, treating or inhibiting allergic diseases such as allergic rhinitis and atopic dermatitis related to the increase of IL-33. Since 4'-demthylnobiletin is one of the metabolic products of nobiletin which is an ingredient of citrus peel and has long been ingested as a food component, the pharmaceuticals, quasi drugs, cosmetics and food and drink compositions of the invention are highly safe and free of side effects.SELECTED DRAWING: Figure 1

Description

  The present invention comprises 4'-demethylnobiletin as an active ingredient and has an interleukin-33 (IL-33) production inhibitory action, thereby producing IL- such as pollinosis, perennial allergic rhinitis and atopic dermatitis. The present invention relates to pharmaceuticals, quasi-drugs, cosmetics, and food and drink compositions having excellent preventive, therapeutic, or inhibitory effects on allergic diseases associated with an increase of 33.

  It is said that hay fever affects about 30% of the population, and allergic rhinitis, including perennial rhinitis caused by house dust and the like, is a major social problem in terms of medical expenses and labor productivity. Also, allergic rhinitis is constitutional and not a disease that can be cured by drugs.

From such a viewpoint, many screenings of substances that suppress the onset of allergy have been performed on plant extracts that can be ingested as food.
For example, β-hexosaminidase is abundant in mast cell granules and is released along with itch-inducing chemical mediators such as histamine when the mast cells are activated (degranulation). Therefore, β-hexosaminidase is frequently used for convenience as an indicator of degranulation, but a number of extracts having an inhibitory effect on it have been reported (for example, see Patent Document 1).

  However, at present, substances and extracts having sufficiently satisfactory improving effects on allergic diseases such as allergic rhinitis such as seasonal hay fever and perennial allergic rhinitis due to house dust, including atopic dermatitis, have not been found. .

  Recently, it has been revealed that IL-33 produced from the epithelium of the nasal mucosa is essential as a pathogenetic mechanism of allergic rhinitis such as hay fever and perennial rhinitis (for example, see Non-Patent Document 1). IL-33 acts on allergy-related cells such as type 2 helper T (Th2) cells, basophils, mast cells, and eosinophils, and transmits allergic signals.

Further, it has been revealed that the production of IL-33 from keratinocytes is promoted in the skin of patients with atopic dermatitis as compared with healthy subjects (for example, see Non-Patent Document 2). Histamine is secreted by -33 activating mast cells.
On the other hand, it has been shown that chronic eczema is induced by eosinophil proliferation through the activation of type II natural lymphocytes by IL-33, leading to the development of atopy (for example, see Non-Patent Document 3). .

  Thus, in the control mechanism of allergic diseases such as hay fever, perennial allergic rhinitis and atopic dermatitis, suppression of IL-33 production is related to the upstream side of degranulation inhibitors from mast cells. Therefore, there is a high possibility that the anti-allergic effect is exerted more effectively on the above-mentioned diseases, and development as a target of a therapeutic drug by a novel mechanism is being promoted.

Patent No. 5403320

Muto Yoko, Allergy, 63 (8): 1119-1125 (2014). Du HY et al., J. Interferon Cytokine Res., 36 (9): 552-62 (2016). Imai, Y. et al., PNAS, 110 (34): 13921-13926 (2013).

  The present invention solves the above problems, contains 4'-demethylnobiletin as an active ingredient, and has an IL-33 production inhibitory action, thereby improving the production of IL-33 such as hay fever, allergic rhinitis, and atopic dermatitis. It is an object of the present invention to develop pharmaceuticals, quasi-drugs, cosmetics, and food and beverage compositions having excellent preventive, therapeutic, or inhibitory effects on allergic diseases associated with an increase.

  Nobiletin, which is a polymethoxyflavonoid, is a flavonoid peculiar to citrus. In recent years, various physiological actions such as cancer prevention, aging inhibition, and anti-atherosclerosis have come to be known.

  The present inventors have clarified that nobiletin as a main component is converted into 4'-demethylnobiletin by fermenting with a specific kind of Aspergillus oryzae using citrus peel containing a large amount of nobiletin. The present inventors have also found that 4'-demethylnobiletin has an excellent memory improving effect (see Japanese Patent No. 5675661).

  The present inventors have further studied the functionality of 4′-demethylnobiletin, and as a result, have an inhibitory effect on the production of IL-33 involved in the development of allergic diseases such as hay fever, allergic rhinitis and atopic dermatitis. Admitted that. Furthermore, the effectiveness was actually recognized by humans, and the present invention was completed.

That is, the interleukin-33 production inhibitor according to the present invention is characterized by containing 4′-demethylnobiletin as an active ingredient.
The pharmaceutical, quasi-drug or cosmetic for preventing, treating or suppressing allergic diseases related to an increase in interleukin-33 according to the present invention is characterized by containing 4'-demethylnobiletin as an active ingredient. I do.
The food and drink composition for preventing or suppressing an allergic disease associated with an increase in interleukin-33 according to the present invention is characterized by containing 4'-demethylnobiletin as an active ingredient.

  The present invention relates to an allergic disease associated with an increase in IL-33 such as hay fever, allergic rhinitis and atopic dermatitis by containing 4'-demethylnobiletin as an active ingredient and having an IL-33 production inhibitory action. It is possible to provide pharmaceuticals, quasi-drugs, cosmetics, and food and beverage compositions having an excellent preventive, therapeutic, or inhibitory effect against

  Moreover, since 4'-demethyl nobiletin can be obtained by converting nobiletin, a citrus peel component, by a koji mold fermentation method, the oral preparation and the food and drink composition of the present invention have abundant food experience and do not have to worry about side effects.

FIG. 4 shows the inhibitory effect of 4′-demethylnobiletin isolate on LPS-induced IL-33 production. The bar graph shows the results of control (no addition), addition of LPS alone, addition of LPS + 4'-demethylnobiletin 5 μM and 10 μM, addition of LPS + nobiletin 5 μM and 10 μM, addition of LPS + tranilast 30 μM, 61 μM and 92 μM from the left. The vertical axis indicates the IL-33 content (pg / mL) in the culture solution. Bars indicate standard deviation. It is a figure which shows the inhibitory effect on LPS-induced IL-33 production of the composition containing 4'-demethylnobiletin. The bar graph shows the results of control (no addition), addition of LPS only, addition of LPS + 4'-demethylnobiletin-containing composition at 5 μg / mL, 10 μg / mL, and 20 μg / mL from the left. The vertical axis indicates the IL-33 content (pg / mL) in the culture solution. Bars indicate standard deviation. It is a figure which shows the inhibitory effect on LPS-induced TNF- (alpha) production of the composition containing 4'- demethyl nobiletin. The bar graph shows the results of control (no addition), addition of LPS only, addition of LPS + 4'-demethylnobiletin-containing composition at 5 μg / mL and 10 μg / mL from the left. The vertical axis indicates the TNF-α content (pg / mL) in the culture solution. Bars indicate standard deviation. It is a figure which shows the inhibitory effect on LPS-induced IL-6 production of the composition containing 4'-demethylnobiletin. The bar graph shows the results of control (no addition), addition of LPS only, addition of LPS + 4'-demethylnobiletin-containing composition at 5 μg / mL and 10 μg / mL from the left. The vertical axis indicates the IL-6 content (pg / mL) in the culture solution. Bars indicate standard deviation.

Hereinafter, embodiments of the present invention will be described in detail.
An embodiment of the present invention is an IL-33 production inhibitor containing 4′-demethylnobiletin as an active ingredient.
Another embodiment of the present invention is a pharmaceutical, quasi-drug, or cosmetic for preventing, treating or suppressing an allergic disease associated with an increase in IL-33, which contains 4'-demethylnobiletin as an active ingredient. Or, it relates to a food or drink composition.

[4'-demethylnobiletin]
Although 4'-demethylnobiletin (Formula 1) is not commercially available, a synthetic product can be used. Alternatively, citrus containing nobiletin, in particular, 4′-demethylnobiletin pure product (isolate) or 4 ′ obtained by koji mold fermentation using pericarp by the method described in Japanese Patent No. 5667561 described above. -A composition containing demethyl nobiletin can be used. The method described in Japanese Patent No. 5675661, which utilizes biotransformation of nobiletin by koji mold fermentation, is more convenient than a synthetic product because it is simpler.

  4'-Demethylnobiletin is also one of the metabolites formed after nobiletin is absorbed in the body. Citrus peel is used as a raw material for sweets such as marmalade and boiled sugar. Mandarin orange (Citrus reticulata), which has a high nobiletin content, has many years of eating experience as a chimpanzee. The product can be taken orally without worry of side effects.

A method for producing 4′-demethylnobiletin and a composition containing the same according to the method described in Japanese Patent No. 5675661 will be described below.
The production method comprises a step of fermenting the following fermentation raw materials with koji mold.

(Fermentation raw materials)
The raw material for koji mold fermentation is the citrus 'fruit' (whole fruit including pericarp, juice, pulp, seeds, etc.) containing the polymethoxyflavonoid nobiletin, especially the nobiletin content and effective waste. From the viewpoint of utilization, it is desirable to use 'peel'.

  In addition, as for the type of citrus, any citrus containing nobiletin can be used, including varieties and strains (for example, Ponkan, Shikuwasha, Tangerine, Tachibana, etc.).

  In addition, as a fermentation raw material, those containing other parts of citrus plants (for example, leaves, buds, stems, flowers, etc.) may be used, but those not including these in terms of the content of nobiletin may be used. It is desirable that

It is desirable to use fresh citrus fruits that have been harvested or collected, and those that have been washed with water, but those that have been dried, frozen, or stored for a long period of time can also be used.
The citrus may be used as it is, but it is preferable to perform a crushing process of chopping, crushing, or crushing.

  The crushing treatment includes a wide range of actions, such as engraving citrus into several pieces, shredding them into small pieces, crushing, crushing, and powdering. Preferably, it can be carried out by making it into a state of being slightly larger than about 1 to several cm.

Furthermore, extracts (extracts and dried products) obtained by extracting nobiletin from these fermentation raw materials in advance, and those isolated as pure nobiletin can also be used.
In addition, it is preferable that these fermentation raw materials are subjected to a heat treatment to sterilize various germs in the raw materials before performing the koji mold fermentation described below.

[Koji mold fermentation process]
Examples of koji molds for fermenting the fermentation raw materials include Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus oryzae lus , Aspergillus saitoi, Aspergillus usamii, Rhizopus filamentous fungi (also known as Lactobacillus). Further, these may be mixed and used.

  Preferably, among the koji molds, when Aspergillus kawachii, Aspergillus awamori, Aspergillus oryzae, and Aspergillus niger, Aspergillus niger is high. Can be obtained at a rate.

  As a method of inoculating the koji mold to the fermentation raw material, the spores of the koji mold can be directly sprinkled on the fermentation raw material and adhered. Alternatively, a medium obtained by preliminarily fermenting the koji mold by liquid culture may be inoculated so as to spread throughout the fermentation raw material.

Microbial fermentation conditions when the koji mold is inoculated to a fermentation raw material are preferably performed under aerobic conditions. Therefore, for example, a container having a bottomed cylindrical shape with a wide bottom and a shallow depth is suitable.
It is preferable to spread the fermentation material evenly on the bottom of such a container so that the contact area with the air is increased.

  The fermentation temperature is preferably 10 to 40 ° C, more preferably 20 to 40 ° C, and still more preferably 25 to 32 ° C, as conditions suitable for the growth of the koji mold. In addition, fermentation in a dark place is preferable as conditions suitable for the growth of the koji mold. Further, it is preferable that the raw material contains sufficient moisture.

The fermentation period of the microbial fermentation for obtaining a large amount of 4'-demethylnobiletin can be 1 to 14 days, preferably 2 to 14 days, more preferably 2 to 10 days, and still more preferably 2 to 14 days. 4 days.
If the fermentation period is less than one day, sufficient microbial fermentation by the koji mold does not progress, so that sufficient 4'-demethylnobiletin cannot be obtained. On the other hand, when the period exceeds 14 days, the decomposition of 4'-demethylnobiletin produced by the microbial conversion proceeds, and the preferable citrus-derived aroma disappears.

In addition, in the koji mold fermentation, nobiletin is demethylated by an enzyme secreted from the koji mold and converted into 4′-demethylnobiletin.
Therefore, instead of performing koji mold fermentation, an enzyme solution containing an enzyme for demethylating nobiletin is obtained by performing solution extraction from the koji mold or a fermentation product obtained after fermentation, and performing an enzyme reaction with the raw material using the enzyme. By obtaining a reaction product, it is also possible to obtain 4′-demethylnobiletin.
Specifically, an enzymatic reaction can be carried out by recovering a water-soluble matter from the fermented product after the koji mold fermentation and using it as a crude enzyme solution.

By performing the above koji mold fermentation, all the nobiletin, which is a polymethoxyflavonoid contained in the citrus raw material, is converted into 4′-demethylnobiletin.
Specifically, the citrus raw material is fermented with Aspergillus oryzae, so that 4′-demethylnobiletin has a high content of about 0.5 to 1.5% by mass (specifically, about 1% by mass) per dry mass. As a result, a fermented product of Aspergillus can be obtained.
Therefore, the fermented product of Aspergillus oryzae obtained here may be used as it is or processed (for example, into pieces, crushed, powdered, dried, etc.) to obtain the IL-33 of the present embodiment. It can be used as a production inhibitor and as an active ingredient of pharmaceuticals, quasi-drugs, cosmetics, or food and drink compositions.

(Solution extraction step)
In view of the purity, in the production of the IL-33 production inhibitor and the pharmaceutical, quasi-drug, cosmetic or food or beverage composition of the present embodiment, a solution is prepared from the fermented product obtained after the koji mold fermentation. It is desirable to perform the extraction to obtain the extract.

  The solution extraction step can be performed directly on the koji mold fermented product.However, the koji mold fermentation product is obtained after further performing any treatment such as shredding, crushing, crushing, and powdering. It is desirable to do it for

As a solvent used in the solution extraction step, water, a buffer, an organic solvent, or a water-containing solvent thereof can be used. Examples of the organic solvent include lower aliphatic alcohols such as ethanol, methanol, isopropanol and butanol, acetone, ethyl acetate, chloroform and the like.
Among these solvents, water, ethanol or hydrated ethanol is particularly preferred in terms of extraction efficiency, ease of handling, and safety.

  When using water-containing ethanol, the extraction efficiency is 4% -demethylnobiletin by performing extraction using water-containing ethanol having a final concentration of 20% or more, more preferably a final concentration of 30% or more (all are v / v). It is preferable because it can improve.

  As the extraction conditions, the solvent is added in an amount of 1 to 20 times, preferably 2 to 10 times (all in a mass ratio) with respect to the raw material (preferably crushed product), and 0 ° C to the boiling point of the solvent. Extraction can be performed by immersion or shaking under conditions, preferably from room temperature to the boiling point of the solvent, for 5 minutes to 1 month, preferably for 20 minutes to 1 week.

The obtained extract can be concentrated to dryness by freeze-drying or drying using an evaporator or the like.
In addition, the solution extraction step can be performed a plurality of times with a plurality of different solvents.

  The extract obtained as described above (the extract or the concentrated dried product) has an excellent inhibitory effect on IL-33 production and prevention, treatment or treatment of allergic diseases such as hay fever, perennial allergic rhinitis, and atopic dermatitis. Since it has an inhibitory effect, it can be used as it is as an active ingredient of the IL-33 production inhibitor of the present embodiment and a pharmaceutical, quasi-drug, cosmetic, or food and drink composition.

(Purification step)
Further, by performing a purification step on the extract, the 4′-demethylnobiletin content can be further increased.
As the purification step, a high-content composition can be obtained by liquid-liquid separation extraction or column purification using silica gel, chemically modified silica gel, activated carbon, a synthetic adsorption resin carrier, or the like. An example of suitable purification conditions is shown below.

First, an extract obtained by hot water extraction was applied to a column of a porous synthetic adsorption resin (specifically, Diaion HP20 (manufactured by Mitsubishi Chemical Corporation)) equilibrated with water, and then subjected to 39 to 41% The liquid eluted with (v / v) ethanol (specifically, 40% (v / v) ethanol) is removed.
Next, 4'-demethylnobiletin was selectively separated by collecting components eluted with 44-46% (v / v) ethanol (specifically, 45% (v / v) ethanol). A 4′-demethylnobiletin-rich composition can be obtained.

  Further, the composition containing 4′-demethylnobiletin obtained as described above is further subjected to ODS column chromatography (specifically, elution with 60% (v / v) methanol), thin-layer chromatography (TLC) (specifically, Specifically, hexane / ethanol (7: 3)) and ODS-HPLC (specifically, a mixed solvent of 33% (v / v) acetonitrile / water) are used to collect the target peak, thereby obtaining 4′- Pure methylnobiletin can be isolated.

  4'-demethylnobiletin obtained as described above is a monodemethyl form of nobiletin demethylated at the 4'-position. 4'-Demethylnobiletin has a higher polarity due to demethylation, and is more soluble in alcohol and water than nobiletin.

In addition, 4'-demethylnobiletin has an excellent inhibitory action on IL-33 production as compared with nobiletin.
Furthermore, 4'-demethylnobiletin has an excellent preventive, therapeutic or inhibitory effect on allergic diseases associated with an increase in IL-33, such as hay fever, perennial allergic rhinitis, and atopic dermatitis.

  Therefore, 4'-demethyl nobiletin is an IL-33 production inhibitor, and excellent prevention against allergic diseases related to an increase in IL-33 such as hay fever, perennial allergic rhinitis, atopic dermatitis, As a therapeutic or inhibitory agent, it can be added to the pharmaceutical, quasi-drug, cosmetic or food or beverage composition of the present embodiment.

(Pharmaceuticals, quasi-drugs, cosmetics and food and beverage compositions)
The present embodiment relates to an IL-33 production inhibitor containing 4'-demethylnobiletin as an active ingredient.
This embodiment comprises 4'-demethylnobiletin as an active ingredient, a pharmaceutical, quasi-drug, cosmetic or food or drink composition for preventing, treating or suppressing an allergic disease associated with an increase in IL-33. About.

The above 4′-demethylnobiletin can be used as a pure product (isolate), a fermentation product obtained by the above-mentioned method, or a crude product thereof (“solution extract”, “high-content composition”, etc.). By mixing with raw materials, it can be used as an active ingredient of pharmaceuticals, quasi-drugs, cosmetics, and food and drink compositions.
Here, the “food and drink composition” includes general food and drink, functional foods, and functional drinks.
Further, the medicine and the quasi-drug may be in any form of an internal preparation and an external preparation.

  The effective amount of 4'-demethylnobiletin when taken orally as an oral preparation or a food or drink composition is 1 mg or more, preferably 5 mg or more per day for an adult weighing 60 kg. -33 production inhibitory action and excellent preventive, therapeutic or inhibitory action against allergic diseases associated with an increase in IL-33 such as hay fever, allergic rhinitis and atopic dermatitis.

  Alternatively, when externally used as an external preparation or cosmetic, the external preparation or cosmetic containing 4'-demethylnobiletin in an amount of 0.001% by mass or more, preferably 0.01% by mass or more, provides an excellent IL-33 production inhibitory effect and pollen. An excellent preventive, therapeutic or inhibitory effect on allergic diseases associated with an increase in IL-33, such as symptom, allergic rhinitis and atopic dermatitis, can be obtained.

  Therefore, by ingesting or applying the pharmaceutical, quasi-drug, cosmetic or food or beverage composition of the present embodiment in a form or ingestion method (number of times, amount) capable of securing the required amount, the above-mentioned pharmacological action can be obtained. It is expected to be. However, it is desirable to appropriately determine the intake and application amount depending on the age, body weight, symptoms, intake schedule, formulation form, and the like of the subject.

In addition, the form of the internal preparation can be, for example, powder, fine granules, granules, and the like. In addition to the form of filling in capsules, the form of a solution dispersed in water, cream, excipients, and the like In the form of a tablet obtained by mixing with a tablet.
When used externally, such as an external preparation or a cosmetic, the form may be a liquid, gel, cream, ointment or the like.

  In the medicine, the quasi-drug or the cosmetic of the present embodiment, besides 4′-demethylnobiletin or a composition containing the same, various carriers, additives, and other medicinal effects within the range of exerting the effects of the present invention. Components and the like may be included.

  In addition, as a form of the food and drink composition, mixed with various food ingredients and additives, for example, biscuits, snacks, gum, chewable tablets, soft drinks, drinks, soups, jellies, candy and the like can do.

  The content of 4′-demethylnobiletin in the internal preparation or the food and drink composition may be an amount that can ensure the above-mentioned effective intake, and specifically, 0.001% by mass or more, preferably 0.01% by mass or more. It can be contained so as to be at least 0.1% by mass, more preferably at least 0.1% by mass. The upper limit may be 20% by mass or less.

  In addition, the above-mentioned food and drink composition, cosmetics, quasi-drugs, and pharmaceuticals can be used both as external and internal skin care.

  Hereinafter, embodiments of the present invention will be described with reference to examples, but the scope of the present invention is not limited thereto.

<Example 1> Preparation of 4'-demethylnobiletin-containing composition According to the method described in Japanese Patent No. 5675661, a nobiletin-converted 4'-demethylnobiletin-containing composition was obtained by fermenting Ponkan peel with Aspergillus oryzae. Prepared.

  That is, 50 kg of Ponkan peel was chopped into pieces and sterilized by steaming. The obtained Ponkan peel was inoculated with Aspergillus awamori (manufactured by Bioc) so as to spread over the entire surface. Fermentation (Koji mold fermentation) was performed aerobically for 5 days in a constant temperature room at 30 ° C., to obtain a Koji mold fermented product.

  180 L of water was added to 30 kg of the obtained koji mold fermented product, and extracted with hot water at 100 ° C. for 2 hours, followed by continuous centrifugation to obtain an extract. The extract is applied to Diaion HP20 (porous synthetic adsorption resin column) equilibrated with water in advance, and after removing non-adsorbed components with 10 L of water, it is further eluted with 10 L of 40% (v / v) ethanol. Components were removed. Next, components eluted with 10 L of 45% (v / v) ethanol were collected. The collected product was concentrated to dryness by a rotary evaporator to obtain a 4'-demethylnobiletin-containing composition (4'-demethylnobiletin content: 32%).

<Example 2> Preparation of 4'-demethylnobiletin pure product 1.5 g of the 4'-demethylnobiletin-containing composition obtained in Example 1 above was dissolved in 20% (v / v) methanol, and the solution was subjected to ODS column chromatography. It was subjected to chromatography (30 g of Wako gel 50C18 was packed in a column having an inner diameter of 20 mm and a length of 30 cm). Components eluted with 40% (v / v) methanol were removed to obtain components eluted with 60% (v / v) methanol.

Then, the obtained component, developing solvent hexane / ethanol 7: Preparative TLC chromatography in three conditions (silica gel 70 pF ₂₅₄ plates Wako, thickness 0.75 mm, manufactured by Wako Pure Chemical) performs, 4'-demethyl Nobile Chin Was collected while confirming using a UV lamp.

  Then, the obtained fraction was applied to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm × 25 cm), and purified with a moving layer of 33% (v / v) acetonitrile to obtain a pure 4′-derivative. 25 mg of methyl nobiletin were obtained.

<Test Example 1> Measurement of IL-33 production inhibitory effect Using the 4'-demethylnobiletin-containing composition obtained in Example 1 and the 4'-demethylnobiletin isolate obtained in Example 2, IL -33 Production inhibitory effect was investigated. RAW264 cells derived from mouse macrophages (RIKEN BioResource Research Center) were used as IL-33 producing cells.

Cells are cultured in a DMEM medium containing 10% fetal bovine serum in a 37 ° C. incubator in the presence of 5% CO ₂ , and passaged every 3 to 4 days by treatment with 0.25% trypsin containing 0.1% EDTA. Was.

RAW264 cells were seeded on a 96-well plate (4 × 10 ⁴ cells / well), and after 5 hours, a composition containing 4′-demethylnobiletin (Example 1 product) or an isolate (Example 2 product) was used. Was added. As a comparative control of 4′-demethylnobiletin, nobiletin or antiallergic agent tranilast (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added. Thirty minutes later, lipopolysaccharide (LPS) was added as an IL-33 production promoter.

  18 hours later, the amount of IL-33 in the culture solution was measured by measuring the absorbance at 450 nm using an ELISA kit (IL-33 Quantikine ELISA Kit, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The inhibitory effect on LPS-induced IL-33 production was evaluated.

First, the inhibitory effect of 4′-demethylnobiletin isolate (the product of Example 2) on IL-33 production was examined.
FIG. 1 shows the inhibitory effect of 4′-demethylnobiletin isolate on LPS-induced IL-33 production. In FIG. 1, the bar graph shows the results of control (no addition), addition of LPS only, addition of LPS + 4′-demethylnobiletin 5 μM and 10 μM, addition of LPS + nobiletin 5 μM and 10 μM, addition of LPS + tranilast 30 μM, 61 μM and 92 μM from the left. The vertical axis indicates the IL-33 content (pg / mL) in the culture solution. Bars indicate standard deviation.

As shown in FIG. 1, 4′-demethylnobiletin (4′-DeNob) exhibited a very strong concentration-dependent IL-33 production inhibitory effect at 5 μM to 10 μM in comparison with nobiletin.
On the other hand, although Tranilast used as a positive control has been used as a therapeutic drug for allergic rhinitis and atopic dermatitis, it has recently been reported that IL-33 production inhibitory action contributes to its mechanism of action ( Hiraide S., et. Al., Eur. J. Pharmacol., 818 (2018): 235-240.).
However, according to the present results, the IL-33 production inhibitory effect of 5 μM of 4′-demethylnobiletin and 61 μM of tranilast were almost the same, indicating that the IL-33 inhibitory effect was 10 times or more stronger than that of tranilast ( (Fig. 1). In the above-mentioned literature, the effect of tranilast is shown at a concentration of 30 μM or more, which is consistent with this result.

Next, the inhibitory effect of the 4′-demethylnobiletin-containing composition (the product of Example 1) on IL-33 production was examined.
FIG. 2 shows the inhibitory effect of the composition containing 4′-demethylnobiletin on LPS-induced IL-33 production. In FIG. 2, the bar graph shows the results of the control (no addition), the addition of LPS only, and the addition of LPS + 4′-demethylnobiletin-containing composition at 5 μg / mL, 10 μg / mL, and 20 μg / mL from the left. The vertical axis indicates the IL-33 content (pg / mL) in the culture solution. Bars indicate standard deviation.
As shown in FIG. 2, the 4′-demethylnobiletin (4′-DeNob) composition also exhibited a significant concentration-dependent IL-33 production inhibitory effect at 5 μg / mL to 20 μg / mL.

  From the above results, 4'-demethyl nobiletin and a composition containing the same show an extremely strong inhibitory action on IL-33 production, whereas nobiletin and tranilast show only a weak inhibitory action on IL-33 production. Do you get it.

<Test Example 2> Measurement of TNF-α and IL-6 production inhibitory effects Therefore, as typical examples of type 1 helper T (Th1) and type 2 helper T (Th2) cytokines, tumor necrosis factor (TNF-α), The inhibitory action of interleukin-6 (IL-6) production was examined.
That is, a composition containing 4′-demethylnobiletin (the product of Example 1) was added as a test substance, and an ELISA kit (Murine TNFα ELISA KIT, manufactured by diaclone) was used to measure TNF-α. The amount of cytokine in the culture solution was measured in the same manner as in Test Example 1 except that an ELISA kit (Mouse IL-6 Assay kit-IBL, manufactured by Takara Bio Inc.) was used for the measurement.

The results are shown in FIGS.
FIG. 3 shows the inhibitory effect of the 4′-demethylnobiletin-containing composition on LPS-induced TNF-α production. In FIG. 3, the bar graph shows the results of the control (no addition), addition of LPS only, addition of LPS + 4′-demethylnobiletin-containing composition 5 μg / mL and 10 μg / mL from the left. The vertical axis indicates the TNF-α content (pg / mL) in the culture solution. Bars indicate standard deviation.

  FIG. 4 shows the inhibitory effect of the 4'-demethylnobiletin-containing composition on LPS-induced IL-6 production. In FIG. 4, the bar graph shows the results of control (no addition), addition of LPS only, addition of LPS + 4′-demethylnobiletin-containing composition at 5 μg / mL and 10 μg / mL from the left. The vertical axis indicates the IL-6 content (pg / mL) in the culture solution. Bars indicate standard deviation.

As shown in FIG. 3, the composition containing 4′-demethylnobiletin showed a weak production inhibitory effect on TNF-α at a concentration of 10 μg / mL.
On the other hand, IL-6 showed a concentration-dependent strong production inhibitory effect at 5 μg / mL to 10 μg / mL.

  From the above results, 4'-demethyl nobiletin and a composition containing the same prevent a decrease in the Th1 / Th2 balance that causes conventionally known allergies, and furthermore, hay fever, allergic rhinitis, It was revealed that IL-33, which causes allergic diseases such as atopic dermatitis, was extremely strongly inhibited.

<Test Example 3> Oral administration test to humans 4'-demethylnobiletin-containing tablets were orally administered to humans, and the efficacy against allergic diseases was examined.

  First, a tablet (Example 3) was prepared using the 4′-demethylnobiletin-containing composition obtained in Example 1 according to the formulation shown in Table 1 below.

(Prescription example: Example 3) 4′-demethylnobiletin-containing (2.5 mg / tablet) tablet

  Subjects were men and women with hay fever and allergic symptoms, and the duration of administration was two weeks to one month. Twelve patients (8 males and 4 females) who took the 4'-demethylnobiletin-containing tablets took two tablets once a day. 4'-demethylnobiletin was contained in 5 mg in 2 tablets. In the placebo group, dextrin was prescribed in place of the composition containing 4'-demethylnobiletin.

Tables 2 and 3 below show the details of the results of the hearing survey of symptoms and sensations.
Subjects included those with perennial allergic rhinitis and atopic dermatitis, in addition to hay fever.
Most of the subjects who took the 4′-demethylnobiletin-containing tablet, except for one (No. 9) who had severe symptoms, experienced some improvement effect. On the other hand, most of the subjects who took the placebo tablet could not feel the improvement effect.

(Table 2) Effect of 4'-demethylnobiletin-containing tablets on subjects with symptoms of allergic rhinitis and atopic dermatitis

(Table 3) Effect of placebo tablets (tablets containing dextrin) on subjects with symptoms of allergic rhinitis and atopic dermatitis

  From the above results, the oral intake of the preparation containing 4′-demethylnobiletin of Example 3 showed an improvement / suppression effect on allergic symptoms such as hay fever, perennial allergic rhinitis, and atopic dermatitis, Effectiveness against allergy was shown.

<Formulation Example 1> Cream for external use Each raw material was blended at the following mass ratio to prepare a cream containing 0.1 mg / g of 4'-demethylnobiletin.

4′-Demethylnobiletin-containing composition (Example 1) 0.03%
Propanediol 6.0%
Palm Dream 5.0%
SEPIGEL305 (gelling agent) 2.0%
86.47% of water
Phenoxyethanol 0.5%

<Prescription example 2> Functional food (supplement)
Each raw material was blended at the following mass ratio, and a 200 mg tablet was produced by a conventional method. This tablet is to be ingested about 7 mg / day as 4 tablets / day, 4'-demethylnobiletin.

4'-Demethylnobiletin-containing composition (Example 1) 5.5%
Dextrin 35.0%
Crystalline cellulose 25.0%
Lactose 18.0%
Whey calcium 14.5%
Sucrose ester 2.0%

<Prescription example 3> Functional beverage Each raw material was blended at the following mass ratio, and a 100 mL / bottle functional beverage was produced by a conventional method. This beverage is to be ingested about 7 mg / day as 4'-demethylnobiletin per bottle.

4'-Demethylnobiletin-containing composition (Example 1) 0.02%
Fructose 6.0%
Malic acid 5.0%
Vitamin C 0.1%
Spice 0.1%
88.78% water

<Formulation Example 4> Oral (medicine)
Each raw material was blended at the following mass ratio, and a 100 mg tablet was produced by a conventional method. This tablet is to be ingested about 8 mg / day as 1 tablet / day, 4'-demethylnobiletin.

4'-demethylnobiletin (Example 2) 8.0%
77.0% crystalline cellulose
Lactose 11.0%
Silicic anhydride 2.0%
Sucrose ester 2.0%

  The present invention relates to an interleukin-33 (IL-33) production inhibitor containing 4'-demethylnobiletin, which is a raw material that has abundant food experience and has no concern for side effects, as an active ingredient raw material, and an allergic rhinitis. It is expected to be used for internal medicines, external preparations, cosmetics, or foods and drinks that have a preventive, therapeutic or suppressive effect on allergic diseases related to an increase in IL-33 such as atopic dermatitis.

Claims (3)

  An interleukin-33 production inhibitor containing 4'-demethylnobiletin as an active ingredient.   A pharmaceutical, quasi-drug or cosmetic for preventing, treating or suppressing an allergic disease associated with an increase in interleukin-33, comprising 4'-demethylnobiletin as an active ingredient. A food or drink composition for preventing or suppressing an allergic disease associated with an increase in interleukin-33, comprising 4'-demethylnobiletin as an active ingredient.