Oral medicines, quasi-drugs, or food and drink compositions for the treatment, improvement, or prevention of xeroderma

Abstract

To develop an oral composition for treating or preventing xeroderma that comprises a food-derived component as an active ingredient, and particularly, provide an internal agent or a food and drink composition for treating or preventing xeroderma involving itch hypersensitivity.SOLUTION: The present invention provides an oral composition for treating or preventing xeroderma that comprises nobiletin and / or 4'-demethylnobiletin as an active ingredient, preferably, a pharmaceutical, a quasi drug or a food and drink composition for treating or preventing xeroderma involving itch hypersensitivity, wherein nobiletin and 4'-demethylnobiletin are derived from fruits of citrus plants having a rich history of use, so that the inventive oral composition comprising them as an active ingredient are highly safe with no risk of side effects.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an oral composition for the treatment or prevention of xeroderma, which contains nobiletin and / or 4'-demethylnobiletin as an active ingredient, and preferably relates to a pharmaceutical, quasi-drug, or food or drink composition for the treatment or prevention of xeroderma accompanied by hyperitchiness. [Background technology]

[0002] The barrier function of the stratum corneum prevents the intrusion of foreign substances from the outside world and prevents the loss of moisture from the body surface. It is known that lipids in the stratum corneum play an important role in maintaining moisture (see, for example, Non-Patent Document 1). However, with age, the sebum and moisture necessary for barrier function decrease, and the skin's moisturizing function declines. This leads to dry skin and itching. In particular, in winter, when the air is dry, elderly people are prone to dry skin below the knees and itching after bathing. Furthermore, a large proportion of people, not limited to the elderly, suffer from itching caused by dry skin due to various reasons such as air conditioning and heating, excessive scrubbing during bathing, and excessive use of soap.

[0003] As described above, dry skin, or dehydrated skin, is prone to itching, and is known as xerosis. If this dry state is left untreated, nerve fibers grow close to the skin surface, causing an itchy hypersensitivity (hypersensitivity to pruritus, alloneosis) reaction even to slight external stimuli such as the friction of clothing (see, for example, Non-Patent Document 2).

[0004] The basic treatment for xerosis and hyper-itchiness is topical moisturizers, mainly heparinoid-containing preparations, urea preparations, and petrolatum. Anti-itch antihistamines and steroids are also used. Antihistamines block the receptors at the ends of nerve fibers that sense itch, and block the receptors to which histamine binds.

[0005] Meanwhile, nobiletin (3',4',5,6,7,8-hexamethoxyflavone), a polymethoxyflavonoid unique to citrus fruits, has recently become known for its various physiological effects, such as cancer prevention, anti-aging, and anti-arteriosclerosis effects (see, for example, Non-Patent Document 3).

[0006] The present inventors have clarified that by using citrus peels, which contain a large amount of nobiletin, and fermenting them with a specific type of koji mold, nobiletin is demethylated and converted to 4'-demethylnobiletin (3',5,6,7,8-pentamethoxy-4'-hydroxyflavone). The present inventors have also found that 4'-demethylnobiletin has an excellent memory-improving effect (see, for example, Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] G. Imokawa, M. Hattori, J.Invest.Dermatol., 84, 282-284 (1985). [Non-patent document 2] Kenji Takamori, Journal of the Japanese Society of Cosmetic Chemistry 38, 92-95 (2014) [Non-patent document 3] A. Minagawa et al., Jpn J. Cancer Res. 92(12):1322-8(2001) [Patent documents]

[0008] [Patent Document 1] Patent No. 5667561 Summary of the Invention [Problem to be solved by the invention]

[0009] However, the above-mentioned topical moisturizers do not provide sufficient therapeutic effects for xerosis or hypersensitivity to itching. Furthermore, only a small proportion of receptors that sense itching are bound to histamine. Therefore, blocking histamine receptors with antihistamines does not stop itching (see, for example, Non-Patent Document 2 and M. Tominaga, K. Takamori (2013) Biol. Pharm. Bull., 36, 1241-1247). Furthermore, steroid drugs have the problem of side effects.

[0010] As described above, there is currently no practical therapeutic agent for xerosis and the associated hyperpruritic skin irritation that is highly effective and free from side effects. Under these circumstances, there is a particular need for the development of an oral agent that is easy to use and highly safe.

[0011] The present invention aims to develop an oral composition for treating or preventing xeroderma, which contains a highly safe food-derived ingredient as an active ingredient, and to provide a pharmaceutical, quasi-drug, or food or beverage composition for preventing or treating xeroderma, particularly when accompanied by hypersensitivity to itching. [Means for solving the problem]

[0012] The present inventors further investigated the functionality of nobiletin and 4'-demethylnobiletin and, as a result, surprisingly discovered that oral administration of nobiletin and 4'-demethylnobiletin exhibits an excellent inhibitory effect on itch, particularly itch hypersensitivity (allonesis), in xeroderma model mice. It was then speculated that, due to this action, nobiletin and 4'-demethylnobiletin are also effective in preventing or treating xeroderma and the itch associated with xeroderma. Based on these findings, the present inventors have completed the present invention.

[0013] That is, the present invention relates to an oral composition for treating or preventing xeroderma, which contains nobiletin and / or 4'-demethylnobiletin as an active ingredient.

[0014] As used herein, "xeroderma" refers to a dry skin condition, including dry skin. The xeroderma may be accompanied by itching and / or hypersensitivity to itching.

[0015] The oral composition may be in the form of a pharmaceutical product, a quasi-drug, or a food or drink composition. [Effects of the Invention]

[0016] The present invention makes it possible to provide an oral composition for the treatment or prevention of xeroderma, which contains nobiletin and / or 4'-demethylnobiletin as an active ingredient, and in particular an oral agent or a food or drink composition for the treatment or prevention of xeroderma accompanied by hyperitchiness.

[0017] Furthermore, nobiletin is a component unique to citrus fruits. 4'-Demethylnobiletin is one of the metabolic products of nobiletin, and can be obtained by bioconversion from nobiletin or the fruits of citrus fruits that contain it in abundance by a koji mold fermentation method. Because these are made from the fruits of citrus fruits, which are plants that are widely eaten, the oral composition for treating or preventing xeroderma of the present invention is highly safe and has no risk of side effects. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a photographic image showing the state of dry skin in AEW-treated mice (Test Example 1). In the figure, (A) represents the AEW treatment + 0.5% CMC administration group, (B) represents the AEW treatment + 0.5% CMC administration group, (C) represents the AEW treatment + nobiletin (NOB) administration group, and (D) represents the AEW treatment + 4'-demethylnobiletin (4'NOB) administration group. [Figure 2] 1 is a graph showing the effect of oral administration of nobiletin and 4'-demethylnobiletin on the dry skin score of AEW-treated mice (Test Example 1). In the figure, the vertical axis represents the dry skin score, and the horizontal axis represents each group. The box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively. [Figure 3]1 is a graph showing the effect of oral administration of nobiletin and 4'-demethylnobiletin on the skin barrier function of AEW-treated mice (Test Example 1). In the figure, (A) shows the distribution of transepidermal water loss (unit: g / m2 / h), and (B) shows the distribution of stratum corneum water content (unit: au). The horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively. [Figure 4] 1 is a graph showing the effect of oral administration of nobiletin and 4'-demethylnobiletin on the scratching behavior of AEW-treated mice (Test Example 1). In the figure, the vertical axis represents the number of scratching behaviors (2 hours), the horizontal axis represents each group, and the box plot represents, from top to bottom, the maximum value, 75%, 50%, 25%, and minimum value, respectively. [Figure 5] 1 is a graph showing the effect of oral administration of nobiletin and 4'-demethylnobiletin on allonesis (itch hypersensitivity) in AEW-treated mice (Test Example 1). In the figure, (A) shows the results of an allonesis assay using 0.07 g of von Frey filaments, and (B) shows the results of an allonesis assay using 0.16 g of von Frey filaments. The vertical axis represents the allonesis score, the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively. [Figure 6] 1 is a graph showing the effect of oral administration of nobiletin and 4'-demethylnobiletin on the amount of spontaneous locomotion in AEW-treated mice (Test Example 1). In the figure, the vertical axis represents the amount of spontaneous locomotion (unit: cm), the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively. [Figure 7] 1 is a graph showing the effect on stratum corneum ceramide synthesis when 4'-demethylnobiletin (DeNOB) is added to a human 3D cultured epidermal model (Test Example 2). In the figure, (A) represents the results for total ceramide, (B) for ceramide EOS, (C) for ceramide NS / NDS, and (D) for ceramide NP. The vertical axis represents the amount of ceramide produced (unit: μg / mg protein), and the bars represent the standard deviation. [Figure 8]1 is a graph showing the effect on stratum corneum ceramide synthesis when a 4'-demethylnobiletin-containing composition (DeNOB extract) is added to a human three-dimensional cultured epidermal model (Test Example 2). In the figure, (A) represents the results for total ceramide, (B) represents the results for ceramide NS / NDS, and (C) represents the results for ceramide NP. The vertical axis represents the amount of ceramide produced (unit: μg / mg protein), and the bars represent the standard deviation. [Figure 9] FIG. 1 shows the ceramide biosynthetic pathway and related genes. [Figure 10] This is a graph showing the effect on ceramide biosynthesis-related genes when 4'-demethylnobiletin (DeNOB) is added to a human three-dimensional cultured epidermal model (Test Example 3). In the figure, (A) shows the results for CERS1, (B) for CERS2, and (C) for UGCG. The vertical axis of the graph shows the ratio of mRNA expression level (relative mRNA expression level) when the mRNA expression level in the untreated control (Control) is set to 1. The bars represent the standard deviation. [Figure 11] 1 is a graph showing the effect on ceramide biosynthesis-related genes when a 4'-demethylnobiletin-containing composition (DeNOB extract) is added to a human three-dimensional cultured epidermal model (Test Example 3). In the figure, (A) shows the results for CERS1, (B) for CERS2, (C) for UGCG, and (D) for SMS2. The vertical axis of the graph shows the ratio of mRNA expression level (relative mRNA expression level) when the mRNA expression level in the untreated control (Control) is set to 1. The bars represent the standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, the embodiments of the present application will be described in detail.

[0020] The present embodiment relates to an oral composition for treating or preventing xeroderma, which contains nobiletin and / or 4'-demethylnobiletin as active ingredients.

[0021] The oral composition according to this embodiment has a preventive or therapeutic effect on xeroderma, preferably xeroderma accompanied by itching, particularly preferably xeroderma accompanied by itching hypersensitivity, and therefore can be made into a pharmaceutical or quasi-drug intended to have these effects.

[0022] Furthermore, since the oral composition of this embodiment has the effect of preventing or improving xeroderma, preferably xeroderma accompanied by itching, and particularly preferably xeroderma accompanied by itching hypersensitivity, it can be made into a food or beverage composition intended to achieve these effects.

[0023] [Nobiletin] Commercially available products can be used as nobiletin (formula I), the active ingredient of this embodiment. Furthermore, nobiletin is contained in large amounts in the 'fruit' of citrus fruits (the entire fruit including the peel, juice, pulp, seeds, etc.). Therefore, nobiletin can be used as a nobiletin-containing composition made from citrus fruits, particularly the 'peel', from the standpoint of the nobiletin content and effective utilization of waste.

[0024] [ka]

[0025] Citrus peels are used as ingredients for sweets such as marmalade and candied fruits, and the peel of mandarin oranges (Citrus reticulata), which has a high nobiletin content, has a long history of being eaten as tangerine, and nobiletin and compositions containing it are extremely safe.

[0026] Furthermore, any variety or lineage of citrus fruit (for example, ponkan, shikuwasha, tangerine, tachibana, etc.) can be used as long as it contains nobiletin.

[0027] The raw material may contain other parts of citrus plants (for example, leaves, buds, stems, flowers, etc.), but it is desirable that the raw material does not contain these parts in terms of the nobiletin content.

[0028] The nobiletin-containing composition may be in the form of the plant itself, or may be one that has been subjected to one or more processing treatments such as shredding, crushing, pulverization, and drying.

[0029] [Solvent extraction] In consideration of purity, in the production of the oral composition of the present embodiment, it is desirable to perform solution extraction on the nobiletin-containing composition to obtain an extract.

[0030] The solvent used in the solution extraction step may be water, a buffer solution, an organic solvent, or a water-containing solvent thereof. Examples of the organic solvent include lower aliphatic alcohols such as ethanol, methanol, isopropanol, and butanol, as well as acetone, ethyl acetate, and chloroform.

[0031] Among these solvents, water, ethanol, or aqueous ethanol is particularly preferred in terms of extraction efficiency, ease of handling, and safety.

[0032] Furthermore, performing extraction using ethanol at a final concentration of 60% or more, preferably 70% or more, and more preferably 90% or more (all by volume) is particularly preferable, as this can suppress the elution of polysaccharides, which are impurities, and improve the extraction efficiency of nobiletin.

[0033] The extraction conditions are as follows: 1 to 50 times, preferably 2 to 15 times (by mass) the amount of the solvent added to the citrus raw material (preferably shredded material), and the mixture is soaked or shaken at a temperature of 0°C to the boiling point of the solvent, preferably room temperature to a temperature below the boiling point of the solvent, for 5 minutes to 1 month, preferably 20 minutes to 1 week.

[0034] The obtained extract can be concentrated to dryness by freeze-drying or drying using an evaporator or the like.

[0035] Furthermore, the solution extraction step can be performed multiple times using multiple different solvents. In particular, when the first extraction is performed using water or a low-concentration aqueous alcohol, the extraction efficiency of nobiletin can be improved by subsequently performing an extraction using ethanol at the specific concentration or higher.

[0036] The extract obtained as described above (the extract liquid or concentrated dry product) can be used as it is as the active ingredient of the oral composition of this embodiment.

[0037] 〔purification〕 Furthermore, by subjecting the extract to a purification step, the purity of nobiletin can be further increased.

[0038] As a purification step, high purity can be achieved by liquid-liquid separation extraction or column purification using silica gel, chemically modified silica gel, activated carbon, synthetic adsorption resin carrier, etc. An example of suitable purification conditions is shown below.

[0039] First, the extract (specifically, the extract obtained by ethanol extraction) is dissolved in a 40% ethanol solution and applied to a column of porous synthetic adsorption resin (specifically, Diaion HP20 [manufactured by Mitsubishi Chemical Corporation]) equilibrated with a 40% ethanol (v / v) solution. Then, components eluted with a 40 to 50% (specifically, 45%) (v / v) ethanol solution are removed. Next, components eluted with 45 to 60% (specifically, 50%) (v / v) ethanol are collected, thereby obtaining a composition with a high nobiletin content.

[0040] Furthermore, the nobiletin-rich composition obtained as described above can be further subjected to ODS column chromatography (specifically, 60% (v / v) methanol elution) and ODS-HPLC (specifically, 40% (v / v) acetonitrile-water mixed solvent), and the target peak can be collected to isolate pure nobiletin.

[0041] Nobiletin, when administered orally, has the effect of improving itching, particularly hypersensitivity to itching, associated with xerosis.

[0042] Therefore, nobiletin can be used as an active ingredient in an oral composition, specifically a pharmaceutical or quasi-drug, for the treatment or prevention of xeroderma, preferably itch, particularly xeroderma accompanied by hypersensitivity to itch.

[0043] Furthermore, nobiletin can be used as an active ingredient in oral compositions, specifically food and drink compositions, for improving or preventing xeroderma, preferably itch, particularly xeroderma accompanied by hypersensitivity to itch.

[0044] [4'-demethylnobiletin] 4'-Demethylnobiletin (Formula II) is not commercially available, but a synthetic product can be used. Alternatively, a pure 4'-demethylnobiletin (isolate) or a 4'-demethylnobiletin-containing composition obtained by koji mold fermentation using citrus fruits containing nobiletin, particularly the peel, by the method of Patent Document 1 described above can be used. The method of Patent Document 1, which utilizes the bioconversion of nobiletin by koji mold fermentation, is more convenient and inexpensive than synthetic products and is therefore preferred.

[0045] [ka]

[0046] 4'-demethylnobiletin is also one of the metabolic products produced after nobiletin is absorbed in the body. As mentioned above, citrus peels have been eaten for many years, so 4'-demethylnobiletin and compositions containing it are extremely safe.

[0047] A method for producing 4'-demethylnobiletin and a composition containing it, based on the method described in Patent Document 1, will be described below.

[0048] [Fermentation ingredients] The raw material for koji fermentation is citrus fruit, which contains the polymethoxyflavonoid nobiletin, and it is particularly desirable to use the peel for the reasons described above. Here, the types of "citrus fruit" and the "fruit" are as described above.

[0049] The fermentation raw material may contain other parts of citrus plants (for example, leaves, buds, stems, flowers, etc.), but it is desirable that the fermentation raw material does not contain these parts in terms of the nobiletin content.

[0050] It is preferable to use the above citrus fruits freshly harvested or washed, but dried, frozen, or long-term stored fruits can also be used.

[0051] Citrus fruits may be used as they are, but it is preferable to subject them to one or more of the following processing: chopping, crushing, or grinding.

[0052] This process includes a wide range of actions, such as roughly chopping the citrus fruits into several pieces, shredding into small pieces, crushing, grinding, powdering, etc. Preferably, this process can be carried out by chopping the citrus fruits into roughly one to several centimeters in size.

[0053] Furthermore, it is also possible to use an extract (extract, dried product) obtained by extracting nobiletin in advance from these fermentation raw materials, or nobiletin isolated as a pure product.

[0054] It is preferable to heat-treat these fermentation raw materials to sterilize any unwanted bacteria in the raw materials before carrying out the koji mold fermentation described below.

[0055] [Koji mold fermentation] Examples of koji molds that can be used to ferment the fermentation raw materials include Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus saitoi, Aspergillus usamii, and Rhizopus filamentous fungi (also known as Rhizopus), and mixtures of these may also be used.

[0056] Of the koji molds, Aspergillus kawachii, Aspergillus awamori, Aspergillus oryzae, and Aspergillus niger are preferably used, whereby 4'-demethylnobiletin can be obtained at a high content.

[0057] The method of inoculating the koji mold into the fermentation raw material can be to sprinkle koji mold spores directly onto the fermentation raw material to attach them to the fermentation raw material, or to inoculate the fermentation raw material with a medium in which the koji mold has been pre-fermented by liquid culture so that the medium is spread throughout the fermentation raw material.

[0058] When the koji mold is inoculated into the fermentation raw material, the microbial fermentation is preferably carried out under aerobic conditions, and therefore a suitable container is, for example, a cylindrical container with a wide bottom and a shallow depth.

[0059] It is advisable to spread the fermentation raw materials evenly on the bottom of such a container so that the contact area with the air becomes large.

[0060] The fermentation temperature is preferably 10 to 40°C, more preferably 20 to 40°C, and even more preferably 25 to 32°C, which are conditions suitable for the growth of the koji mold. In addition, fermentation is preferably carried out in a dark place, which is a condition suitable for the growth of the koji mold. Furthermore, it is preferable that the raw materials contain sufficient moisture.

[0061] The fermentation period for microbial fermentation to obtain a large amount of 4'-demethylnobiletin can be 1 to 14 days, preferably 2 to 14 days, more preferably 2 to 10 days, and even more preferably 2 to 4 days.

[0062] If the fermentation period is less than one day, the microbial fermentation by the koji mold hardly progresses, and sufficient 4'-demethylnobiletin cannot be obtained. Conversely, if the fermentation period exceeds 14 days, decomposition of the 4'-demethylnobiletin produced by microbial conversion progresses, and the desirable aroma derived from citrus fruits is lost.

[0063] Furthermore, in the koji mold fermentation, nobiletin is demethylated by an enzyme secreted from the koji mold, converting it to 4'-demethylnobiletin.

[0064] Therefore, instead of performing koji mold fermentation, it is also possible to obtain 4'-demethylnobiletin by performing solution extraction from the koji mold or the fermented product obtained after fermentation to obtain an enzyme solution containing an enzyme that demethylates nobiletin, and then performing an enzymatic reaction with the raw material using the enzyme to obtain a reaction product.

[0065] Specifically, a water soluble matter is collected from the fermented product after the koji mold fermentation and used as a crude enzyme solution to carry out the enzyme reaction.

[0066] By carrying out the above-mentioned koji mold fermentation, all of the nobiletin, which is a polymethoxyflavonoid contained in the citrus raw material, is converted to 4'-demethylnobiletin.

[0067] Specifically, by fermenting the citrus raw material with koji mold, it is possible to obtain a koji mold fermentation product with a high content of 4'-demethylnobiletin, approximately 0.5 to 1.5 mass % (specifically, approximately 1 mass %) per dry weight.

[0068] Therefore, the koji mold fermentation product obtained here can be used as an active ingredient in the oral composition of this embodiment either in the form in which it is obtained or after processing (e.g., by pulverizing, crushing, powdering, drying, etc.).

[0069] [Solution extraction] In consideration of the purity of 4'-demethylnobiletin, in producing the oral composition of the present embodiment, it is desirable to obtain an extract by performing solution extraction from the fermentation product obtained after the koji mold fermentation.

[0070] The solution extraction step can be performed directly on the koji mold fermentation product, but is preferably performed on the koji mold fermentation product after it has been subjected to one or more of processing steps such as shredding, crushing, grinding, powdering, and drying.

[0071] The solvent used in the solution extraction step is the same as in the case of nobiletin described above. In particular, performing extraction using ethanol at a final concentration of 55% or more, preferably 60% or more, and more preferably 80% or more (all by volume) is preferable because it can suppress the elution of polysaccharides, which are impurities, and improve the extraction efficiency of 4'-demethylnobiletin.

[0072] The extraction conditions are the same as those for nobiletin described above. The obtained extract can be lyophilized or dried using an evaporator or the like to obtain a concentrated, dried product.

[0073] Furthermore, the solution extraction step can be performed multiple times using multiple different solvents. In particular, if the first extraction is performed with water or a low-concentration aqueous alcohol, the extraction efficiency of 4'-demethylnobiletin can be improved by subsequently performing an extraction using ethanol at the specific concentration or higher.

[0074] The extract obtained as described above (the extract liquid or concentrated dry product) can be used as it is as the active ingredient of the oral composition of this embodiment.

[0075] 〔purification〕 Furthermore, by subjecting the extract to a purification step, the purity of 4'-demethylnobiletin can be further increased.

[0076] As a purification step, high purity can be achieved by liquid-liquid separation extraction or column purification using silica gel, chemically modified silica gel, activated carbon, synthetic adsorption resin carrier, etc. An example of suitable purification conditions is shown below.

[0077] First, the extract (specifically, the extract obtained by ethanol extraction) is dissolved in a 30% (v / v) ethanol solution and applied to a column of porous synthetic adsorption resin (specifically, Diaion HP20 [manufactured by Mitsubishi Chemical Corporation]) equilibrated with a 30-40% (specifically, 35%) (v / v) ethanol solution. Then, components eluted with a 39-43% (specifically, 43%) (v / v) ethanol solution are removed. Next, components eluted with a 44-46% (specifically, 45%) (v / v) ethanol solution are collected, thereby obtaining a composition with a high content of 4'-demethylnobiletin.

[0078] Furthermore, the 4'-demethylnobiletin-rich composition obtained as described above can be further subjected to ODS column chromatography (specifically, 60% (v / v) methanol elution) and ODS-HPLC (specifically, 38% (v / v) acetonitrile-water mixed solvent), and the target peak can be collected to isolate pure 4'-demethylnobiletin.

[0079] The 4'-demethylnobiletin obtained as described above is a monodemethyl form of nobiletin in which the 4'-position has been demethylated. 4'-demethylnobiletin becomes more polar due to demethylation, and has superior solubility in alcohol and water compared to nobiletin.

[0080] Oral administration of 4'-demethylnobiletin has the effect of improving xeroderma and / or the associated itching, particularly itch hypersensitivity. Here, the mechanism by which itch hypersensitivity is suppressed as observed in Test Example 1 described below is thought to be due to the inhibition of the penetration of nerve fibers into the epidermis, which is induced by dryness.

[0081] On the other hand, as shown in Test Examples 2 and 3 described below, 4'-demethylnobiletin also has the effect of promoting stratum corneum ceramide synthesis, and is therefore expected to have an effect of improving xeroderma and / or associated itching, particularly hyperitchiness, by improving the skin barrier function or suppressing a decline in the skin barrier function.

[0082] Thus, 4'-demethylnobiletin is thought to be effective in treating or preventing xeroderma from the perspectives of both inhibiting the invasion of nerve fibers into the epidermis and improving skin barrier function.

[0083] Therefore, 4'-demethylnobiletin can be used as an active ingredient in an oral composition, specifically a pharmaceutical or quasi-drug, for the treatment or prevention of xeroderma, preferably itch, particularly xeroderma accompanied by hypersensitivity to itch.

[0084] In addition, 4'-demethylnobiletin can be used as an active ingredient in oral compositions, specifically food and drink compositions, for improving or preventing xeroderma, preferably itch, particularly xeroderma accompanied by hypersensitivity to itch.

[0085] [Oral composition for treating or preventing xeroderma] The therapeutic or preventive oral composition of this embodiment is characterized by containing nobiletin and / or 4'-demethylnobiletin as active ingredients.

[0086] Specific examples of the oral composition include pharmaceuticals and quasi-drugs for the treatment or prevention of xeroderma, preferably xeroderma accompanied by itching, particularly xeroderma accompanied by itching hypersensitivity; and food and beverage compositions for the improvement or prevention of xeroderma, preferably xeroderma accompanied by itching, particularly xeroderma accompanied by itching hypersensitivity.

[0087] Nobiletin and 4'-demethylnobiletin can be used as active ingredients in the oral composition, pharmaceutical product, quasi-drug, or food and beverage composition of the present embodiment by mixing them with various raw materials as the compositions obtained in the above steps ('composition directly containing the fermentation product,' 'solution extract,' 'high-content composition') or 'isolates'.

[0088] Herein, the term "food and drink composition" includes general food and drink, functional food and drink, and functional drink.

[0089] The pharmaceuticals and quasi-drugs are preferably in the form of oral preparations.

[0090] The effective intake amounts of nobiletin and 4'-demethylnobiletin as oral agents or food and beverage compositions are each 1 mg or more, and preferably 5 mg or more, per day for an adult weighing 60 kg. By orally taking the above amounts, an excellent inhibitory effect on itching and itch hypersensitivity induced by dry skin is obtained, and an improving effect on skin barrier function based on the promoting effect of ceramide synthesis in the stratum corneum is obtained, thereby obtaining an excellent therapeutic, ameliorating, or preventive effect on xeroderma and associated itching, particularly itch hypersensitivity.

[0091] Therefore, it is expected that the above-mentioned pharmacological effects can be obtained by ingesting the oral composition, pharmaceutical product, quasi-drug, or food and beverage composition of this embodiment in a form and by an ingestion method (frequency, amount) that can ensure this required amount. However, it is desirable to appropriately determine the intake amount depending on the age, weight, symptoms, ingestion schedule, formulation form, etc. of the subject.

[0092] Furthermore, the contents of nobiletin and 4'-demethylnobiletin in an oral composition, a pharmaceutical product, a quasi-drug, or a food and drink composition may be any content that ensures the above-mentioned necessary intake amounts, and specifically, they can be contained so that they are 0.001% by mass or more, preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. The upper limit can be 20% by mass or less.

[0093] The oral compositions, pharmaceuticals, and quasi-drugs may be in the form of, for example, powder, fine granules, granules, etc., and may be filled into capsules or may be in the form of a solution dispersed in water, a cream, or a tablet obtained by mixing with excipients, etc.

[0094] The oral composition, pharmaceutical product, and quasi-drug of the present embodiment may contain various carriers, additives, other medicinal ingredients, and the like, in addition to nobiletin, 4'-demethylnobiletin, or a composition containing thereof, as long as the effects of the present invention are achieved.

[0095] Furthermore, food and beverage compositions can be prepared by mixing with various food ingredients and additives to form, for example, biscuits, snacks, gum, chewable tablets, soft drinks, drinks, soups, jellies, candies, etc. [Example]

[0096] Hereinafter, embodiments of the present invention will be described with reference to examples, but the scope of the present invention is not limited thereto. In the following, "%" means "% by mass" unless otherwise specified.

[0097] Example 1 Preparation of nobiletin-containing composition A nobiletin-containing composition was prepared by extraction from Ponkan orange peel as follows.

[0098] That is, 8 kg of Ponkan peel was chopped into small pieces, 80 L of 90% (v / v) ethanol was added, and the extract was extracted at 80°C for 1 hour to obtain an extract. The extract was applied to a Diaion HP20 (porous synthetic adsorption resin column) that had been equilibrated with 40% (v / v) ethanol in advance, and after removing non-adsorbed components with 6 L of 45% (v / v) ethanol, the components eluted with 12 L of 50% (v / v) ethanol were recovered. The recovered material was concentrated to dryness using a rotary evaporator to obtain a composition containing 19% nobiletin.

[0099] Example 2: Preparation of nobiletin isolate The nobiletin-containing composition obtained in Example 1 was purified as follows, and nobiletin was isolated.

[0100] That is, 3 g of the nobiletin-containing composition was dissolved in 40% (v / v) methanol and subjected to ODS column chromatography (a column with an inner diameter of 40 mm and a length of 20 cm packed with 120 g of Wako Gel 50C18). The component eluted with 40% (v / v) methanol was removed, and the component eluted with 60% (v / v) methanol was obtained.

[0101] The obtained fraction was then subjected to a preparative HPLC column (Mightysil RP-18GP Aqua 250-20 (5 μm), manufactured by Kanto Chemical Co., Inc.), and 35 mg of pure nobiletin was obtained using a mobile phase of 40% (v / v) acetonitrile.

[0102] Example 3 Preparation of a 4'-demethylnobiletin-containing composition According to the method described in Patent Document 1, a composition containing 4'-demethylnobiletin, a nobiletin conversion product, was prepared by fermenting Ponkan peel with Aspergillus oryzae.

[0103] Specifically, 30 kg of Ponkan peel was chopped into small pieces and sterilized by steaming. The resulting Ponkan peel was inoculated with Aspergillus awamori (manufactured by Bioc Co., Ltd.) so that it was distributed throughout the peel. Then, aerobically fermented (koji mold fermentation) in a thermostatic chamber at 30°C for 4 days to obtain a koji mold fermented product.

[0104] 75 L of 80% (v / v) ethanol was added to 6 kg of the obtained koji mold fermentation product, and the extract was extracted at 80°C for 1 hour to obtain an extract. The extract was applied to a Diaion HP20 (porous synthetic adsorption resin column) that had been previously equilibrated with 40% (v / v) ethanol, and after removing non-adsorbed components with 3 L of 43% (v / v) ethanol, the components eluted with 10 L of 45% (v / v) ethanol were recovered. The recovered material was concentrated to dryness using a rotary evaporator to obtain a composition containing 20% ​​4'-demethylnobiletin.

[0105] Example 4: Preparation of 4'-demethylnobiletin isolate The 4'-demethylnobiletin-containing composition obtained in Example 3 was purified as follows to isolate 4'-demethylnobiletin.

[0106] That is, 6 g of the 4'-demethylnobiletin-containing composition was dissolved in 30% (v / v) methanol and subjected to ODS column chromatography (120 g of Wako Gel 50C18 packed in a column with an inner diameter of 40 mm and a length of 20 cm). The component eluted with 40% (v / v) methanol was removed, and the component eluted with 60% (v / v) methanol was obtained.

[0107] The obtained fraction was then subjected to a preparative HPLC column (Mightysil RP-18 GP Aqua 250-20 (5 μm), manufactured by Kanto Chemical Co., Inc.), and 122 mg of pure 4′-demethylnobiletin was obtained using a mobile phase of 38% (v / v) acetonitrile.

[0108] <Test Example 1> Oral administration test using xeroderma model mice Using a xeroderma model mouse created by repeated application of acetone / ether / water (AEW) (hereinafter referred to as "AEW treatment"), the effectiveness of nobiletin and 4'-demethylnobiletin (oral administration) on skin barrier function, itch behavior, and dry skin-induced alloneosis (itch hypersensitivity) was examined.

[0109] (method) The generation of xeroderma model mice and alloknesis assay were performed based on previous literature (Akiyama T. et al., Mouse Model of Touch-Evoked Itch (Alloknesis), J. Invest. Dermatol. (2012) 132, 1886-1891; Bourane S. et al., Gate control of mechanical itch by a subpopulation of spinal cord interneurons, Science (2015) 350(6260):550-554).

[0110] Three days before the start of AEW treatment, the backs of C57BL6 / J mice were shaved. Xeroderma model mice were created by applying AEW treatment to the shaved skin (twice daily, morning and evening, for seven days). AEW treatment consisted of a 1:1 volumetric mixture of acetone (A) and diethyl ether (E) soaked in cotton and applied to the shaved area for 15 seconds. Next, sterile water was soaked in cotton and applied to the shaved area for 30 seconds (AEW group). The control group received sterile water for 45 seconds (W group).

[0111] As the test substance, 25 mg / kg body weight of nobiletin (prepared in Example 2) or 25 mg / kg body weight of 4'-demethylnobiletin (prepared in Example 4) was orally administered to the mice once daily using a disposable oral probe (manufactured by Fuchigami Kikai Co., Ltd.) before the AEW treatment. Mice in the control group were orally administered 100 μL / 10 g body weight of 0.5% CMC (carboxymethylcellulose) instead of the test substance.

[0112] After the final AEW treatment, the skin barrier function of the xeroderma model mice was evaluated by measuring transepidermal water loss (TEWL) and stratum corneum hydration (SC hydration) at the shaved area. The skin condition at the shaved area was evaluated using a skin dryness score of 3 points for each of four indices: redness / bleeding, crusting / dryness, edema, and abrasions / tissue loss, for a total of 12 points.

[0113] Sixteen hours after the final AEW treatment, scratching behavior was measured for 2 hours using the next-generation scratching behavior quantification system "SCLABA®-Real" (Noveltec) to evaluate spontaneous scratching behavior induced by dry skin. Based on the data obtained by SCLABA®-Real, spontaneous locomotion was measured using SCLABA®-Real Tracking software.

[0114] Finally, we evaluated itch hypersensitivity by performing an allonesis assay, in which a slight mechanical stimulus was applied to dry skin using von Frey filaments and whether scratching behavior occurred immediately after the stimulus.

[0115] As a pretreatment, one AEW-treated (or W-treated) mouse was placed per cage and allowed to acclimate for at least 1 hour. A von Frey filament with a bending force of 0.07 g or 0.16 g was pressed perpendicularly to the posterior neck of the mouse (AEW-treated or W-treated site) three times with an interval of at least 5 seconds between each. Each of these three tactile stimuli constituted one set, and 10 sets were performed. The average interval between sets was approximately 7 minutes and 30 seconds. The allonesis score was calculated by assigning 1 point to the number of times the mouse exhibited scratching behavior at the stimulation site immediately after each of the 30 tactile stimuli (3 times x 10 sets).

[0116] The test groups were as follows: (1) W-treated mice + 0.5% CMC administration group (n=11) (2) AEW-treated mice + 0.5% CMC administration group (n=11) (3) AEW-treated mice + 25 mg / kg nobiletin administration group (n=11) (4) AEW-treated mice + 25 mg / kg 4'-demethylnobiletin administration group (n=10)

[0117] (result) The results are shown in Figures 1 to 5. Figure 1 is a photographic image showing the state of dry skin in mice in each group after the end of AEW treatment. In Figure 1, (A) represents the W treatment + 0.5% CMC administration group, (B) represents the AEW treatment + 0.5% CMC administration group, (C) represents the AEW treatment + nobiletin (NOB) administration group, and (D) represents the AEW treatment + 4'-demethylnobiletin (4'NOB) administration group. Figure 2 is a graph showing the distribution of dry skin scores in mice in each group. In Figure 2, the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively. Note that ** and *** indicate significant differences compared to the W + 0.5% CMC group at p<0.01 and p<0.001, respectively.

[0118] A comparison of the appearance and dry skin scores with the AEW + 0.5% CMC group showed that repeated application of AEW significantly induced dry skin in the AEW + 0.5% CMC group (Figures 1 and 2). On the other hand, oral administration of nobiletin and 4'-demethylnobiletin had no effect on the dry skin caused by AEW treatment (Figure 2).

[0119] Figure 3 shows the transepidermal water loss (unit: g / m) of mice in each group. 2 3A and 3B are graphs showing the distribution of (A) stratum corneum moisture content (unit: au) and (B) the distribution of stratum corneum moisture content (unit: au). In Figure 3, the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values. **, ***, and **** indicate significant differences compared to the W+0.5% CMC group at p<0.01, p<0.001, and p<0.0001, respectively.

[0120] Skin dryness was evaluated using two parameters: transepidermal water loss (TEWL) and stratum corneum hydration (SC hydration). Compared to the control W + 0.5% CMC group, the AEW + 0.5% CMC group showed a significant increase in TEWL values ​​and a decrease in SC hydration. This confirmed that AEW treatment disrupted the skin barrier function and induced dry skin. Oral administration of nobiletin and 4'-demethylnobiletin did not significantly suppress TEWL and SC hydration, parameters of dry skin, but a tendency for these parameters to improve was observed (Figure 3).

[0121] As will be described in detail later, 4'-demethylnobiletin has been shown to promote the synthesis of stratum corneum ceramides; however, in this test example, stratum corneum ceramides were repeatedly removed by AEW treatment, so it is presumed that oral administration of 4'-demethylnobiletin would be unlikely to have any effect.

[0122] Figure 4 is a graph showing the distribution of the number of scratching behaviors (over 2 hours) in each group of mice. In Figure 4, the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively.

[0123] To evaluate spontaneous itch behavior caused by dry skin, scratching behavior was measured for 2 hours using SCLABA®-Real. The results showed a tendency for spontaneous itch behavior caused by dry skin to increase in the AEW + 0.5% CMC group compared to the W + 0.5% CMC group (no significant difference). Furthermore, a tendency for the number of spontaneous itch behaviors caused by dry skin to decrease in the 4'-demethylnobiletin group compared to the AEW + 0.5% CMC group was observed (Figure 4).

[0124] Figure 5 shows the results of the allonesis assay for mice in each group. In Figure 5, (A) shows the distribution of allonesis scores when 0.07 g von Frey filaments were used, and (b) shows the distribution of allonesis scores when 0.16 g von Frey filaments were used. The horizontal axis represents each group, and the box plots show the maximum, 75%, 50%, 25%, and minimum values, from top to bottom. *, ***, and **** indicate significant differences compared to the AEW + 0.5% CMC group at p<0.05, p<0.001, and p<0.0001, respectively.

[0125] To examine the effects of oral administration of nobiletin and 4'-demethylnobiletin on dry skin-induced allonesis (hyperitchiness), an allonesis assay was performed using von Frey filaments with bending forces of 0.07g and 0.16g. As a result, the allonesis score was significantly higher in the AEW + 0.5% CMC group compared to the W + 0.5% CMC group, confirming the development of dry skin-induced allonesis. Allonesis in the AEW + 0.5% CMC group was significantly suppressed by oral administration of nobiletin and 4'-demethylnobiletin at both (A) 0.07g and (B) 0.16g (Figure 5).

[0126] Figure 6 is a graph showing the distribution of spontaneous locomotion (unit: cm) of mice in each group. In Figure 6, the horizontal axis represents each group, and the box plots represent, from top to bottom, the maximum, 75%, 50%, 25%, and minimum values, respectively.

[0127] Finally, the spontaneous locomotion of the mice administered nobiletin and 4'-demethylnobiletin was measured using SCLABA (registered trademark)-Real Tracking software, and the distance traveled by each mouse (over 2 hours) was calculated to examine whether the test substances had a sedative effect. As a result, no significant difference was observed in the spontaneous locomotion between the AEW + 0.5% CMC group and the nobiletin or 4'-demethylnobiletin administration groups (Figure 6).

[0128] In this test example, the effects of oral administration of nobiletin and 4'-demethylnobiletin on the itch and allonesis (itch hypersensitivity) associated with xeroderma were examined using AEW-treated xeroderma model mice. As a result, oral administration of nobiletin and 4'-demethylnobiletin significantly suppressed allonesis associated with xeroderma (Figure 5). Furthermore, oral administration of nobiletin and 4'-demethylnobiletin showed a tendency to improve skin barrier function and spontaneous itch behavior associated with xeroderma (Figures 3 and 4).

[0129] From the above, it is suggested that oral administration of nobiletin and 4'-demethylnobiletin exerts therapeutic and preventive effects on xeroderma and the itching and itch hypersensitivity associated with xeroderma.The mechanism of suppression of itch hypersensitivity observed in Figure 5 is thought to be due to the inhibition of the penetration of nerve fibers into the epidermis, which is induced by dryness.

[0130] Example 5 Preparation of 4'-demethylnobiletin-containing composition and isolate In the same manner as in Example 3, 10 kg of Ponkan peel was fermented with koji mold to prepare a composition containing 21% of the nobiletin conversion product 4'-demethylnobiletin.

[0131] 2 g of the obtained 4'-demethylnobiletin-containing composition was dissolved in 20% (v / v) methanol and subjected to ODS column chromatography (a 20 mm inner diameter, 30 cm long column packed with 30 g of Wako Gel 50C18). The component eluted with 40% (v / v) methanol was removed, and the component eluted with 60% (v / v) methanol was obtained.

[0132] The resulting components were then subjected to preparative TLC chromatography (silica gel 70PF) using a developing solvent of hexane / ethanol = 7:3. 254 A Wako plate (film thickness 0.75 mm, manufactured by Wako Pure Chemical Industries) was used, and the fractions containing 4'-demethylnobiletin were collected while checking using a UV lamp.

[0133] The obtained fraction was then applied to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm×25 cm), and 20 mg of pure 4′-demethylnobiletin was obtained using a mobile phase of 37% (v / v) acetonitrile.

[0134] <Test Example 2> Effect of 4'-demethylnobiletin on stratum corneum ceramide synthesis Pure 4'-demethylnobiletin or a composition containing 4'-demethylnobiletin prepared in Example 5 was added to a human three-dimensional cultured epidermal model (stratum corneum model), and the effect on stratum corneum ceramide synthesis was examined.

[0135] (method) A LabCyte EPI-MODEL 6D (J-TEC) was used as a human 3D cultured epidermal model. After opening the epidermal model, it was cultured in the accompanying dedicated assay medium ([EPI-MODEL][CORNEA-MODEL] (J-TEC)) for one day. Thereafter, the model was cultured in a medium prepared by dissolving 4'-demethylnobiletin (10 μM) or a composition containing 4'-demethylnobiletin (4.5, 9, 18 μg / mL) in the above assay medium, and the tissue was collected on the sixth day of culture. The medium was changed every other day. As a control, a similar culture was performed using an assay medium supplemented with 0.1% (volume ratio) DMSO instead of 4'-demethylnobiletin or the composition containing 4'-demethylnobiletin.

[0136] The amount of ceramide produced in the stratum corneum was evaluated for total ceramide, ceramide EOS, ceramide NS / NDS (total of NS and NDS), and ceramide NP (number of samples: n = 3). The quantification method for each ceramide is as follows.

[0137] The tissue collected on day 6 of culture was treated with trypsin to separate the stratum corneum. The resulting stratum corneum was washed multiple times with PBS and then disrupted for a set period of time using a glass homogenizer containing an extract (chloroform:methanol:PBS = 1:2:0.8 (volume ratio)). After disruption, the stratum corneum was transferred to a test tube, vortexed for 20 minutes, and then centrifuged to separate the precipitate and supernatant. The precipitate was subjected to protein quantification using the BCA method and used to calculate the amount of ceramide per weight of stratum corneum protein.

[0138] Separately, the extract (chloroform:PBS = 1:1 (volume ratio)) was added to the supernatant, and the mixture was vortexed for 20 minutes, followed by centrifugation to separate the layers. The lower layer was collected using a glass syringe, and the solvent was completely removed by drying with nitrogen. After that, a predetermined amount of a chloroform-methanol mixture (chloroform:methanol = 2:1 (volume ratio)) was added and vortexed until dissolved to prepare the sample solution.

[0139] Ceramide quantification was performed using silica gel thin-layer chromatography (TLC) with reference to "Experimental Methods for the Development of Functional Cosmetic Materials - In Vitro / Cell / Tissue Culture" (edited by Genji Imokawa, 2007, CMC Publishing). The non-hydroxy ceramide and hydroxy ceramide used as standard substances were purchased from Matreya, LLC. Ceramide III and Ceramide VI were provided by Evonik Industries AG (Essen), and Ceramide TIC-001 was provided by Takasago International Corporation. Each standard substance was dissolved in a chloroform-methanol mixture (chloroform:methanol = 2:1 (volume ratio)) to a concentration of 0.2 mg / mL to prepare the standard solution.

[0140] A predetermined amount of sample solution and standard solution is applied to a silica gel plate (HPTLC glass plate, silica gel 60 F 254(Merck)) and developed with a developing solvent (chloroform:methanol:acetic acid = 190:9:1 (volume ratio)). The removed TLC plate was air-dried at room temperature and then developed again with a developing solvent (chloroform:methanol:acetic acid = 197:2:1 (volume ratio)). The removed TLC plate was air-dried at room temperature, then sprayed with a mixture of 10% copper sulfate and 8% phosphoric acid (mass ratio) and heated at 180°C for 6 minutes to develop the spots. The developed TLC plate was scanned and analyzed using Image J (Wayne Rasband), and each ceramide was quantified based on the density and size of each spot.

[0141] In addition, non-hydroxy ceramide was used as the standard substance for the quantification of ceramide NS / NDS. Hydroxy ceramide was used as the standard substance for the quantification of ceramide AS. Ceramide III was used as the standard substance for the quantification of ceramide NP. Ceramide VI was used as the standard substance for the quantification of ceramide AP. Ceramide TIC-001 was used as the standard substance for the quantification of ceramide NDS. Total ceramide was calculated as the total amount of ceramides EOS, NS, NDS, NP, EOH, AS, NH, AP, and AH, including the ceramides determined above.

[0142] (result) The results are shown in Figures 7 and 8. Figure 7 is a graph showing the effect (after 6 days) on stratum corneum ceramide synthesis when 4'-demethylnobiletin (DeNOB) was added to a human 3D cultured epidermal model. In Figure 7, (A) represents the results for total ceramide, (B) represents ceramide EOS, (C) represents ceramide NS / NDS, and (D) represents ceramide NP. The vertical axis represents the amount of ceramide produced (unit: μg / mg protein), the bars represent the standard deviation, and * and ** represent significant differences at p<0.05 and p<0.01, respectively, compared to the untreated control.

[0143] The addition of 4'-demethylnobiletin significantly increased total ceramide by 1.54 times, ceramide EOS by 1.50 times, NS / NDS by 1.59 times, and ceramide NP by 1.57 times (Figure 7).

[0144] Figure 8 is a graph showing the effect on stratum corneum ceramide synthesis (after 6 days) when a composition containing 4'-demethylnobiletin (DeNOB) was added to a human 3D cultured epidermal model. In Figure 8, (A) shows the results for total ceramide, (B) shows the results for ceramide NS / NDS, and (C) shows the results for ceramide NP. The vertical axis shows the amount of ceramide produced (unit: μg / mg protein), the bars show the standard deviation, and * indicates a significant difference at p<0.05 compared to the untreated control.

[0145] Addition of the 4'-demethylnobiletin-containing composition significantly increased total ceramide by 1.36 times, NS / NDS by 1.34 times, and ceramide NP by 1.40 times (Figure 8).

[0146] (Conclusion) From the above, it was clarified that 4'-demethylnobiletin and compositions containing 4'-demethylnobiletin have the effect of promoting ceramide synthesis in the stratum corneum. This suggests that 4'-demethylnobiletin may improve the skin barrier function and exert therapeutic and preventive effects on xeroderma.

[0147] <Test Example 3> Effect of 4'-demethylnobiletin on ceramide synthase gene expression Pure 4'-demethylnobiletin or a composition containing 4'-demethylnobiletin prepared in Example 5 was added to a human three-dimensional cultured epidermal model (stratum corneum model), and the effect on stratum corneum ceramide synthase gene expression was examined. Figure 9 is a diagram showing the ceramide biosynthesis pathway and related genes. In this test example, ceramide synthesis-related genes, ceramide synthase 1 (CERS1), 2 (CERS2), glucosylceramide synthase (UGCG), and sphingomyelin synthase 2 (SMS2), were examined.

[0148] (method) A human three-dimensional cultured epidermal model was cultured in the same manner as in Test Example 2 above, and epidermal cells were extracted by trypsin treatment from the tissue collected on day 3 of culture after the addition of the substance to be evaluated. Here, the amount of 4'-demethylnobiletin added to the assay medium was 10 μM, and the amount of the 4'-demethylnobiletin-containing composition added was 9.18 μg / mL. As a control, culture was carried out in the same manner using an assay medium to which 0.1% (volume ratio) DMSO had been added instead of 4'-demethylnobiletin or the 4'-demethylnobiletin-containing composition.

[0149] Total RNA was extracted from the epidermal cells using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized from the total RNA using the Rever TraAce qPCR RT kit (Toyobo). The resulting cDNA was then used to quantify the gene expression levels of ceramide biosynthesis-related enzymes (CERS1, CERS2, UGCG, and SMS2) by real-time RT-PCR. For real-time RT-PCR, TB Green Fast qPCR Mix (TaKaRa) was used, and the primers for CERS1, CERS2, UGCG, and SMS2 had the sequences shown in Table 1.

[0150] GAPDH was used as an internal standard. Real-time RT-PCR was performed according to established methods, and the expression levels of CERS1, CERS2, UGCG, and SMS2 mRNA were determined as a ratio to the expression level of GAPDH mRNA, which was the internal standard. The expression rates of CERS1, CERS2, UGCG, and SMS2 were calculated as the ratio of the mRNA expression level in the 4'-demethylnobiletin or 4'-demethylnobiletin-containing composition added group to the mRNA expression level in the control (DMSO 0.1%) (number of samples: n = 3).

[0151] [Table 1]

[0152] (result) The results are shown in Figures 10 and 11. Figure 10 is a graph showing the effect on ceramide-related genes (after 3 days) when 4'-demethylnobiletin (DeNOB) was added to a human 3-dimensional cultured epidermal model. In Figure 10, (A) shows the results for CERS1, (B) for CERS2, and (C) for UGCG. The vertical axis of the graph shows the ratio of mRNA expression level (relative mRNA expression level) when the mRNA expression level in the no-addition control (Control) is set to 1. The bars show the standard deviation, and * indicates a significant difference compared to the no-addition control (Control) at p<0.05. The addition of 4'-demethylnobiletin showed a significant increase in the expression levels of CERS1, CERS2, and UGCG (Figure 10).

[0153] Figure 11 is a diagram showing the effect on ceramide-related genes when a 4'-demethylnobiletin-containing composition (DeNOB extract) is added to a human three-dimensional cultured epidermal model. In Figure 11, (A) represents the results for CERS1, (B) represents CERS2, (C) represents UGCG, and (D) represents SMS2. The vertical axis of the graph represents the ratio of mRNA expression level (relative mRNA expression level) when the mRNA expression level in the no-addition control (Control) is set to 1. The bars represent standard deviation, and # and * represent a trend at p<0.1 and a significant difference at p<0.05, respectively, compared to the no-addition control (Control). The addition of a 4'-demethylnobiletin-containing composition resulted in a significant increase in the expression levels of CERS1, CERS2, UGCG, and SMS2 (Figure 11).

[0154] (Conclusion) From the above, it was demonstrated that the addition of 4'-demethylnobiletin or a composition containing 4'-demethylnobiletin to cultured cells enhanced ceramide synthase activity, thereby promoting ceramide synthesis in the stratum corneum. This suggests that 4'-demethylnobiletin may improve skin barrier function and exert therapeutic and preventive effects for xeroderma.

[0155] Example 6: Oral formulation Prescription example 1 The raw materials were blended in the amounts shown below, and a nobiletin blend preparation (300 mg tablet) was produced by a conventional method. Nobiletin-containing composition (preparation of Example 1) 50 mg Dextrin 160mg Crystalline cellulose, maltitol, sucrose fatty acid ester 90mg

[0156] Prescription example 2 The raw materials were blended in the amounts shown below, and a 4'-demethylnobiletin blend preparation (300 mg tablet) was produced by a conventional method. 4'-demethylnobiletin-containing composition (preparation of Example 3) 50 mg Dextrin 160mg Crystalline cellulose, maltitol, sucrose fatty acid ester 90mg

[0157] Prescription example 3 The raw materials were blended in the amounts shown below, and a nobiletin blend preparation (300 mg tablet) was produced by a conventional method. Nobiletin (Example 2 preparation) 5 mg Dextrin 205mg Crystalline cellulose, maltitol, sucrose fatty acid ester 90mg

[0158] Prescription example 4 The raw materials were blended in the amounts shown below, and a 4'-demethylnobiletin blend preparation (300 mg tablet) was produced by a conventional method. 4'-demethylnobiletin (preparation of Example 4) 5 mg Dextrin 205mg Crystalline cellulose, maltitol, sucrose fatty acid ester 90mg

[0159] Example 7: Production example of food and drink composition The ingredients were blended in the amounts shown below, and a nobiletin-blended beverage (180 mL) was produced by a conventional method. Nobiletin-containing composition (preparation of Example 1) 0.025 g 5g apple juice concentrate Lactose 2g High fructose corn syrup 5g Acidulant (citric acid) 0.3g Sweetener (sucralose) 0.01g Preservative (sodium benzoate) 0.03g Water Residual

[0160] A 4'-demethylnobiletin-containing beverage (180 mL) was produced in the same manner as above, except that the same amount of 4'-demethylnobiletin-containing composition (prepared product of Example 3) was used instead of the nobiletin-containing composition in the above beverage.

[0161] The embodiments and examples of the present invention have been described in detail above with reference to the drawings, but the specific configurations are not limited to these, and design changes that do not deviate from the gist of the present invention are included in the present invention.

[0162] For example, in the above examples, the effect of improving xeroderma by nobiletin and 4'-demethylnobiletin extracted and purified from Ponkan orange peel or a composition containing these has been described, but the present invention is not limited to this, and oral compositions for treating or preventing xeroderma containing nobiletin and 4'-demethylnobiletin as active ingredients, extracted from any citrus fruit containing nobiletin instead of Ponkan orange, can also be included in the present invention. [Industrial Applicability]

[0163] The present invention contains nobiletin and 4'-demethylnobiletin, which are highly safe raw materials, as active ingredients, and thereby has an anti-scratch effect on xeroderma and the associated hyperitchiness, and is expected to be used in pharmaceuticals, quasi-drugs, food and drink compositions, and the like for the treatment or prevention of xeroderma and the associated hyperitchiness.

Claims

1. An oral pharmaceutical or quasi-drug for the treatment or prevention of xeroderma accompanied by itching hypersensitivity due to its effect of improving skin barrier function, which contains nobiletin and / or 4'-demethylnobiletin as active ingredients.

2. A food and beverage composition for improving or preventing xerosis accompanied by itching hypersensitivity due to the effect of improving skin barrier function, comprising nobiletin and / or 4'-demethylnobiletin as active ingredients.

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