Composition for external use on the skin for promoting ceramide synthesis in the stratum corneum

Abstract

To provide skin external compositions exerting the effects of moisturizing skin, of preventing or improving skin dryness, or of preventing or improving skin diseases associated with deterioration of a skin barrier function, by a horny layer ceramide synthesis-promoting action, using food-derived ingredients as active ingredients.SOLUTION: Provided are skin external compositions for promoting horny layer ceramide synthesis, containing 4'-demethylnobiletin as an active ingredient, preferably, pharmaceuticals, quasi-drugs, or cosmetics for moisturizing the skin, for preventing or improving skin dryness, or for preventing or improving diseases associated with deterioration of a skin barrier function. Since 4'-demethylnobiletin can be obtained by converting nobiletin, which is an ingredient of citrus peel, by a fermentation method of fungus Aspergillus oryzae, the skin external composition of the present invention is highly safe and has no concern about side effects.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a topical skin composition for promoting stratum corneum ceramide synthesis, which contains 4'-demethylnobiletin as an active ingredient, and which is used to moisturize the skin by promoting stratum corneum ceramide synthesis, to prevent or alleviate dry skin, or to prevent or treat diseases associated with a decrease in skin barrier function. [Background technology]

[0002] The skin has various functions, of which the most important are the barrier function that prevents the intrusion of harmful substances from the outside world and the moisturizing function of the skin surface that prevents the loss of moisture from the body surface. However, with aging, the barrier function and moisturizing function decline, making the skin more susceptible to dryness and skin problems due to the intrusion of harmful substances and microorganisms (see, for example, Non-Patent Document 1).

[0003] It is also known that the amount of ceramide is significantly reduced in the skin and non-skin areas of patients with atopic dermatitis compared to healthy individuals (see, for example, Non-Patent Document 1). Furthermore, it has been revealed that senile xerosis caused by aging is not only due to a decrease in sebum, as has been conventionally known, but also to a decrease in ceramide (see, for example, Non-Patent Document 2).

[0004] To date, compounds that have been reported to promote stratum corneum ceramide synthesis in cell systems include phytosphingosine, which is ceramide with an acyl group removed (see, for example, Patent Document 1), and tiliroside, a strawberry seed extract (see, for example, Non-Patent Document 3). However, the effects of these stratum corneum ceramide synthesis promoters when applied to actual human skin have not been fully verified, and none are currently in practical use.

[0005] On the other hand, nobiletin, a polymethoxyflavonoid, is a flavonoid unique to citrus fruits, and in recent years has become known for its various physiological effects, including cancer prevention, anti-aging, and anti-arteriosclerosis.

[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, one of the main components, is converted to 4'-demethylnobiletin. The present inventors have also found that 4'-demethylnobiletin has an excellent memory-improving effect (see, for example, Patent Document 2). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Imokawa G, et al.,J Invest Dermatol,96,523-526(1991) [Non-patent document 2] Akimoto K, et al., J Dermatol (Tokyo), 20, 1-6 (1993) [Non-patent document 3] Takeda S, et al., PLoS ONE 13(10):e0205061(2018) [Patent documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2020-7270 [Patent Document 2] Patent No. 5667561 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a composition for external use on the skin that contains a highly safe food-derived ingredient as an active ingredient and, by promoting stratum corneum ceramide synthesis, exerts a skin moisturizing effect, a preventive or ameliorative effect on dry skin, or a preventive or therapeutic effect on diseases associated with a decrease in skin barrier function. [Means for solving the problem]

[0010] The present inventors further investigated the functionality of 4'-demethylnobiletin and, as a result, surprisingly discovered that 4'-demethylnobiletin has the effect of promoting stratum corneum ceramide synthesis. They also found that, due to this effect, applying 4'-demethylnobiletin to the skin is effective in moisturizing the skin and preventing dryness. They also speculated that applying 4'-demethylnobiletin to the skin would be effective in preventing or treating diseases associated with a decrease in skin barrier function, which is caused by a decrease in stratum corneum ceramide. Based on these findings, the present inventors have completed the present invention.

[0011] That is, the present invention relates to an external skin composition for promoting ceramide synthesis in the stratum corneum, which contains 4'-demethylnobiletin as an active ingredient.

[0012] The above-mentioned topical skin composition for promoting stratum corneum ceramide synthesis can be made into a pharmaceutical, quasi-drug, or cosmetic for moisturizing the skin, preventing or improving dry skin, or preventing or treating diseases associated with a decrease in skin barrier function. [Effects of the Invention]

[0013] The present invention makes it possible to provide a composition for external use on the skin that contains 4'-demethylnobiletin as an active ingredient, thereby promoting stratum corneum ceramide synthesis when applied to the skin, and exerting a skin moisturizing effect, an effect of preventing or alleviating dry skin, and an effect of preventing or treating diseases associated with a decrease in skin barrier function.

[0014] Since 4'-demethylnobiletin can be obtained by converting nobiletin, a component of citrus peel, through a koji mold fermentation method, the topical skin composition for promoting ceramide synthesis in the stratum corneum of the present invention is highly safe and has no risk of side effects. [Brief explanation of the drawings]

[0015] [Figure 1]This figure shows the effect of adding 4'-demethylnobiletin (DeNOB) to a human three-dimensional cultured epidermal model on stratum corneum ceramide synthesis (Example 3). In the figure, (A) shows the results for total ceramide, (B) for ceramide EOS, (C) for ceramide NS / NDS, and (D) for ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate standard deviation, and * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the untreated control. [Figure 2] This 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 (Example 3). In the figure, (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 of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate standard deviation, and * indicates a significant difference at p<0.05 compared to the untreated control (Control). [Figure 3] FIG. 1 shows the ceramide biosynthetic pathway and related genes. [Figure 4] This is a diagram showing the effect on ceramide biosynthesis-related genes when 4'-demethylnobiletin (DeNOB) is added to a human three-dimensional cultured epidermal model (Example 4). 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 no-addition control (Control) is set to 1. The bars show the standard deviation, and * indicates a significant difference at p<0.05 compared to the no-addition control (Control). [Figure 5]1 shows 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 (Example 4). 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 no-addition control (Control) is set to 1. Bars indicate standard deviation, and # and * indicate a trend at p<0.1 and a significant difference at p<0.05, respectively, compared to the no-addition control (Control). [Figure 6] This is a diagram showing the effect of a lotion containing 4'-demethylnobiletin on stratum corneum moisture content in a test of the skin moisturizing effect of continuous use in humans (Example 5). In the figure, (A) shows the measured stratum corneum moisture content value, and (B) shows the difference in stratum corneum moisture content value. The vertical axis of the graph shows stratum corneum moisture content (unit: au). Bars indicate standard deviation, and * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the additive-free placebo group. [Figure 7] This is a graph showing the effect of a lotion containing 4'-demethylnobiletin on transepidermal water loss in a skin moisturizing effect test using the lotion continuously on humans (Example 5). In the figure, (A) shows the measured transepidermal water loss value, (B) shows the transepidermal water loss difference value, and (C) shows the relative transepidermal water loss value. The vertical axis of the graph shows the transepidermal water loss (unit: g / h / m2). The bars show the standard deviation, and ** and *** indicate significant differences at p<0.01 and p<0.001, respectively, compared to the additive-free placebo group. [Figure 8] This is a diagram showing the effect of a lotion containing 4'-demethylnobiletin on skin elasticity in a human skin moisturizing test after continuous use (Example 5). In the figure, (A) shows the measured skin elasticity value, and (B) shows the difference in skin elasticity value. The vertical axis of the graph shows skin elasticity (unit: au). Bars show standard deviation, and † indicates a trend with p<0.1 compared to the additive-free placebo group. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, the embodiments of the present application will be described in detail. The present embodiment relates to an external skin composition for promoting ceramide synthesis in the stratum corneum, which contains 4'-demethylnobiletin as an active ingredient.

[0017] The topical skin composition for promoting stratum corneum ceramide synthesis according to this embodiment has, when applied to the skin, a skin moisturizing effect, an effect of preventing or improving dry skin, or an effect of preventing or treating diseases associated with a decrease in skin barrier function, and therefore can be made into a pharmaceutical, quasi-drug, or cosmetic intended to have these effects.

[0018] [4'-demethylnobiletin] Although 4'-demethylnobiletin (Formula 1) is not commercially available, a synthetic product can be used. Alternatively, a pure 4'-demethylnobiletin product (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 2 described above can be used. The method of Patent Document 2, which utilizes the bioconversion of nobiletin by koji mold fermentation, is preferable to synthetic products because it is simpler and less expensive.

[0019] [ka]

[0020] 4'-demethylnobiletin is also one of the metabolites produced after nobiletin is absorbed in the body. Citrus peel is used as an ingredient in sweets such as marmalade and candied fruits, and mandarin oranges (Citrus reticulata), which have a high nobiletin content, have a long history of being eaten as tangerine, and 4'-demethylnobiletin and compositions containing it are extremely safe.

[0021] A method for producing 4'-demethylnobiletin and a composition containing it, which is based on the method described in Patent Document 2, will be described below.

[0022] [Fermentation ingredients] The raw material for koji fermentation is the 'fruit' of citrus fruits (the entire fruit including the peel, juice, pulp, seeds, etc.), which contains the polymethoxyflavonoid nobiletin. However, it is particularly desirable to use the 'peel' from the standpoint of nobiletin content and effective utilization of waste materials.

[0023] Furthermore, any variety or lineage of citrus fruit (for example, ponkan, shikuwasha, tangerine, tachibana, etc.) can be used as long as it contains nobiletin.

[0024] 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.

[0025] The above citrus fruits are preferably harvested and collected fresh, or washed, but dried, frozen, or long-term stored fruits can also be used.

[0026] Citrus fruits may be used in their original form, but it is preferable to chop, crush or grind them.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] [Koji mold fermentation] Examples of koji mold 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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 citrus-derived aroma is lost.

[0038] Furthermore, in the koji mold fermentation, nobiletin is demethylated by an enzyme secreted from the koji mold, converting it to 4'-demethylnobiletin.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Therefore, the koji mold fermentation product obtained here can be used as an active ingredient in the topical skin composition for promoting stratum corneum ceramide synthesis of this embodiment, either in the form in which it is obtained or after processing (e.g., by pulverizing, crushing, powdering, drying, etc.).

[0044] [Solution extraction] In terms of purity, in the production of the topical skin composition for promoting stratum corneum ceramide synthesis of this embodiment, it is desirable to obtain an extract by performing solution extraction from the fermented product obtained after the koji mold fermentation.

[0045] 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 further processing such as shredding, crushing, grinding, or powdering.

[0046] 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.

[0047] Among these solvents, water, ethanol, or aqueous ethanol is particularly preferred in terms of extraction efficiency, ease of handling, and safety.

[0048] Furthermore, performing extraction using ethanol at a final concentration of 55% or more, preferably 60% or more, and more preferably 80% or more is particularly preferable because it can suppress the elution of polysaccharides as impurities and improve the extraction efficiency of 4'-demethylnobiletin.

[0049] 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 raw material (preferably the crushed material), and the raw material 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.

[0050] The obtained extract can be concentrated to dryness by freeze-drying or drying using an evaporator or the like.

[0051] 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.

[0052] The extract obtained as described above (the extract liquid or concentrated dried product) can be used as it is as an active ingredient in the external skin composition for promoting ceramide synthesis in the stratum corneum of this embodiment.

[0053] 〔purification〕 Furthermore, the purity can be further increased by subjecting these to a purification step.

[0054] 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.

[0055] First, the extract (specifically, the extract obtained through ethanol extraction) is applied to a column of porous synthetic adsorption resin (specifically, Diaion HP20 [manufactured by Mitsubishi Chemical Corporation]) equilibrated with 33 to 41% ethanol (specifically, 40% ethanol). Then, components eluted with 39 to 43% ethanol (specifically, 43% ethanol) are removed. Next, components eluted with 44 to 46% ethanol (specifically, 45% ethanol) are collected, thereby obtaining a composition with a high content of 4'-demethylnobiletin.

[0056] Furthermore, the 4'-demethylnobiletin-rich composition obtained as described above can be further subjected to ODS column chromatography (specifically, 60% methanol elution), thin layer chromatography (TLC) (specifically, hexane / ethanol [7:3]), or ODS-HPLC (specifically, a 37% [V / V] acetonitrile-water mixed solvent), and the target peak can be collected to isolate pure 4'-demethylnobiletin.

[0057] 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.

[0058] Furthermore, 4'-demethylnobiletin has an excellent effect of promoting stratum corneum ceramide synthesis, and therefore when applied to the skin, it has an excellent moisturizing effect, as well as the effect of preventing, treating, or improving diseases associated with a decrease in skin barrier function.

[0059] Here, the "diseases associated with impaired skin barrier function" are not particularly limited, but examples thereof include psoriasis, dry skin, xerosis, atopic dermatitis, and the like.

[0060] Therefore, 4'-demethylnobiletin can be used as an active ingredient in an external skin composition for promoting ceramide synthesis in the stratum corneum.

[0061] Furthermore, due to its effect of promoting stratum corneum ceramide synthesis, 4'-demethylnobiletin can be used as an active ingredient in external skin compositions, preferably pharmaceuticals, quasi-drugs, or cosmetics, for moisturizing the skin, preventing or improving dry skin, or preventing or treating diseases associated with a decrease in skin barrier function.

[0062] [External skin composition for promoting stratum corneum ceramide synthesis, pharmaceuticals, quasi-drugs, and cosmetics] The external skin composition for promoting stratum corneum ceramide synthesis of this embodiment is characterized by containing 4'-demethylnobiletin as an active ingredient.

[0063] In addition, specific examples of topical skin compositions for promoting stratum corneum ceramide synthesis include pharmaceuticals, quasi-drugs, and cosmetics for moisturizing the skin, preventing or improving dry skin, or preventing or treating diseases associated with a decrease in skin barrier function.

[0064] The above-mentioned 4'-demethylnobiletin can be used as an active ingredient in a topical skin composition for promoting stratum corneum ceramide synthesis, a pharmaceutical product, a quasi-drug, or a cosmetic product by mixing it with various raw materials as each of the compositions obtained in the above steps ('composition directly containing the fermentation product,' 'solution extract,' 'high-content composition') or as an 'isolate'.

[0065] The effective intake amount of 4'-demethylnobiletin is 0.001% by mass or more, preferably 0.01% by mass or more, of 4'-demethylnobiletin. By applying a topical skin composition containing this 4'-demethylnobiletin to the skin at least once a day, preferably 1 to 3 times a day, an excellent effect of promoting ceramide synthesis in the stratum corneum and a moisturizing effect on the skin can be obtained, and excellent preventive, therapeutic or ameliorative effects can be obtained for diseases associated with dry skin and a decrease in skin barrier function.

[0066] Therefore, it is expected that the above-mentioned pharmacological effects can be obtained by applying the topical skin composition for promoting stratum corneum ceramide synthesis, pharmaceutical product, quasi-drug, or cosmetic of this embodiment to the skin in a form and in a method of ingestion (frequency, amount) that ensures this required amount. However, it is desirable to appropriately determine the intake amount and application amount depending on the subject's age, weight, symptoms, ingestion schedule, formulation form, etc.

[0067] Furthermore, the content of 4'-demethylnobiletin in a topical skin composition, pharmaceutical, quasi-drug, or cosmetic for promoting stratum corneum ceramide synthesis may be any content that ensures the above-mentioned necessary intake amount, but specifically, it can be contained so that it is 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.

[0068] The external skin composition for promoting stratum corneum ceramide synthesis, pharmaceuticals, quasi-drugs or cosmetics may be in the form of, for example, a liquid, gel, cream or ointment.

[0069] The topical skin composition for promoting stratum corneum ceramide synthesis, pharmaceutical product, quasi-drug, or cosmetic of the present embodiment may contain, in addition to 4'-demethylnobiletin or a composition containing it, various carriers, additives, other medicinal ingredients, and the like, as long as the effects of the present invention are achieved. [Example]

[0070] The present invention will be described below with reference to examples, but the scope of the present invention is not limited to these examples.

[0071] Example 1 Preparation of a 4'-demethylnobiletin-containing composition According to the method described in Patent Document 2, a composition containing 4'-demethylnobiletin, a nobiletin conversion product, was prepared by fermenting Ponkan peel with Aspergillus oryzae.

[0072] Specifically, 10 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.

[0073] 75 L of 80% (v / v) ethanol was added to 6 kg of the obtained koji mold fermentation product, and an extract was obtained by extracting at 100°C for 1 hour. 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 21% 4'-demethylnobiletin.

[0074] Example 2: Preparation of 4'-demethylnobiletin isolate 2 g of the 4'-demethylnobiletin-rich composition obtained above was dissolved in 20% (v / v) methanol and subjected to ODS column chromatography (a column with an inner diameter of 20 mm and a length of 30 cm 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.

[0075] 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.

[0076] 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.

[0077] Example 3: Effect on stratum corneum ceramide synthesis using a human three-dimensional cultured epidermal model Pure 4'-demethylnobiletin or a composition containing 4'-demethylnobiletin was added to a human three-dimensional cultured epidermal model (stratum corneum model), and the effect on stratum corneum ceramide synthesis was examined.

[0078] A LabCyte EPI-MODEL 6D (J-TEC) was used as a human three-dimensional 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, preparation of Example 2) or a 4'-demethylnobiletin-containing composition (4.5, 9, 18 μg / mL, preparation of Example 1) 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 to which 0.1% (volume ratio) DMSO had been added instead of 4'-demethylnobiletin or the 4'-demethylnobiletin-containing composition.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] A predetermined amount of sample solution and standard solution was 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.

[0084] 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 sum of ceramides EOS, NS, NDS, NP, EOH, AS, NH, AP, and AH, including the ceramides determined above.

[0085] The results are shown in Figures 1 and 2. Figure 1 is a diagram showing the effect of adding 4'-demethylnobiletin (DeNOB) to a human three-dimensional cultured epidermis model on stratum corneum ceramide synthesis (after 6 days) (Example 3).

[0086] In Figure 1, (A) shows the results for total ceramide, (B) for ceramide EOS, (C) for ceramide NS / NDS, and (D) for ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate standard deviation, and * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the untreated control.

[0087] The addition of 4'-demethylnobiletin (preparation of Example 2) 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 1).

[0088] FIG. 2 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 three-dimensional cultured epidermis model (Example 3).

[0089] In Figure 2, (A) shows the results for total ceramide, (B) for ceramide NS / NDS, and (C) for ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate standard deviation, and * indicates a significant difference (p<0.05) compared to the untreated control.

[0090] Addition of the 4'-demethylnobiletin-containing composition (preparation of Example 1) significantly increased total ceramide by 1.36 times, NS / NDS by 1.34 times, and ceramide NP by 1.40 times (Figure 2).

[0091] From the above, it was revealed that 4'-demethylnobiletin and compositions containing 4'-demethylnobiletin have the effect of promoting ceramide synthesis in the stratum corneum.

[0092] Example 4: Effect on ceramide synthase gene expression using a human three-dimensional cultured epidermal model Pure 4'-demethylnobiletin or a composition containing 4'-demethylnobiletin was added to a human 3-dimensional cultured epidermal model (stratum corneum model), and the effect on stratum corneum ceramide synthase gene expression was examined. Figure 3 is a diagram showing the ceramide biosynthesis pathway and related genes. In this example, ceramide synthesis-related genes, ceramide synthase 1 (CERS1), 2 (CERS2), glucosylceramide synthase (UGCG), and sphingomyelin synthase 2 (SMS2), were examined.

[0093] A human three-dimensional cultured epidermal model was cultured in the same manner as in Example 3 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 (prepared in Example 2) added to the assay medium was 10 μM, and the amount of the 4'-demethylnobiletin-containing composition (prepared in Example 1) 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.

[0094] 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 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.

[0095] 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).

[0096] [Table 1]

[0097] The results are shown in Figures 4 and 5. Figure 4 is a diagram showing the effect on ceramide-related genes (after 3 days) when 4'-demethylnobiletin (DeNOB) was added to a human three-dimensional cultured epidermal model. In Figure 4, (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. Bars indicate standard deviation, and * indicates a significant difference compared to the no-addition control (Control) at p<0.05. The addition of 4'-demethylnobiletin (prepared in Example 2) showed a significant increase in the expression levels of CERS1, CERS2, and UGCG (Figure 4).

[0098] Figure 5 shows the effect on ceramide-related genes when a composition containing 4'-demethylnobiletin (DeNOB) is added to a human three-dimensional cultured epidermal model. In Figure 5, (A) shows the results for CERS1, (B) shows CERS2, (C) shows UGCG, and (D) shows 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. Bars indicate standard deviation, and # and * indicate a trend at p<0.1 and a significant difference at p<0.05, respectively, compared to the untreated control (Control). Addition of a composition containing 4'-demethylnobiletin (prepared in Example 1) resulted in a significant increase in the expression levels of CERS1, CERS2, UGCG, and SMS2 (Figure 5).

[0099] 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.

[0100] Example 5: Test of moisturizing effect of lotion containing 4'-demethylnobiletin on human skin after repeated use Ten subjects were asked to apply 0.25 mL of the test product (a lotion containing 0.005% (mass ratio) of 4'-demethylnobiletin prepared in Example 2) to one half of their face, and 0.25 mL of a control placebo product (a lotion not containing 4'-demethylnobiletin) to the other half twice a day, morning and evening. The condition and changes in the test area were measured and evaluated three times: before the start of the test, after two weeks of continuous use, and after four weeks of continuous use. Table 2 below shows the lotion compositions of the test product and placebo product.

[0101] That is, on each of the three observation days, the subjects washed their faces with a commercially available cleanser and facial wash, and then spent approximately 20 minutes resting in a room maintained at a temperature of 21.0±0.3°C and a relative humidity of 45.0±2.0%. After the skin had acclimatized to the test environment, the stratum corneum moisture content, transepidermal water loss, and skin elasticity were measured by instruments as follows to evaluate the effects of moisturizing the skin and improving dryness.

[0102] Stratum corneum moisture content: Measurements were taken three times using a Corneometer CM825 (Courage+Khazaka, Germany), and the average value was used as the stratum corneum moisture content (unit: au) of the subject.

[0103] Transepidermal water loss: Measured using a Tewameter TM300 (Courage+Khazaka, Germany), and the measured value was converted into the subject's transepidermal water loss (unit: g / h / m 2 ) was decided.

[0104] Skin elasticity: Measured twice using a Cutometer CT580 (probe opening 2.0 mm, negative pressure 150 mbar, Courage+Khazaka, Germany), and the average value of "R7" was used as the skin elasticity (unit: au) of the subject.

[0105] [Table 2]

[0106] The results are shown in Figures 6 and 7. Figure 6 is a diagram showing the effect of a lotion containing 4'-demethylnobiletin on stratum corneum moisture content in a human skin moisturizing test with continuous use. In Figure 6, (A) shows the measured stratum corneum moisture content, and (B) shows the difference in stratum corneum moisture content. The vertical axis of the graph shows stratum corneum moisture content (unit: au). Bars indicate standard deviation, and * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the additive-free placebo group.

[0107] The measured values ​​of stratum corneum moisture content after 2 and 4 weeks of continuous use, as well as the difference between before and after the test, were significantly higher in the test product group than in the placebo group (Figure 6(A), Figure 6(B)). This suggests that the continuous use of a lotion containing 4'-demethylnobiletin promoted ceramide synthesis in the stratum corneum, resulting in better skin condition.

[0108] FIG. 7 is a graph showing the effect of a lotion containing 4'-demethylnobiletin on transepidermal water loss in a human skin moisturizing test after continuous use. In FIG. 7, (A) shows the measured transepidermal water loss, (B) shows the transepidermal water loss difference value, and (C) shows the relative transepidermal water loss value. The vertical axis of the graph shows the transepidermal water loss (unit: g / h / m 2 ) The bars indicate the standard deviation, and ** and *** indicate significant differences at p<0.01 and p<0.001, respectively, compared to the placebo group.

[0109] In measurements of transepidermal water loss after four weeks of continuous use, the test product group showed significantly lower values ​​than the placebo group (Figure 7(A)). Furthermore, in the difference in transepidermal water loss before and after the test and the relative values ​​after two and four weeks of continuous use, the test product group showed significantly lower values ​​than the placebo group (Figures 7(B), 7(C)). From the above, it is believed that the continuous use of a lotion containing 4'-demethylnobiletin promoted ceramide synthesis in the stratum corneum, improving the skin's barrier function.

[0110] Figure 8 is a graph showing the effect of a lotion containing 4'-demethylnobiletin on skin elasticity in a human skin moisturizing test after continuous use. In Figure 8, (A) shows the measured skin elasticity value, and (B) shows the difference in skin elasticity value. The vertical axis of the graph shows skin elasticity (unit: au). Bars show standard deviation, and † indicates a trend with p<0.1 compared to the additive-free placebo group.

[0111] The test product group showed higher values, with a tendency to be higher, compared to the placebo group in terms of the measured skin elasticity values ​​after four weeks of continuous use and the difference between the values ​​before and after the test (Figure 8(A), Figure 8(B)). From this, it appears that the improvement in the skin's barrier function (see Figure 7) resulting from the continuous use of a lotion containing 4'-demethylnobiletin is reflected in the firmness of the skin.

[0112] The above results indicate that the continued use of a lotion containing 4'-demethylnobiletin has the effect of moisturizing the skin, improving dryness, improving skin elasticity (firmness), and improving the skin's barrier function.

[0113] Example 6: Formulation example of a skin external composition Formulation Example 1 Preparation of cream The following amounts of each ingredient were blended to produce a cream containing 4'-demethylnobiletin. Olive squalane 30ml 5g beeswax 5ml of 1,3-propanediol solution containing 100mg of 4'-demethylnobiletin 15ml purified water

[0114] Formulation Example 2 Preparation of ointment The raw materials were blended in the amounts shown below to produce an ointment containing 4'-demethylnobiletin. Olive squalane 30ml 5g beeswax 3 drops of tea tree essential oil 5ml of 1,3-propanediol solution containing 100mg of 4'-demethylnobiletin

[0115] Formulation Example 3: Preparation of lotion The raw materials were blended in the amounts shown below to produce a lotion containing 4'-demethylnobiletin. 7g glycerin 5ml of 1,3-propanediol solution containing 20mg of 4'-demethylnobiletin 86ml water Carbopol 0.1g

[0116] Formulation Example 4 Preparation of lotion The raw materials were blended in the amounts shown below to produce a lotion containing 4'-demethylnobiletin. 200ml water urea 5g 1ml glycerin 1ml of 1,3-propanediol solution containing 20mg of 4'-demethylnobiletin

[0117] Formulation Example 5 Preparation of Sunscreen The raw materials were blended in the amounts shown below to produce a sunscreen containing 4'-demethylnobiletin. 30ml of squalane 5g beeswax Fine particle titanium dioxide 7g 1ml of 1,3-propanediol solution containing 20mg of 4'-demethylnobiletin Purified water 70ml [Industrial Applicability]

[0118] The present invention contains 4'-demethylnobiletin, a highly safe raw material, as an active ingredient, and has the effect of promoting stratum corneum ceramide synthesis when applied to the skin. It is expected that the composition will be used particularly in cosmetics, quasi-drugs, pharmaceuticals, etc. as a topical skin composition for moisturizing the skin, preventing and improving dry skin, and preventing, treating, and improving diseases associated with a decrease in skin barrier function.

Claims

[Claim 1] The composition for external application to the skin for promoting ceramide synthesis in the stratum corneum contains 4'-demethylnobiletin as an active ingredient, and is a quasi-drug or cosmetic for moisturizing the skin and preventing or improving dry skin.

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