JP2022077501A - Skin external composition for promoting horny layer ceramide synthesis - Google Patents

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

The present invention is a composition for external use on the skin for promoting the synthesis of stratum corneum ceramide containing 4'-demethylnoviretin as an active ingredient, for moisturizing the skin by promoting the synthesis of stratum corneum, for preventing or improving the dryness of the skin, or. The present invention relates to an external composition for skin for the prevention or treatment of diseases associated with a decrease in skin barrier function.

The skin has various functions, but the most important is the barrier function that prevents the invasion of harmful substances from the outside world and the moisturizing function of the skin surface that prevents the loss of water from the body surface. However, with aging, the barrier function and the moisturizing function deteriorate, and skin troubles are likely to occur due to the dryness of the skin and the invasion of harmful substances / microorganisms (see, for example, Non-Patent Document 1).

In addition, it is known that the amount of ceramide in the dermatological and non-dermatological parts of patients with atopic dermatitis is significantly reduced as compared with healthy subjects (see, for example, Non-Patent Document 1). Furthermore, it has been clarified that age-related senile xerosis is caused not only by the conventionally known decrease in sebum but also by the decrease in ceramide (see, for example, Non-Patent Document 2).

So far, as compounds having an effect of promoting the synthesis of ceramide in the cell system, phytosphingosine in which the acyl group is removed from the ceramide (see, for example, Patent Document 1) and chiliroside of strawberry seed extract (see, for example, Non-Patent Document 3). ) Etc. have been reported. However, the effect of the ceramide synthesis promoter for the stratum corneum when applied to human skin has not been sufficiently verified, and none of them has been practically used.

On the other hand, nobiletin, which is a polymethoxyflavonoid, is a flavonoid peculiar to citrus fruits, but in recent years, various physiological actions such as cancer prevention, aging suppression, and anti-arteriosclerosis have become known.

The present inventors have clarified that nobiletin, which is one of the main components, is converted to 4'-demethylnobiletin by fermenting with a specific type of aspergillus using the peel of citrus fruits containing a large amount of nobiletin. .. The present inventors have also found that 4'-demethylnobiletin has an excellent memory-improving effect (see, for example, Patent Document 2).

Imokawa G, et al., J Invest Dermatol, 96,523-526 (1991) Akimoto K, et al., J Dermatol (Tokyo), 20,1-6 (1993) Takeda S, et al., PLoS ONE 13 (10): e0205061 (2018)

Japanese Unexamined Patent Publication No. 2020-7270 Japanese Patent No. 5667561

The present invention contains a highly safe ingredient derived from food as an active ingredient, and has a moisturizing effect on the skin, a preventive or ameliorating effect on the dryness of the skin, or a prevention or treatment of a disease associated with a decrease in the skin barrier function by promoting the synthesis of ceramide in the stratum corneum. It is an object of the present invention to provide an effective composition for external use on the skin.

As a result of further studies on the functionality of 4'-demethylnobiletin, the present inventors have surprisingly found that 4'-demethylnobiletin has a ceramide synthesis promoting effect on the stratum corneum. .. Then, it was found that when 4'-demethylnobiletin is applied to the skin due to this action, it is effective in moisturizing and preventing dryness of the skin. In addition, it was speculated that the application of 4'-demethylnobiletin to the skin is also effective for the prevention or treatment of diseases associated with the deterioration of the skin barrier function due to the decrease in the ceramide in the stratum corneum. Based on these findings, the present inventors have completed the present invention.

That is, the present invention relates to a composition for external use on the skin for promoting the synthesis of stratum corneum ceramide containing 4'-demethylnobiletin as an active ingredient.

The above-mentioned external composition for promoting the synthesis of stratum corneum ceramide is a pharmaceutical product, a non-medicinal product or a cosmetic for moisturizing the skin, preventing or improving the dryness of the skin, or preventing or treating a disease associated with a decrease in the skin barrier function. Can be.

The present invention promotes the synthesis of stratum corneum ceramide when applied to the skin by containing 4'-demethylnoviretin as an active ingredient, and has a moisturizing effect on the skin, a preventive or ameliorating effect on the dryness of the skin, and a decrease in the skin barrier function. It is possible to provide a composition for external use on the skin, which has a preventive or therapeutic effect on the diseases associated with the disease.

Since 4'-demethylnobiletin can be obtained by converting nobiletin, which is a component of citrus peel, by the fermentation method of aspergillus, the external composition for promoting stratum corneum ceramide synthesis of the present invention is highly safe and has no concern about side effects.

It is a figure which shows the effect on the stratum corneum ceramide synthesis when 4'-demethylnobiletin (DeNOB) is added to the human 3D culture epidermis model (Example 3). In the figure, (A) shows the results of total ceramide, (B) shows the results of ceramide EOS, (C) shows the results of ceramide NS / NDS, and (D) shows the results of ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate the standard deviation, and * and ** indicate that there is a significant difference at p <0.05 and p <0.01, respectively, as compared with the control without addition (Control). It is a figure which shows the effect on the stratum corneum ceramide synthesis when 4'-demethylnobiletin-containing composition (DeNOB extract) is added to the human 3D culture epidermis model (Example 3). In the figure, (A) shows the results of total ceramide, (B) shows the results of ceramide NS / NDS, and (C) shows the results of ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). Bars indicate standard deviation, and * indicates a significant difference at p <0.05 compared to the control without additives. It is a figure which shows the ceramide biosynthesis pathway and related genes. It is a figure which shows the effect on the ceramide biosynthesis-related gene when 4'-demethylnobiletin (DeNOB) is added to the human 3D culture epidermis model (Example 4). In the figure, (A) shows the result of CERS1, (B) shows the result of CERS2, and (C) shows the result of 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 control without addition (Control) is 1. Bars indicate standard deviation and * indicates a significant difference at p <0.05 compared to the control without additives. It is a figure which shows the effect on the ceramide biosynthesis-related gene when 4'-demethylnobiletin-containing composition (DeNOB extract) is added to the human 3D culture epidermis model (Example 4). In the figure, (A) shows the results of CERS1, (B) shows the results of CERS2, (C) shows the results of UGCG, and (D) shows the results of 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 control without addition (Control) is 1. The bars indicate the standard deviation, and # and * indicate a tendency at p <0.1 and a significant difference at p <0.05, respectively, as compared with the control without addition (Control). It is a figure which shows the effect on the water content of the stratum corneum in the human continuous skin moisturizing effect test of the 4'-demethylnobiletin-containing cosmetic solution (Example 5). In the figure, (A) shows the measured value of the water content of the stratum corneum, and (B) shows the difference value of the water content of the stratum corneum. The vertical axis of the graph shows the water content of the stratum corneum (unit: a.u.). The bars indicate the standard deviation, and * and ** indicate that there is a significant difference at p <0.05 and p <0.01, respectively, as compared with the placebo group without the addition. It is a figure which shows the effect on the transdermal water evaporation amount in the human continuous skin moisturizing effect test of the 4'-demethylnobiletin-containing cosmetic solution (Example 5). In the figure, (A) shows the measured value of percutaneous water evaporation, (B) shows the difference value of percutaneous water evaporation, and (C) shows the relative value of percutaneous water evaporation. The vertical axis of the graph shows the amount of transepidermal water loss (unit: g / h / m ² ). Bars indicate standard deviation, and ** and *** show significant differences at p <0.01 and p <0.001, respectively, compared to the placebo group without additives. It is a figure which shows the effect on the skin elasticity in the human continuous skin moisturizing test of the 4'-demethylnobiletin-containing cosmetic solution (Example 5). In the figure, (A) shows the measured value of skin elasticity, and (B) shows the difference value of skin elasticity. The vertical axis of the graph shows skin elasticity (unit: a.u.). Bars indicate standard deviation and † tends to be p <0.1 compared to the placebo group without additives.

Hereinafter, embodiments of the present application will be described in detail.
The present embodiment relates to a composition for external use on the skin for promoting the synthesis of stratum corneum ceramide containing 4'-demethylnobiletin as an active ingredient.

The external composition for promoting stratum corneum ceramide synthesis according to the present embodiment has a moisturizing effect on the skin, a preventive or ameliorating effect on the dryness of the skin, or a preventive or therapeutic effect on diseases associated with a decrease in the skin barrier function when applied to the skin. Therefore, it can be a drug, a non-medicinal product, or a cosmetic for the purpose of these effects.

[4'-demethylnobiletin]
Although 4'-demethylnobiletin (formula 1) is not commercially available, synthetic products can be used. Alternatively, a 4'-demethylnobiletin pure product (isolated) or a 4'-demethylnobiletin-containing composition obtained by fermentation of citrus fruits containing nobiletin, particularly aspericarp, by the method of Patent Document 2 described above. Things can be used. The method of Patent Document 2 utilizing the biological conversion of nobiletin by fermentation of Jiuqu is more convenient and cheaper than the synthetic product, and is therefore suitable.

4'-demethylnobiletin is also one of the metabolites produced after nobiletin is absorbed in the body. Citrus peel is used as a raw material for sweets such as marmalade and sugar boiled, and mandarin orange (Citrus reticulata), which has a high nobiletin content, has many years of eating experience as a chimpi, and 4'-demethylnobiletin and its composition. Is very safe.

A method for producing 4'-demethylnobiletin and a composition containing the same according to the method described in Patent Document 2 will be described below.

[Fermentation raw material]
The raw material for fermenting aspergillus is the'fruit'of citrus fruits containing nobiletin, which is a polymethoxyflavonoid (whole fruit including peel, juice, pulp, seeds, etc.), but in particular, the content of nobiletin and the effectiveness of waste From the viewpoint of utilization, it is desirable to use'pericarp'.

Further, as the type of citrus fruits, any varieties and strains (for example, ponkan, shikuwasha, tangerine, tachibana, etc.) can be used as long as they are citrus fruits containing nobiletin.

As the fermentation raw material, those containing other parts of the citrus plant (for example, leaves, buds, stems, flowers, etc.) may be used, but those containing nobiletin in terms of the content of nobiletin may be used. Is desirable.

As the citrus fruits, it is preferable to use harvested / collected raw citrus fruits or washed citrus fruits, but dried, frozen, long-term stored citrus fruits and the like can also be used.

In addition, citrus fruits may be used as they are, but it is desirable to perform any treatment of chopping, crushing, or crushing.

The process involves a wide range of actions, such as chopping citrus fruits into small pieces, chopping them into small pieces, crushing them, crushing them, and making them into powder. It can be preferably carried out by making it into a state of being chopped into large pieces of about 1 to several cm.

Furthermore, extracts (extracts, dried products) obtained by extracting nobiletin in advance from these fermentation raw materials, or those isolated as pure nobiletin can also be used.

It is preferable that these fermentation raw materials are heat-treated to sterilize various germs in the raw materials before the fermentation of Jiuqu, which will be described later.

[Jiuqu fermentation]
Examples of aspergillus that fertilize the fermented raw material include Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, and Aspergillus soya. , Aspergillus saitoi, Aspergillus usamii, Aspergillus filamentous fungus (also known as Aspergillus), and the like can be used. Further, these may be mixed and used.

Of the aspergillus, 4'-demethylnovilletin is preferably used with Aspergillus kawachii, Aspergillus awamori, Aspergillus oryzae, and Aspergillus niger. It can be obtained with a high content.

As a method of inoculating the fermented raw material with the aspergillus, spores of the aspergillus can be directly sprinkled on the fermented raw material and attached. In addition, a medium obtained by pre-fermenting the Jiuqu bacterium by liquid culture may be inoculated so as to spread over the entire fermentation raw material.

As the microbial fermentation conditions for inoculating the aspergillus into the fermentation raw material, it is desirable to carry out under aerobic conditions. Therefore, for example, a container having a bottomed cylindrical bottom and a shallow depth is suitable.

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

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

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, still more preferably 2 to. 4 days.

When this fermentation period is less than one day, sufficient 4'-demethylnobiletin cannot be obtained because the microbial fermentation by the aspergillus is hardly progressing. On the contrary, if it exceeds 14 days, the decomposition of 4'-demethylnobiletin produced by microbial conversion proceeds, and the preferable aroma derived from citrus fruits disappears.

Further, in the fermentation of Jiuqu, nobiletin is demethylated by an enzyme secreted from Jiuqu and converted into 4'-demethylnobiletin.

Therefore, instead of fermenting aspergillus, a solution is extracted from the aspergillus or the fermented product obtained after fermentation to obtain an enzyme solution containing an enzyme that demethylates nobiletin, and the enzyme is used to carry out an enzymatic reaction with the raw material. It is also possible to obtain 4'-demethylnobiletin by obtaining the reaction product.

Specifically, the enzymatic reaction can be carried out by recovering the water-dissolved product from the fermented product after the fermentation of Jiuqu and using it as a crude enzyme solution.

By performing the above-mentioned fermentation of Jiuqu, all nobiletin, which is a polymethoxyflavonoid contained in the citrus raw material, is converted to 4'-demethyl nobiletin.

Specifically, by fermenting the citrus raw material with Jiuqu, 4'-demethylnobiletin has a high content of about 0.5 to 1.5% by mass (specifically, about 1% by mass) per dry weight. A fermented product of Jiuqu can be obtained.

Therefore, the fermented aspergillus fermented product obtained here may be used as it is or processed (for example, shredded, crushed, powdered, dried, etc.) to form the stratum corneum ceramide of the present embodiment. It can be used as an active ingredient of an external composition for promoting synthesis.

[Solution extraction]
In view of purity, in the production of the external composition for promoting the synthesis of stratum corneum ceramide of the present embodiment, it is possible to obtain an extract by extracting a solution from the fermented product obtained after the fermentation of Jiuqu. desirable.

The solution extraction step can be performed directly on the fermented aspergillus, but it is obtained after the fermented aspergillus is further subjected to any treatment such as fragmentation, crushing, crushing, and powdering. It is desirable to do it for the fermented food.

As the solvent used in the solution extraction step, water, a buffer solution, an organic solvent, or a water-containing solvent thereof can be used. Examples of the organic solvent include lower fatty alcohols such as ethanol, methanol, isopropanol and butanol, acetone, ethyl acetate, chloroform and the like.

Among these solvents, water, ethanol or hydrous ethanol is particularly preferable in terms of extraction efficiency, ease of handling and safety.

Further, in particular, by performing extraction using ethanol having a final concentration of 55% or more, preferably a final concentration of 60% or more, and more preferably 80% or more, elution of polysaccharides, which are impurities, can be suppressed. '-Preferably because it can improve the extraction efficiency of demethylnobiletin.

As extraction conditions, the solvent is added in an amount of 1 to 50 times, preferably 2 to 15 times (both by mass ratio) to the raw material (preferably fragmented product), and the temperature is 0 ° C. to the boiling point of the solvent. Extraction is possible by immersion or shaking under conditions, preferably at room temperature to below the boiling point of the solvent, for 5 minutes to 1 month, preferably 20 minutes to 1 week.

The obtained extract can be made into a concentrated dry solid by freeze-drying or drying using an evaporator or the like.

Further, the solution extraction step can be performed a plurality of times with a plurality of different solvents. In particular, when the first extraction is performed with water or a low-concentration hydrous alcohol, the extraction efficiency of 4'-demethylnobiletin can be improved by performing the extraction with ethanol having a specific concentration or higher. can.

The extract (extract or concentrated dry matter) obtained as described above can be used as it is as an active ingredient of the composition for external skin for promoting the synthesis of stratum corneum ceramide of the present embodiment.

〔purification〕
Further, by performing a purification step on these, the purity can be further increased.

As a purification step, high purification can be performed by liquid-liquid separation extraction or column purification using silica gel, chemically modified silica gel, activated carbon, a synthetic adsorption resin carrier or the like. An example of suitable purification conditions is shown below.

First, a porous synthetic adsorption resin (specifically, Diaion HP20) in which an extract (specifically, an extract that has undergone ethanol extraction) is equilibrated with 33 to 41% ethanol (specifically, 40% ethanol). [Manufactured by Mitsubishi Chemical Co., Ltd.]) to be used in the column. Then, the component eluted with 39 to 43% ethanol (specifically, 43% ethanol) is removed. Next, a composition having a high content of 4'-demethylnobiletin can be obtained by recovering the components eluted with 44 to 46% ethanol (specifically, 45% ethanol).

Further, the 4'-demethylnoviretin high content composition obtained as described above is further subjected to ODS column chromatography (specifically, 60% methanol elution) and thin layer chromatography (TLC) (specifically, hexane). / Ethanol [7: 3]), ODS-HPLC (specifically, 37% [V / V] acetonitrile / water mixed solvent), and the target peak was collected to obtain pure 4'-demethylnobiletin. The article can be isolated.

The 4'-demethylnobiletin obtained as described above is a monodemethyl form of nobiletin in which the 4'position is demethylated. 4'-demethylnobiletin becomes more polar due to demethylation and is more soluble in alcohol and water than nobiletin.

In addition, 4'-demethylnobiletin has an excellent effect of promoting the synthesis of ceramide in the stratum corneum, so that it has an excellent moisturizing effect when applied to the skin and a preventive, therapeutic or ameliorating effect on diseases associated with a decrease in skin barrier function. It has.

Here, the "disease associated with the deterioration of the skin barrier function" is not particularly limited, and examples thereof include psoriasis, dry skin, xerosis, and atopic dermatitis.

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

In addition, 4'-demethylnoviretin is used for moisturizing the skin, preventing or ameliorating dryness of the skin, or for preventing or treating diseases associated with deterioration of the skin barrier function due to its ceramide synthesis promoting action. It can be used as an active ingredient in compositions, preferably pharmaceuticals, non-pharmaceutical products or cosmetics.

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

In addition, as the external composition for promoting the synthesis of ceramide in the stratum corneum, specifically, a pharmaceutical product for moisturizing the skin, preventing or improving the dryness of the skin, or preventing or treating a disease associated with a decrease in the skin barrier function. , Non-pharmaceutical products or cosmetics.

The above-mentioned 4'-demethylnobiletin can be used as various compositions obtained in the above steps ('compositions directly containing a fermented product',' solution extracts',' high-containing compositions') and'isolated products'. By mixing with the raw material, it can be used as an active ingredient of an external composition for promoting the synthesis of stratum corneum ceramide, a pharmaceutical product, a non-pharmaceutical product, or a cosmetic.

As an effective intake of 4'-demethylnoviletin, a composition for external use on the skin containing 0.001% by mass or more, preferably 0.01% by mass or more of 4'-demethylnoviletin is once or more once a day, preferably 1 When applied to the skin up to 3 times / day, excellent ceramide synthesis promoting action and skin moisturizing action can be obtained, and excellent prevention, treatment or ameliorating action for diseases associated with dry skin and deterioration of skin barrier function. Can be obtained.

Therefore, by applying the external composition for promoting stratum corneum ceramide synthesis, pharmaceuticals, quasi-drugs, or cosmetics of the present embodiment to the skin in a form and ingestion method (number of times, amount) that can secure this required amount. , It is expected that the above-mentioned pharmacological action can be obtained. However, it is desirable to appropriately determine the intake amount and application amount according to the age, weight, symptoms, intake schedule, formulation form, etc. of the subject.

In addition, the content of 4'-demethylnobiletin in the external composition for promoting the synthesis of stratum corneum ceramide, pharmaceuticals, quasi-drugs, or cosmetics may be such that the required intake can be guaranteed. However, specifically, it can be contained so as to be 0.001% by mass or more, preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. Further, as the upper limit, 20% by mass or less can be mentioned.

The form of the external composition for promoting the synthesis of ceramide in the stratum corneum, the drug, the quasi-drug, or the cosmetic may be, for example, a liquid, a gel, a cream, an ointment, or the like.

In addition to 4'-demethylnobiletin or a composition containing the same, the composition for external skin for promoting the synthesis of stratum corneum ceramide, pharmaceuticals, quasi-drugs or cosmetics of the present embodiment is within the range in which the effect of the present invention is exhibited. Therefore, various carriers, additives, other medicinal ingredients and the like may be contained.

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

<Example 1> Preparation of 4'-demethylnobiletin-containing composition A nobiletin-converted product 4'-demethylnobiletin-containing composition was prepared by fermenting ponkan peel with aspergillus according to the method described in Patent Document 2. ..

That is, 10 kg of ponkan peel was chopped into small pieces and sterilized by steaming. The obtained ponkan peel was inoculated with Aspergillus awamori (manufactured by Bioc Co., Ltd.) so as to spread throughout. Then, the fermentation treatment (jiuqu fermentation) was carried out aerobically for 4 days in a constant temperature room at 30 ° C. to obtain a fermented aspergillus.

To 6 kg of the obtained fermented aspergillus, 75 L of 80% (v / v) ethanol was added, and the extract was extracted at 100 ° C. for 1 hour to obtain an extract. The extract was subjected to Diaion HP20 (porous synthetic adsorption resin column) equilibrated with 40% (v / v) ethanol in advance, and the non-adsorbed component was removed with 3 L of 43% (v / v) ethanol. The components eluted with 10 L of 45% (v / v) ethanol were recovered. The recovered product was concentrated to dryness on a rotary evaporator to obtain a composition containing 21% of 4'-demethylnobiletin.

<Example 2> Preparation of 4'-demethylnobiletin isolate 2 g of the 4'-demethylnobiletin high-containing composition obtained above was dissolved in 20% (v / v) methanol and subjected to ODS column chromatography (ODS column chromatography). A column having an inner diameter of 20 mmφ and a length of 30 cm was packed with 30 g of Wako gel 50C18). The component eluted with 40% (v / v) methanol was removed to obtain a component eluted with 60% (v / v) methanol.

Next, the obtained components were subjected to preparative TLC chromatography (silica gel 70PF ₂₅₄ plate Wako, film thickness 0.75 mm, manufactured by Wako Pure Chemical Industries, Ltd.) under the condition of developing solvent hexane / ethanol = 7: 3, and 4'-de. Fractions containing methylnobiletin were collected while checking using a UV lamp.

Then, the obtained fraction was subjected to a preparative HPLC column (TSK GEL ODS, manufactured by Tosoh Corporation, 4.6 mm × 25 cm), and a pure 4'- was added to a moving layer of 37% (v / v) acetonitrile. 20 mg of demethylnobiletin was obtained.

<Example 3> Effect on stratum corneum ceramide synthesis using a human three-dimensional cultured epidermis model A human three-dimensional cultured epidermis model (horny layer model) using a 4'-demethylnobiletin pure product or a 4'-demethylnobiletin-containing composition. ), And the effect on the synthesis of ceramide in the stratum corneum was investigated.

As a human three-dimensional cultured epidermis model, LabCyte EPI-MODEL 6D (J-TEC) was used. The epidermis model was cultured in the attached dedicated assay medium ([EPI-MODEL] [CORNEA-MODEL] (J-TEC)) for 1 day after opening. Then, 4'-demethylnobiletin (10 μM, Example 2 preparation) or 4'-demethylnobiletin-containing composition (4.5, 9, 18 μg / mL, Example 1 preparation) was added to the above assay medium. Was cultured in the medium in which Nobiletin was dissolved, and the tissue was collected on the 6th day of the culture. The medium was exchanged every other day. As a control, the assay medium supplemented with 0.1% (volume ratio) of DMSO instead of the 4'-demethylnobiletin or 4'-demethylnobiletin-containing composition was similarly cultured.

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 (sample number: n = 3). The method for quantifying each ceramide is as follows.

The tissue collected on the 6th day of culture was treated with trypsin to separate the stratum corneum. The obtained stratum corneum was washed with PBS multiple times, and then the stratum corneum was crushed for a certain period of time with a glass homogenizer containing an extract (chloroform: methanol: PBS = 1: 2: 0.8 (volume ratio)). After crushing, the stratum corneum was transferred to a test tube, shaken with a vortex for 20 minutes, and then the precipitate and the supernatant were separated by centrifugation. The precipitate was quantified by the BCA method and used to calculate the amount of ceramide per weight of stratum corneum protein.

On the other hand, an extract (chloroform: PBS = 1: 1 (volume ratio)) was added to the above supernatant, shaken with a vortex for 20 minutes, and then the layers were separated by centrifugation. Only the lower layer is recovered with a glass syringe, the solvent is completely removed with nitrogen dry solid, and then a predetermined amount of chloroform / methanol mixed solution (chloroform: methanol = 2: 1 (volume ratio)) is added and vortexed until dissolved. Therefore, it was used as a sample solution.

For the quantification of ceramide, refer to "Experimental method for developing functional cosmetic materials-in vitro / cell / tissue culture-" (supervised by Genji Imokawa, 2007, CMC Publishing), and silica gel thin layer chromatography ( TLC) was used. The non-hydroxy ceramide and ceramide used as standard substances were purchased from Matreya and LLC, Ceramide III and Ceramide VI were purchased from Evonik Industries AG (Essen), and Ceramide Takasago International Corporation (Essen). rice field. Each standard substance was dissolved in a mixed solution of chloroform / methanol (chloroform: methanol = 2: 1 (volume ratio)) so as to be 0.2 mg / mL, and used as a standard solution.

A predetermined amount of the sample solution and the standard solution were donated 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, sprayed with a mixed solution of 10% copper sulfate and 8% phosphoric acid (each mass ratio), and heated at 180 ° C. for 6 minutes to develop a spot color. After scanning, the colored TLC plate was analyzed by Image J (Wayne Rasband), and each ceramide was quantified based on the concentration and size of each spot.

In the quantification of ceramide NS / NDS, non-hydroxy ceramide was used as a standard substance. Hydroxy ceramide was used as a standard substance for the quantification of ceramide AS. In the quantification of ceramide NP, Ceramide III was used as a standard substance. Ceramide VI was used as a standard substance in the quantification of ceramide AP. In the quantification of ceramide NDS, Ceramide TIC-001 was used as a standard substance. In the quantification of total ceramide, it was calculated as the total amount of ceramide EOS, NS, NDS, NP, EOH, AS, NH, AP and AH including the ceramide obtained above.

The results are shown in FIGS. 1 and 2. FIG. 1 is a diagram showing the effect (after 6 days) on stratum corneum ceramide synthesis when 4'-demethylnobiletin (DeNOB) was added to a human three-dimensional cultured epidermis model (Example 3).

In FIG. 1, (A) shows the results of total ceramide, (B) shows the results of ceramide EOS, (C) shows the results of ceramide NS / NDS, and (D) shows the results of ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). The bars indicate the standard deviation, and * and ** indicate that there is a significant difference at p <0.05 and p <0.01, respectively, as compared with the control without addition (Control).

By adding 4'-demethylnobiletin (Example 2 preparation), total ceramide is 1.54 times, ceramide EOS is 1.50 times, NS / NDS is 1.59 times, and ceramide NP is 1.57 times. And each increased significantly (Fig. 1).

FIG. 2 is a diagram showing the effect (after 6 days) on the ceramide synthesis of the stratum corneum when the composition containing 4'-demethylnobiletin (DeNOB) was added to the human 3D cultured epidermis model (Example 3). ).

In FIG. 2, (A) shows the results of total ceramide, (B) shows the results of ceramide NS / NDS, and (C) shows the results of ceramide NP. The vertical axis of the graph shows the amount of ceramide produced (unit: μg / mg protein). Bars indicate standard deviation and * indicates a significant difference at p <0.05 compared to the control without additives.

With the addition of the 4'-demethylnobiletin-containing composition (Example 1 preparation product), total ceramide increased 1.36 times, NS / NDS increased 1.34 times, and ceramide NP increased 1.40 times. (Fig. 2).

From the above, the effect of promoting the synthesis of ceramide in the stratum corneum of the 4'-demethylnobiletin and 4'-demethylnobiletin-containing compositions was clarified.

<Example 4> Effect on ceramide synthase gene expression using a human three-dimensional cultured epidermis model A human three-dimensional cultured epidermis model (horny layer) using a 4'-demethylnoviretin pure product or a 4'-demethylnoviretin-containing composition. It was added to the model) and its effect on the expression of the stratum corneum ceramide synthase gene was investigated. FIG. 3 is a diagram showing a ceramide biosynthesis pathway and related genes. In this example, ceramide synthase 1 (CERS1), 2 (CERS2), glucosylceramide synthase (UGCG), and sphingomyelin synthase 2 (SMS2) were examined as ceramide synthesis-related genes.

A human three-dimensional cultured epidermis model was cultured in the same manner as in Example 3, and epidermal cells were taken out by trypsin treatment from the tissues collected on the third day of culture after the addition of the evaluation substance. Here, the amount of 4'-demethylnobiletin (Example 2 preparation) added to the assay medium is 10 μM, and the amount of the 4'-demethylnobiletin-containing composition (Example 1 preparation) added is 9,18 μg / mL. And said. As a control, the assay medium supplemented with 0.1% (volume ratio) of DMSO instead of the 4'-demethylnobiletin or 4'-demethylnobiletin-containing composition was similarly cultured.

Total RNA was extracted from the obtained epidermal cells using RNeasy Mini Kit (Qiagen), and cDNA was synthesized from total RNA using River TraAce qPCR RT kit (TOYOBO). Next, using the obtained cDNA, the gene expression levels of ceramide biosynthesis-related enzymes (CERS1, CERS2, UGCG and SMS2) were quantified by the real-time RT-PCR method. For the real-time RT-PCR method, TB Green Fast qPCR Mix (TaKaRa) was used, and the primers of CERS1, CERS2, UGCG and SMS2 were those having the base sequences shown in Table 1.

GAPDH was used as an internal standard. The operation of real-time RT-PCR was performed according to a predetermined method, and the expression level of CERS1, CERS2, UGCG and SMS2 mRNA was determined as a ratio to the expression level of GAPDH mRNA, which is an internal standard. The expression rates of CERS1, CERS2, UGCG and SMS2 are the ratio of the mRNA expression level of the 4'-demethylnobiletin or 4'-demethylnobiletin-containing composition-added group to the mRNA expression level of the control (DMSO 0.1%). Calculated (number of samples: n = 3).

The results are shown in FIGS. 4 and 5. FIG. 4 is a diagram showing the effect (after 3 days) on ceramide-related genes when 4'-demethylnobiletin (DeNOB) is added to a human three-dimensional cultured epidermis model. In FIG. 4, (A) shows the result of CERS1, (B) shows the result of CERS2, and (C) shows the result of 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 control without addition (Control) is 1. Bars indicate standard deviation, and * indicates a significant difference at p <0.05 compared to the control without additives. The addition of 4'-demethylnobiletin (Example 2 preparation) showed a significant increase in the expression levels of CERS1, CERS2, and UGCG (FIG. 4).

FIG. 5 is a diagram showing the effect on ceramide-related genes when a 4'-demethylnobiletin (DeNOB) -containing composition is added to a human three-dimensional cultured epidermis model. In FIG. 5, (A) shows the results of CERS1, (B) shows the results of CERS2, (C) shows the results of UGCG, and (D) shows the results of 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 control without addition (Control) is 1. The bars indicate the standard deviation, and # and * indicate a tendency at p <0.1 and a significant difference at p <0.05, respectively, as compared with the control without addition (Control). The addition of the 4'-demethylnobiletin-containing composition (Example 1 preparation) showed a significant increase in the expression levels of CERS1, CERS2, UGCG, and SMS2 (FIG. 5).

From the above, it is confirmed that the addition of the 4'-demethylnobiletin or 4'-demethylnobiletin-containing composition to the cultured cells enhanced the ceramide synthase activity, thereby promoting the ceramide synthesis in the stratum corneum. Was done.

<Example 5> Human continuous skin moisturizing effect test of 4'-demethylnobiletin-containing cosmetic solution The test product (4'-demethylnobiletin prepared in Example 2) was applied to the half face of the face of 10 subjects. A lotion containing 0.005% (mass ratio)) and a placebo product (a lotion containing no 4'-demethylnoviretin) as a control were continuously used twice a day in the morning and evening, 0.25 mL each. .. The condition and changes of the test site were measured and evaluated three times before the start of the test, after continuous use for 2 weeks, and after continuous use for 4 weeks. Table 2 below shows the composition of the test product and the placebo product.

That is, the subject was kept in a room where the temperature was 21.0 ± 0.3 ° C. and the relative humidity was 45.0 ± 2.0% after washing the face with a commercially available cleansing and washing pigment every three observation days described above. After spending a rest period of about 20 minutes in the test environment and acclimatizing the skin to the test environment, measure the skin moisture content, transepidermal moisture evaporation, and skin elasticity as follows to improve skin moisturization and dryness. The effect was evaluated.

The water content of the stratum corneum: The water content of the stratum corneum was measured three times by Corneometer CM825 (Courage + Khazaka, Germany), and the average value was taken as the water content of the stratum corneum (unit: a.u.) Of the subject.

-Transdermal water evaporation amount: Measured by Tewameter TM300 (Courage + Khazaka, Germany), and the measured value was taken as the percutaneous water evaporation amount (unit: g / h / m ² ) of the subject.

-Skin elasticity: Measured twice with a Cutometer CT580 (probe opening 2.0 mm, negative pressure 150 mbar, Course + Khazaka, Germany), and the average value of "R7" is the skin elasticity of the subject (unit: a. u.).

The results are shown in FIGS. 6 and 7. FIG. 6 is a diagram showing the effect of a 4'-demethylnobiletin-containing cosmetic solution on the water content of the stratum corneum in a human continuous skin moisturizing test. In FIG. 6, (A) shows the measured value of the water content of the stratum corneum, and (B) shows the difference value of the water content of the stratum corneum. The vertical axis of the graph shows the water content of the stratum corneum (unit: a.u.). The bars indicate the standard deviation, and * and ** indicate that there is a significant difference at p <0.05 and p <0.01, respectively, as compared with the placebo group without the addition.

In the measured values of the water content of the stratum corneum after continuous use after 2 weeks and 4 weeks, and the difference values before and after the test, the test product-applied group showed significantly higher values than the placebo group (FIG. 6 (A), FIG. 6 (B)). From this, it was considered that the continuous use of the 4'-demethylnobiletin-containing cosmetic solution promoted the synthesis of ceramide in the stratum corneum and made the skin more smooth.

FIG. 7 is a diagram showing the effect of a 4'-demethylnobiletin-containing cosmetic solution on the amount of transepidermal water loss in a human continuous skin moisturizing test. In FIG. 7, (A) shows the measured value of percutaneous water evaporation, (B) shows the difference value of percutaneous water evaporation, and (C) shows the relative value of percutaneous water evaporation. The vertical axis of the graph shows the amount of transepidermal water loss (unit: g / h / m ² ). Bars indicate standard deviation, and ** and *** show significant differences at p <0.01 and p <0.001, respectively, compared to the placebo group without additives.

In the measurement of the amount of transepidermal water loss after continuous use for 4 weeks, the test product-applied group showed a significantly lower value than the placebo group (FIG. 7 (A)). In addition, the difference value and relative value of the transdermal water evaporation amount after continuous use for 2 weeks and 4 weeks before and after the test showed significantly lower values in the test product application group than in the placebo group (FIG. 7 (B). ), FIG. 7 (C)). From the above, it was considered that the continuous use of the 4'-demethylnobiletin-containing cosmetic solution promoted the synthesis of ceramide in the stratum corneum and improved the barrier function of the skin.

FIG. 8 is a diagram showing the effect of a 4'-demethylnobiletin-containing cosmetic solution on skin elasticity in a human continuous skin moisturizing test. In FIG. 8, (A) shows the measured value of skin elasticity, and (B) shows the difference value of skin elasticity. The vertical axis of the graph shows skin elasticity (unit: a.u.). Bars indicate standard deviation and † tends to be p <0.1 compared to the placebo group without additives.

In the measured values of skin elasticity after continuous use for 4 weeks and the difference values before and after the test, the test product-applied group showed higher values with a tendency difference than the placebo group (FIGS. 8 (A) and 8 (B)). )). From this, it is considered that the improvement of the barrier function of the skin (see FIG. 7) due to the continuous use of the 4'-demethylnobiletin-containing cosmetic solution appeared in the firmness of the skin.

From the above results, continuous use of 4'-demethylnobiletin-containing cosmetic solution has the effects of improving skin moisturization and dryness, improving skin elasticity (tension), and improving skin barrier function. Was shown.

<Example 6> Example of prescription of external composition for skin Formulation Example 1 Preparation of cream Each raw material was blended in the following amounts to produce a cream containing 4'-demethylnobiletin.
Olive squalane 30ml
Beeswax 5g
4'-Demethylnobiletin 100 mg containing 1,3-propanediol solution 5 ml
Purified water 15ml

Formulation Example 2 Preparation of ointment Each raw material was blended in the following amounts to produce an ointment containing 4'-demethylnobiletin.
Olive squalane 30ml
Beeswax 5g
3 drops of tea tree essential oil
4'-Demethylnobiletin 100 mg containing 1,3-propanediol solution 5 ml

Formulation Example 3 Preparation of lotion Each raw material was blended in the following amounts to produce a lotion containing 4'-demethylnobiletin.
Glycerin 7g
4'-Demethylnobiletin 20 mg containing 1,3-propanediol solution 5 ml
86 ml of water
Carbo-mer 0.1g

Formulation Example 4 Preparation of cosmetic water Each raw material was blended in the following amounts to produce a lotion containing 4'-demethylnobiletin.
200 ml of water
Urea 5g
Glycerin 1ml
1 ml of 1,3-propanediol solution containing 20 mg of 4'-demethylnobiletin

Formulation Example 5 Preparation of sunscreen agent Each raw material was blended in the following amounts to produce a sunscreen preparation containing 4'-demethylnobiletin.
Squalene 30ml
Beeswax 5g
Fine particle titanium dioxide 7g
1 ml of 1,3-propanediol solution containing 20 mg of 4'-demethylnobiletin
Purified water 70ml

The present invention has a stratum corneum ceramide synthesis promoting effect when applied to the skin by containing 4'-demethylnoviletin, which is a highly safe raw material, as an active ingredient, for moisturizing the skin and drying the skin. It is expected to be used as an external composition for prevention / improvement and for prevention / treatment / improvement of diseases associated with deterioration of skin barrier function, particularly for cosmetics, non-medicinal products, pharmaceuticals and the like.

Claims (2)

A composition for external use on the skin for promoting the synthesis of stratum corneum ceramide containing 4'-demethylnobiletin as an active ingredient. The promotion of stratum corneum ceramide synthesis according to claim 1, which is a drug, a non-medicinal product or a cosmetic for moisturizing the skin, preventing or improving the dryness of the skin, or preventing or treating a disease associated with a decrease in the skin barrier function. External composition for skin.

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