Claudin-1 gene expression promoter

Abstract

To provide a claudin-1 gene expression promoter.SOLUTION: A claudin-1 gene expression promoter contains one or two or more types of extracts from Sargassum horneri, Undaria pinnatifida, Patiria pectinifera and Angelica furcijuga as active ingredients. The claudin-1 gene expression promoter has the effect of enhancing a tight junction barrier function, thus enhancing a skin barrier function.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a claudin-1 gene expression promoter comprising a component having an effect of promoting claudin-1 gene expression. More specifically, the present invention relates to a claudin-1 gene expression promoter containing one or more active ingredients selected from extracts obtained from Akamoku, Mekabu, Asterina pectinifera, and Hyuugatouki. [Background technology]

[0002] In recent years, various functions have been required for topical skin preparations, and new substances and new methods have been investigated and developed. One of these is that ingredients in topical skin preparations used for moisturizing purposes are required to be as effective as or more effective than conventionally known active ingredients, or even safer.

[0003] One of the important functions of the skin is its barrier function. This function maintains homeostasis, protects the body from physical and chemical stimuli and microorganisms, and prevents leakage of moisture and essential components from the body. Barrier functions include physical barriers, chemical barriers, and immunological barriers. It has been discovered that the tight junctions present in the stratum corneum, the outermost layer of the epidermis, and its inner granular layer, play a major role in the physical barrier.

[0004] The stratum corneum has a barrier structure (stratum corneum barrier) composed of keratinocytes, which are epidermal cells enucleated in the final stage of keratinization, and the intercellular lipids that fill the spaces between them. This stratum corneum barrier separates the air environment from the body's internal fluid environment, protecting the cells inside from damage caused by dryness and external forces. Tight junctions in the granular layer are composed of four-transmembrane proteins, such as claudins and occludin, and the membrane-enhancing protein ZO-1. These membrane-spanning molecules form particles that connect adjacent cells, which then polymerize into string-like structures, forming a continuous, mesh-like structure that surrounds the cell periphery. This structure acts as a barrier that selectively restricts the movement of substances through the intercellular space (Non-Patent Document 1). A weakened skin barrier function can cause pathological symptoms such as atopic dermatitis, as well as make skin problems such as dry skin, sensitive skin, and rough skin more likely to occur, so controlling the skin barrier function is also an important cosmetic issue (Non-Patent Document 2).

[0005] To date, various approaches have been proposed for improving skin barrier function, both from the inside and outside of the skin, such as topical application of ceramide or ceramide analogs alone or in combination with other intercellular lipid components such as cholesterol or fatty acids, and methods for promoting the synthesis of intercellular lipids such as ceramide in the epidermis (Non-Patent Document 2). Skin barrier function improvers that focus on tight junctions include claudin production promoters and occludin production promoters containing extracts of specific plants or royal jelly (Patent Document 1), and a claudin production promoter whose active ingredient is a pearl solubilizer (Patent Document 2). However, the development of ingredients that focus on tight junction barrier function is still insufficient, and the development of new, more effective substances is desired. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-256244 [Patent Document 2] Japanese Patent Application Publication No. 2015-091781 [Non-patent literature]

[0007] [Non-Patent Document 1] COSMETIC STAGE Vol.11,No.1:61-66(2016) [Non-patent document 2] Chemistry and Biology Vol.46, No.2:135-141(2008) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a novel claudin-1 gene expression promoter that enhances the tight junction barrier function and improves the skin barrier function by promoting the expression of tight junctions. [Means for solving the problem]

[0009] In order to solve these problems, the present inventors conducted an intensive search for components that promote the expression of claudin genes, and as a result, discovered that extracts obtained from Sargassum horneri, Mekabu, Starfish pectinifera and Angelica acutiloba effectively function as promoters of claudin-1 gene expression, leading to the completion of the present invention.

[0010] That is, the present invention relates to the following inventions. <1> A claudin-1 gene expression promoter containing one or more active ingredients selected from extracts obtained from Akamoku, Mekabu, Pectinifera starfish, and Hyuugatouki. <2> The above-mentioned wherein the active ingredient is an extract obtained from Akamoku or Mekabu. <1> The claudin-1 gene expression promoter described in [Effects of the Invention]

[0011] The present invention provides a novel claudin-1 gene expression promoter. The claudin-1 gene expression promoter of the present invention contains extracts of naturally derived Sargassum serrata, Mekabu serrata, Starfish pectinifera pectinifera, and Angelica acutiloba as active ingredients, and is expected to effectively prevent and improve skin conditions such as dry skin, rough skin, atopic dermatitis, and various infectious diseases. DETAILED DESCRIPTION OF THE INVENTION

[0012] The following describes in detail the embodiments of the present invention. However, the description of the constituent elements described below is an example (representative example) of an embodiment of the present invention, and the present invention is not limited to the following content as long as it does not exceed the gist of the present invention.

[0013] The claudin-1 gene expression promoter of the present invention contains extracts obtained from Sargassum serrata, Mekabu, Starfish pectinifera, and Angelica acutiloba as active ingredients. That is, the claudin-1 gene expression promoter of the present invention contains at least one selected from extracts of Sargassum serrata, Mekabu, Starfish pectinifera, and Angelica acutiloba (hereinafter sometimes referred to as "extracts").

[0014] Claudins are one of the main proteins involved in the formation of tight junctions at intercellular junctions. To date, 27 members of the claudin family have been reported in humans and mice. Claudin-1 knockout mice die shortly after birth due to dehydration, a subcutaneously administered tracer for intercellular mass transport does not penetrate above the granular layer in normal mice, but penetrates above this layer in the epidermis of claudin-1 knockout mice, and claudin-1 expression is reduced in patients with atopic dermatitis. These findings suggest that claudin-1 plays a key role in the formation of the barrier in the granular layer of the epidermis.

[0015] Next, the raw materials for the active ingredients of the claudin-1 gene expression promoter of the present invention, namely, Sargassum serrata, Mekabu, Starfish pectinifera and Angelica acutiloba, and their extracts (extracts) will be specifically described.

[0016] (1) Akamoku is a seaweed of the Sargassaceae family, and its scientific name is Sargassum horneri. The Akamoku extract used in the present invention is an extract obtained by extracting Akamoku with water and purifying it using cellulase as appropriate during the extraction process.

[0017] (2) Mekabu is the sporophyll of wakame seaweed. Wakame is a seaweed of the Polytrichum genus, and its scientific name is Undaria pinnatifida. It consists of vegetative leaves and sporophyll, and the vegetative leaves are generally eaten. Mekabu refers to the sporophyll, which is the reproductive body formed at the bottom of the wakame stem. The mekabu extract used in the present invention is an extract obtained by extracting the reproductive body (sporophyll) of mature wakame seaweed with water.

[0018] (3) The starfish Pectinifera is a starfish of the family Pectiniferidae in the class Asteroidea, and its scientific name is Patiria pectinifera. The starfish extract used in the present invention is an extract obtained by extracting the starfish Pectinifera with water and purifying it using a protease as appropriate during the extraction process.

[0019] (4) Hyuugatouki is a plant of the Umbelliferae family, and its scientific name is Angelica furcijuga. The Hyuugatouki extract of the present invention is an extract obtained by extracting the above-ground parts of Hyuugatouki, particularly preferably the leaves and stems, with a 1,3-butylene glycol solution.

[0020] Next, the methods for producing the extracts (extracts) of Akamoku, Mekabu, Asterina pectinifera, and Angelica acutiloba will be described in detail.

[0021] <Akamoku extract> The term "Sargassum horneri extract" used in the present invention refers to an extract obtained by extracting Sargassum horneri with water under certain conditions. A suitable method for producing the Sargassum horneri extract, which is one of the active ingredients of the present invention, is described below. A 1.25% aqueous solution of sodium citrate is added to the raw material, Akamoku, in a ratio of Akamoku (weight):1.25% aqueous solution of sodium citrate (weight) of 1:2 to 1:100, and the mixture is stirred at a temperature of 5 to 90°C for 1 to 48 hours for extraction. The pH of this extract is adjusted to 3.0 to 6.0, and a cellulolytic enzyme is added to decompose the cellulosic material, followed by filtration. Impurities that adversely affect quality are dissolved and removed by adding 1 to 5 times the weight of ethanol to the filtrate, and a precipitate (of Akamoku) is obtained by centrifugation or other methods. The resulting precipitate is dissolved in water and purified by re-filtration to obtain the Akamoku extract of the present invention. In this extraction step, it is preferable to carry out an enzyme treatment to further improve the extraction efficiency of the active ingredient, and cellulase is preferably used as the enzyme.

[0022] <Wedaka extract> The mekabu (Undaria pinnatifida) extract of the present invention refers to an extract obtained by extracting mekabu with water under certain conditions. A preferred method for producing the mekabu extract, which is the active ingredient of the present invention, is described below. A 1% aqueous solution of sodium citrate is added to the raw material, Mekabu, in a ratio of Mekabu (by weight):1% aqueous solution of sodium citrate (by weight) of 1:1 to 1:100, and the mixture is stirred for 5 hours or more for extraction at a temperature of 40 to 60°C. This extract is then heated at approximately 120°C for 10 to 40 minutes, filtered, and purified to obtain the Mekabu extract of the present invention.

[0023] <Starfish extract> The starfish (Patiria pectinifera) extract of the present invention refers to an extract obtained by extracting the starfish with water under certain conditions. A suitable method for producing the starfish extract, which is the active ingredient of the present invention, is described below. First, water is added to the starfish (raw material) at a ratio of starfish (weight):water (weight) of 1:1 to 1:100, and extraction is carried out at room temperature for about 10 to 24 hours, preferably about 1 to 3 hours. The filtrate is then adjusted to pH 3.0 to 5.0 and filtered. Next, the pH of this filtrate is adjusted to pH 8.0, protease is added to decompose the protease, and the pH is further adjusted to pH 6.0 and ultrafiltered to obtain the starfish extract of the present invention. It is preferable to use live starfish (Patiria pectinifera) as the starting material in order to suppress the peculiar odor.

[0024] <Hyugatouki extract> The "Hyugatouki (Angelica furcijuga) extract" of the present invention refers to an extract obtained by extracting Hyugatouki with a 1,3-butylene glycol solution under certain conditions. A suitable method for producing the Hyugatouki extract, the active ingredient of the present invention, is described below. The 1,3-butylene glycol solution is added to the raw material, Hyuuga Touki, in a ratio of Hyuuga Touki (weight):1,3-butylene glycol solution (weight) of 1:1 to 1:100, and the mixture is stirred and extracted at 40 to 80°C for 1 to 3 hours, followed by filtration and purification to obtain the Hyuuga Touki extract of the present invention. The starting material, Hyuugatouki (Angelica furcijuga), is preferably used in the form of above-ground parts, particularly leaves and stems.

[0025] The extracts of Sargassum horneri, Mekabu, Starfish pectinifera and Angelica acutiloba of the present invention (hereinafter simply referred to as "extracts of Sargassum horneri, etc." or "extracts of Sargassum horneri, etc.") have the effect of promoting claudin-1 gene expression, and by promoting the expression of tight junctions, strengthen the tight junction barrier function and have the effect of enhancing skin barrier function.

[0026] Among the extracts of Sargassum horneri and the like according to the present invention, extracts of Sargassum horneri and Mekabu are highly effective in promoting the expression of the claudin-1 gene and can be preferably used.

[0027] The content of the extract of Sargassum horneri etc. in the claudin-1 gene expression promoter of the present invention is not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the extract of Sargassum horneri etc. may be contained in the claudin-1 gene expression promoter by one kind or by two or more kinds. Furthermore, the extract of Sargassum horneri etc. may contain other components within the scope of the present invention, such as physiological saline for adjusting the concentration of the extract.

[0028] Furthermore, the claudin-1 gene expression promoter of the present invention can be used in any known form as a quasi-drug or cosmetic, such as a cream, lotion, emulsion, essence, or pack, as long as it is suitable for topical application, without any particular limitation. [Example]

[0029] The present invention will now be described with reference to examples. However, the disclosure of these examples is intended to illustrate preferred embodiments of the present invention and is not intended to limit the present invention in any way.

[0030] <Production Example 1> Production of Akamoku extract To 10 kg of Akamoku, 500 g of trisodium citrate dihydrate and 39.5 kg of purified water were added, and the mixture was extracted by stirring at 50°C for 5 hours, and the pH was adjusted to 4.5 with 10% lactic acid. Next, the mixture was enzymatically hydrolyzed using 0.004% by weight of cellulase (manufactured by MP Biomedicals, trade name: CELLULASE) by stirring at 50°C for 24 hours, after which the enzyme was inactivated at 105°C for 10 minutes and filtered. Two times the weight of ethanol was added to the filtrate, and the resulting precipitate was dissolved in 50 kg of purified water and filtered to obtain Akamoku extract.

[0031] <Production Example 2> Production of wakame seaweed extract 5 kg of sliced ​​Mekabu was added with 320 g of trisodium citrate dihydrate and 32.7 kg of purified water, and the mixture was extracted by stirring at 50°C for 5 hours. 28 kg of purified water was then added and the mixture was heated at 120°C for 35 minutes. The extract was then cooled, and 0.3% by weight of activated carbon was added. The mixture was stirred at room temperature for 30 minutes and then filtered. 0.5% by weight of phenoxyethanol and 3% by weight of 1,3-butylene glycol were added to the filtrate and stirred. This liquid was then filtered to obtain Mekabu extract.

[0032] <Production Example 3> Production of starfish extract 20 kg of purified water was added to 10 kg of live starfish, and the mixture was immersed at room temperature for 3 hours for extraction. The pH of the extract was then adjusted to 4.0 with 10% lactic acid and filtered. The pH of the resulting filtrate was adjusted to 8.0 with 5% sodium hydroxide solution, and enzymatically hydrolyzed with Actinase (registered trademark; Kaken Pharmaceutical Co., Ltd., product name: Actinase E) by stirring at 50°C for 3 hours. The liquid was then cooled, the pH was adjusted to 6.0 with 10% lactic acid, and ultrafiltered to obtain a starfish extract.

[0033] <Production Example 4> Production of Hyuugatouki extract 40 kg of 30% 1,3-butylene glycol solution was added to 2.0 kg of dried ground Hyuugatouki leaves and stems, and the mixture was extracted by stirring at 60°C for 3 hours and then filtered. Activated carbon (0.1% by weight based on the total weight of the filtrate) was added, stirred at room temperature for 30 minutes, and then filtered to obtain Hyuugatouki extract.

[0034] <Production Example 5> Production of peony extract 44 kg of purified water was added to 1.76 kg of crushed peony root, and the mixture was extracted by stirring at 40°C for 3 hours, followed by ultrafiltration and resin treatment to obtain a peony extract. <Production Example 6> Production of Job's Tears Extract 45 kg of purified water was added to 4.5 kg of crushed Job's tears seeds from which the seed coat had been removed, and the mixture was extracted by stirring at 40° C. for 3 hours, followed by ultrafiltration to obtain a Job's tears extract.

[0035] <Production Example 7> Production of kabosu extract To prepare the kabosu extract, 34 kg of kabosu juice was filtered, and activated carbon was added in an amount of 0.1% by weight based on the total weight of the obtained filtrate. The mixture was stirred at room temperature for 30 minutes and then filtered to obtain an extract.

[0036] Example 1 The extracts obtained in the above <Production Example 1> to <Production Example 4> were used as samples to examine the promoting effect on claudin-1 gene expression by the following test method.

[0037] (1) Test method HaCaT cells (human epidermal keratinocyte cell line) were placed in a 6-cm dish at 2.5 × 10 5 The cells were seeded and cultured in 10% FBS-containing D-MEM medium at 5% CO2 and 37°C until they reached 70-80% confluence. After confirming that the cells were 70-80% confluent, the medium was replaced with FBS-free D-MEM and cultured for approximately 24 hours, followed by a sample-containing FBS-free D-MEM culture and further culture for 24 hours. Thereafter, the cells were washed with PBS(-) and recovered using a cell lysis solution, ISOGEN II (Nippon Gene Co., Ltd.) Water was added to this cell lysis solution, which was then centrifuged to recover the supernatant. The collected supernatant was precipitated with ethanol and washed to isolate total RNA, and cDNA was synthesized using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo Co., Ltd.). Using the synthesized cDNA as a template, real-time PCR was performed using primers for the claudin-1 gene (Invitrogen) listed in the Sequence Listing (SEQ ID NOs: 3 and 4) and TB Green Premix Ex Taq II (Takara Bio Inc.), and gene expression was compared using the comparative Ct method (ΔΔCt method). The GAPDH primers listed in the Sequence Listing (SEQ ID NOs: 1 and 2) were used as an internal standard. From the results obtained, the claudin-1 gene expression rate (%) of each sample was calculated as the claudin-1 gene expression promotion rate (%), assuming that the claudin-1 gene expression rate without sample addition was 100%.

[0038] (2) Test results The test results are shown in Table 1.

[0039] [Table 1]

[0040] As can be seen from Table 1, it was confirmed that extracts of Akamoku, Mekabu, Pectinifera starfish, and Hyuugatouki have the effect of promoting claudin-1 gene expression.

[0041] (Comparative Example 1) The claudin-1 gene expression promoting activity of the extracts of peony, Job's tears, and kabosu obtained in the above <Production Examples 5> to <Production Examples 7> was investigated using the test method of Example 1. The test results are shown in Table 2.

[0042] [Table 2]

[0043] As can be seen from Table 2, the extracts of peony, Job's tears, and kabosu did not show any clear promoting effect of 105% or more on claudin-1 gene expression. [Industrial Applicability]

[0044] The claudin-1 gene expression promoter of the present invention is expected to promote the expression of claudin-1, thereby strengthening the tight junction barrier function and thereby enhancing the skin barrier function, thereby preventing and improving skin problems such as dry skin, sensitive skin, and rough skin. Furthermore, the claudin-1 gene expression promoter of the present invention contains naturally derived extracts as active ingredients, and is therefore highly safe.

Claims

[Claim 1] A claudin-1 gene expression promoter characterized by containing as an active ingredient an extract of Hyuugatouki obtained by extracting the leaves and stems with a 1,3-butylene glycol solution.

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