JP2024042188A - OCCLUDIN mRNA EXPRESSION PROMOTER, CLAUDIN-1 mRNA EXPRESSION PROMOTER, CLAUDIN-4 mRNA EXPRESSION PROMOTER, TIGHT JUNCTION PROTEIN-1 mRNA EXPRESSION PROMOTER, TIGHT JUNCTION PROTEIN-2 mRNA EXPRESSION PROMOTER AND BARRIER FUNCTION ENHANCER - Google Patents
OCCLUDIN mRNA EXPRESSION PROMOTER, CLAUDIN-1 mRNA EXPRESSION PROMOTER, CLAUDIN-4 mRNA EXPRESSION PROMOTER, TIGHT JUNCTION PROTEIN-1 mRNA EXPRESSION PROMOTER, TIGHT JUNCTION PROTEIN-2 mRNA EXPRESSION PROMOTER AND BARRIER FUNCTION ENHANCER Download PDFInfo
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Abstract
Description
本発明は、オクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤に関するものである。 The present invention relates to an occludin mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, a tight junction protein-1 mRNA expression promoter, a tight junction protein-2 mRNA expression promoter, and a barrier function enhancer. It is.
生体においてその内外を隔てる構造の一つに、上皮細胞から構成される上皮組織がある。上皮組織は、物質透過を制御するバリア機能を有しており、これにより生体において外界とは異なる内部環境を作り上げている。このようなバリア機能は、主に細胞間の接着により形成されるが、かかる接着の一つがタイトジャンクション（以下「ＴＪ」という場合がある。）である。ＴＪは、隣接する上皮細胞同士を密着させるだけでなく、細胞と細胞との隙間をシールすることで物質の透過を制御する細胞間接着構造である。ＴＪを構成しているのは、細胞膜タンパク質であるクローディン（ＣＬＤＮ）やオクルディン（ＯＣＬＮ）、裏打ちタンパク質であるタイトジャンクションプロテイン－１（ＴＪＰ－１，ＺＯ－１（Zonulaoccludens-1））やタイトジャンクションプロテイン－２（ＴＪＰ－２，ＺＯ－２（Zonulaoccludens-2））等であり、これらのタンパク質はＴＪストランドの骨格を構成し、ＴＪのバリア機能を制御すると考えられている（非特許文献１参照）。 One of the structures that separates the inside and outside of a living body is epithelial tissue composed of epithelial cells. Epithelial tissue has a barrier function that controls substance permeation, and thereby creates an internal environment in living organisms that is different from the external environment. Such a barrier function is mainly formed by adhesion between cells, and one such adhesion is a tight junction (hereinafter sometimes referred to as "TJ"). TJs are intercellular adhesion structures that not only bring adjacent epithelial cells into close contact with each other, but also control the permeation of substances by sealing the gaps between cells. TJs are composed of cell membrane proteins claudin (CLDN) and occludin (OCLN), lining proteins tight junction protein-1 (TJP-1, ZO-1 (Zonulaoccludens-1)), and tight junction proteins. Protein-2 (TJP-2, ZO-2 (Zonulaoccludens-2)), etc., and these proteins constitute the skeleton of TJ strands and are thought to control the barrier function of TJs (see Non-Patent Document 1). ).
現在まで、クローディンとしては２０種以上のクローディン分子が報告され、クローディンファミリーが形成されている。これらのクローディンが組織特異的な発現パターンを示すことが分かっており、表皮においてはクローディン－１（ＣＬＤＮ－１）及びクローディン－４（ＣＬＤＮ－４）が発現している。クローディン－４は、粘膜上皮においても多く発現しており、身体の内外で異物の侵入を防ぐバリアとして機能している。 To date, more than 20 types of claudin molecules have been reported, forming the claudin family. It is known that these claudins exhibit tissue-specific expression patterns, and claudin-1 (CLDN-1) and claudin-4 (CLDN-4) are expressed in the epidermis. Claudin-4 is also highly expressed in mucosal epithelium, and functions as a barrier to prevent foreign substances from entering inside and outside the body.
クローディン、オクルディン、タイトジャンクションプロテイン－１、タイトジャンクションプロテイン－２等の発現が何らかの原因で減少した場合、ＴＪの機能低下を引き起こす。例えば、消化管においてＴＪの機能が低下すると、食物アレルゲンや病原性微生物等が体内へ侵入してしまい、炎症性腸疾患や各種感染症等の一因になると考えられる。また、従来、皮膚のバリア機能は角質層のみが担っていると考えられていたが、近年、表皮顆粒層に存在するＴＪの構成タンパク質を遺伝子レベルで欠損させると皮膚のバリア機能が崩壊することが見いだされ、ＴＪも皮膚のバリア機能に重要な役割を担うと考えられるようになっている（非特許文献２参照）。クローディン、オクルディン、タイトジャンクションプロテイン－１、タイトジャンクションプロテイン－２等の発現が何らかの原因で減少した場合、ＴＪの構造的な破壊が起こり、物質の透過バリアとして機能しなくなることによって、乾燥肌、荒れ肌、アトピー性皮膚炎や各種感染症等の皮膚症状の一因になると考えられる。 If the expression of claudin, occludin, tight junction protein-1, tight junction protein-2, etc. decreases for some reason, TJ function decreases. For example, when TJ function in the gastrointestinal tract decreases, food allergens, pathogenic microorganisms, and the like may invade the body, which is considered to be a contributing factor to inflammatory bowel disease and various infectious diseases. In addition, it was previously thought that the skin's barrier function was solely carried out by the stratum corneum, but in recent years, it has been shown that the skin's barrier function collapses when the constituent proteins of TJ, which are present in the granular layer of the epidermis, are deleted at the genetic level. has been discovered, and TJs are now considered to play an important role in the barrier function of the skin (see Non-Patent Document 2). If the expression of claudin, occludin, tight junction protein-1, tight junction protein-2, etc. decreases for some reason, structural destruction of TJs occurs and they no longer function as a permeation barrier for substances, resulting in dry skin, It is thought to be a contributing factor to skin symptoms such as rough skin, atopic dermatitis, and various infections.
そのため、クローディン、オクルディン、タイトジャンクションプロテイン－１、タイトジャンクションプロテイン－２の産生促進等を通じてＴＪの機能を強化することで、上皮組織におけるバリア機能を強化し、消化管においては炎症性腸疾患や食物アレルギー、各種感染症等を予防又は改善することができ、一方表皮においては乾燥肌、荒れ肌、アトピー性皮膚炎や各種感染症等の皮膚症状を予防又は改善することができると考えられる。クローディン産生促進作用及びオクルディン産生促進作用を有するものとして、例えば、アスパラサスリネアリス抽出物（特許文献１参照）等が知られている。また、タイトジャンクションプロテイン－１産生促進作用及びタイトジャンクションプロテイン－２産生促進作用を有するものとして、例えば、アスチルビン（特許文献２参照）等が知られている。 Therefore, by strengthening TJ function by promoting the production of claudin, occludin, tight junction protein-1, and tight junction protein-2, the barrier function in epithelial tissues is strengthened, and in the gastrointestinal tract, inflammatory bowel disease and Food allergies, various infectious diseases, etc. can be prevented or improved, and on the other hand, in the epidermis, it is thought that skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infectious diseases can be prevented or improved. For example, Aspalathus linearis extract (see Patent Document 1) is known as having an effect of promoting claudin production and an effect of promoting occludin production. In addition, astilbin (see Patent Document 2), for example, is known as having an effect of promoting the production of tight junction protein-1 and tight junction protein-2.
本発明は、安全性の高い天然物の中からオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用及びバリア機能亢進作用を有する物質を見出し、それを有効成分とするオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤を提供することを目的とする。 The present invention provides occludin mRNA expression promoting effects, claudin-1 mRNA expression promoting effects, claudin-4 mRNA expression promoting effects, tight junction protein-1 mRNA expression promoting effects, and tight junction protein-2 mRNA expression effects from highly safe natural products. An occludin mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, a tight junction protein-1 mRNA expression promoter, which has been found to have a promoting effect and a barrier function-enhancing effect, and uses the substance as an active ingredient. The purpose of the present invention is to provide an agent for promoting tight junction protein-2 mRNA expression and an agent for enhancing barrier function.
上記課題を解決するため、本発明のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、β－１，３－グルカンを有効成分として含有する。 In order to solve the above problems, the present invention provides an occludin mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, a tight junction protein-1 mRNA expression promoter, a tight junction protein-2 mRNA expression promoter, and The barrier function enhancer contains β-1,3-glucan as an active ingredient.
本発明によれば、優れたオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用及びバリア機能亢進作用を有し、安全性の高いオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤を提供することができる。 According to the present invention, it is possible to provide a highly safe occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancer that have excellent occludin mRNA expression promoting effects, claudin-1 mRNA expression promoting effects, claudin-4 mRNA expression promoting effects, tight junction protein-1 mRNA expression promoting effects, tight junction protein-2 mRNA expression promoting effects, and barrier function enhancing effects.
本発明の実施の形態について詳細に説明する。
本実施形態に係るオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、いずれも、β－１，３－グルカンを有効成分として含有する。
Embodiments of the present invention will be described in detail.
The occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancer according to the present embodiment are Both contain β-1,3-glucan as an active ingredient.
本実施形態における有効成分としてのβ－１，３－グルカンは、グルコースがβ１，３結合のみで連結してなる１本の糖鎖（又は糖鎖構造）を主鎖として有するものであればよく、直鎖状のものに限らず、分枝鎖を有するものであってもよい。β－１，３－グルカンは合成により得られるものであってもよいが、β－１，３－グルカンを含有する植物、菌類、藻類等（以下「植物等」という場合がある。）の抽出物から単離・精製することにより得られるものであってもよい。なお、β－１，３－グルカンを含有する植物等の抽出物には、β－１，３－グルカンを含有する植物等を抽出原料として得られる抽出液、当該抽出液の希釈液若しくは濃縮液、当該抽出液を乾燥して得られる乾燥物、又はこれらの粗精製物若しくは精製物等が含まれる。 The β-1,3-glucan used as the active ingredient in this embodiment may be one having as a main chain a sugar chain (or sugar chain structure) formed by connecting glucose only through β1,3 bonds. , is not limited to a straight chain, and may have a branched chain. β-1,3-glucan may be obtained by synthesis, but extraction from plants, fungi, algae, etc. (hereinafter sometimes referred to as “plants, etc.”) containing β-1,3-glucan It may also be obtained by isolation and purification from a substance. Note that extracts of plants containing β-1,3-glucan include extracts obtained from plants containing β-1,3-glucan as raw materials for extraction, and diluted or concentrated solutions of the extracts. , a dried product obtained by drying the extract, or a crudely purified or purified product thereof.
β－１，３－グルカンを含有する植物等の抽出物は、植物等の抽出に一般に用いられている方法によって得ることができる。β－１，３－グルカンを含有する植物等としては、例えば、レイシ（霊芝，学名：Ganoderma lucidum（Fr．）Karst．）、シイタケ（学名：Lentinula edodes）、スエヒロタケ（学名：Schizophyllum commune）、マイタケ（学名：Grifola frondosa）、ハナビラタケ（学名：Sparassis crispa）、ミドリムシ（学名：Euglena gracilis）等が挙げられる。 Extracts of plants, etc. containing β-1,3-glucan can be obtained by methods commonly used for extracting plants, etc. Examples of plants containing β-1,3-glucan include Reishi (scientific name: Ganoderma lucidum (Fr.) Karst.), Shiitake (scientific name: Lentinula edodes), Suehirotake (scientific name: Schizophyllum commune), Examples include maitake (scientific name: Grifola frondosa), Hanabiratake (scientific name: Sparassis crispa), and Euglena gracilis (scientific name: Euglena gracilis).
レイシ（Ganoderma lucidum（Fr．）Karst．）は、サルノコシカケ科（Polyporaceae）に属する担子菌類である。抽出原料として使用し得るレイシの構成部位は特に制限されず、目的に応じて適宜選定することができるが、特に子実体を用いるのが好ましい。 Ganoderma lucidum (Fr.) Karst. is a basidiomycete belonging to the family Polyporaceae. The constituent parts of Ganoderma that can be used as the extraction raw material are not particularly limited and can be appropriately selected depending on the purpose, but it is particularly preferable to use the fruiting body.
シイタケ（Lentinula edodes）は、キシメジ科シイタケ属に属する食用の担子菌類であり、日本、朝鮮、台湾、中国に分布し、栽培されており、これらの地域から容易に入手可能である。抽出原料として使用し得る部位は特に制限されず、目的に応じて適宜選定することができるが、子実体が好ましい。 Shiitake mushrooms (Lentinula edodes) are edible basidiomycetes belonging to the genus Shiitake in the family Asteraceae, and are distributed and cultivated in Japan, Korea, Taiwan, and China, and are easily available from these regions. The part that can be used as an extraction raw material is not particularly limited and can be appropriately selected depending on the purpose, but fruiting bodies are preferred.
スエヒロタケ（Schizophyllum commune）は、スエヒロタケ科スエヒロタケ属に属する担子菌類である。抽出原料として使用し得る部位は特に制限されず、目的に応じて適宜選定することができるが、子実体が好ましい。 Schizophyllum commune is a basidiomycete belonging to the family Schizophyllum, genus Schizophyllum. The part that can be used as an extraction raw material is not particularly limited and can be appropriately selected depending on the purpose, but fruiting bodies are preferred.
マイタケ（Grifola frondosa）は、マイタケ科マイタケ属に属する食用の担子菌類であり、暖温帯から温帯北部にかけて分布し、栽培されており、これらの地域から容易に入手可能である。抽出原料として使用し得る部位は特に制限されず、目的に応じて適宜選定することができるが、子実体が好ましい。 Maitake (Grifola frondosa) is an edible basidiomycete belonging to the genus Maitake of the family Maitake family, and is distributed and cultivated from the warm temperate zone to the northern temperate zone, and is easily available from these regions. The part that can be used as an extraction raw material is not particularly limited and can be appropriately selected depending on the purpose, but fruiting bodies are preferred.
ハナビラタケ（Sparassis crispa）は、ハナビラタケ科ハナビラタケ属に属する担子菌類である。抽出原料として使用し得る部位は特に制限されず、目的に応じて適宜選定することができるが、子実体が好ましい。 Sparassis crispa is a basidiomycete belonging to the family Sparassis, genus Sparassis. The part that can be used as an extraction raw material is not particularly limited and can be appropriately selected depending on the purpose, but fruiting bodies are preferred.
ミドリムシ（Euglena gracilis）は、ユーグレナ科ミドリムシ属に属する生物である。抽出原料として使用し得る部位は特に制限されず、目的に応じて適宜選定することができる。 Euglena gracilis is an organism belonging to the family Euglenaceae and the genus Euglena. The part that can be used as an extraction raw material is not particularly limited, and can be appropriately selected depending on the purpose.
例えば、β－１，３－グルカンを含有する植物等の抽出物は、抽出原料をそのまま抽出溶媒による抽出に供することにより、又は抽出原料を乾燥した後、そのまま若しくは粗砕機を用いて粉砕し、抽出溶媒による抽出に供することにより得られ得る。抽出原料の乾燥は天日で行ってもよいし、通常使用される乾燥機を用いて行ってもよい。また、グルコシダーゼ、アミラーゼ等を用いた酵素処理、ヘキサン等の非極性溶媒による脱脂等の前処理を施してから抽出原料として使用してもよい。当該前処理を行うことにより、植物等の極性溶媒による抽出処理を効率よく行うことができる。 For example, an extract of a plant containing β-1,3-glucan can be obtained by subjecting the extraction raw material to extraction with an extraction solvent as it is, or by drying the extraction raw material and then pulverizing it as is or using a coarse crusher. It can be obtained by subjecting it to extraction with an extraction solvent. The extraction raw material may be dried in the sun or may be dried using a commonly used dryer. Alternatively, it may be used as an extraction raw material after being subjected to pretreatment such as enzyme treatment using glucosidase, amylase, etc., or defatting with a nonpolar solvent such as hexane. By performing the pretreatment, extraction treatment using a polar solvent such as plants can be efficiently performed.
抽出溶媒としては、極性溶媒を使用するのが好ましく、例えば、水、水と親水性有機溶媒との混合液等が挙げられ、これらを室温又は溶媒の沸点以下の温度で使用することが好ましい。 As the extraction solvent, it is preferable to use a polar solvent, such as water, a mixture of water and a hydrophilic organic solvent, etc., and it is preferable to use these at room temperature or a temperature below the boiling point of the solvent.
抽出溶媒として使用し得る水としては、例えば、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等の他、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧の調整、緩衝化等が含まれる。したがって、本実施形態において抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。 Examples of water that can be used as an extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and those obtained by subjecting these to various treatments. Examples of treatments applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, water that can be used as an extraction solvent in this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
抽出溶媒として使用し得る親水性有機溶媒としては、例えば、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１～５の低級脂肪族アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３－ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２～５の多価アルコール等が挙げられる。 Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; - Polyhydric alcohols having 2 to 5 carbon atoms, such as butylene glycol, propylene glycol, and glycerin.
水と親水性有機溶媒との混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には水１０容量部に対して低級脂肪族アルコール１～９０容量部を混合することが好ましい。水と低級脂肪族ケトンとの混合液を使用する場合には、水１０容量部に対して低級脂肪族ケトン１～４０容量部を混合することが好ましい。水と多価アルコールとの混合液を使用する場合には、水１０容量部に対して多価アルコール１～９０容量部を混合することが好ましい。 When a mixture of water and a hydrophilic organic solvent is used as an extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when using a mixed solution of water and lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of the lower aliphatic alcohol to 10 parts by volume of water. When using a mixed solution of water and a lower aliphatic ketone, it is preferable to mix 1 to 40 parts by volume of the lower aliphatic ketone to 10 parts by volume of water. When using a mixed solution of water and polyhydric alcohol, it is preferable to mix 1 to 90 parts by volume of polyhydric alcohol to 10 parts by volume of water.
抽出処理は、抽出原料に含まれる可溶性成分を抽出溶媒に溶出させ得る限り特殊な抽出方法を採用する必要はなく、室温又は還流加熱下で抽出することができる。例えば、抽出溶媒を満たした処理槽に抽出原料を投入し、必要に応じて撹拌しながら、３０分～４時間静置して可溶性成分を溶出した後、濾過して固形物を除去することにより抽出物を得ることができる。得られた抽出液から抽出溶媒を留去するとペースト状の濃縮物が得られ、この濃縮物をさらに乾燥することにより乾燥物が得られる。 In the extraction process, there is no need to employ a special extraction method as long as the soluble components contained in the extraction raw material can be eluted into the extraction solvent, and extraction can be performed at room temperature or under reflux heating. For example, by putting the extraction raw material into a processing tank filled with an extraction solvent, stirring if necessary, leaving it to stand for 30 minutes to 4 hours to elute the soluble components, and then filtering to remove solids. Extracts can be obtained. A paste-like concentrate is obtained by distilling off the extraction solvent from the obtained extract, and a dry product is obtained by further drying this concentrate.
以上のようにして得られた抽出液、当該抽出液の濃縮物又は当該抽出液の乾燥物からβ－１，３－グルカンを単離・精製する方法は、特に限定されるものではなく、常法により行うことができる。例えば、植物等の抽出物を、シリカゲルやアルミナ等の多孔質物質、スチレン－ジビニルベンゼン共重合体やポリメタクリレート等の多孔性樹脂等を用いたカラムクロマトグラフ法によりβ－１，３－グルカンを得ることができる。 The method for isolating and purifying β-1,3-glucan from the extract obtained as described above, the concentrate of the extract, or the dried product of the extract is not particularly limited and can be carried out by a conventional method. For example, β-1,3-glucan can be obtained from an extract of a plant or the like by a column chromatography method using a porous substance such as silica gel or alumina, or a porous resin such as a styrene-divinylbenzene copolymer or polymethacrylate.
さらに、カラムクロマトグラフィーにより得られた画分を、ＯＤＳを用いた逆相シリカゲルクロマトグラフィー、再結晶、液－液向流抽出、イオン交換樹脂を用いたカラムクロマトグラフィー等の任意の有機化合物精製手段を用いて精製してもよい。 Furthermore, the fraction obtained by column chromatography can be purified by any organic compound purification method such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc. may be used for purification.
以上のようにして得られるβ－１，３－グルカンは、オクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を有しているため、それらの作用を利用してオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤の有効成分として用いられ得る。 The β-1,3-glucan obtained as described above has an effect of promoting occludin mRNA expression, an effect of promoting claudin-1 mRNA expression, an effect of promoting claudin-4 mRNA expression, an effect of promoting tight junction protein-1 mRNA expression, and an effect of promoting expression of tight junction protein-1 mRNA. -2 mRNA expression promoting effect, these effects can be utilized to produce occludin mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, tight junction protein-1 mRNA expression promoters, and It can be used as an active ingredient of a tight junction protein-2 mRNA expression promoter.
また、β－１，３－グルカンは、上記作用を利用してバリア機能亢進剤の有効成分として用いられ得る。ここで、β－１，３－グルカンが有するバリア機能亢進作用は、オクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用又はタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用に基づいて発揮されるのが好ましい。ただし、β－１，３－グルカンが有するバリア機能亢進作用は、上記作用に基づいて発揮されるバリア機能亢進作用に限定されるものではない。 Further, β-1,3-glucan can be used as an active ingredient of a barrier function enhancer by utilizing the above action. Here, the barrier function enhancing effect of β-1,3-glucan is an effect of promoting occludin mRNA expression, an effect of promoting claudin-1 mRNA expression, an effect of promoting claudin-4 mRNA expression, an effect of promoting tight junction protein-1 mRNA expression, or an effect of promoting tight junction protein-1 mRNA expression. It is preferably exerted based on the action of promoting junction protein-2 mRNA expression. However, the barrier function enhancing effect of β-1,3-glucan is not limited to the barrier function enhancing effect exerted based on the above-mentioned effects.
本実施形態に係るオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、β－１，３－グルカンのみからなるものであってもよいし、β－１，３－グルカンを製剤化したものであってもよい。 The occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancer according to this embodiment may consist of only β-1,3-glucan, or may be a formulation of β-1,3-glucan.
β－１，３－グルカンは、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、液状等の任意の剤形に製剤化することができる。この際、助剤としては、例えば、賦形剤、安定剤、矯臭剤等を用いることができる。β－１，３－グルカンは、他の組成物（例えば、皮膚化粧料等）に配合して使用することができるほか、軟膏剤、外用液剤、貼付剤等として使用することができる。 β-1,3-glucan can be formulated into any desired dosage form such as powder, granules, or liquid according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. can be converted into In this case, as the auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent, etc. can be used. β-1,3-glucan can be used in combination with other compositions (eg, skin cosmetics, etc.), and can also be used as ointments, external solutions, patches, etc.
なお、本実施形態に係るオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、必要に応じて、オクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用又はタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を有する他の天然抽出物を配合して有効成分として用いることができる。 In addition, the occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancement agent according to the present embodiment The agent has an occludin mRNA expression promoting effect, a claudin-1 mRNA expression promoting effect, a claudin-4 mRNA expression promoting effect, a tight junction protein-1 mRNA expression promoting effect, or a tight junction protein-2 mRNA expression promoting effect, as necessary. Natural extracts of can be blended and used as active ingredients.
本実施形態のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤は、β－１，３－グルカンが有するオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用又はタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を通じて、上皮組織におけるタイトジャンクションの形成を促すことでバリア機能を亢進し、炎症性腸疾患や食物アレルギー、消化管から感染する各種感染症等；乾燥肌、荒れ肌、アトピー性皮膚炎等の皮膚症状や各種感染症等を予防、治療又は改善することができる。ただし、本実施形態のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、これらの用途以外にもオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用又はタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を発揮することに意義のあるすべての用途に用いることができる。 The occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of the present embodiment are β-1,3 - Tight junctions in epithelial tissues are enhanced through the effect of glucan on promoting occludin mRNA expression, claudin-1 mRNA expression, claudin-4 mRNA expression, tight junction protein-1 mRNA expression, or tight junction protein-2 mRNA expression. Enhances barrier function by promoting the formation of inflammatory bowel disease, food allergies, and various infections transmitted from the gastrointestinal tract; prevents skin conditions such as dry skin, rough skin, and atopic dermatitis, and various infections. , can be treated or ameliorated. However, the occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancer of the present embodiment In addition to these uses, it exerts an effect of promoting occludin mRNA expression, an effect of promoting claudin-1 mRNA expression, an effect of promoting claudin-4 mRNA expression, an effect of promoting tight junction protein-1 mRNA expression, or an effect of promoting tight junction protein-2 mRNA expression. It can be used for any purpose of particular significance.
本実施形態のバリア機能亢進剤は、有効成分であるβ－１，３－グルカンが有するバリア機能亢進作用を通じて、上皮組織におけるバリア機能を亢進することができ、例えば、消化管におけるバリア機能を亢進し、炎症性腸疾患や食物アレルギー、消化管から感染する各種感染症等を予防、治療又は改善することができる。また、表皮におけるバリア機能及び水分保持機能を高め、乾燥肌、荒れ肌、アトピー性皮膚炎等の皮膚症状や各種感染症等を予防、治療又は改善することができる。ただし、本実施形態のバリア機能亢進剤は、これらの用途以外にもバリア機能亢進作用を発揮することに意義のあるすべての用途に用いることができる。 The barrier function enhancer of this embodiment can enhance the barrier function in epithelial tissue through the barrier function enhancing effect of the active ingredient β-1,3-glucan, and can, for example, enhance the barrier function in the digestive tract and prevent, treat or ameliorate inflammatory bowel disease, food allergies, and various infectious diseases transmitted through the digestive tract. It can also enhance the barrier function and moisture retention function of the epidermis and prevent, treat or ameliorate skin conditions such as dry skin, rough skin, and atopic dermatitis, as well as various infectious diseases. However, the barrier function enhancer of this embodiment can be used in all applications where it is meaningful to exert a barrier function enhancing effect in addition to these applications.
また、本実施形態のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤又はバリア機能亢進剤は、優れたオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を有するため、例えば、皮膚外用剤又は経口組成物に配合するのに好適である。この場合に、β－１，３－グルカンをそのまま皮膚外用剤又は経口組成物に配合してもよいし、β－１，３－グルカンから製剤化したオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤又はバリア機能亢進剤を配合してもよい。 The occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, or barrier function enhancer of the present embodiment has excellent occludin mRNA expression promoting action, claudin-1 mRNA expression promoting action, claudin-4 mRNA expression promoting action, tight junction protein-1 mRNA expression promoting action, and tight junction protein-2 mRNA expression promoting action, and is therefore suitable for incorporation, for example, into a skin topical agent or oral composition. In this case, β-1,3-glucan may be directly incorporated into a skin topical agent or oral composition, or an occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, or barrier function enhancer formulated from β-1,3-glucan may be incorporated.
ここで、皮膚外用剤としては、その区分に制限はなく、経皮的に使用される皮膚化粧料、医薬部外品、医薬品等を幅広く含むものであり、具体的には、例えば、軟膏、クリーム、乳液、美容液、ローション、パック、ファンデーション、リップクリーム、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディシャンプー等が挙げられる。 Here, there are no restrictions on the category of external skin preparations, and they include a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally, and specifically include, for example, ointments, Examples include creams, milky lotions, serums, lotions, packs, foundations, lip balms, bath salts, hair tonics, hair lotions, soaps, and body shampoos.
皮膚外用剤におけるβ－１，３－グルカンの配合量は、皮膚外用剤の種類に応じて適宜調整することができるが、好適な配合率は、０．０００１～１０質量％であり、特に好適な配合率は、０．００１～１質量％である。 The amount of β-1,3-glucan in the external skin preparation can be adjusted as appropriate depending on the type of external skin preparation, but the preferred blending ratio is 0.0001 to 10% by mass, and particularly suitable The compounding ratio is 0.001 to 1% by mass.
経口組成物とは、人の健康に危害を加えるおそれが少なく、通常の社会生活において、経口又は消化管投与により摂取されるものをいい、行政区分上の食品、医薬品、医薬部外品等の区分に制限されるものではない。したがって、本実施形態における「経口組成物」は、経口的に摂取される一般食品、飼料、健康食品、保健機能食品（特定保健用食品、栄養機能食品、機能性表示食品）、医薬部外品、医薬品等を幅広く含むものである。本実施形態における経口組成物は、当該経口組成物又はその包装に、β－１，３－グルカンが有する好ましい作用を表示することのできる経口組成物であることが好ましく、保健機能食品（特定保健用食品、栄養機能食品、機能性表示食品）、医薬部外品又は医薬品であることが特に好ましい。 Oral compositions refer to those that have little risk of harming human health and are ingested orally or through gastrointestinal administration in normal social life, and are classified as foods, drugs, quasi-drugs, etc. under administrative classification. It is not limited to classification. Therefore, the "oral composition" in this embodiment refers to general foods, feeds, health foods, foods with health claims (foods for specified health uses, foods with nutritional function claims, foods with functional claims), and quasi-drugs that are orally ingested. , including a wide range of pharmaceuticals, etc. The oral composition in this embodiment is preferably an oral composition that can display the favorable effects of β-1,3-glucan on the oral composition or its packaging, It is particularly preferable that the food product is a food for use in food, a food with nutritional function claims, a food with functional claims), a quasi-drug, or a pharmaceutical.
経口組成物におけるβ－１，３－グルカンの配合量は、使用目的、症状、性別等を考慮して適宜変更することができるが、添加対象となる経口組成物の一般的な摂取量を考慮して、成人１日あたりの抽出物摂取量が約１～１０００ｍｇになるようにするのが好ましい。なお、添加対象経口組成物が顆粒状、錠剤状又はカプセル状の場合、β－１，３－グルカンの添加量は、添加対象経口組成物に対して通常０．０００１～１０質量％であり、好ましくは０．００１～１質量％ である。 The amount of β-1,3-glucan in the oral composition can be changed as appropriate, taking into consideration the purpose of use, symptoms, gender, etc., but the amount of β-1,3-glucan added may be changed as appropriate, taking into account the general intake of the oral composition to which it is added. Preferably, the intake amount of the extract is about 1 to 1000 mg per day for an adult. In addition, when the oral composition to be added is in the form of granules, tablets, or capsules, the amount of β-1,3-glucan added is usually 0.0001 to 10% by mass with respect to the oral composition to be added, Preferably it is 0.001 to 1% by mass.
また、本実施形態のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤又はバリア機能亢進剤は、優れたオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を有するので、これらの作用機構に関する研究のための試薬としても好適に利用することができる。 Further, the occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, or barrier function enhancer of the present embodiment. has excellent occludin mRNA expression promoting effects, claudin-1 mRNA expression promoting effects, claudin-4 mRNA expression promoting effects, tight junction protein-1 mRNA expression promoting effects, and tight junction protein-2 mRNA expression promoting effects. It can also be suitably used as a reagent for research on mechanisms.
なお、本実施形態のオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤又はバリア機能亢進剤は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物に対して適用することもできる。 Note that the occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, or barrier function enhancer of the present embodiment Although these are preferably applied to humans, they can also be applied to animals other than humans as long as the respective effects are achieved.
以下、試験例等を挙げて本発明をさらに詳細に説明するが、本発明は下記の試験例等に何ら限定されるものではない。なお、下記の試験例においては、試料としてβ－１，３－グルカン（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）を使用した。 Hereinafter, the present invention will be explained in more detail with reference to test examples and the like, but the present invention is not limited to the following test examples and the like. In the following test examples, β-1,3-glucan (manufactured by Sigma-Aldrich) was used as a sample.
［試験例１］オクルディン（ＯＣＬＮ）ｍＲＮＡ発現促進作用試験
正常ヒト新生児表皮角化細胞（ＮＨＥＫ）を、正常ヒト表皮角化細胞増殖培地（ＫＧＭ）を用いて培養した後、トリプシン処理により回収した。回収した細胞を１．５×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＫＧＭで希釈した後、２４ウェルプレートに１ウェル当たり５００μＬずつ播種し、２４時間培養した。培養終了後、２０μＭコルチゾールを含有するＫＧＭ（以下「コルチゾール含有ＫＧＭ」という。）に培地を交換し、その後２４時間ごとに新たなコルチゾール含有ＫＧＭに交換しながら、７２時間培養した。培養後に培養液を廃棄し、ＫＧＭで溶解した試料溶液（試料濃度は下記表１を参照）を各ウェルに２５０μＬ添加し、最終濃度１．５ｍＭ Ｃａ２＋及び２０μＭコルチゾールとなるように調整したＫＧＭを各ウェルに２５０μＬ加え、７２時間培養した。
[Test Example 1] Occludin (OCLN) mRNA expression promoting effect test Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM) and then collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 1.5×10 5 cells/mL, then seeded at 500 μL per well in a 24-well plate, and cultured for 24 hours. After the culture was completed, the medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM"), and then cultured for 72 hours while replacing with new cortisol-containing KGM every 24 hours. After culturing, discard the culture medium, add 250 μL of sample solution dissolved in KGM (see Table 1 below for sample concentration) to each well, and add KGM adjusted to a final concentration of 1.5 mM Ca 2+ and 20 μM cortisol. 250 μL was added to each well and cultured for 72 hours.
培養後、培養液を廃棄し、ＩＳＯＧＥＮ II（ＮＩＰＰＯＮ ＧＥＮＥ社製）にてｔｏｔａｌ ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１５０ｎｇ／μＬになるようにｔｏｔａｌ ＲＮＡを調製した。 After culturing, the culture solution was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared at 150 ng/μL.
ｔｏｔａｌ ＲＮＡを鋳型とし、オクルディンｍＲＮＡ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出は、リアルタイムＰＣＲ装置（Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標) Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ III，タカラバイオ社製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ，タカラバイオ社製）及びＴＢ Ｇｒｅｅｎ(登録商標) Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（タカラバイオ社製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。オクルディンｍＲＮＡの発現量をＧＡＰＤＨ ｍＲＮＡの発現量で補正し、補正値を算出した。 Using total RNA as a template, the expression levels of occludin mRNA and GAPDH mRNA, which was an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green ( Registered trademark) Fast A two-step real-time RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of occludin mRNA was corrected by the expression level of GAPDH mRNA, and a correction value was calculated.
得られた補正値から、下記式によりオクルディンｍＲＮＡ発現促進率（％）を算出した。
オクルディンｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
From the obtained correction value, the occludin mRNA expression promotion rate (%) was calculated using the following formula.
Occludin mRNA expression promotion rate (%) = A/B x 100
In the formula, A represents the "correction value when adding the sample" and B represents the "correction value when the sample is not added."
The results are shown in Table 1.
［試験例２］クローディン－１（ＣＬＤＮ－１）ｍＲＮＡ発現促進作用試験
正常ヒト新生児表皮角化細胞（ＮＨＥＫ）を、正常ヒト表皮角化細胞増殖培地（ＫＧＭ）を用いて培養した後、トリプシン処理により回収した。回収した細胞を１．５×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＫＧＭで希釈した後、２４ウェルプレートに１ウェル当たり５００μＬずつ播種し、２４時間培養した。培養終了後、２０μＭコルチゾールを含有するＫＧＭ（以下「コルチゾール含有ＫＧＭ」という。）に培地を交換し、その後２４時間ごとに新たなコルチゾール含有ＫＧＭに交換しながら、７２時間培養した。培養後に培養液を廃棄し、ＫＧＭで溶解した試料溶液（試料濃度は下記表１を参照）を各ウェルに２５０μＬ添加し、最終濃度１．５ｍＭ Ｃａ２＋及び２０μＭコルチゾールとなるように調整したＫＧＭを各ウェルに２５０μＬ加え、７２時間培養した。
[Test Example 2] Claudin-1 (CLDN-1) mRNA expression promoting effect test After culturing normal human neonatal epidermal keratinocytes (NHEK) using normal human epidermal keratinocyte growth medium (KGM), trypsin Recovered through processing. The collected cells were diluted with KGM to a cell density of 1.5×10 5 cells/mL, then seeded at 500 μL per well in a 24-well plate, and cultured for 24 hours. After the culture was completed, the medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM"), and then cultured for 72 hours while replacing with new cortisol-containing KGM every 24 hours. After culturing, discard the culture medium, add 250 μL of sample solution dissolved in KGM (see Table 1 below for sample concentration) to each well, and add KGM adjusted to a final concentration of 1.5 mM Ca 2+ and 20 μM cortisol. 250 μL was added to each well and cultured for 72 hours.
培養後、培養液を廃棄し、ＩＳＯＧＥＮ II（ＮＩＰＰＯＮ ＧＥＮＥ社製）にてｔｏｔａｌ ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１５０ｎｇ／μＬになるようにｔｏｔａｌ ＲＮＡを調製した。 After culturing, the culture solution was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared at 150 ng/μL.
ｔｏｔａｌ ＲＮＡを鋳型とし、クローディン－１ｍＲＮＡ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出は、リアルタイムＰＣＲ装置（Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標) Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ III，タカラバイオ社製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ，タカラバイオ社製）及びＴＢ Ｇｒｅｅｎ(登録商標) Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（タカラバイオ社製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。クローディン－１ｍＲＮＡの発現量をＧＡＰＤＨ ｍＲＮＡの発現量で補正し、補正値を算出した。 Using total RNA as a template, the expression levels of claudin-1 mRNA and GAPDH mRNA, which was an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green ( Registered trademark) Fast A two-step real-time RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of claudin-1 mRNA was corrected by the expression level of GAPDH mRNA, and a correction value was calculated.
得られた補正値から、下記式によりクローディン－１ｍＲＮＡ発現促進率（％）を算出した。
クローディン－１ｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
From the obtained correction value, the claudin-1 mRNA expression promotion rate (%) was calculated using the following formula.
Claudin-1 mRNA expression promotion rate (%) = A/B x 100
In the formula, A represents the "correction value when adding the sample" and B represents the "correction value when the sample is not added."
The results are shown in Table 1.
［試験例３］クローディン－４（ＣＬＤＮ－４）ｍＲＮＡ発現促進作用試験
正常ヒト新生児表皮角化細胞（ＮＨＥＫ）を、正常ヒト表皮角化細胞増殖培地（ＫＧＭ）を用いて培養した後、トリプシン処理により回収した。回収した細胞を１．５×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＫＧＭで希釈した後、２４ウェルプレートに１ウェル当たり５００μＬずつ播種し、２４時間培養した。培養終了後、２０μＭコルチゾールを含有するＫＧＭ（以下「コルチゾール含有ＫＧＭ」という。）に培地を交換し、その後２４時間ごとに新たなコルチゾール含有ＫＧＭに交換しながら、７２時間培養した。培養後に培養液を廃棄し、ＫＧＭで溶解した試料溶液（試料濃度は下記表１を参照）を各ウェルに２５０μＬ添加し、最終濃度１．５ｍＭ Ｃａ２＋及び２０μＭコルチゾールとなるように調整したＫＧＭを各ウェルに２５０μＬ加え、７２時間培養した。
[Test Example 3] Claudin-4 (CLDN-4) mRNA expression promoting effect test After culturing normal human neonatal epidermal keratinocytes (NHEK) using normal human epidermal keratinocyte growth medium (KGM), trypsin Recovered through processing. The collected cells were diluted with KGM to a cell density of 1.5×10 5 cells/mL, then seeded at 500 μL per well in a 24-well plate, and cultured for 24 hours. After the culture was completed, the medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM"), and then cultured for 72 hours while replacing with new cortisol-containing KGM every 24 hours. After culturing, discard the culture medium, add 250 μL of sample solution dissolved in KGM (see Table 1 below for sample concentration) to each well, and add KGM adjusted to a final concentration of 1.5 mM Ca 2+ and 20 μM cortisol. 250 μL was added to each well and cultured for 72 hours.
培養後、培養液を廃棄し、ＩＳＯＧＥＮ II（ＮＩＰＰＯＮ ＧＥＮＥ社製）にてｔｏｔａｌ ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１５０ｎｇ／μＬになるようにｔｏｔａｌ ＲＮＡを調製した。 After culturing, the culture solution was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared at 150 ng/μL.
ｔｏｔａｌ ＲＮＡを鋳型とし、クローディン－４ｍＲＮＡ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出は、リアルタイムＰＣＲ装置（Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標) Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ III，タカラバイオ社製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ，タカラバイオ社製）及びＴＢ Ｇｒｅｅｎ(登録商標) Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（タカラバイオ社製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。クローディン－４ｍＲＮＡの発現量をＧＡＰＤＨ ｍＲＮＡの発現量で補正し、補正値を算出した。 Using total RNA as a template, the expression levels of claudin-4 mRNA and GAPDH mRNA, which was an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green ( Registered trademark) Fast A two-step real-time RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of claudin-4 mRNA was corrected by the expression level of GAPDH mRNA, and a correction value was calculated.
得られた補正値から、下記式によりクローディン－４ｍＲＮＡ発現促進率（％）を算出した。
クローディン－４ｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
From the obtained correction value, the claudin-4 mRNA expression promotion rate (%) was calculated using the following formula.
Claudin-4 mRNA expression promotion rate (%) = A/B x 100
In the formula, A represents the "correction value when adding the sample" and B represents the "correction value when the sample is not added."
The results are shown in Table 1.
［試験例４］タイトジャンクションプロテイン－１（ＴＪＰ－１）ｍＲＮＡ発現促進作用試験
正常ヒト新生児表皮角化細胞（ＮＨＥＫ）を、正常ヒト表皮角化細胞増殖培地（ＫＧＭ）を用いて培養した後、トリプシン処理により回収した。回収した細胞を１．５×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＫＧＭで希釈した後、２４ウェルプレートに１ウェル当たり５００μＬずつ播種し、２４時間培養した。培養終了後、２０μＭコルチゾールを含有するＫＧＭ（以下「コルチゾール含有ＫＧＭ」という。）に培地を交換し、その後２４時間ごとに新たなコルチゾール含有ＫＧＭに交換しながら、７２時間培養した。培養後に培養液を廃棄し、ＫＧＭで溶解した試料溶液（試料濃度は下記表１を参照）を各ウェルに２５０μＬ添加し、最終濃度１．５ｍＭ Ｃａ２＋及び２０μＭコルチゾールとなるように調整したＫＧＭを各ウェルに２５０μＬ加え、７２時間培養した。
[Test Example 4] Tight junction protein-1 (TJP-1) mRNA expression promoting effect test After culturing normal human neonatal epidermal keratinocytes (NHEK) using normal human epidermal keratinocyte growth medium (KGM), Collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 1.5×10 5 cells/mL, then seeded at 500 μL per well in a 24-well plate, and cultured for 24 hours. After the culture was completed, the medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM"), and then cultured for 72 hours while replacing with new cortisol-containing KGM every 24 hours. After culturing, discard the culture medium, add 250 μL of sample solution dissolved in KGM (see Table 1 below for sample concentration) to each well, and add KGM adjusted to a final concentration of 1.5 mM Ca 2+ and 20 μM cortisol. 250 μL was added to each well and cultured for 72 hours.
培養後、培養液を廃棄し、ＩＳＯＧＥＮ II（ＮＩＰＰＯＮ ＧＥＮＥ社製）にてｔｏｔａｌ ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１５０ｎｇ／μＬになるようにｔｏｔａｌ ＲＮＡを調製した。 After culturing, the culture solution was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared at 150 ng/μL.
ｔｏｔａｌ ＲＮＡを鋳型とし、タイトジャンクションプロテイン－１ｍＲＮＡ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出は、リアルタイムＰＣＲ装置（Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標) Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ III，タカラバイオ社製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ，タカラバイオ社製）及びＴＢ Ｇｒｅｅｎ(登録商標) Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（タカラバイオ社製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。タイトジャンクションプロテイン－１ｍＲＮＡの発現量をＧＡＰＤＨ ｍＲＮＡの発現量で補正し、補正値を算出した。 Using total RNA as a template, the expression levels of tight junction protein-1 mRNA and GAPDH mRNA, which was an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green ( Registered trademark) Fast A two-step real-time RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of tight junction protein-1 mRNA was corrected by the expression level of GAPDH mRNA, and a correction value was calculated.
得られた補正値から、下記式によりタイトジャンクションプロテイン－１ｍＲＮＡ発現促進率（％）を算出した。
タイトジャンクションプロテイン－１ｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
From the obtained correction value, the tight junction protein-1 mRNA expression promotion rate (%) was calculated using the following formula.
Tight junction protein-1 mRNA expression promotion rate (%) = A/B x 100
In the formula, A represents the "correction value when adding the sample" and B represents the "correction value when the sample is not added."
The results are shown in Table 1.
［試験例５］タイトジャンクションプロテイン－２（ＴＪＰ－２）ｍＲＮＡ発現促進作用試験
正常ヒト新生児表皮角化細胞（ＮＨＥＫ）を、正常ヒト表皮角化細胞増殖培地（ＫＧＭ）を用いて培養した後、トリプシン処理により回収した。回収した細胞を１．５×１０５ｃｅｌｌｓ／ｍＬの細胞密度になるようにＫＧＭで希釈した後、２４ウェルプレートに１ウェル当たり５００μＬずつ播種し、２４時間培養した。培養終了後、２０μＭコルチゾールを含有するＫＧＭ（以下「コルチゾール含有ＫＧＭ」という。）に培地を交換し、その後２４時間ごとに新たなコルチゾール含有ＫＧＭに交換しながら、７２時間培養した。培養後に培養液を廃棄し、ＫＧＭで溶解した試料溶液（試料濃度は下記表１を参照）を各ウェルに２５０μＬ添加し、最終濃度１．５ｍＭ Ｃａ２＋及び２０μＭコルチゾールとなるように調整したＫＧＭを各ウェルに２５０μＬ加え、７２時間培養した。
[Test Example 5] Tight junction protein-2 (TJP-2) mRNA expression promoting effect test After culturing normal human neonatal epidermal keratinocytes (NHEK) using normal human epidermal keratinocyte growth medium (KGM), Collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 1.5×10 5 cells/mL, then seeded at 500 μL per well in a 24-well plate, and cultured for 24 hours. After the culture was completed, the medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM"), and then cultured for 72 hours while replacing with new cortisol-containing KGM every 24 hours. After culturing, discard the culture medium, add 250 μL of sample solution dissolved in KGM (see Table 1 below for sample concentration) to each well, and add KGM adjusted to a final concentration of 1.5 mM Ca 2+ and 20 μM cortisol. 250 μL was added to each well and cultured for 72 hours.
培養後、培養液を廃棄し、ＩＳＯＧＥＮ II（ＮＩＰＰＯＮ ＧＥＮＥ社製）にてｔｏｔａｌ ＲＮＡを抽出し、波長２６０ｎｍにおける吸光度からＲＮＡ量を計算し、１５０ｎｇ／μＬになるようにｔｏｔａｌ ＲＮＡを調製した。 After culturing, the culture solution was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared at 150 ng/μL.
ｔｏｔａｌ ＲＮＡを鋳型とし、タイトジャンクションプロテイン－２ｍＲＮＡ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出は、リアルタイムＰＣＲ装置（Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標) Ｒｅａｌ Ｔｉｍｅ Ｓｙｓｔｅｍ III，タカラバイオ社製）を用いて、ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ，タカラバイオ社製）及びＴＢ Ｇｒｅｅｎ(登録商標) Ｆａｓｔ ｑＰＣＲ Ｍｉｘ（タカラバイオ社製）による２ステップリアルタイムＲＴ－ＰＣＲ反応により行った。タイトジャンクションプロテイン－２ｍＲＮＡの発現量をＧＡＰＤＨ ｍＲＮＡの発現量で補正し、補正値を算出した。 Using total RNA as a template, the expression levels of tight junction protein-2 mRNA and GAPDH mRNA, which was an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green ( Registered trademark) Fast A two-step real-time RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of tight junction protein-2 mRNA was corrected by the expression level of GAPDH mRNA, and a correction value was calculated.
得られた補正値から、下記式によりタイトジャンクションプロテイン－２ｍＲＮＡ発現促進率（％）を算出した。
タイトジャンクションプロテイン－２ｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
From the obtained correction value, the tight junction protein-2 mRNA expression promotion rate (%) was calculated using the following formula.
Tight junction protein-2 mRNA expression promotion rate (%) = A/B x 100
In the formula, A represents the "correction value when adding the sample" and B represents the "correction value when the sample is not added."
The results are shown in Table 1.
表１に示すように、β－１，３－グルカンは、優れたオクルディンｍＲＮＡ発現促進作用、クローディン－１ｍＲＮＡ発現促進作用、クローディン－４ｍＲＮＡ発現促進作用、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進作用及びタイトジャンクションプロテイン－２ｍＲＮＡ発現促進作用を有することが確認された。 As shown in Table 1, β-1,3-glucan has an excellent effect of promoting occludin mRNA expression, claudin-1 mRNA expression, claudin-4 mRNA expression, tight junction protein-1 mRNA expression, and tight junction protein-1 mRNA expression. It was confirmed that it has the effect of promoting junction protein-2 mRNA expression.
本実施形態に係るオクルディンｍＲＮＡ発現促進剤、クローディン－１ｍＲＮＡ発現促進剤、クローディン－４ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－１ｍＲＮＡ発現促進剤、タイトジャンクションプロテイン－２ｍＲＮＡ発現促進剤及びバリア機能亢進剤は、炎症性腸疾患や食物アレルギー、消化管から感染する各種感染症等；乾燥肌、荒れ肌、アトピー性皮膚炎等の皮膚症状や各種感染症等の予防、治療又は改善等に大きく貢献することができる。 The occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, tight junction protein-2 mRNA expression promoter, and barrier function enhancer according to the present embodiment are , inflammatory bowel disease, food allergies, various infectious diseases transmitted from the gastrointestinal tract, etc.; can greatly contribute to the prevention, treatment, or improvement of skin conditions such as dry skin, rough skin, atopic dermatitis, and various infectious diseases. can.
Claims (6)
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