JP7579469B1 - CDP choline production promoter - Google Patents
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Abstract
【課題】表皮再生を促進する成分を取得することを目的とする。
【解決手段】 本発明者らは、ＣＤＰコリンが、表皮再生促進、ケラチノサイトの遊走促進、創傷治癒、細胞膜の増強、皮膚バリア機能の向上といった作用を発揮することを見出し、ＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、表皮再生促進剤、ケラチノサイトの遊走促進剤、創傷治癒の促進剤、ケラチノサイトの細胞膜の増強剤、又は皮膚バリア機能促進剤を提供する。また、ＣＤＰコリン産生促進剤として、リンデンエキスを提供する。
【選択図】図１
The objective is to obtain a component that promotes epidermal regeneration.
[Solution] The present inventors have found that CDP choline exerts effects such as promoting epidermal regeneration, promoting keratinocyte migration, wound healing, enhancing cell membranes, and improving skin barrier function, and provide a CDP choline production promoter, or an epidermal regeneration promoter, a keratinocyte migration promoter, a wound healing promoter, a keratinocyte cell membrane enhancer, or a skin barrier function promoter that contains CDP choline. In addition, the present inventors provide linden extract as a CDP choline production promoter.
[Selected Figure] Figure 1
Description
ＣＤＰコリンの産生促進の技術分野に関する。ＣＤＰコリンの産生を促進することで、表皮再生が促進され、ケラチノサイトの遊走促進、創傷治癒、細胞膜の増強、皮膚バリア機能の向上などの作用を発揮することができる。 This relates to the technical field of promoting the production of CDP choline. By promoting the production of CDP choline, epidermal regeneration can be promoted, and effects such as promoting keratinocyte migration, wound healing, strengthening cell membranes, and improving skin barrier function can be achieved.
ＣＤＰコリンは、ヌクレオチドの一つであるシチジン二リン酸（ＣＤＰ）にリン酸エステル結合を介してコリンが結合した物質である。ＣＤＰコリンは、ホスファチジルコリンの生合成経路における中間体の一つであり、またスフィンゴミエリン生合成の中間体の一つでもある。ＣＤＰコリンは認知機能の改善作用を有すること、神経保護作用を有することが報告されている（特許文献１：特開２０２３－０１１８００号公報、特許文献２：特開２０２２－１０７６３１号公報）、国内では医療用医薬品として、また海外ではサプリメントとしても販売されている。また、ＣＤＰコリンは、血管内皮細胞に作用し、タイトジャンクション因子であるＺＯ－１及びオクルーディンの発現に影響することが報告されている（非特許文献１：ＰＬｏＳ Ｏｎｅ． ２０１３）。 CDP choline is a substance in which choline is bound to cytidine diphosphate (CDP), a nucleotide, via a phosphate ester bond. CDP choline is one of the intermediates in the biosynthetic pathway of phosphatidylcholine and also one of the intermediates in the biosynthesis of sphingomyelin. CDP choline has been reported to have cognitive function improving effects and neuroprotective effects (Patent Document 1: JP 2023-011800 A, Patent Document 2: JP 2022-107631 A), and is sold as a medical drug in Japan and as a supplement overseas. It has also been reported that CDP choline acts on vascular endothelial cells and affects the expression of tight junction factors ZO-1 and occludin (Non-Patent Document 1: PLoS One. 2013).
セイヨウシナノキから得られるリンデンエキスは、エラスターゼ活性阻害、ヒアルロニダーゼ活性阻害、コラゲナーゼ活性阻害、ＳＯＤ様作用、ＤＰＰＨラジカル消去作用、ＰＧＥ２産生抑制、ＭＭＰ１活性阻害、ＡＧＥｓ生成抑制、エストロゲン様作用などの様々な効能を発揮することが知られている。これらの活性は主に真皮層に作用するものであり、たるみの改善、コラーゲン産生促進、美白などの様々な効能を目的に化粧料に配合される他、ハーブティーやサプリメントなどの食品としても用いられている。一方、表皮細胞への効果についての報告は少なく、創傷治癒に対する効果も不明であった。 Linden extract obtained from the western linden tree is known to exert various effects, such as elastase activity inhibition, hyaluronidase activity inhibition, collagenase activity inhibition, SOD-like action, DPPH radical scavenging action, PGE2 production inhibition, MMP1 activity inhibition, AGEs production inhibition, and estrogen-like action. These activities mainly act on the dermis layer, and it is incorporated into cosmetics for various purposes such as improving sagging, promoting collagen production, and whitening, and is also used as a food such as herbal tea and supplements. However, there have been few reports on its effect on epidermal cells, and its effect on wound healing was unknown.
表皮再生を促進する成分を取得することを目的とする。 The aim is to obtain ingredients that promote epidermal regeneration.
本発明者らは、培養ケラチノサイトに対してＣＤＰコリンを添加することにより、タイトジャンクション関連タンパク質の発現が増大し、またスクラッチアッセイにおいてケラチノサイトの遊走を促進することすることを見出した。このようにＣＤＰコリンによる、表皮再生効果に着目し、ＣＤＰコリンの体内での産生を促進することで、表皮に対し作用する成分をスクリーニングしたところ、驚くべきことに、リンデンエキスが、ケラチノサイトにおいてＣＤＰコリンの産生を増大することを見出し、本発明に至った。
そこで、本発明は以下に関する：
［１] リンデンエキスを含む、ＣＤＰコリン産生促進剤。
［２］ 前記ＣＤＰコリン産生促進剤が、ケラチノサイトにおけるＣＤＰコリン産生を促進する、項目１に記載のＣＤＰコリン産生促進剤。
［３］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、表皮再生促進剤。
［４］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、ケラチノサイトの遊走促進剤。
［５］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、創傷治癒の促進剤。
［６］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、ケラチノサイトの細胞膜の増強剤。
［７］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンを含む、皮膚バリア機能促進剤。
［８］ 項目１に記載のＣＤＰコリン産生促進剤、又はＣＤＰコリンが配合された、経皮又は経口投与用の組成物。
［９］ 化粧用組成物又は医薬組成物である、項目８に記載の組成物。
［１０］ 項目１に記載のＣＤＰコリン産生促進剤を含む食品。
［１１］創口を有する対象の創傷部にリンデンエキスを含むＣＤＰコリン産生促進剤を適用することを含む、創傷治癒方法。
［１２］肌荒れを含む対象に、リンデンエキスを含むＣＤＰコリン産生促進剤を適用することを含む、美容方法。
［１３］創傷治癒において使用するための、リンデンエキス。
［１４］ＣＤＰコリンの産生を促進することを介して、創傷が治癒される、項目１３に記載のリンデンエキス。
［１５］ＣＤＰコリンの産生が促進され、それにより表皮再生促進、ケラチノサイトの遊走促進、ケラチノサイトの細胞膜の増強、又は皮膚バリア機能の改善される、項目１４に記載のリンデンエキス。
［１６］創傷治癒において使用するための、ＣＤＰコリン。
［１７］表皮再生促進、ケラチノサイトの遊走促進、ケラチノサイトの細胞膜の増強、又は皮膚バリア機能の改善を介して、創傷が治癒される、項目１６に記載のＣＤＰコリン。
［１８］ＣＤＰコリン産生促進剤を製造のための、リンデンエキスの使用。
［１９］表皮再生促進剤、ケラチノサイト遊走促進剤、又は創傷治癒の促進剤の製造のための、リンデンエキス又はＣＤＰコリンの使用。
The present inventors found that the addition of CDP-choline to cultured keratinocytes increases the expression of tight junction-associated proteins and promotes the migration of keratinocytes in scratch assays. Thus, focusing on the epidermal regeneration effect of CDP-choline, they screened for ingredients that act on the epidermis by promoting the production of CDP-choline in the body, and surprisingly found that Linden Extract increases the production of CDP-choline in keratinocytes, leading to the present invention.
Thus, the present invention relates to:
[1] A CDP choline production promoter containing linden extract.
[2] The CDP choline production promoter according to
[3] An epidermal regeneration promoter comprising the CDP choline production promoter according to
[4] A keratinocyte migration promoter comprising the CDP choline production promoter according to
[5] A wound healing promoter comprising the CDP choline production promoter according to
[6] An agent for enhancing the cell membrane of keratinocytes, comprising the CDP-choline production promoter according to
[7] A skin barrier function promoter comprising the CDP choline production promoter according to
[8] A composition for transdermal or oral administration, which contains the CDP choline production enhancer or CDP choline according to
[9] The composition according to item 8, which is a cosmetic composition or a pharmaceutical composition.
[10] A food comprising the CDP choline production promoter according to
[11] A wound healing method comprising applying a CDP choline production promoter containing linden extract to a wound area of a subject having a wound.
[12] A cosmetic method comprising applying a CDP choline production promoter containing linden extract to a subject having rough skin.
[13] Linden extract for use in wound healing.
[14] The linden extract according to item 13, which promotes the production of CDP-choline and thereby heals wounds.
[15] The linden extract according to item 14, which promotes production of CDP-choline, thereby promoting epidermal regeneration, promoting keratinocyte migration, strengthening the cell membrane of keratinocytes, or improving skin barrier function.
[16] CDP-choline for use in wound healing.
[17] The CDP choline according to item 16, which heals wounds through promotion of epidermal regeneration, promotion of keratinocyte migration, strengthening of keratinocyte cell membranes, or improvement of skin barrier function.
[18] Use of linden extract for the production of a CDP choline production promoter.
[19] Use of linden extract or CDP choline for the manufacture of an epidermal regeneration promoter, a keratinocyte migration promoter, or an agent for promoting wound healing.
リンデンエキスは、ＣＤＰコリンの産生を促進する。ＣＤＰコリンの産生促進を介して、表皮再生、ケラチノサイトの遊走促進、創傷治癒、細胞膜の増強、皮膚バリア機能の改善に寄与する。 Linden extract promotes the production of CDP choline. By promoting the production of CDP choline, it contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function.
本発明は、リンデンエキスを含む、ＣＤＰコリン産生促進剤に関する。ＣＤＰコリンは、ケラチノサイトで産生促進されると、表皮再生、ケラチノサイトの遊走促進、創傷治癒、細胞膜の増強、皮膚バリア機能の改善に寄与する。 The present invention relates to a CDP choline production promoter that contains linden extract. When the production of CDP choline is promoted in keratinocytes, it contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function.
ＣＤＰコリンは、以下の式：
ＣＤＰコリン産生促進剤は、適用された際に、任意の細胞、特にケラチノサイトにおいてＣＤＰコリンの産生を促進する成分をいう。ケラチノサイトにおいて、ＣＤＰコリンの産生が促進することにより、表皮再生、ケラチノサイトの遊走促進、創傷治癒、細胞膜の増強、皮膚バリア機能の改善に寄与する。 A CDP choline production promoter is a component that, when applied, promotes the production of CDP choline in any cell, particularly keratinocytes. Promoting the production of CDP choline in keratinocytes contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function.
皮膚は創傷を受けると、まず出血が生じる。時間経過と共に、血液が凝固し瘡蓋を生じる。瘡蓋の内部では、創傷部周囲のケラチノサイトが増殖し、遊走することで傷口をふさぐと共に、角層分化を生じ、分化と共にタイトジャンクションを形成する。タイトジャンクションを形成することで、皮膚バリア機能が向上し、表皮が再生される。 When the skin is wounded, bleeding occurs first. Over time, the blood coagulates and a scab forms. Inside the scab, keratinocytes around the wound proliferate and migrate to seal the wound, while also differentiating into the stratum corneum and forming tight junctions as they differentiate. The formation of tight junctions improves the skin's barrier function and regenerates the epidermis.
ケラチノサイトは、通常、基底層において分裂を繰り返しており、分裂した細胞が、表層へと移動する際に分化し、有棘層、顆粒層、角質層を形成する。一方、皮膚に傷害が生じた場合、受傷部周辺のケラチノサイト及びその前駆細胞である表皮幹細胞は、受傷部位に遊走を開始する。遊走は、一例として、低酸素状態により誘導され、瘡蓋等で外の空気と遮断された内部のケラチノサイトが遊走を促進する。ケラチノサイトの遊走能は培養細胞において、スクラッチアッセイを行うことで測定することができる。スクラッチアッセイは、コンフルエントに培養したディッシュ表面の一部領域の細胞を掻きとったのちに、かかる領域へ遊走してきた細胞に基づき遊走能を決定する手法である。ＣＤＰコリンを添加した培養物では、遊走能が高いことが示された（図４Ａ及びＢ）。これにより、本発明は、ＣＤＰコリンを含む、ケラチノサイトにおけるケラチノサイトの遊走促進剤、創傷治癒促進剤又は表皮再生促進剤に関していてもよい。 Keratinocytes usually divide repeatedly in the basal layer, and when the divided cells migrate to the surface layer, they differentiate to form the spinous layer, granular layer, and stratum corneum. On the other hand, when an injury occurs to the skin, keratinocytes around the injured area and their precursor cells, epidermal stem cells, begin to migrate to the injured area. Migration is induced, for example, by a hypoxic state, and migration is promoted by internal keratinocytes that are blocked from the outside air by a scab or the like. The migration ability of keratinocytes can be measured by performing a scratch assay in cultured cells. The scratch assay is a method in which cells in a part of the surface of a dish cultured to confluence are scraped off, and then the migration ability is determined based on the cells that have migrated to that part. It was shown that the culture to which CDP-choline was added had a high migration ability (Figures 4A and B). Thus, the present invention may relate to a keratinocyte migration promoter, wound healing promoter, or epidermal regeneration promoter in keratinocytes, which contains CDP-choline.
タイトジャンクションは、角層の顆粒層の細胞同士で形成され、タイトジャンクションが形成されることで、皮膚バリア機能を発揮する。これにより、皮膚内部の成分や水分が失われるのを妨げるとともに、皮膚外部からのアレルゲンや病原体の侵入を防ぐことができる。タイトジャンクションに関わるタイトジャンクション因子として、クローディン、オクルーディン、ＺＯファミリータンパク質（ＺＯ－１～ＺＯ－３）などが知られている。これらのタンパク質の発現が増大することで、タイトジャンクションがより強固になり、皮膚バリア機能が亢進する。また、タイトジャンクションはケラチノサイトのみならず、上皮細胞系、例えば腸管上皮細胞、血管内皮細胞などでも形成される。ＣＤＰコリンは、低酸素下において血管内皮細胞におけるタイトジャンクション因子の発現を亢進することが知られている（非特許文献１）。本発明者らにより、ＣＤＰコリンは、低酸素下においてケラチノサイトにおけるタイトジャンクション因子の発現を亢進することが示されており（図２Ａ～Ｃ）、また、低酸素条件で培養したケラチノサイトにおいて、ＣＤＰコリンにより細胞膜に局在するオクルーディンの発現が亢進することが示されている（図３Ａ及びＢ）。これにより、本発明は、ＣＤＰコリンを含む、ケラチノサイトにおけるタイトジャンクション形成促進剤、又は皮膚バリア機能亢進剤ともいうことができる。 Tight junctions are formed between cells in the granular layer of the stratum corneum, and the formation of tight junctions exerts the skin barrier function. This prevents the loss of components and moisture inside the skin, and also prevents the invasion of allergens and pathogens from the outside of the skin. Claudins, occludins, ZO family proteins (ZO-1 to ZO-3), etc. are known as tight junction factors involved in tight junctions. Increased expression of these proteins strengthens tight junctions, enhancing skin barrier function. Tight junctions are formed not only in keratinocytes, but also in epithelial cell systems, such as intestinal epithelial cells and vascular endothelial cells. CDP-choline is known to enhance the expression of tight junction factors in vascular endothelial cells under hypoxic conditions (Non-Patent Document 1). The present inventors have shown that CDP-choline enhances the expression of tight junction factors in keratinocytes under hypoxic conditions (FIGS. 2A-C), and have also shown that CDP-choline enhances the expression of occludin localized in the cell membrane in keratinocytes cultured under hypoxic conditions (FIGS. 3A and B). Thus, the present invention can be said to be a promoter of tight junction formation in keratinocytes, or a skin barrier function enhancer, which contains CDP-choline.
リンデンエキスは、シナノキ科シナノキ（Ｔｉｌｌｉａ）属に属する植物から抽出されたエキスである。シナノキ科シナノキ属に属する植物としては、アメリカボダイジュ（Ｔ． ａｍｅｒｉｃａｎａ Ｌ．）、ナツボダイジュ（Ｔ． ｐｌａｔｙｐｈｙｌｌｏｓ Ｓｃｏｐ．）、フユボダイジュ（Ｔ． ｃｏｒｄａｔａ Ｍｉｌｌ．）、及びその交配種、例えばセイヨウシナノキ（Ｔｉｌｉａ × ｅｕｒｏｐａｅａ）などが挙げられる。リンデンエキスは、西洋菩提樹抽出液、フユボダイジュエキスともいうこともある。これらの植物エキスは、植物体、特に芽、葉、花、枝、幹、根、実、及び種からなる群から選ばれる少なくとも１の部位を、植物エキス製造のための一般的な方法により得ることができる。一例として、花又は新芽をそのまま又は乾燥させて抽出溶媒とともに常温又は加熱して浸漬または加熱還流した後、濾過し、濃縮して得ることができる。抽出溶媒としては、通常抽出に用いられる溶媒であれば任意に用いることができ、例えば、水性溶媒、例えば水、生理食塩水、リン酸緩衝液、ホウ酸緩衝液、あるいは有機溶媒、例えばエタノール、プロピレングリコール、１，３－ブチレングリコール、グリセリン等のアルコール類、含水アルコール類、クロロホルム、ジクロルエタン、四塩化炭素、アセトン、酢酸エチル、ヘキサン等を、それぞれ単独あるいは組み合わせて用いることができる。好ましくは、溶媒として水とアルコール、例えば１，３－ブチレングリコールの混合溶媒が使用される。上記溶媒で抽出して得られた抽出物をそのまま、あるいは例えば凍結乾燥などにより濃縮したエキスを使用でき、また必要であれば吸着法、例えばイオン交換樹脂を用いて不純物を除去したものや、ポーラスポリマー（例えばアンバーライトＸＡＤ－２）のカラムにて吸着させた後、所望の溶媒で溶出し、さらに濃縮したものも使用することができる。リンデンエキスは、市販のものを使用することもできる。より具体的に、水、アルコール又はそれらの混合溶液で抽出することで調製することができる。アルコールとしては、エタノール、プロピレングリコール、又はブチレングリコールが用いられる。より好ましくは、水とアルコールの任意の割合の混合液、例えば１０：９０～９０：１０の混合液、好ましく３０：７０～７０：３０、さらに好ましくは５０：５０の混合液により抽出されうる。より好ましくは水と１，３‐ブチレングリコールの混合物が用いられる。リンデンエキスは、化粧料に、０．００５％～０．１％の濃度で配合され、０．０１％～０．０５％がより好ましい。 Linden extract is an extract extracted from a plant belonging to the genus Tillia of the family Tilia. Examples of plants belonging to the genus Tillia of the family Tilia include T. americana L., T. platyphyllos Scop., T. cordata Mill., and hybrids thereof, such as Tilia x europaea. Linden extract is also called linden extract or tilia extract. These plant extracts can be obtained by a general method for producing plant extracts from at least one part of the plant body, particularly selected from the group consisting of buds, leaves, flowers, branches, trunks, roots, fruits, and seeds. As an example, the extract can be obtained by immersing or refluxing the flowers or buds as they are or after drying with an extraction solvent at room temperature or at high temperature, filtering, and concentrating. As the extraction solvent, any solvent normally used for extraction can be used. For example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, and glycerin, water-containing alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc. can be used alone or in combination. Preferably, a mixed solvent of water and alcohol, such as 1,3-butylene glycol, is used as the solvent. The extract obtained by extraction with the above solvent can be used as it is, or can be concentrated by, for example, freeze-drying, and if necessary, it can be used after removing impurities using an adsorption method, for example, an ion exchange resin, or after adsorption on a column of porous polymer (e.g., Amberlite XAD-2), eluted with a desired solvent, and further concentrated. Linden extract can also be used as a commercially available product. More specifically, it can be prepared by extraction with water, alcohol, or a mixture thereof. As the alcohol, ethanol, propylene glycol, or butylene glycol is used. More preferably, it can be extracted with a mixture of water and alcohol in any ratio, for example, a mixture of 10:90 to 90:10, preferably a mixture of 30:70 to 70:30, and even more preferably a mixture of 50:50. More preferably, a mixture of water and 1,3-butylene glycol is used. Linden extract is blended in the cosmetic at a concentration of 0.005% to 0.1%, more preferably 0.01% to 0.05%.
リンデンエキスは、培養ケラチノサイトにおいて、ＣＤＰコリンの産生を促進することができ（図１Ａ及びＢ）、それにより、リンデンエキスはＣＤＰコリンの産生促進剤として使用することができる。さらに、ＣＤＰコリンは、ケラチノサイトにおいてクローディン、オクルーディン、ＺＯ－１の発現を促進することができ（図２Ａ～Ｃ、図３Ａ及びＢ）、さらにスクラッチアッセイにおいて、ケラチノサイトの遊走を促進する（図４Ａ及びＢ）。したがって、リンデンエキスは、ＣＤＰコリンの産生を介した作用を発揮することもできる。これにより、本発明は、リンデンエキスを含む表皮再生促進剤、ケラチノサイトの遊走促進剤、創傷治癒促進剤、細胞膜の増強剤、皮膚バリア機能の改善剤に関していてもよい。 Linden extract can promote the production of CDP-choline in cultured keratinocytes (FIGS. 1A and 1B), and thus linden extract can be used as a promoter of CDP-choline production. Furthermore, CDP-choline can promote the expression of claudin, occludin, and ZO-1 in keratinocytes (FIGS. 2A-C, 3A and 3B), and further promotes keratinocyte migration in a scratch assay (FIGS. 4A and 4B). Thus, linden extract can also exert an effect via the production of CDP-choline. Thus, the present invention may relate to an epidermal regeneration promoter, a keratinocyte migration promoter, a wound healing promoter, a cell membrane enhancer, and a skin barrier function improver, which contain linden extract.
本発明のＣＤＰコリンの産生促進剤、表皮再生促進剤、ケラチノサイトの遊走促進剤、創傷治癒促進剤、細胞膜の増強剤、皮膚バリア機能の改善剤は、それぞれ化粧料、医薬品又は医薬部外品に配合されてもよく、また、食品、例えばサプリメントなどの栄養補助食品、機能性表示食品に配合されてもよい。食品は、食品組成物ということもでき、肌荒れ、創傷、掻き傷、皮膚バリア機能低下に悩む対象に投与されうる。これらの薬剤は、経口、又は非経口、例えば経皮投与されてもよい。経皮投与される場合、皮膚外用剤に剤形することができる。皮膚外用剤は、皮膚に適用可能であれば特に限定されず、例えば、溶液状、乳化状、固形状、半固形状、粉末状、粉末分散状、水－油二層分離状、水－油－粉末三層分離状、軟膏状、ゲル状、エアゾール状、ムース状、スティック状等、任意の剤型が適用できる。皮膚外用剤に剤形される場合には、皮膚外用剤に通常用いられる基剤、及び賦形剤、例えば保存剤、乳化剤、ｐＨ調整剤などが用いられてもよい。また、創傷部を覆う貼布剤として剤形してもよい。化粧料に配合する場合、化粧水、乳液、美容液、クリーム、ローション、パック、エッセンス、ジェル等の顔用又は体用の化粧料や、ファンデーション、化粧下地、コンシーラー等のメーキャップ化粧料、さらには浴用剤などに配合することができる。本発明の成分を含む化粧品、医薬品及び医薬部外品を用いることにより、細胞、特にケラチノサイトにおいてＣＤＰコリンの産生促進を介して、表皮再生促進、ケラチノサイトの遊走促進、創傷治癒促進、細胞膜の増強、皮膚バリア機能の改善からなる群から選ばれる少なくとも１の作用を発揮することができる。 The CDP choline production promoter, epidermal regeneration promoter, keratinocyte migration promoter, wound healing promoter, cell membrane enhancer, and skin barrier function improver of the present invention may each be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, and may also be incorporated into foods, such as nutritional supplements such as supplements, and functional foods. Foods can also be called food compositions, and can be administered to subjects suffering from rough skin, wounds, scratches, and reduced skin barrier function. These drugs may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into topical skin preparations. The topical skin preparation is not particularly limited as long as it is applicable to the skin, and any formulation can be applied, such as a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a skin topical preparation, a base and excipients, such as a preservative, an emulsifier, and a pH adjuster, that are commonly used in skin topical preparations may be used. The composition may also be formulated into a patch to cover a wound. When incorporated into cosmetics, the composition may be incorporated into face or body cosmetics such as lotions, milky lotions, beauty essences, creams, lotions, packs, essences, and gels, makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. By using cosmetics, medicines, and quasi-drugs containing the components of the present invention, at least one action selected from the group consisting of promoting epidermal regeneration, promoting keratinocyte migration, promoting wound healing, strengthening cell membranes, and improving skin barrier function can be achieved through the promotion of CDP-choline production in cells, particularly keratinocytes.
本発明のＣＤＰコリン産生促進剤、表皮再生促進剤、ケラチノサイトの遊走促進剤、創傷治癒促進剤、細胞膜の増強剤、及び皮膚バリア機能の改善剤は、それぞれ所望の効果、例えば皮膚バリア機能改善、皮膚再生能促進を発揮させる観点で任意に濃度を選択することができる。皮膚外用剤として配合する観点からは、本発明に係るエキスは、０．０００５％～０．５％で配合することができる。効果を十分に発揮させる観点から、好ましくは０．００１％以上で配合でき、さらに好ましくは０．００５％以上で配合することができる。強いにおいを避ける観点から、好ましくは０．１％以下で配合することができ、さらに好ましくは０．０５％以下で配合することができる。 The CDP choline production promoter, epidermal regeneration promoter, keratinocyte migration promoter, wound healing promoter, cell membrane enhancer, and skin barrier function improver of the present invention can be arbitrarily selected in concentration from the viewpoint of exerting the desired effect, for example, improving skin barrier function and promoting skin regeneration ability. From the viewpoint of formulation as a skin external preparation, the extract of the present invention can be formulated at 0.0005% to 0.5%. From the viewpoint of fully exerting the effect, it can be formulated preferably at 0.001% or more, and more preferably at 0.005% or more. From the viewpoint of avoiding a strong odor, it can be formulated preferably at 0.1% or less, and more preferably at 0.05% or less.
本発明のさらに別の態様では、本発明はＣＤＰコリンの産生促進を介して、表皮再生促進、ケラチノサイトの遊走促進、創傷治癒促進、細胞膜の増強、皮膚バリア機能の改善するための方法に関していてもよい。より具体的に、本発明の方法は、本発明に係るＣＤＰコリンを含む組成物、又はリンデンエキスを含む組成物を対象の皮膚に適用する工程を含む方法にも関する。かかる方法は、治療方法であってもよいし、非治療的な美容方法であってもよい。このような対象は、表皮再生促進、ケラチノサイトの遊走促進、創傷治癒促進、細胞膜の増強、皮膚バリア機能の改善を望む対象であり、一例として肌荒れ又は皮膚炎症、掻き傷に悩まされる対象に投与されうる。 In yet another aspect of the present invention, the present invention may relate to a method for promoting epidermal regeneration, promoting keratinocyte migration, promoting wound healing, strengthening cell membranes, and improving skin barrier function through promoting the production of CDP choline. More specifically, the method of the present invention also relates to a method comprising a step of applying a composition containing CDP choline according to the present invention or a composition containing linden extract to the skin of a subject. Such a method may be a therapeutic method or a non-therapeutic cosmetic method. Such a subject is a subject who desires to promote epidermal regeneration, promote keratinocyte migration, promote wound healing, strengthen cell membranes, and improve skin barrier function, and as an example, the composition may be administered to a subject suffering from rough skin, skin inflammation, or scratches.
本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 All documents referred to in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and are not intended to limit the scope of the present invention. The scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.
実施例１：メタボローム解析
ヒト上皮細胞用増殖培地（ＣＥＬＬｎＴＥＣ社）に播種されたヒトケラチノサイト（クラボウ）の培養物にリンデンエキス（香栄興業株式会社）を０．１％添加して６時間培養した。培養後培地を除去し、メタノール溶液を添加して攪拌し、さらに、Ｍｉｌｌｉ－Ｑ 水を加えて撹拌し、遠心分離（２，３００×ｇ、４℃、５分）を行った。限外ろ過チューブ（ウルトラフリーＭＣ ＰＬＨＣＣ，ＨＭＴ，遠心式フィルターユニット５ｋＤａ）により限外ろ過処理を実施後、ろ液を乾固させ、再びＭｉｌｌｉ－Ｑ 水に溶解して測定に供した。１試料あたり１．２６×１０6の細胞を用い、Ｎ＝５でメタボローム測定を実施した。
Example 1: Metabolomic Analysis A culture of human keratinocytes (Kurabo) seeded in a growth medium for human epithelial cells (CELLnTEC) was added with 0.1% linden extract (Koei Kogyo Co., Ltd.) and cultured for 6 hours. After the culture, the medium was removed, a methanol solution was added and stirred, and Milli-Q water was added and stirred, followed by centrifugation (2,300 x g, 4 ° C, 5 minutes). After ultrafiltration using an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa), the filtrate was dried and dissolved in Milli-Q water again for measurement. Metabolomic measurement was performed using 1.26 x 10 6 cells per sample, N = 5.
代謝物の検出
キャピラリー電気泳動（Agilent CE system）と質量分析計（ＭＳ）を組み合わせたＣＥ－ＭＳシステムにより陽イオン性代謝物質及び陰イオン性代謝物質を網羅的に検出した。検出されたピークから自動積分ソフトウェアを用いてシグナル／ノイズ（Ｓ／Ｎ）比が３以上のピークを自動抽出し、質量電荷比（ｍ／ｚ）、ピーク面積値、泳動時間 (Migration time: MT) を得た。シグナル/ノイズ （Ｓ／Ｎ） 比が３以上のピークを自動抽出し、質量電荷比（ｍ／ｚ）、ピーク面積値、泳動時間 (Migration time: MT) を得た。 ｍ／ｚ とＭＴ の値をもとにＨＭＴ代謝物質ライブラリに登録された全物質との照合、検索を行い、代謝物を同定した。結果を図１（Ａ）に示す。リンデンエキスの添加により産生量が増大した成分を黒文字で記載し、産生量が減少した成分を灰色の文字で記載した。ＣＤＰコリンに着目し、産生量の変化を示した（図１（Ｂ））。
Detection of Metabolites Cationic and anionic metabolites were comprehensively detected by a CE-MS system combining capillary electrophoresis (Agilent CE system) and a mass spectrometer (MS). Peaks with a signal/noise (S/N) ratio of 3 or more were automatically extracted from the detected peaks using automatic integration software, and mass/charge ratios (m/z), peak area values, and migration time (MT) were obtained. Peaks with a signal/noise (S/N) ratio of 3 or more were automatically extracted, and mass/charge ratios (m/z), peak area values, and migration time (MT) were obtained. Based on the m/z and MT values, the metabolites were identified by matching and searching with all substances registered in the HMT metabolite library. The results are shown in Figure 1 (A). Components whose production increased with the addition of linden extract are written in black letters, and components whose production decreased are written in gray letters. Focusing on CDP choline, the change in production is shown (Figure 1 (B)).
実施例２：ＣＤＰ－コリンの添加による影響
ヒト上皮細胞用増殖培地（ＣＥＬＬｎＴＥＣ社）に播種後、ヒト上皮細胞用分化培地（ＣＥＬＬｎＴＥＣ社）中で培養されたヒトケラチノサイト（クラボウ）の培養物にＣＤＰコリン（Sigma-Aldrich、最終濃度０．１ｍＭ）を添加して１％酸素濃度で２４時間培養後、トータルＲＮＡを抽出し、ＡｆｆｉｎｉｔｙＳｃｒｉｐｔ ＱＰＣＲ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Agilent Technologies）を用いてｃＤＮＡを調製した。ｑＰＣＲは、ＬｉｇｈｔＣｙｃｌｅｒ（登録商標）ＦａｓｔＳｔａｒｔ ＤＮＡ ＭａｓｔｅｒＰＬＵＳ ＳＹＢＲ Ｇｒｅｅｎ Ｉ及びＬｉｇｈｔＣｙｃｌｅｒ（Roche Molecular Systems,Inc、Pleasanton）を用いた。クラウディン、オクルーディン、ＺＯ－１の遺伝子発現を、下記のプライマーを用いて測定し、添付の説明書に従って、ＧＡＰＤＨを標準としてΔＣｔ法により数値化した。結果を図２に示す。
細胞免疫染色
ヒト上皮細胞用培地（ＣＥＬＬｎＴＥＣ社）に播種後、ヒト上皮細胞用分化培地（ＣＥＬＬｎＴＥＣ社）中で培養されたヒトケラチノサイト（クラボウ）にＣＤＰコリン（最終濃度０．１ｍＭ）を添加して１％酸素濃度で２４時間培養後、メタノール固定した。４％スキムミルクでブロッキングした後、抗オクルディンモノクローナル抗体（Invitrogen）と４℃で一晩反応させ、最後にＡｌｅｘａ Ｆｌｕｏｒ ５９４標識抗マウスＩｇＧ抗体（Invitrogen）と反応させて、共焦点顕微鏡ＬＳＭ８８０（Leica）により検出した。得られた画像の蛍光強度について、ＩｍａｇｅＪ画像解析ソフトウェアを用いて定量した。結果を図３（Ａ）及び（Ｂ）に示す。
Cell immunostaining After seeding in a human epithelial cell medium (CELLnTEC), human keratinocytes (Kurabo) cultured in a human epithelial cell differentiation medium (CELLnTEC) were added with CDP choline (final concentration 0.1 mM) and cultured at 1% oxygen concentration for 24 hours, followed by methanol fixation. After blocking with 4% skim milk, the cells were reacted with anti-occludin monoclonal antibody (Invitrogen) overnight at 4°C, and finally reacted with Alexa Fluor 594-labeled anti-mouse IgG antibody (Invitrogen) and detected by a confocal microscope LSM880 (Leica). The fluorescence intensity of the obtained images was quantified using ImageJ image analysis software. The results are shown in Figures 3(A) and (B).
スクラッチアッセイ
Ｉｎｃｕｃｙｔｅ（登録商標）９６―Ｗｅｌｌ Ｓｃｒａｔｃｈ Ｗｏｕｎｄ Ｃｅｌｌ Ｍｉｇｒａｔｉｏｎ ａｎｄ Ｉｎｖａｓｉｏｎ Ａｓｓａｙｓモジュール（Sartorius）の説明書に従って行った。ヒトケラチノサイトを Ｉｎｃｕｃｙｔｅ（登録商標） Ｉｍａｇｅｌｏｃｋ ９６－Ｗｅｌｌ Ｐｌａｔｅ （Sartorius）上でコンフルエントになるまで培養後、Ｉｎｃｕｃｙｔｅ（登録商標） ９６－Ｗｅｌｌ Ｗｏｕｎｄｍａｋｅｒ Ｔｏｏｌ（Sartorius）を用いて、一定の幅で細胞を掻き取ってウェル上に溝を作成し、ＣＤＰコリン（最終濃度０．１ｍＭ）を添加して１％酸素濃度で３６時間培養し、ウェル上の細胞のない領域が埋められる様子をインキュサイトライブセル解析システムにより解析した。結果を図４（Ａ）に示す。スクラッチ箇所における細胞密度を計算してしました（図４（Ｂ））。
Scratch assay was performed according to the instructions for the Incucyte® 96-Well Scratch Wound Cell Migration and Invasion Assays module (Sartorius). After culturing human keratinocytes on an Incucyte® Imagelock 96-Well Plate (Sartorius) until confluent, cells were scraped off at a certain width using the Incucyte® 96-Well Woundmaker Tool (Sartorius) to create grooves on the wells, and CDP-choline (final concentration 0.1 mM) was added and cultured at 1% oxygen concentration for 36 hours. The state in which the cell-free areas on the wells were filled was analyzed using the Incucyte Live Cell Analysis System. The results are shown in FIG. 4(A). The cell density at the scratch site was calculated (Figure 4(B)).
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