JP2011093830A - Skin barrier function improving agent - Google Patents
Skin barrier function improving agent Download PDFInfo
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- JP2011093830A JP2011093830A JP2009248016A JP2009248016A JP2011093830A JP 2011093830 A JP2011093830 A JP 2011093830A JP 2009248016 A JP2009248016 A JP 2009248016A JP 2009248016 A JP2009248016 A JP 2009248016A JP 2011093830 A JP2011093830 A JP 2011093830A
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- trpv
- trpv4
- ligand
- action
- skin
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Abstract
Description
本発明は、皮膚バリア機能改善用の組成物に関し、詳しくは、温度感受性受容体（TRPV：Transient receptor potential vanilloid）リガンドを含有する組成物に関する。
The present invention relates to a composition for improving skin barrier function, and more particularly, to a composition containing a temperature sensitive receptor (TRPV) ligand.
皮膚には、熱や痛みなどの外部刺激を生体内に伝える働きに加え、水分の蒸散を防いだり、外部刺激や物理的刺激から体内を保護するバリア機能が存する。皮膚バリア機能は、生体の機能維持に重要な働きをしているが、温度、湿度、皮膚の過度の摩擦、生活リズムの変調、ストレスなどにより機能が低下することが知られており、この様な原因により、皮膚の乾燥が進み、刺激に対し過敏になり、刺激を受け易くなる。さらに、この状態が進めば、体外からの細菌や有害物質の侵入による直接的な障害、アレルギ−反応などを引き起こし、アトピ−性皮膚炎や皮脂欠乏性湿疹などの皮膚障害の発症に繋がる。また、皮膚は、人目に触れ、見た目の美しさに直接影響するため、多くの女性にとって大きな関心事であり、肌状態を健康に保つために、皮膚バリア機能改善剤（例えば、特許文献１を参照）、肌荒れ改善剤（例えば、特許文献２を参照）、セラミド合成促進剤（例えば、特許文献３を参照）等を含有する皮膚外用剤等の様々な試みがなされている。
In addition to the function of transmitting external stimuli such as heat and pain to the living body, the skin has a barrier function that prevents the evaporation of moisture and protects the body from external stimuli and physical stimuli. The skin barrier function plays an important role in maintaining the functions of the living body, but it is known that the function decreases due to temperature, humidity, excessive skin friction, lifestyle rhythm modulation, stress, etc. Due to various causes, the skin becomes dry, becomes hypersensitive to irritation, and is susceptible to irritation. Furthermore, if this state progresses, it causes direct damage due to invasion of bacteria and harmful substances from outside the body, allergic reaction, etc., leading to the onset of skin disorders such as atopic dermatitis and sebum-deficient eczema. In addition, since skin touches human eyes and directly affects the beauty of appearance, it is a major concern for many women. In order to keep the skin condition healthy, a skin barrier function improving agent (for example,
１９８９年にショウジョウバエの光受容器異常変異株の原因遺伝子として同定された非選択性陽イオンチャネルのTRP（Transient reｃeptor potential）チャネルは、アミノ酸配列の相同性により少なくとも９種類のサブファミリ−に分類されている。ＴＲＰチャネルに属する温度感受性受容体は、初めてＴＲＰＶ１の分子実体が解明され、現在に至るまで、哺乳類では、低温から高温までそれぞれの温度刺激により開口する９種類の温度感受性受容体（TRPV１、ＴＲＰＶ２、ＴＲＰＶ３、ＴＲＰＶ４、ＴＲＰＭ２、ＴＲＰＭ４、ＴＲＰＭ５、ＴＲＰＭ８、ＴＲＰＡ１）が報告されている。TRPV４は、当初、低侵透圧で活性化される浸透圧感受性受容体として発見され、感覚神経、視床下部、皮膚、腎臓、肺、内耳などの様々な組織で発現し、浸透圧刺激、機械刺激による痛みや炎症性の痛みの伝達に重要な役割を果たしている。また、細胞膜貫通型カルシウム流入チャネルとして機能するTRPV４は、前記の物理的な刺激に加え、内在性カンナビノイド、アラキドン酸代謝物、４α−ホルボ−ルエステルなどの化合物によっても活性化される。この様に、発現する組織や刺激応答の多様性から多くの生物活性を示すことが考えられ、刺激及び痛みの認識、自閉症をはじめとする神経疾患症状に深く関与する。また、これら以外の生物活性としては、TRPV４欠損マウスにおいて体重増加、総脂肪量及び内臓脂肪量の有意な抑制作用（例えば、特許文献４を参照）等のエネルギ−代謝関連の活性が報告されている。また、表皮細胞に存在するTRPV４に関する研究により、ＴＲＰＶは、水分蒸散センサ−としての機能を有し、４α−ホルボ−ルエステル（４α−PDD）による活性化により、水分蒸散を抑制すること、皮膚バリア機能を亢進することが報告されている（例えば、非特許文献１、非特許文献２を参照）。
The non-selective cation channel TRP (Transient Receptor Potential) channel, which was identified in 1989 as the causative gene of a Drosophila photoreceptor mutant, is classified into at least nine subfamilies based on amino acid sequence homology. ing. The temperature sensitive receptors belonging to the TRP channel have been elucidated for the first time as the molecular entity of TRPV1. Until now, in mammals, nine types of temperature sensitive receptors (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, TRPA1) have been reported. TRPV4 was first discovered as an osmotic-sensitive receptor activated by low osmotic pressure and expressed in various tissues such as sensory nerve, hypothalamus, skin, kidney, lung, and inner ear. It plays an important role in the transmission of pain caused by stimulation and inflammatory pain. TRPV4 that functions as a transmembrane calcium influx channel is also activated by compounds such as endogenous cannabinoids, arachidonic acid metabolites, and 4α-phorbol esters in addition to the above physical stimulation. Thus, it is considered that many biological activities are exhibited from the variety of expressed tissues and stimulation responses, and deeply involved in neurological disease symptoms such as stimulation and pain recognition and autism. In addition, as biological activities other than these, energy-metabolism-related activities such as weight gain, significant suppression of total fat mass and visceral fat mass (eg, see Patent Document 4) have been reported in TRPV4-deficient mice. Yes. In addition, TRPV4, which is present in epidermal cells, has a function as a moisture transpiration sensor. TRPV4 suppresses moisture transpiration by activation with 4α-phorbol ester (4α-PDD). It has been reported that the function is enhanced (for example, see Non-Patent
TJ及びAJは、細胞の周囲にベルト状に存在し、隣り合った細胞同士を密着させることにより隙間を塞ぐと共に、連続的に細胞を繋ぎ止める細胞間接着構造体である。皮膚組織において、TJは、顆粒層の表皮細胞に存在しており、水や物質が細胞間隙を透過するのを防ぎ、皮膚バリア機能を維持するために重要な役割を果たしている（例えば、非特許文献３を参照）。また、AJの正常な形成がTJの形成に重要な役割を果たすことも知られている。しかしながら、TJ及びAJなどの細胞間接着構造体とTRPVの相互作用に関しては、これまで全く知られていなかった。このため、TRPVとＴＪ及びＡＪなどの細胞間接着構造体との相互作用、取り分け、ＴＲＰＶ４の活性化によりTJ及び／又はAJの形成が促進される作用を有する成分には、新たな作用機序を有する皮膚バリア機能改善剤として相加又は加乗効果が期待出来る。 TJ and AJ are cell-cell adhesion structures that exist in the form of a belt around cells, close gaps by adhering adjacent cells together, and continuously connect cells. In skin tissue, TJ is present in the epidermal cells of the granule layer and plays an important role in preventing water and substances from penetrating the cell gap and maintaining the skin barrier function (for example, non-patented Reference 3). It is also known that normal formation of AJ plays an important role in TJ formation. However, until now, nothing has been known about the interaction between TRPV and TRPV such as TJ and AJ. For this reason, there is a new mechanism of action for a component having an action that promotes the formation of TJ and / or AJ by the interaction of TRPV with intercellular adhesion structures such as TJ and AJ, and activation of TRPV4. An additive or additive effect can be expected as a skin barrier function-improving agent.
TRPV、特に、TＲＰＶ４に作用する物質としては、ビフェニル誘導体（例えば、特許文献２を参照）、１，４−ジアミン誘導体（例えば、特許文献３を参照）等のＴＲＰＶ４アゴニスト、更には、ピペリジン及びピロリジン誘導体、チオフェン誘導体（例えば、特許文献４を参照）のＴＲＰＶ４アゴニスト（例えば、特許文献５、特許文献６、特許文献７を参照）が、疼痛、間接リュウマチ、神経障害、痛覚過敏などの症状に緩和、改善に有効であることが知られている。しかしながら、ＴＲＰＶ４アゴニストに皮膚バリア機能改善作用が存することは、４α−ホルボ−ルエステルに（４α−PDD）よる活性化により、水分蒸散を抑制すること、皮膚バリア機能を亢進することが報告されている（例えば、非特許文献１、非特許文献２を参照）のみであり、その作用機序は解明されていなかった。また、細胞間接着構造体のTJ及びAJとTRPVに共に作用する成分、取り分け、表皮に存在するTRPV４を活性化することよりTJ及び／又はAJの形成を促進する作用を有する成分が、皮膚バリア機能を向上させ、肌荒れ改善に有効であることも知られていなかった。
TRPV, in particular, TRPV4 acts as TRPV4 agonists such as biphenyl derivatives (see, for example, Patent Document 2) and 1,4-diamine derivatives (see, for example, Patent Document 3), piperidine and pyrrolidine. Derivatives, thiophene derivatives (see, for example, Patent Document 4) TRPV4 agonists (see, for example, Patent Document 5, Patent Document 6, and Patent Document 7) alleviate symptoms such as pain, indirect rheumatism, neuropathy, and hyperalgesia It is known to be effective for improvement. However, it has been reported that the TRPV4 agonist has an effect of improving the skin barrier function, that the activation by 4α-phorbol ester (4α-PDD) suppresses water transpiration and enhances the skin barrier function. (See, for example,
本発明は、この様な状況下において為されたものであり、皮膚バリア機能改善に好適な、新規なTRPVリガンドを含有する組成物を提供することにある。 The present invention has been made under such circumstances, and an object thereof is to provide a composition containing a novel TRPV ligand suitable for improving the skin barrier function.
この様な実情に鑑みて、本発明者等は、皮膚バリア機能改善に好適な、組成物を求めて、鋭意、努力を重ねた結果、新規なTRPVリガンドを含有する組成物が、その様な特性を備えていることを見出した、更に、詳細な検討を加えた結果、この様なTRPVリガンドとしては、特に、TRPVを活性化するアゴニスト作用を有するリガンドが好ましく、この様な成分としては、ミソハギ科サルスベリ属、又は、アカネ科カギカズラ属に属する植物より得られる植物抽出物にTRPV活性化作用が存することを見出し、発明を完成させるに至った。本発明は、以下に示す通りである。
＜１＞ ミソハギ科サルスベリ属に属する植物、アカネ科カギカズラ属に属する植物より得られる抽出物よりなる温度感受性受容体（TRPV：Transient receptor potential vanilloid）リガンド。
＜２＞ 前記ミソハギ科サルスベリ属に属する植物、アカネ科カギカズラ属に属する植物が、ミソハギ科サルスベリ属オオバナサルスベリ、アカネ科カギカズラ属ガンビ−ルであることを特徴とする、＜１＞に記載のTRPVリガンド。
＜３＞ 前記TRPVが、TRPV４であることを特徴とする、＜１＞又は＜２＞に記載のTRPVリガンド。
＜４＞ TRPVリガンドの作用が、TRPV４アゴニスト作用であることを特徴とする＜１＞〜＜３＞の何れか一項に記載のTRPVリガンド。
＜５＞ TRPVリガンドは、表皮細胞におけるタイトジャンクション（TJ：Tight-junction）及び／又はアドヘレンスジャンクション（AJ：Adherens-junction）の形成促進作用を有することを特徴とする、＜１＞〜＜４＞の何れか一項に記載のTRPVリガンド。
＜６＞ ＜１＞〜＜５＞の何れか一項に記載のTRPVリガンドを含有することを特徴とする、皮膚外用剤。
＜７＞ 皮膚バリア機能改善用であることを特徴とする、＜６＞に記載の皮膚外用剤。
＜８＞ 化粧料、医薬部外品であることを特徴とする、＜６＞又は＜７＞の何れか一項に記載の皮膚外用剤。
＜９＞ ミソハギ科サルスベリ属、又は、アカネ科カギカズラ属に属する植物の抽出物を含有する、皮膚バリア機能改善のための化粧料の製造方法であって、ミソハギ科サルスベリ属、又は、アカネ科カギカズラ属の植物の植物体を極性溶媒で抽出し、所望により、溶媒除去、分画精製を行い、得られた抽出物のTRPVに対するリガンド作用を調べ、リガンド作用を有する抽出物、その溶媒除去又はその分画精製物を抽出候補として選択し、かかる抽出物候補に関し、表皮のTJ及び／又はAJへの作用を調べ、TJ及び／又はAJの形成促進作用を有するものを選択し、化粧品に配合することを特徴とする、皮膚バリア機能向上のための化粧料の製造方法。
In view of such circumstances, the present inventors sought and sought for a composition suitable for improving the skin barrier function, and as a result of intensive efforts, a composition containing a novel TRPV ligand has been developed. As a result of further detailed investigation, it was found that such a TRPV ligand, in particular, a ligand having an agonistic action to activate TRPV is preferable, and as such a component, The present inventors have found that a plant extract obtained from a plant belonging to the genus Crape myraceae or the genus Rubiaceae is found to have a TRPV activating action, and completed the invention. The present invention is as follows.
<1> A temperature-sensitive receptor (TRPV) ligand consisting of an extract obtained from a plant belonging to the genus Sarsbergi, a plant belonging to the genus Rubiaceae.
<2> The TRPV according to <1>, wherein the plant belonging to the genus Cryptaceae, the plant belonging to the genus Rubiaceae is the genus Abyssum, Gambir of the genus Rubiaceae Ligand.
<3> The TRPV ligand according to <1> or <2>, wherein the TRPV is TRPV4.
<4> The TRPV ligand according to any one of <1> to <3>, wherein the action of the TRPV ligand is a TRPV4 agonistic action.
<5> The TRPV ligand has an action of promoting the formation of tight junctions (TJ: Tight-junction) and / or adherence junctions (AJ: Adherens-junction) in epidermal cells. 4> The TRPV ligand according to any one of 4>.
<6> An external preparation for skin, comprising the TRPV ligand according to any one of <1> to <5>.
<7> The external preparation for skin according to <6>, which is for improving skin barrier function.
<8> The external preparation for skin according to any one of <6> or <7>, which is a cosmetic or a quasi-drug.
<9> A method for producing a cosmetic for improving the skin barrier function, comprising an extract of a plant belonging to the genus Cranaceae or Aceraceae, The plant body of the genus plant is extracted with a polar solvent, and if necessary, the solvent is removed, fractional purification is performed, and the ligand action of the obtained extract on TRPV is examined. Select a fractionated purified product as an extraction candidate, examine the effect of the epidermis on TJ and / or AJ, select one that has a TJ and / or AJ formation promoting effect, and add it to cosmetics. A method for producing a cosmetic material for improving the skin barrier function.
本発明によれば、皮膚バリア機能改善に好適な、新規なTRPVリガンドを含有する組成物を提供することが出来る。
ADVANTAGE OF THE INVENTION According to this invention, the composition containing a novel TRPV ligand suitable for skin barrier function improvement can be provided.
＜本発明の組成物の必須成分であるTRPVリガンド＞
TRPチャネルは、低温から高温までそれぞれの温度刺激により開口する９種類の温度感受性受容体（TRPV１、TRPV２、TRPV３、TRPV４、ＴＲＰＭ２、ＴＲＰＭ４、ＴＲＰＭ５、ＴＲＰＭ８、ＴＲＰA１）が知られている。これらの内、ＴＲＰＶ１、TRPV３、TRPV４は、表皮細胞に存在することが報告されている。ＴＲＰＶは、細胞膜貫通型のカルシウムイオンチャネルの分子実体であり、細胞内カルシウムイオン濃度を調整することにより、皮膚バリア機能に深く関与するとされている。特に、TRPV１及びTRPV４は、水分蒸散、皮膚バリア機能に深く関与していることが示唆されている。しかしながら、その作用機序は、詳細には解明されていない。本発明の組成物は、TRPVリガンドを含有することを特徴とする。本発明におけるTRPVリガンドは、TRPVに直接的又は間接的に作用するリガンドであれば、特段の限定なく適応出来る。前記TRPVリガンドの内、より好ましいものとしては、TRPV４に特異的に作用するリガンドが好適に例示出来る。また、TRPV、取り分け、TRPV４に作用するリガンドとしては、アゴニスト、アンタゴニスト、パ−シャルアゴニスト、インバ−スアゴニスト等のリガンドが好適に例示出来るが、これらの内、好ましいものとしては、アゴニスト、パ−シャルアゴニスト等の受容体を活性化する作用を有するリガンドが好ましい。前記の直接的又は間接的なTRPV活性化作用を有するリガンドは、細胞間脂質及び表皮細胞内におけるカルシウムイオン濃度を調整し、皮膚バリア機能を向上させる。また、本発明においては、TRPV４活性化作用を有するリガンドは、TRPV４に対し直接的又は間接的に作用することによりTJ及び／又はAJの形成促進作用を発揮し、皮膚バリア機能を向上させる。このため、TRPV４活性化作用を介しTJ及び／又はAJ形成促進作用を発揮する物質、若しくは、ＴＲＰＶ４活性化作用とＴＪ及び／又はＡＪ形成促進作用を有する成分を組み合わせることにより、相加又は相乗的な皮膚バリア機能向上作用が発現することが期待される。
<TRPV ligand which is an essential component of the composition of the present invention>
Nine types of temperature-sensitive receptors (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, and TRPA1) that are opened by a temperature stimulus from a low temperature to a high temperature are known as TRP channels. Of these, TRPV1, TRPV3, and TRPV4 have been reported to be present in epidermal cells. TRPV is a molecular entity of a transmembrane calcium ion channel, and is considered to be deeply involved in the skin barrier function by adjusting the intracellular calcium ion concentration. In particular, it has been suggested that TRPV1 and TRPV4 are deeply involved in moisture transpiration and skin barrier function. However, the mechanism of action has not been elucidated in detail. The composition of the present invention is characterized by containing a TRPV ligand. The TRPV ligand in the present invention can be applied without particular limitation as long as it is a ligand that directly or indirectly acts on TRPV. Among the TRPV ligands, a ligand that specifically acts on TRPV4 can be preferably exemplified. Further, as ligands acting on TRPV, particularly, TRPV4, ligands such as agonists, antagonists, partial agonists, inverse agonists and the like can be preferably exemplified. Among these, preferred are agonists, A ligand having an action of activating a receptor such as a char agonist is preferred. The ligand having a direct or indirect TRPV activation action adjusts the concentration of calcium ions in intercellular lipids and epidermal cells and improves the skin barrier function. Further, in the present invention, a ligand having TRPV4 activating action exerts a TJ and / or AJ formation promoting action by acting directly or indirectly on TRPV4 and improves the skin barrier function. Therefore, an additive or synergistic effect is obtained by combining a substance that exhibits TJ and / or AJ formation promoting action through TRPV4 activation action, or a component having TRPV4 activation action and TJ and / or AJ formation promoting action. It is expected that the effect of improving the skin barrier function is exhibited.
本発明の皮膚バリア機能向上作用を有する成分の鑑別方法におけるTRPVの活性化作用の評価方法としては、TRPVの活性化作用が評価可能な方法であれば、特段の限定なく適応することが出来るが、特に、細胞内カルシウムイオン濃度を指標としたTRPVの活性化作用の評価方法が好ましい。また、前記TRPVの活性化作用の評価方法の内、TRPV４の活性化作用を評価する方法が好ましい。これは、TRPV４が、表皮細胞に存することが確認されており、皮膚バリア機能への関与が期待されるためである。前記TRPVの活性化作用を評価する方法の内、さらに好ましいものとしては、TRPV４の発現が確認された細胞、より好ましくは、TRPV４を過剰発現した細胞における細胞内カルシウムイオン濃度を指標としてTRPV４の活性化作用を判別する方法が好ましい。TRPV４の活性化作用を判別するための指標となる細胞内カルシウムイオン濃度を測定する方法としては、細胞内のカルシウムイオン濃度を測定する方法であれば特段の限定なく適応することが出来るが、操作の簡便性、測定感度及び精度などの面から、例えば、Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M.: Drosophila painless is a Ca2+-requiring channel activated by noxious heat.: J Neurosci.， ２００８ Oct １；２８（４０）：９９２９−３８に記載のカルシウムイメ−ジング法又はパッチクランプ法が好ましい。 As an evaluation method of TRPV activation in the method for identifying a component having an action of improving skin barrier function according to the present invention, any method capable of evaluating the activation of TRPV can be applied without particular limitation. In particular, a method for evaluating the activation effect of TRPV using the intracellular calcium ion concentration as an index is preferable. Of the methods for evaluating the activation effect of TRPV, a method for evaluating the activation effect of TRPV4 is preferred. This is because TRPV4 has been confirmed to exist in epidermal cells and is expected to be involved in the skin barrier function. Among the methods for evaluating the activation effect of TRPV, more preferably, the activity of TRPV4 is measured using the intracellular calcium ion concentration in cells in which TRPV4 expression is confirmed, more preferably in cells overexpressing TRPV4 as an index. A method for discriminating the chemical action is preferred. As a method for measuring intracellular calcium ion concentration as an index for discriminating the activation action of TRPV4, any method can be used without particular limitation as long as it is a method for measuring intracellular calcium ion concentration. From the aspects of simplicity, measurement sensitivity and accuracy, for example, Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M .: Drosophila painless is a Ca 2+ -requiring channel activated by noxious heat .: J Neurosci., 2008 Oct 1: Calcium imaging method or patch clamp method described in 28 (40): 9929-38 is preferable.
本発明のTRPV活性化作用を有する成分を評価するために用いる細胞としては、TRPV発現が確認されている細胞であれば、特段の限定なく適応することが出来るが、測定感度及び精度の面から、TRPV過剰発現細胞を使用することがより好ましい。特に、ＴＲＰＶ１過剰発現細胞の作製に関しては、例えば、特開２００９−０８２０５３号公報に記載の方法に従い、また、ＴＲＰＶ４過剰発現細胞の作製に関しては、前記方法を基に、後記の方法によりＴＲＰＶ４過剰発現細胞を作製することが出来る。 As a cell used for evaluating the component having TRPV activation action of the present invention, any cell having TRPV expression confirmed can be applied without particular limitation, but from the viewpoint of measurement sensitivity and accuracy. More preferably, TRPV overexpressing cells are used. In particular, for the production of TRPV1 overexpressing cells, for example, according to the method described in JP-A-2009-082053, and for the production of TRPV4 overexpressing cells, TRPV4 overexpression is carried out by the method described below based on the above method. Cells can be made.
本発明におけるTRPV４活性化作用の評価に使用するTRPV４の発現が確認されている細胞に関しては、TRPV４を発現している細胞であれば特段の限定なく適応出来、より好ましいものとしては、TRPV４発現ベクタ−を宿主細胞に導入することによりTRPVを過剰発現させた細胞が好ましい。また、TRPV４を過剰発現させる宿主細胞としては、細菌の細胞、植物細胞、動物細胞、昆虫細胞などが挙げられ、より好ましくは、HEK２９３細胞、CHO細胞、COS-７細胞、NIH３T３細胞等が好適に例示出来る。前記宿主細胞としては、TRPV４発現ベクタ−を取り込み、効率的にTRPV４が発現され、且つ、培養が容易であることが好ましい。また、TRPV４発現ベクタ−の作製においては、TRPV４をコ−ドするcＤＮＡであれば特段の限定なく使用することが出来、より好ましいものとしては、哺乳動物細胞用ベクタ−であるｐｃＤＮＡ３（インビトロジェン社製）にＴＲＰＶ４をコ−ドした核酸をライゲ−トしたものが好適に例示出来る。前記ＴＲＰＶ４をコ−ドした核酸としては、全ての塩基配列をコ−ドしたものを、例えば、m-RNAを抽出し、これに逆転写酵素を作用させｃDNAとしたものを使用することも出来るし、TRPV４主要機能をコ−ドした部分のみを適切なプライマ−を選択し、PCRにより増幅させて、これを用いてライゲ−トすることも出来る。特に好ましいものとしては、「配列表１に示すオリゴヌクレオチドが例示出来、かかる配列のオリゴヌクレオチドは、配列番号２（配列表２）と配列番号３（配列表３）のプライマーを用いて抽出されたDNA或いは、cDNAをPCR反応に付すことにより、得ることができる。
Regarding cells in which the expression of TRPV4 used for evaluation of the TRPV4 activation action in the present invention is confirmed, any cell expressing TRPV4 can be applied without particular limitation, and more preferable is a TRPV4 expression vector. Cells in which TRPV is overexpressed by introducing-into a host cell are preferred. Examples of host cells overexpressing TRPV4 include bacterial cells, plant cells, animal cells, insect cells, etc. More preferably, HEK293 cells, CHO cells, COS-7 cells, NIH3T3 cells and the like are suitable. It can be illustrated. As the host cell, it is preferable that a TRPV4 expression vector is taken in, TRPV4 is efficiently expressed, and culture is easy. Further, in the preparation of the TRPV4 expression vector, any cDNA can be used as long as it is a cDNA encoding TRPV4. More preferable is a cDNA cell vector pcDNA3 (manufactured by Invitrogen). And a nucleic acid obtained by ligating a nucleic acid obtained by coding TRPV4. As the nucleic acid encoded with TRPV4, for example, a nucleic acid obtained by encoding all the nucleotide sequences, for example, an mRNA extracted from this and reacted with reverse transcriptase to form a cDNA can also be used. It is also possible to select an appropriate primer only for the portion where the TRPV4 main function is coded, amplify it by PCR, and use this to ligate. As an especially preferable example, “the oligonucleotide shown in
本発明のＴＲＰＶ活性化作用の評価に使用することが出来る前記ＴＲＰＶ４を過剰発現する細胞は、当該細胞におけるＴＲＰＶ４の機能発現、蛋白質レベルでの発現、蛍光物質の共導入などを指標とし、選択することが出来る。前記ＴＲＰＶ４をコ−ドする核酸が発現可能に宿主細胞に導入されている細胞の選択には、適切な選択培地を用いることが出来る。例えば、ＤＭＥＭやＲＰＭＩ培地にＦＢＳ等の増殖因子を加えたものなどが好適に例示出来る。 The cells overexpressing TRPV4 that can be used for evaluation of the TRPV activation action of the present invention are selected based on the function expression of TRPV4 in the cells, expression at the protein level, co-introduction of a fluorescent substance, and the like as indices. I can do it. An appropriate selection medium can be used for selection of cells in which the nucleic acid encoding TRPV4 has been introduced into a host cell so that it can be expressed. For example, a material obtained by adding a growth factor such as FBS to DMEM or RPMI medium can be suitably exemplified.
前記TRPV４を過剰発現する細胞の培養に用いられる培地としては、当該TRPV４を過剰発現する細胞が生育するのに適した成分、例えば、グルコ−ス、アミノ酸、ペプトン、ビタミン、細胞増殖促進因子（例えば、細胞成長因子、ホルモン、結合タンパク質、細胞接着因子、脂質）、血清（例えば、ＦＢＳ、ＦＣＳなど）、塩化カルシウム、塩化マグネシウムなどを成分とする培地であればよい。前記培地は、市販されている培地であってもよい。前記TRPV４を過剰発現する細胞の培養に用いられる培地としては、かかる細胞に適した培地であればよく、特に限定されないが、ＭＥＭ培地、ＤＭＥＭ培地、ＲＰＭＩ １６４０培地などが挙げられる。例えば、用いられる宿主細胞がＨＥＫ２９３細胞である場合、高グルコ−ス及び１０質量％ＦＢＳ含有ＤＭＥＭ培地などが用いられる。 Examples of the medium used for culturing cells that overexpress TRPV4 include components suitable for growth of cells overexpressing TRPV4, such as glucose, amino acids, peptone, vitamins, cell growth promoting factors (for example, , Cell growth factors, hormones, binding proteins, cell adhesion factors, lipids), serum (eg, FBS, FCS, etc.), calcium chloride, magnesium chloride, etc. The medium may be a commercially available medium. The medium used for culturing the cells overexpressing TRPV4 is not particularly limited as long as it is a medium suitable for such cells, and examples thereof include MEM medium, DMEM medium, and RPMI 1640 medium. For example, when the host cell used is HEK293 cells, a DMEM medium containing high glucose and 10% by mass FBS is used.
本発明におけるTRPVリガンドは、TRPV、取り分け、表皮細胞に存在するＴＲＰＶ４を活性化することにより、ＴＪ及び／又はＡＪの形成を促進することにより皮膚バリア機能向上作用を発現する。本発明におけるＴＲＰＶ（特に、ＴＲＰＶ４）と細胞間接着構造体のＴＪ及び／又はＡＪとの皮膚バリア機能向上に関する相互作用は、以下の試験結果により確認される。即ち、ＴＲＰＶ４リガンドである４α−PDD（４α−ホルボ−ルエステル）によるTRPV４活性化により、TJ及び／又はAJ形成促進が確認された。即ち、TRPV４活性化作用を有する成分は、TJ及び／又はAJ形成促進により皮膚バリア機能向上作用を奏しているということが出来る。 The TRPV ligand in the present invention activates TRPV, particularly, TRPV4 present in epidermal cells, and thereby promotes the formation of TJ and / or AJ, thereby expressing a skin barrier function improving action. The interaction regarding the improvement of the skin barrier function between TRPV (particularly TRPV4) and TJ and / or AJ of the intercellular adhesion structure in the present invention is confirmed by the following test results. That is, TRJ4 activation by TRPV4 ligand 4α-PDD (4α-phorbol ester) was confirmed to promote TJ and / or AJ formation. That is, it can be said that the component having TRPV4 activating action exhibits an action of improving skin barrier function by promoting TJ and / or AJ formation.
本発明のTRPVリガンドによるTJ及び／又はAJの形成促進作用を評価する方法としては、例えば、非特許文献３に記載された３次元表皮モデル又は生体皮膚を用いたビオチン標識拡散トレ−サ−による細胞間物質移動確認法、培養細胞シ−トを用いたFITC-dextran細胞間物質透過試験法、フリ−ズフラクチャ−法によるTJストランド観察によるストランドの出来具合で機能を類推する方法、更には、特開２００７−１７４９３１号公報に記載の経上皮細胞電気抵抗値（TER：transepithelical electronic resistance）を測定する方法等が例示出来るが、操作の簡便性や測定精度等の面から、FITC-Dextran透過性試験法、経上皮細胞電気抵抗値（TER値）を測定する方法が好適に例示出来る。
As a method for evaluating the TJ and / or AJ formation promoting action by the TRPV ligand of the present invention, for example, a three-dimensional epidermis model described in
かかるTJ及び／又はAJの形成促進作用の評価方法によれば、支持体上で構築した表皮角化細胞層膜における細胞間物質移動の程度を、細胞間物質透過性又は経上皮細胞電気抵抗値（TER値）の測定などにより確認し、細胞間物質移動の程度が、被験物質非存在下に比して、被験物質存在下で抑制された場合、表皮角化細胞層膜のTJ及び／又はAJが緻密に構築されていると判断され、これによってin vivoの皮膚に適応した場合には、角層又は表皮顆粒層の物質透過抑制作用が向上し、以って、皮膚バリア機能が向上されると判定される。 According to the method for evaluating the formation promoting action of TJ and / or AJ, the degree of intercellular mass transfer in the epidermal keratinized cell layer membrane constructed on the support is determined based on the intercellular substance permeability or transepithelial cell electrical resistance value. (TER value) confirmation, etc., when the degree of intercellular mass transfer is suppressed in the presence of the test substance compared to the absence of the test substance, TJ and / or When it is judged that AJ is densely constructed and applied to the skin in vivo, the substance permeation inhibitory action of the stratum corneum or epidermal granule layer is improved, thereby improving the skin barrier function. It is determined.
＜試験例１：TRPVリガンドを用いたＴＥＲ値測定試験＞
凍結正常ヒト表皮角化細胞（NHEK）（倉敷紡績株式会社製）を解凍し、０．１５mM−カルシウムイオン含有培養液（Humedia−KG２：倉敷紡績株式会社製）にて、３７℃、５％二酸化炭素気流下にて培養した。Millicell Tissue Culture Plate（ミリポア社製）にトランズウェル（Corning社製、３４６０-Clear）をセットし、上層０．５ mＬ、下層１．５ mＬの前記培養液を入れ、前記トランズウェル上層に前記NHEKを２．５×１０５cells／cm２で播種し、３７℃、５％二酸化炭素気流下にて２４時間培養した。前記NHEKが１２０％コンフルエントまで増殖したことを確認し、１．５mM 塩化カルシウム添加Humedia−KG２培地に交換し、３３℃、５％二酸化炭素気流下にて４８〜７２時間培養して、表皮角化細胞層膜を構築した。その後、１０μM ４α−PDD（SIGMA社製）、３mM Camphor（control、SIGMA社製）又はメタノ−ル（vehicle、和光純薬株式会社製）をそれぞれ添加した１．５mM 塩化カルシウム添加Humedia−KG２培地に交換し、TRPV４／TRPV３不活性温度領域である２４℃、５％二酸化炭素気流下にて培養した。各物質を含有する培地に交換後、０、３、６、９時間後にTER値を測定した。TER値は、サンプルをクリ−ンベンチ内で３０分間馴化した後、Millicell ERS（Millipore会製）を用いて測定を行った。結果を図１に示す。
<Test Example 1: TER value measurement test using TRPV ligand>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed and cultured at 37 ° C., 5% in 0.15 mM-calcium ion-containing culture medium (Humedia-KG2: Kurashiki Boseki Co., Ltd.). Culturing was performed under a carbon stream. Set a Transwell (Corning, 3460-Clear) on a Millicell Tissue Culture Plate (Millipore), put 0.5 mL of the upper layer and 1.5 mL of the lower layer into the upper layer of the Transwell. Was seeded at 2.5 × 10 5 cells / cm 2 and cultured at 37 ° C. in a 5% carbon dioxide stream for 24 hours. Confirmed that NHEK has grown to 120% confluence, replaced with Humid-KG2 medium supplemented with 1.5 mM calcium chloride, cultured at 33 ° C. under 5% carbon dioxide stream for 48 to 72 hours, and keratinized A cell layer membrane was constructed. Then, to 10 mM 4α-PDD (manufactured by SIGMA), 3 mM Camphor (control, manufactured by SIGMA) or methanol (vehicle, manufactured by Wako Pure Chemical Industries, Ltd.) and 1.5 mM calcium chloride added Humedia-KG2 medium, respectively. The cells were exchanged and cultured in a TRPV4 / TRPV3 inactive temperature range of 24 ° C. in a 5% carbon dioxide stream. The TER value was measured after 0, 3, 6, and 9 hours after changing to the medium containing each substance. The TER value was measured using Millicell ERS (manufactured by Millipore Association) after acclimating the sample for 30 minutes in the clean bench. The results are shown in FIG.
皮膚バリア機能に深く関与する表皮細胞においては、TRPV３及びTRPV４の特異的な発現が確認されている。図１の結果より、TRPV４不活性温度領域において、TRPV４リガンドである４α−PDD存在下で培養したNHEKは、非存在下で培養したNHEKに比して、統計学的に有意なTER値の上昇が確認された。一方、TRPV３リガンドのCamphor存在下で培養したNHEKにおいては、非存在下に比して、TER値の上昇は認められなかった。TER値の上昇、即ち、TJ及び／又はAJ形成促進による皮膚バリア機能向上作用は、TRPV４の特異的な活性化により発揮されることが示された。 Specific expression of TRPV3 and TRPV4 has been confirmed in epidermal cells that are deeply involved in the skin barrier function. From the results of FIG. 1, in the TRPV4 inactive temperature region, NHEK cultured in the presence of TRPV4 ligand 4α-PDD has a statistically significant increase in TER value compared to NHEK cultured in the absence. Was confirmed. On the other hand, in the NHEK cultured in the presence of the TRPV3-ligand Camphor, no increase in TER value was observed compared to the absence. It was shown that the increase in TER value, that is, the action of improving skin barrier function by promoting TJ and / or AJ formation is exhibited by specific activation of TRPV4.
＜試験例２：TRPVリガンドを用いたFITC−Dextran物質透過試験＞
前記のTER値測定試験において、測定に使用した培地交換後６時間後のサンプルの培地を除去した。前記トランズウェル上層に０．５ mLのApical Buffer（１．４５mM CaCl２、１０mM glucose、１ mg／mL FITC−Dextranを含有するＰＢＳ）、下層に１．５ mＬのBasolateral Bufer（１．４５mM CaCl２、１０mM glucoseを含有するＰＢＳ）を添加し、３時間培養した。Basolateral Bufferを回収した後、９６well plateにApical Bufferにより標準曲線群（１００ mg/mＬ〜０ mg/mＬ）を作製する。回収したBasolateral Bufferを９６wellに２００mＬずつ添加した後、分光光度計（励起４８５／５３５nm）にて測定した。結果を図２に示す。
<Test Example 2: FITC-Dextran substance permeation test using TRPV ligand>
In the TER value measurement test, the medium of the sample 6 hours after the medium exchange used for measurement was removed. 0.5 mL of Apical Buffer (1.45 mM CaCl 2 , 10 mM glucose, PBS containing 1 mg / mL FITC-Dextran) is placed on the upper layer of the Transwell, and 1.5 mL of Basolateral Bufer (1.45 mM CaCl 2 is placed on the lower layer. PBS containing 10 mM glucose) was added and cultured for 3 hours. After collecting the Basolateral Buffer, a standard curve group (100 mg / mL to 0 mg / mL) is prepared with Apical Buffer on a 96-well plate. The recovered Basolateral Buffer was added to each 96 ml in 200 mL, and then measured with a spectrophotometer (excitation 485/535 nm). The results are shown in FIG.
図２の結果より、TRPV４温度不活性化領域において、ＴＲＰＶ４リガンドである４α−PDD存在下で培養したNHEKは、非存在下で培養したNHEKに比して、統計学的に有意なFITC−Dextran透過量の抑制が確認された。一方、TRPV３リガンドのCamphor存在下においては、FITC−Dextran透過量の抑制は認められなかった。FITC−Dextran透過量の抑制、即ち、TJ及び／又はAＪの形成促進による皮膚バリア機能向上作用は、TRPV４の特異的な活性化により発揮されることが示された。 From the results shown in FIG. 2, in the TRPV4 temperature inactivation region, NHEK cultured in the presence of TRPV4 ligand 4α-PDD is statistically significant compared to NHEK cultured in the absence. Suppression of permeation amount was confirmed. On the other hand, suppression of FITC-Dextran permeation was not observed in the presence of Camphor of the TRPV3 ligand. It was shown that the suppression of FITC-Dextran permeation, that is, the skin barrier function improving action by promoting the formation of TJ and / or AJ is exhibited by specific activation of TRPV4.
＜試験例３：si−RNA トランスフェクション試験＞
凍結正常ヒト表皮角化細胞（NHEK）（倉敷紡績株式会社製）を解凍し、サブコンフルエントになるまで培養した後、トリプシンEDTAにより剥離、回収し、６ well plateに７．５×１０５cells／wellで播種し、３７℃、５％二酸化炭素気流下にて２時間培養した。また、以下の手順に従い、si−RNAトランスフェクション（volumeは1 well分で記載）を行った。５００μＬ Opti-MEMに、５μＬの１０μM si−RNA（On TargetTplus SMARTpool L-004195-00-0005、Ｈｕｍａｎ TRPV4、NM １４７２０４、Target Sequence：配列表４、配列表５、配列表６及び配列表７の配列を有するsi−RNAの混合物）を加え、次に１５μＬのHiperFectを加えてボルテックスにより混合した後、室温で１０分間静置した。ウェルに滴下し緩やかに拡散させた後、３７℃、５％二酸化炭素気流下で２４時間培養した後、１．５mM 塩化カルシウム含有−Humedia−KG２培地に交換し、経時的にTER値及びFITC-Dextran物質透過性を測定した。また、si−RNAによるTRPV４ノックダウン効率は、Hs TRPV４ 1 SG QuantiTect Primer Assay（Qiagen: QT00077217、配列非公開）をプライマ−として、RT-PCRを行い、TRPV４ mRNA発現量を求め、これにより確認した。結果を図３〜図６に示す。尚、これらのsi−RNAのトランスフェクション試験は、市販のデザイン済みsi−RNA ON−TARGET plus SMART pool Human TRPV４（Thermo scientific社製）を使用し、行った。
<Test Example 3: si-RNA transfection test>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed and cultured until they become subconfluent, then detached and collected with trypsin EDTA, and 7.5 × 10 5 cells / well on a 6-well plate. The cells were seeded in a well and cultured at 37 ° C. in a 5% carbon dioxide stream for 2 hours. Moreover, si-RNA transfection (volume is described in 1 well) was performed according to the following procedure. In 500 μL Opti-MEM, 5 μL of 10 μM si-RNA (On TargetTplus SMARTpool L-004195-00-0005, Human TRPV4, NM 147204, Target Sequence: Sequences 4, 4, 5, and 7) The mixture was then mixed with vortex and then allowed to stand at room temperature for 10 minutes. After dripping into the well and diffusing gently, the cells were cultured for 24 hours at 37 ° C. in a 5% carbon dioxide stream, then replaced with 1.5 mM calcium chloride-containing-Humedia-KG2, and the TER value and FITC- Dextran material permeability was measured. Moreover, TRPV4 knockdown efficiency by si-RNA was confirmed by performing RT-PCR using Hs TRPV41 SG QuantiTect Primer Assay (Qiagen: QT00077217, sequence not disclosed) as a primer to determine the expression level of TRPV4 mRNA. . The results are shown in FIGS. In addition, the transfection test of these si-RNA was performed using commercially available si-RNA ON-TARGET plus SMART pool Human TRPV4 (Thermo scientific company make).
図３及び図４の結果より、NHEKにおけるsi−RNAトランスフェクションにより、極めて高いノックダウン効率でTRPV４ mRNAの発現を抑制し、その抑制作用は、TRPV４に特異的であった。また、図５及び図６の結果により、NHEKに比して、TRPV４をノックダウンしたNHEK（TRPV４／KD）は、統計学的に有意なTER値の抑制が確認された。さらに、TRPV４／KDは、統計学的に有意なFITC-Dextran透過性の増加が確認された。 From the results of FIG. 3 and FIG. 4, expression of TRPV4 mRNA was suppressed with extremely high knockdown efficiency by si-RNA transfection in NHEK, and the inhibitory action was specific to TRPV4. In addition, from the results shown in FIGS. 5 and 6, it was confirmed that NHEK (TRPV4 / KD), in which TRPV4 was knocked down, had a statistically significant suppression of the TER value as compared with NHEK. Furthermore, TRPV4 / KD was confirmed to have a statistically significant increase in FITC-Dextran permeability.
前記の試験１〜３の結果は、TRPV、取り分け、TRPV４を活性化することにより、TJ及び／又はAJの形成が促進され、皮膚バリア機能が向上することを示している。この様に、TRPVリガンド作用を有する成分は、TJ及び／又はAJの形成促進作用を介し、皮膚バリア機能向上作用を発揮し、皮膚バリア機能改善剤として有用である。
The results of the
本発明のＴＲＰＶリガンドとしては、単純な化学物質、動植物由来の抽出物が好適に例示出来、かかる成分を唯１種のみ含有することも出来るし、２種以上を組み合わせて含有させることも出来る。ここで、本発明の動植物由来の抽出物とは、動物又は植物由来の抽出物、具体的には、抽出物自体、抽出物の画分、精製した画分、抽出物乃至は画分、精製物の溶媒除去物の総称を意味する。かかる成分の内、好ましいものとしては、ミソハギ科サルスベリ属、アカネ科カギカズラ属に属する植物より得られる抽出物が好適に例示出来、より好ましくは、ミソハギ科サルスベリ属オオバナサルスベリ、アカネ科カギカズラ属ガンビ−ルより得られる植物抽出物が好適に例示出来る。ミソハギ科サルスベリ属オオバナサルスベリは、熱帯アジア原産の常緑小高木であり、日本においては、沖縄及び九州などで多く栽培されている。また、オオバナサルスベリの葉を糖尿病薬に用いるほか、葉を乾燥させることによりバナバ茶を製造し、漢方、サプリメント等として使用されている。アカネ科カギカズラ属ガンビ−ルは、マラッカ海峡沿岸地方を原産とする植物であり、ガンビ−ルの葉より得られる乾燥水製エキスをアセンヤク（阿仙薬）と呼び、抗線溶活性作用、抗血栓作用、抗腫瘍作用などが知られている。 As the TRPV ligand of the present invention, simple chemical substances and extracts derived from animals and plants can be preferably exemplified, and only one kind of such components can be contained, or two or more kinds can be contained in combination. Here, the animal or plant-derived extract of the present invention is an animal or plant-derived extract, specifically, the extract itself, a fraction of the extract, a purified fraction, an extract or a fraction, purification. This is a general term for the product from which the solvent is removed. Among these components, preferable examples include extracts obtained from plants belonging to the genus Cranaceae and Rubiaceae, and more preferable are extracts of Cranalis genus C., Rubiaceae ganbi- A plant extract obtained from the above can be suitably exemplified. The giant crape myrtle is a evergreen small Takagi native to tropical Asia. In Japan, it is cultivated in Okinawa and Kyushu. In addition to the use of the leaf of the giant crape myrtle as an antidiabetic drug, banaba tea is produced by drying the leaves and used as a traditional Chinese medicine or supplement. The Rubiaceae genus Gambir is a plant native to the coast of the Malacca Strait. The action, the antitumor action, etc. are known.
本発明における前記の植物より得られる抽出物は、日本においては、自生又は生育された植物、漢方生薬原料等として販売される日本産のものを用い抽出物を作製することも出来るし、丸善株式会社などの植物抽出物を取り扱う会社より販売されている市販の抽出物を購入し、使用することも出来る。前記植物より得られる抽出物の作製に用いる植物部位には、特段の限定がなされず、全草を用いることが出来るが、勿論、植物体、地上部、根茎部、木幹部、葉部、茎部、花穂、花蕾等の部位のみを使用することも可能である。抽出に際し、植物体などの抽出に用いる部位は、予め、粉砕或いは細切して抽出効率を向上させるように加工することが好ましい。抽出物製造においては、植物体等の抽出に用いる部位乃至はその乾燥物１質量に対して、溶媒を１〜３０質量部加え、室温であれば数日間、沸点付近の温度であれば数時間浸漬する。浸漬後は、室温まで冷却し、所望により不溶物を除去した後、溶媒を減圧濃縮するなどにより除去することが出来る。しかる後、シリカゲルやイオン交換樹脂を充填したカラムクロマトグラフィ−などで分画精製し、所望の抽出物を得ることが出来る。 In the present invention, the extract obtained from the above-mentioned plant in Japan can be produced using a plant grown in Japan or sold in Japan as a herbal medicine raw material, etc. Commercial extracts sold by companies that handle plant extracts such as companies can be purchased and used. The plant part used for producing the extract obtained from the plant is not particularly limited, and whole grass can be used. Of course, the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, and the stem are used. It is also possible to use only parts such as parts, flower spikes and flower buds. At the time of extraction, it is preferable that a part used for extraction of a plant body or the like is processed in advance so as to improve extraction efficiency by crushing or chopping. In the production of extracts, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a part used for extraction of a plant or the like, or a dry product thereof. Immerse. After the immersion, the solution can be cooled to room temperature, insoluble matter can be removed if desired, and then the solvent can be removed by concentration under reduced pressure. Thereafter, fractionation and purification can be performed by column chromatography packed with silica gel or an ion exchange resin to obtain a desired extract.
前記抽出溶媒としては、極性溶媒が好ましく、水、エタノ−ル、イソプロピルアルコ−ル、ブタノ−ルなどのアルコ−ル類、１，３−ブタンジオ−ル、ポリプロピレングリコ−ルなどの多価アルコ−ル類、アセトン、メチルエチルケトンなどのケトン類、ジエチルエ−テル、テトラヒドロフランなどのエ−テル類から選択される１種乃至は２種以上が好適に例示出来る。 The extraction solvent is preferably a polar solvent, and alcohols such as water, ethanol, isopropyl alcohol, and butanol, and polyvalent alcohols such as 1,3-butanediol and polypropylene glycol. One or two or more selected from ketones such as alcohols, acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran can be preferably exemplified.
本発明のＴＲＰＶリガンドは、皮膚外用剤全量に対し、０．００００１質量％〜１０質量％、より好ましくは、０．０００１質量％〜５質量％、さらに好ましくは、０．００１質量〜３質量％含有させることが好ましい。これは、ＴＲＰＶリガンドの含有量が、少なすぎるとＴＲＰＶ活性化作用、並びに、ＴＲＰＶ活性化によるＴＪ及び／又はＡＪの形成促進作用を介した皮膚バリア機能向上作用が発揮されず、多すぎても、効果が頭打ちになり、この系の自由度を損なう場合が存するためである。また、かかる成分は、ＴＲＰＶ、取り分け、ＴＲＰＶ４が存在する表皮細胞への作用、標的部位への集積性及び選択性に優れ、高い安全性及び安定性を有するために、医薬、食品、化粧料などへの使用が好ましい。 The TRPV ligand of the present invention is 0.00001% by mass to 10% by mass, more preferably 0.0001% by mass to 5% by mass, and further preferably 0.001% by mass to 3% by mass with respect to the total amount of the external preparation for skin. It is preferable to contain. This is because if the content of the TRPV ligand is too small, the TRPV activating action and the skin barrier function improving action through the TJ and / or AJ formation promoting action by the TRPV activation are not exhibited. This is because there are cases where the effect reaches a peak and the degree of freedom of this system is impaired. In addition, since such components are excellent in TRPV, especially, action on epidermal cells in which TRPV4 is present, accumulation on target sites and selectivity, and high safety and stability, pharmaceuticals, foods, cosmetics, etc. Use in is preferred.
＜本発明のＴＲＰＶリガンドを含有する皮膚外用剤＞
本発明の皮膚外用剤は、必須成分としてＴＲＰＶリガンドを含有することを特徴とする。本発明におけるＴＲＰＶリガンドは、ＴＲＰＶに直接的又は間接的に作用するリガンドであれば、特段の限定なく使用することが出来る。前記TRPVリガンドの内、より好ましいものとしては、TRPV４リガンドが好適に例示出来る。また、TRPV、取り分け、TRPV４に作用するリガンドとしては、アゴニスト、パ−シャルアゴニスト等の受容体を活性化する作用を有するリガンドが好ましい。本発明のＴＲＰＶリガンドは、単純な化学物質、動植由来の抽出物とその分画生精製物などの混合精製物のいずれでもよい。本発明の皮膚外用剤には、前記ＴＲＰＶリガンドを、唯１種含有させることも出来るし、２種以上を組み合わせて含有させることも出来る。本発明の皮膚外用剤は、ＴＲＰＶリガンドを配合することにより、TRPVを活性化させ、TJ及び／又はAJの形成を促進し、皮膚バリア機能向上効果を発揮する。
<Skin external preparation containing TRPV ligand of the present invention>
The external preparation for skin of the present invention is characterized by containing a TRPV ligand as an essential component. The TRPV ligand in the present invention can be used without particular limitation as long as it is a ligand that directly or indirectly acts on TRPV. Among the above-mentioned TRPV ligands, TRPV4 ligand can be preferably exemplified as a more preferable one. Moreover, as a ligand which acts on TRPV, especially TRPV4, a ligand having an action of activating a receptor such as an agonist or a partial agonist is preferable. The TRPV ligand of the present invention may be a simple chemical substance or a mixed purified product such as an extract derived from animals and plants and a fractionated purified product thereof. The skin external preparation of the present invention can contain only one TRPV ligand, or a combination of two or more thereof. The external preparation for skin of the present invention activates TRPV by adding a TRPV ligand, promotes the formation of TJ and / or AJ, and exhibits an effect of improving the skin barrier function.
本発明の皮膚外用剤においては、前記必須成分以外に、通常化粧料で使用される任意成分を含有することが出来る。この様な任意成分としては、スクワラン、ワセリン、マイクロクリスタリンワックスなどの炭化水素類、ホホバ油、カルナウバワックス、オレイン酸オクチルドデシルなどのエステル類、オリ−ブ油、牛脂、椰子油などのトリグリセライド類、ステアリン酸、オレイン酸、レチノイン酸などの脂肪酸、オレイルアルコ−ル、ステアリルアルコ−ル、オクチルドデカノ−ル等の高級アルコ−ル、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、アルキルベタイン塩等の両性界面活性剤類、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ソルビタン脂肪酸エステル、脂肪酸モノグリセライド、これらのポリオキシエチレン付加物、ポリオキシエチレンアルキルエ−テル、ポリオキシエチレン脂肪酸エステル等の非イオン界面活性剤類、ポリエチレングリコ−ル、グリセリン、１，３−ブタンジオ−ル等の多価アルコ−ル類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等を含有することができる。製造は、常法に従い、これらの成分を処理することにより、困難なく、為しうる。 In the skin external preparation of the present invention, in addition to the essential components, optional components usually used in cosmetics can be contained. Such optional ingredients include hydrocarbons such as squalane, petrolatum, microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow, coconut oil, etc. , Fatty acids such as stearic acid, oleic acid, retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol, octyldodecanol, anionic surface activity such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate Agents, amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, polyoxyethylenes Nonionic surfactants such as fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin, 1,3-butanediol, thickening / gelling agents, antioxidants, UV absorbers, Coloring agents, preservatives, powders and the like can be contained. Manufacture can be done without difficulty by treating these components according to conventional methods.
これらの必須成分、任意成分を常法に従って処理し、ロ−ション、乳液、エッセンス、クリ−ム、パック化粧料、洗浄料などに加工することにより、本発明の皮膚外用剤は製造できる。皮膚に適応させることの出来る剤型であれば、いずれの剤型でも可能であるが、有効成分が皮膚に浸透して効果を発揮することから、皮膚への馴染みの良い、ロ−ション、乳液、クリ−ム、エッセンスなどの剤型がより好ましい。 The skin external preparation of this invention can be manufactured by processing these essential components and arbitrary components according to a conventional method and processing them into lotions, emulsions, essences, creams, pack cosmetics, cleansing agents and the like. As long as the dosage form can be adapted to the skin, any dosage form is possible, but since the active ingredient penetrates the skin and exerts its effect, the lotion and emulsion that are well-familiar with the skin , Cream, essence and the like are more preferable.
以下に、実施例をあげて、本発明について更に詳細に説明を加えるが、本発明がかかる実施例にのみ、限定されないことは言うまでもない。 Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited to such examples.
＜本発明のTRPVリガンド作用を有する植物抽出物の製造方法＞
本発明の植物抽出物（ミツハギ科サルスベリ属オオバナサルスベリ、アカネ科カギカズラ属ガンビ−ルより得られる抽出物）は、医薬部外品原料規格２００６に従い製造することも出来るし、丸善株式会社等により市販されている植物抽出物を購入し、使用することも出来る。本発明においては、丸善株式会社より購入した植物抽出物を使用した。
<Method for producing plant extract having TRPV ligand action of the present invention>
The plant extract of the present invention (extract obtained from the honey bee family Crape myrtle, Rubiaceae genus Gambir) can be produced according to the quasi-drug raw material standard 2006, and is commercially available from Maruzen Co., Ltd. It is also possible to purchase and use plant extracts. In the present invention, a plant extract purchased from Maruzen Co., Ltd. was used.
＜試験例１：ヒトTRPV４を発現する細胞の作製＞
下記の実験手順に従い、ヒトTRPV４発現細胞を作製した。ヒトTRPV４をコ−ドするｃＤＮＡ〔配列表１：（ＧｅｎＢａｎｋアクセッション番号：ＮＭ ０２１６２５）の９０bp〜２７０５bpのポリヌクレオチド〕を、配列表2及び配列表3のオリゴヌクレオチドをプライマ−として、ＰＣＲを行い、増幅し、哺乳動物細胞用ベクタ−である商品名：ｐｃＤＮＡ３（インビトロジェン社製）のMCSのBamHIサイト及びXhoIサイトに挿入し、ヒトTRPV４発現ベクタ−を得た。得られたヒトTRPV４発現ベクタ−（０．５μｇ相当量）とDsRed（０．１μｇ相当量）、プラスリ−ジェント（商品名、カタログ番号：１１５１４−０１５、インビトロジェン社製）６μＬ、ＯＰＴＩ−ＭＥＭ（登録商標）Ｉ Ｒｅｄｕｃｅｄ−Ｓｅｒｕｍ Ｍｅｄｉｕｍ（カタログ番号：１１０５８０２１、インビトロジェン社製）１００μＬとを混合し、混合物１を得た。また、リポフェクタミン（登録商標、カタログ番号：１８３２４−０１２、インビトロジェン社製） ４μＬとＯＰＴＩ−ＭＥＭ １００μＬとを混合し、混合物２を得た。一方、ＨＥＫ２９３細胞（５×１０５個／直径３５ｍｍシャ−レ）を１０質量％ＦＢＳ含有ＤＭＥＭ培地にて、３７℃、５％二酸化炭素気流下にて７０％のコンフルエントまで培養した。その後、得られた細胞に、前記混合物１と混合物２との混合物を添加した。これにより、ＨＥＫ２９３細胞に前記ヒトＴＲＰＶ４発現ベクタ−を導入し、ＴＲＰＶ４発現細胞を得た。また、同様の方法で、ヒトＴＲＰＶ４を挿入していないベクタ−を作製し、これをＨＥＫ２９３細胞に導入してMock 細胞を作製した。
<Test Example 1: Production of cells expressing human TRPV4>
Human TRPV4-expressing cells were prepared according to the following experimental procedure. PCR was performed using cDNA encoding human TRPV4 [SEQ ID NO: 1 (GenBank Accession No: NM 021625) 90 bp to 2705 bp polynucleotide] as oligonucleotides of SEQ ID NO: 2 and SEQ ID NO: 3. The product was amplified and inserted into the MCS BamHI site and XhoI site of the product name: pcDNA3 (manufactured by Invitrogen), which is a vector for mammalian cells, to obtain a human TRPV4 expression vector. Human TRPV4 expression vector (0.5 μg equivalent) and DsRed (0.1 μg equivalent), positive reagent (trade name, catalog number: 11514-015, manufactured by Invitrogen) 6 μL, OPTI-MEM (registered) (Trademark) I Reduced-Serum Medium (catalog number: 11058021, manufactured by Invitrogen) 100 μL was mixed to obtain a
＜ＴＲＰＶ活性化作用評価１：本発明における植物抽出物のカルシウムイメ−ジング法によるＴＲＰＶ４活性化作用評価＞
本発明の植物抽出物を、溶液A〔組成：１４０mM NaCl、５mM KCl、２mM MgCl２、２mM CaCl２、１０mM グルコ−ス、１０mM HEPES、（ｐＨ７．４）〕に添加し、試験液を作製した。各試験液中における植物抽出物の濃度は、０．１質量％とした。前記の実施例２に従い作製されたＴＲＰＶ４発現細胞もしくはMock細胞を、１〜２０μｇ／ｍｌのＦＵＲＡ ２−ＡＭ（インビトロジェン社製）を含む１０％ＦＢＳ含有ＤＭＥＭ培地中で６０〜９０分間インキュベ−ション（３７℃、５％二酸化炭素気流下）して、TRPV４発現細胞もしくはMock細胞にＦＵＲＡ ２−ＡＭを導入した。ＦＵＲＡ ２−ＡＭ導入後のTRPV４発現細胞もしくはMock細胞を、カルシウムイメ−ジング装置倒立顕微鏡のチャンバ−（RC-26G; Warner Instruments）に入れ、溶液Aにて洗浄した。続いて、前記試験液をチャンバ−に入れて循環させながら、励起波長３４０ｎｍでの蛍光強度と励起波長３８０ｎｍでの蛍光強度とを測定した。その後、前記試験液を用いた場合の励起波長３４０ｎｍでの蛍光強度と励起波長３８０ｎｍでの蛍光強度との蛍光強度比（３４０ｎｍでの蛍光強度／３８０ｎｍでの蛍光強度）を算出した。細胞内カルシウムイオン濃度の変化（カルシウムイオン濃度の増加）に対する被験物質による亢進作用は、IpLabソフトウェア（Scanalytics社製）により解析した。また、陽性対照としてTRPV４リガンドである４α−ホルボ−ルエステル（４α-PDD）を用いて同様の評価を実施した。結果を図７〜図９に示す。
<TRPV activation effect evaluation 1: TRPV4 activation effect evaluation by the calcium imaging method of the plant extract in the present invention>
The plant extract of the present invention was added to solution A [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, 10 mM HEPES (pH 7.4)] to prepare a test solution. . The concentration of the plant extract in each test solution was 0.1% by mass. Incubation of TRPV4-expressing cells or Mock cells prepared according to Example 2 above in a DMEM medium containing 10% FBS containing 1-20 μg / ml FURA 2-AM (manufactured by Invitrogen) for 60-90 minutes ( FURA 2-AM was introduced into TRPV4-expressing cells or Mock cells. The TRPV4-expressing cells or Mock cells after introduction of FURA 2-AM were placed in a chamber (RC-26G; Warner Instruments) of an inverted microscope of calcium imaging apparatus and washed with solution A. Subsequently, the fluorescence intensity at an excitation wavelength of 340 nm and the fluorescence intensity at an excitation wavelength of 380 nm were measured while circulating the test solution in a chamber. Thereafter, a fluorescence intensity ratio (fluorescence intensity at 340 nm / fluorescence intensity at 380 nm) between the fluorescence intensity at the excitation wavelength of 340 nm and the fluorescence intensity at the excitation wavelength of 380 nm when the test solution was used was calculated. The enhancing effect of the test substance on the change in intracellular calcium ion concentration (increased calcium ion concentration) was analyzed by IpLab software (manufactured by Scanalytics). Further, the same evaluation was performed using 4α-phorbol ester (4α-PDD) which is a TRPV4 ligand as a positive control. The results are shown in FIGS.
図７〜図９の結果より、陽性対照の４α-PDDは、顕著な細胞内カルシウムイオン濃度の増加が認められ、この評価系の客観性が確認された。また、本発明の植物抽出物は、TRPV４発現細胞において顕著な細胞内カルシウムイオン濃度の増加が確認された。一方、Mock細胞においては確認されなかった（図１０及び１１）。さらに、カルシウムイオンフリ−の溶液B〔組成：１４０mM NaCl、５mM KCl、２mM MgCl２、５ｍM EGTA、１０mM グルコ−ス、１０mM HEPES、（ｐＨ７．４）〕に本発明の植物抽出物としてアセンヤク抽出物を添加したところ、ＴＲＰＶ４発現細胞における細胞内カルシウムイオン濃度の変化は確認されず（図１２及び１３）、Mock細胞においても確認されなかった（図１４及び図１５）。このことから、アセンヤクエキスによる細胞内カルシウムイオン濃度の増加作用は、ＴＲＰＶ４を介した細胞外からのカルシウムイオン流入であることがわかる。 From the results of FIGS. 7 to 9, the positive control 4α-PDD showed a marked increase in intracellular calcium ion concentration, confirming the objectivity of this evaluation system. In addition, the plant extract of the present invention was confirmed to have a marked increase in intracellular calcium ion concentration in TRPV4-expressing cells. On the other hand, it was not confirmed in Mock cells (FIGS. 10 and 11). Furthermore, the Acacia yak extract as a plant extract of the present invention in calcium ion free solution B [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 5 mM EGTA, 10 mM glucose, 10 mM HEPES, (pH 7.4)] Was added, no change in intracellular calcium ion concentration in TRPV4-expressing cells was confirmed (FIGS. 12 and 13), and no confirmation was observed in Mock cells (FIGS. 14 and 15). From this, it can be seen that the increase effect of intracellular calcium ion concentration by Acacia yak extract is influx of calcium ions from the outside via TRPV4.
＜TJ及びAJの形成促進作用評価１：本発明における植物抽出物のTER値測定＞
前記の試験例１に記載の試験方法に従い、本発明の植物抽出物のＴＥＲ値を測定した。結果を図１６に示す。図１６において、縦軸は、ＴＥＲ値（Ωｍ２）、横軸は、測定時間を表す。図１６の結果より、本発明の植物抽出物は、顕著なTER値の増加を示す、ＴＪ及び／又はＡＪの機能促進作用が認められた。
<Evaluation of TJ and AJ formation promoting action 1: TER value measurement of plant extract in the present invention>
According to the test method described in Test Example 1, the TER value of the plant extract of the present invention was measured. The results are shown in FIG. In FIG. 16, the vertical axis represents the TER value (Ωm 2 ), and the horizontal axis represents the measurement time. From the results of FIG. 16, the plant extract of the present invention was found to have a TJ and / or AJ function promoting action showing a marked increase in TER value.
＜TJ及びAJの形成促進作用評価２：本発明における植物抽出物のFITC-Dextran物質透過試験＞
前記の試験例２に記載の試験方法に従い、本発明の植物抽出物のFITC-Dextran物質透過性を評価した。結果を図１７に示す。図１７の結果より、本発明の植物抽出物は、顕著なFITC-Dextran物質透過性の抑制が認められ、ＴＪ及び／又はＡＪの形成促進作用が認められた。
<Evaluation of TJ and AJ formation promoting action 2: FITC-Dextran substance permeation test of plant extract in the present invention>
According to the test method described in Test Example 2 above, the FITC-Dextran substance permeability of the plant extract of the present invention was evaluated. The results are shown in FIG. From the results shown in FIG. 17, the plant extract of the present invention was found to significantly suppress FITC-Dextran substance permeability and to promote the formation of TJ and / or AJ.
図１６及び図１７に示した結果より、本発明の植物抽出物は、前記のTJ及び／又はAJの形成促進作用評価（ＴＥＲ値測定及びFITC-Dextran物質透過試験）において、共にTJ及びAJ形成促進作用を示した。前記結果より、本発明の植物抽出物は、TRPV活性化作用、並びに、TJ及び／又はAJの形成促進作用を有し、TRPV活性化作用を介するTJ及び／又はAJの形成促進作用による皮膚バリア機能改善効果を有する。
From the results shown in FIG. 16 and FIG. 17, the plant extract of the present invention has both TJ and AJ formation in the TJ and / or AJ formation promoting activity evaluation (TER value measurement and FITC-Dextran substance permeation test). It showed a promoting effect. From the above results, the plant extract of the present invention has a TRPV activation action and a TJ and / or AJ formation promoting action, and a skin barrier by the TJ and / or AJ formation promoting action via the TRPV activation action. Has a function improvement effect.
＜本発明の組成物＞
＜本発明の皮膚バリア機能向上作用を有する成分を含有する組成物（皮膚外用剤）の製造＞
表１に示す処方に従って、本発明の皮膚バリア機能向上作用を有する成分を含有する組成物（皮膚外用剤）であるロ−ション化粧料を作製した。即ち、処方成分を８０℃で攪拌し、可溶化し、しかる後に、攪拌下冷却して、ロ−ション化粧料（化粧料１又は化粧料２）を得た。また、同様の操作を行い本発明の組成物（皮膚外用剤）の「皮膚バリア機能向上作用を有する成分」を「水」に置換した比較例１も作製した。
<Composition of the present invention>
<Manufacture of a composition (skin external preparation) containing a component having an action of improving the skin barrier function of the present invention>
According to the formulation shown in Table 1, a lotion cosmetic, which is a composition (external preparation for skin) containing a component having an action of improving the skin barrier function of the present invention, was prepared. That is, the prescription ingredients were stirred at 80 ° C., solubilized, and then cooled with stirring to obtain a lotion cosmetic (cosmetic 1 or cosmetic 2). In addition, the same operation was performed to prepare Comparative Example 1 in which the “component having an action of improving skin barrier function” of the composition of the present invention (external preparation for skin) was replaced with “water”.
＜本発明の皮膚外用剤の肌荒れ改善試験＞
パネラ−を使用し、化粧料１、化粧料２、比較例１に付いて、テ−プストリッピングによって作成した肌荒れモデルでの、肌荒れ改善作用を評価した。即ち、左右の前腕に３cm×５cmの部位を４つずつ作成し、テ−プストリッピングを各部位１５回行い、経皮的散逸水分量（TEWL）をインテグラル社製の「テヴァメ−タ−」で計測した。その後、一日一度検体を５０μL塗布し、この作業を６日間続け、７日目に再度TEWLを計測した。最初の日のTEWL値から７日目のTEWL値を減じ、最初の日のTEWL値で除し、１００を乗じてTEWL改善率（％）を算出した。ｎ数は１５とした。結果を表３に示す。これより、本発明の皮膚外用剤は肌荒れ改善作用に優れることがわかる。
<Skin roughness improvement test of the external preparation for skin of the present invention>
Using a panel, the rough skin improving effect of the rough skin model prepared by tape stripping was evaluated for
本発明は、化粧料などの皮膚外用剤に応用出来る。 The present invention can be applied to an external preparation for skin such as cosmetics.
Claims (9)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013035808A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Ltbp-4 production promotor |
| JP2013035807A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Elastic-fiber formation promotor |
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2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013035808A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Ltbp-4 production promotor |
| JP2013035807A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Elastic-fiber formation promotor |
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