JP7500380B2 - Screening method for agents for preventing and improving skin photoaging - Google Patents
Screening method for agents for preventing and improving skin photoaging Download PDFInfo
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- JP7500380B2 JP7500380B2 JP2020168210A JP2020168210A JP7500380B2 JP 7500380 B2 JP7500380 B2 JP 7500380B2 JP 2020168210 A JP2020168210 A JP 2020168210A JP 2020168210 A JP2020168210 A JP 2020168210A JP 7500380 B2 JP7500380 B2 JP 7500380B2
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Description
特許法第３０条第２項適用 （１）掲載年月日 令和２年９月１０日 （２）掲載アドレス ｈｔｔｐ：／／ｉｆｓｃｃ２０２０．ｃｏｍ／ｓｃｉｅｎｔｉｆｉｃ＿ｐｒｏｇｒａｍ．ｈｔｍｌArticle 30, paragraph 2 of the Patent Act applies. (1) Publication date: September 10, 2020. (2) Publication address: http://ifscc2020. com/scientific_program. html
本発明は、光老化に起因する皮膚の硬化、皮膚弾力性の低下を抑制することにより、皮膚のシワ、タルミの形成、ハリの低下を予防又は改善することができる物質のスクリーニング方法に関する。 The present invention relates to a method for screening substances that can prevent or improve the formation of wrinkles, sagging skin, and loss of firmness by inhibiting hardening of the skin and loss of skin elasticity caused by photoaging.
皮膚は、加齢に伴い老化して弾力を喪失し、ハリの低下や、シワ、タルミ等の形成といった変化を生じる。特に顔面など慢性的な紫外線の影響を受けやすい部位では、顕著にシワやタルミが形成する。これら紫外線暴露部で起こる皮膚老化は特に光老化と呼ばれ、この防止および改善は皮膚外用剤研究者にとって、最も大きな課題の一つである。 As we age, the skin loses elasticity and undergoes changes such as loss of firmness and the formation of wrinkles and sagging. Wrinkles and sagging are particularly prominent in areas that are chronically exposed to ultraviolet rays, such as the face. Skin aging that occurs in areas exposed to ultraviolet rays is called photoaging, and preventing and improving this is one of the biggest challenges for researchers of topical skin products.
皮膚の支持組織である真皮はコラーゲンからなる膠原線維、エラスチンからなる弾性線維等の細胞外基質が主となり構成され、膠原線維は皮膚の強度、弾性線維は皮膚の柔軟性や弾力性に寄与する。光老化部真皮では、コラーゲンの減少、エラスチンの異常沈着（線維肥厚や無定形塊蓄積など）が生じていることが知られており、これら線維の異常は皮膚の強度や柔軟性の喪失をもたらす。特に異常沈着したエラスチンは、光老化部位の皮膚の弾力性の低下および真皮の硬度の増加に寄与する可能性が報告されている（非特許文献１、２）。 The dermis, which is the supporting tissue of the skin, is mainly composed of extracellular matrix such as collagen fibers made of collagen and elastic fibers made of elastin, with collagen fibers contributing to the strength of the skin and elastic fibers contributing to the flexibility and resilience of the skin. It is known that a decrease in collagen and abnormal deposition of elastin (such as fiber thickening and accumulation of amorphous masses) occurs in the dermis of photoaged areas, and these fiber abnormalities lead to a loss of strength and flexibility of the skin. In particular, it has been reported that abnormally deposited elastin may contribute to a decrease in the elasticity of the skin and an increase in the hardness of the dermis in photoaged areas (Non-Patent Documents 1 and 2).
エラスチンは、マトリックスメタロプロテアーゼ（ＭＭＰ）や好中球エラスターゼ、線維芽細胞エラスターゼ等の細胞外基質分解酵素により切断・分解されることが知られている。しかしながら、光老化部では細胞外基質分解酵素の発現量が増加しているにも関わらず、エラスチンの異常沈着が生じていることが報告されている（非特許文献３、４）。光老化部でこの異常沈着が生じるメカニズムはこれまで明らかになっていなかった。 It is known that elastin is cleaved and degraded by extracellular matrix degrading enzymes such as matrix metalloproteinases (MMPs), neutrophil elastase, and fibroblast elastase. However, it has been reported that abnormal deposition of elastin occurs in photoaged areas, despite increased expression of extracellular matrix degrading enzymes (Non-Patent Documents 3 and 4). The mechanism by which this abnormal deposition occurs in photoaged areas has not yet been elucidated.
一方で従来、シワ、タルミの形成、ハリ低下等を予防又は改善する光老化予防改善剤としては、コラーゲン、エラスチンを増加させる又はそれらの分解を抑制することを目的としたものが用いられてきた（特許文献１―４）。特に、エラスチンを対象にしたものに於いては、エラスターゼによるエラスチンの分解を抑制する方法が広く利用されている。これは、エラスチンの分解抑制により皮膚内のエラスチン量を増加させ、光老化部位で生じた弾力性の低下を補うことを目的としたものである。また、この方法はエラスチン分解産物の異常沈着も抑制できる可能性がある。しかしながら、すでに変性して異常沈着してしまったエラスチン自体に対する効力は報告されておらず、従来の技術では、変性エラスチンによる異常沈着を抑制する手段は知られていなかった。つまり、従来技術だけでは光老化部位で生じるエラスチンの異常沈着を十分に抑制出来ず、またそれに伴う光老化症状の進行に対しても根本的に予防および改善することは出来ていなかった。 On the other hand, conventionally, photoaging prevention and improvement agents that prevent or improve wrinkles, sagging, loss of firmness, etc. have been used that aim to increase collagen and elastin or inhibit their decomposition (Patent Documents 1-4). In particular, in those that target elastin, a method of inhibiting the decomposition of elastin by elastase is widely used. This is intended to increase the amount of elastin in the skin by inhibiting the decomposition of elastin, and to compensate for the loss of elasticity that occurs in photoaging sites. This method may also be able to inhibit the abnormal deposition of elastin decomposition products. However, there has been no report of its effectiveness against elastin itself that has already been denatured and abnormally deposited, and in conventional technology, there was no known means of inhibiting the abnormal deposition of denatured elastin. In other words, conventional technology alone has not been able to fully inhibit the abnormal deposition of elastin that occurs in photoaging sites, and has also not been able to fundamentally prevent or improve the progression of photoaging symptoms associated with it.
他方、タンパク質のニトロ化修飾は、生体内で発生した活性窒素種によって生じるタンパク質翻訳後修飾のひとつであり、タンパク質を構成する芳香族アミノ酸のチロシン、トリプトファンの残基中のベンゼン環にニトロ基が付与されたものである。ニトロ化反応はアミノ酸中のベンゼン環が、活性窒素種により形成されるニトロニウムイオン（ＮＯ２ ＋）や二酸化窒素ラジカルなどと求電子置換反応をおこすことで生じる（非特許文献５）。生体内に存在する多くのタンパク質中のトリプトファンの含有率はチロシンのそれよりもはるかに小さく、タンパク質のニトロ化修飾は主にチロシン残基に生じると考えられている（非特許文献６）。タンパク質にニトロ化修飾が生じると、酵素やチロシンキナーゼ型受容体の機能低下を引き起こすことで、細胞機能に影響を及ぼすことが知られる（非特許文献７）。また、タンパク質中のニトロチロシンは動脈硬化や脳虚血疾患などで蓄積することが知られており、これらの疾患に関与することが報告されている（非特許文献８）。ニトロチロシンは皮膚中にも存在することが知られており、特に角層中に存在するニトロチロシンは皮膚色と関連する（特許文献５）。しかしながら光老化部皮膚でのエラスチンの異常沈着との関係については知られていなかった。 On the other hand, nitration of proteins is one of the post-translational modifications of proteins caused by reactive nitrogen species generated in vivo, in which a nitro group is added to the benzene ring in the residues of aromatic amino acids tyrosine and tryptophan that constitute proteins. Nitration occurs when the benzene ring in an amino acid undergoes an electrophilic substitution reaction with nitronium ions (NO 2 + ) formed by reactive nitrogen species or nitrogen dioxide radicals (Non-Patent Document 5). The content of tryptophan in many proteins present in the body is much lower than that of tyrosine, and it is believed that nitration of proteins occurs mainly in tyrosine residues (Non-Patent Document 6). It is known that nitration of proteins affects cell function by causing a decrease in the function of enzymes and tyrosine kinase receptors (Non-Patent Document 7). It is also known that nitrotyrosine in proteins accumulates in arteriosclerosis and cerebral ischemic diseases, and it has been reported that it is involved in these diseases (Non-Patent Document 8). Nitrotyrosine is also known to exist in the skin, and nitrotyrosine present in the stratum corneum in particular is associated with skin color (Patent Document 5). However, the relationship with abnormal deposition of elastin in photoaged skin was unknown.
このような状況下、本発明者は光老化皮膚症状の予防改善には、エラスチンの異常沈着の抑制が重要であるとの認識に至った。そこで本発明者は、光老化部皮膚でエラスチン異常沈着が生じる機序についての考察を行った。光老化部では、細胞外基質分解酵素の発現量が増加しているにも関わらずエラスチンの異常沈着が生じているが、この現象は、何らかの原因により増加した分解酵素の機能が正常に発揮されず、エラスチンが正常に分解されないために生じると考えた。具体的には、紫外線暴露によりエラスチンに何れかの変性が生じ本来であれば増加しているはずの分解酵素による分解が抑制されているとの発想に至った。 Under these circumstances, the inventor came to the realization that inhibiting abnormal deposition of elastin is important for preventing and improving photoaging skin symptoms. The inventor then considered the mechanism by which abnormal deposition of elastin occurs in photoaged skin. In photoaged areas, abnormal deposition of elastin occurs despite increased expression of extracellular matrix degrading enzymes, and it was believed that this phenomenon occurs because, for some reason, the increased degrading enzymes do not function normally, preventing elastin from being broken down normally. Specifically, the inventor came to the idea that some kind of denaturation occurs in elastin due to exposure to ultraviolet light, suppressing decomposition by degrading enzymes that would normally be increased.
そこで、本発明者は光老化部皮膚でのエラスチンの分解抑制に係る新たな因子を探索したところ、光老化部皮膚で異常沈着しているエラスチンの構成アミノ酸にはニトロ化修飾が生じていることを見出した。さらに鋭意検討の結果、ニトロ化エラスチンは凝集を促進する特性があり、エラスターゼによる分解に対して抵抗性を獲得すること、さらには硬度が増加し、弾力性が低下することを見出した。 The inventors therefore searched for new factors involved in inhibiting the decomposition of elastin in photoaged skin, and discovered that the constituent amino acids of elastin abnormally deposited in photoaged skin are modified by nitration. As a result of further intensive investigations, they found that nitrated elastin has the property of promoting aggregation, acquires resistance to decomposition by elastase, and furthermore, increases hardness and decreases elasticity.
つまり、光老化部では積年の紫外線照射によりエラスチンにニトロ化修飾等の変性が生じており、これがエラスチンの凝集を惹起するとともに分解酵素によるエラスチンの分解低下をもたらし、結果、光老化部でエラスチンの異常沈着が引き起こされることを見出した。さらには、光老化皮膚でのエラスチンの硬化等の物性変化や、弾力性低下等の機能低下の一要因であることを突き止めた。 In other words, they found that in photoaged areas, long-term exposure to UV rays causes denaturation of elastin through nitration and other modifications, which induces elastin aggregation and reduces the decomposition of elastin by degrading enzymes, resulting in abnormal deposition of elastin in photoaged areas. They also found that this is one of the factors behind changes in the physical properties of elastin in photoaged skin, such as hardening, and functional decline, such as reduced elasticity.
さらに本発明者は、ニトロ化のみならず糖化、カルボニル化修飾等のタンパク質翻訳後修飾をうけたエラスチンは、エラスターゼによる分解に対して抵抗性を獲得することを確認した。 Furthermore, the inventors confirmed that elastin that has undergone post-translational protein modifications, such as nitration, glycation, and carbonylation, acquires resistance to degradation by elastase.
以上の新知見から、変性したエラスチンの分解を促進、又は変性エラスチンの生成自体を抑制することにより、光老化部位に生じるエラスチンの異常沈着を抑制でき、皮膚の硬化および皮膚弾力性の低下、それに伴うシワ、タルミの形成、ハリの低下等を予防し又はこれらを改善することができると確信し、本発明に至った。 Based on these new findings, we believe that by promoting the decomposition of denatured elastin or inhibiting the production of denatured elastin itself, it is possible to inhibit the abnormal deposition of elastin in photoaging sites, and prevent or improve skin hardening and loss of skin elasticity, as well as the associated formation of wrinkles, sagging, and loss of firmness, which led to the invention.
光老化部で生じるエラスチンの異常沈着を抑制し、皮膚の硬化、皮膚弾力性の低下を抑制、光老化に伴う皮膚のシワ、タルミの形成、ハリの低下等を予防し又はこれらを改善することができる物質のスクリーニング方法を提供することを課題とする。 The objective of the present invention is to provide a method for screening substances that can suppress abnormal deposition of elastin in photoaged areas, suppress hardening of the skin and loss of skin elasticity, and prevent or improve skin wrinkles, sagging, loss of firmness, etc., associated with photoaging.
変性エラスチンの分解促進又は生成抑制を指標とすることで上記課題を解決した。 The above problem was solved by using the promotion of decomposition or inhibition of production of denatured elastin as an indicator.
本発明は、以下のスクリーニング方法を提供するものである。
〔１〕変性エラスチン量を指標とした光老化予防改善剤のスクリーニング方法
〔２〕変性エラスチン量を指標としたシワ、タルミの形成、ハリ低下の予防改善剤のスクリーニング方法
〔３〕変性エラスチン量を指標とした皮膚の弾力性低下抑制剤及び／又は皮膚の硬化抑制剤のスクリーニング方法
〔４〕変性エラスチン量を指標としたエラスチンの異常沈着抑制剤のスクリーニング方法
〔５〕前記変性エラスチン量の指標が、変性エラスチンの分解促進である〔１〕乃至〔４〕のいずれか１項に記載のスクリーニング方法
〔６〕前記変性エラスチンの分解促進が、変性エラスチンの直接分解及び／又は細胞外基質分解酵素による分解の促進である〔５〕に記載のスクリーニング方法
〔７〕前記変性エラスチン量の指標が、変性エラスチンの生成抑制である〔１〕乃至〔４〕のいずれか１項に記載のスクリーニング方法
〔８〕前記変性が、ニトロ化修飾に起因するものである〔１〕乃至〔７〕のいずれか１項に記載のスクリーニング方法
The present invention provides the following screening methods.
[1] A method for screening an agent for preventing and improving photoaging using the amount of denatured elastin as an index; [2] A method for screening an agent for preventing and improving wrinkles, sagging, and loss of firmness using the amount of denatured elastin as an index; [3] A method for screening an agent for suppressing loss of skin elasticity and/or an agent for suppressing skin hardening using the amount of denatured elastin as an index; [4] A method for screening an agent for suppressing abnormal elastin deposition using the amount of denatured elastin as an index; [5] A screening method according to any one of [1] to [4], wherein the index for the amount of denatured elastin is promotion of decomposition of denatured elastin; [6] A screening method according to [5], wherein the promotion of decomposition of denatured elastin is promotion of direct decomposition of denatured elastin and/or promotion of decomposition by extracellular matrix decomposing enzymes; [7] A screening method according to any one of [1] to [4], wherein the index for the amount of denatured elastin is inhibition of production of denatured elastin; [8] A screening method according to any one of [1] to [7], wherein the denaturation is caused by nitration modification.
本発明によれば、変性エラスチンの分解の促進又は生成の抑制を指標に用いることにより、光老化に伴うエラスチンの異常沈着を抑制する新たな物質のスクリーニング方法が提供される。加えて、皮膚の硬度の増加、皮膚弾力性の低下も抑制することができるため、それに伴うシワ、タルミの形成、ハリの低下を予防し、これらを改善する新たな物質のスクリーニング方法が提供される。 The present invention provides a method for screening for new substances that suppress the abnormal deposition of elastin associated with photoaging by using the promotion of decomposition or inhibition of production of denatured elastin as an indicator. In addition, it is possible to suppress an increase in skin hardness and a decrease in skin elasticity, thereby providing a method for screening for new substances that prevent and improve the formation of wrinkles, sagging, and loss of firmness associated with these.
本発明で用いるエラスチンは、特に限定されないが、例えばトロポエラスチンタンパク質或いはエラスチンタンパク質が挙げられる。これらは天然物でも人工的に作製したものでも良く、天然物としては、ヒト、ラット、ウシ、ウサギ、サケ等を含む脊椎動物から抽出されたものを用いることができ、人工的に作製したものとしては、上記生物由来のトロポエラスチン遺伝子を大腸菌や酵母等の微生物細胞にトランスフェクションし産生させた、或いは大腸菌やコムギ胚芽、加えて培養細胞などの抽出液中の酵素を用いた無細胞系にて産生させたものを用いることができる。エラスチンはタンパク質として存在する状態でも良いし、複数のエラスチンが架橋して構成された線維状の構造物であっても良く、例えば上記生物から回収される弾性線維やエラウニン線維等の弾性系線維のほか、人工的に架橋操作を行い線維状構造物に成形したエラスチンファイバーなどの状態であってもよい。更に、これらのタンパク質或いは線維状のエラスチンはそのまま用いることも出来るし、加水分解や酵素分解等の操作を加えて断片化した状態のものを用いても良い。またこれらのエラスチンには、分解を評価するために色素や蛍光物質などの化学標識が施されていても良い。 The elastin used in the present invention is not particularly limited, and examples thereof include tropoelastin protein and elastin protein. These may be natural products or artificially produced products. Natural products include those extracted from vertebrates including humans, rats, cows, rabbits, salmon, etc. Artificial products include those produced by transfecting tropoelastin genes derived from the above-mentioned organisms into microbial cells such as E. coli or yeast, or those produced in a cell-free system using enzymes in extracts from E. coli, wheat germ, and cultured cells. Elastin may exist as a protein, or may be a fibrous structure formed by crosslinking multiple elastins. For example, it may be elastic fibers such as elastic fibers and elaunin fibers recovered from the above-mentioned organisms, or elastin fibers artificially crosslinked to form a fibrous structure. Furthermore, these proteins or fibrous elastins may be used as they are, or may be fragmented by hydrolysis, enzymatic decomposition, or the like. These elastins may be chemically labeled with dyes, fluorescent substances, etc. to evaluate decomposition.
本発明の変性とは、エラスチンに生じる変性を指しており、例えばニトロ化修飾、糖化修飾、カルボニル化修飾等の翻訳後修飾の付加や、ミスフォールディングによる立体構造の変化、或いは他タンパク質・分子との異常結合、これらを原因とする凝集物の形成などが挙げられる。本願では、エラスチンが上記の変性のうちいずれか１種以上の変性を有すエラスチンを「変性エラスチン」と呼ぶ。 The term "denaturation" as used herein refers to modifications that occur in elastin, including, for example, the addition of post-translational modifications such as nitration, glycation, and carbonylation, changes in three-dimensional structure due to misfolding, or abnormal binding with other proteins and molecules, and the formation of aggregates due to these. In this application, elastin that has undergone one or more of the above modifications is referred to as "denatured elastin."
エラスチンに変性を生じさせる変性処理の方法としては、特に限定されない。例えばエラスチンに試薬を添加することによりニトロ化修飾や酸化、糖化、カルボニル化といった翻訳後修飾を生じさせる方法のほか、エラスチンに熱処理を加えて熱変性を生じさせる方法、酸又はアルカリの添加により構造的な変性を生じさせる方法、紫外線照射や太陽光の暴露により光変性を生じさせる方法、経年により加齢変性を生じさせる方法等を用いることができる。これらの処理により、ニトロ化修飾、糖化修飾、カルボニル化修飾等の翻訳後修飾の付加や、ミスフォールディングによる立体構造の変化、或いは他のエラスチンやタンパク質・分子との異常結合、またこれらを原因とした凝集物の形成、などに代表される変化をエラスチンに生じさせることができる。もっとも、エラスチンに直接変性処理を行うだけでなく、エラスチンの産生元であるヒト、ラット、ウシ、ウサギ、サケ等を含む脊椎動物や、大腸菌や酵母等の微生物細胞に対し、これらの生物が生きているうちに上述の変性処理、例えば翻訳後修飾を生じさせる試薬等を与えることができることは言うまでもない。 There are no particular limitations on the method of denaturation treatment that causes denaturation of elastin. For example, in addition to a method of causing post-translational modifications such as nitration, oxidation, glycation, and carbonylation by adding a reagent to elastin, a method of causing thermal denaturation by subjecting elastin to heat treatment, a method of causing structural denaturation by adding an acid or alkali, a method of causing photodenaturation by exposure to ultraviolet light or sunlight, and a method of causing age-related denaturation over time can be used. These treatments can cause changes in elastin, such as the addition of post-translational modifications such as nitration, glycation, and carbonylation, changes in three-dimensional structure due to misfolding, abnormal binding with other elastin, proteins, and molecules, and the formation of aggregates due to these. However, it goes without saying that in addition to directly denaturing elastin, the above-mentioned denaturation treatment, such as a reagent that causes post-translational modification, can be given to vertebrate cells including humans, rats, cows, rabbits, and salmon, which are the source of elastin, and microbial cells such as E. coli and yeast, while these organisms are still alive.
エラスチンに翻訳後修飾を生じさせるために用いるニトロ化修飾試薬としては、公知のものを用いることができ、例えばペルオキシナイトライト等の活性窒素種を直接用いるほか、試験系中に活性窒素種を発生させることができる化合物群を混合して用いても良い。例えば、一酸化窒素とスーパーオキシドアニオンを混合する、又はミエロペルオキシダーゼ（ＭＰＯ）とＮａＮＯ２、過酸化水素等と混合することによる。加えて、ニトロニウムイオンを発生させるテトラニトロメタンや硝酸などの試薬等を用いることもできる。このほか、塩化ニトロイルやニトロソペルオキシカルボキシレート、またアジ化ナトリウムおよびカタラーゼ等の試薬を用いることもできる。ニトロ化修飾自体は公知の手法を用いることができる。上記試薬の利用により、エラスチン中に含まれるチロシンやトリプトファン、フェニルアラニン、システイン残基にニトロ基が導入され、ニトロチロシンやジニトロチロシン、ニトロトリプトファン、ニトロフェニルアラニン、ジニトロフェニルアラニン、ニトロシステイン等を含むエラスチンが形成される。 The nitration modification reagent used to cause post-translational modification of elastin can be a known one, for example, active nitrogen species such as peroxynitrite can be directly used, or a group of compounds capable of generating active nitrogen species in a test system can be mixed and used. For example, nitric oxide can be mixed with superoxide anion, or myeloperoxidase (MPO) can be mixed with NaNO 2 , hydrogen peroxide, etc. In addition, reagents such as tetranitromethane and nitric acid that generate nitronium ions can be used. In addition, reagents such as nitroyl chloride, nitrosoperoxycarboxylate, sodium azide, and catalase can be used. The nitration modification itself can be performed by a known method. By using the above reagents, nitro groups are introduced into tyrosine, tryptophan, phenylalanine, and cysteine residues contained in elastin, and elastin containing nitrotyrosine, dinitrotyrosine, nitrotryptophan, nitrophenylalanine, dinitrophenylalanine, nitrocysteine, etc. is formed.
エラスチンに翻訳後修飾を生じさせるために用いる酸化試薬としては公知のものを用いることができ、例えば過酸化水素や次亜塩素酸、スーパーオキサイドやヒドロキシラジカルを直接用いるほか、試験系中に活性酸素種を発生させることができる化合物群を混合して用いても良い。また、糖化試薬としては公知のものを用いることができ、例えばリボースやグルコース、グリセルアルデヒドやグリオキサールを用いることができる。カルボニル化試薬としては例えばアクロレインやヒドロキシノネナール等を用いることができる。 The oxidizing reagent used to cause post-translational modification of elastin can be a known one, such as hydrogen peroxide, hypochlorous acid, superoxide, or hydroxyl radical, which can be used directly, or a mixture of compounds capable of generating reactive oxygen species in the test system. The glycation reagent can be a known one, such as ribose, glucose, glyceraldehyde, or glyoxal. The carbonylation reagent can be acrolein, hydroxynonenal, or the like.
本発明で用いる変性エラスチンは、上述のような操作を行い、人為的にエラスチンに変性処理を加えたものを用いることができほか、上述のように変性処理を行わずとも、すでに変性が生じているエラスチンも変性エラスチンとして用いることができる。すでに変性が生じているエラスチンとしては、例えば長期間紫外線にさらされた上記生物から抽出したエラスチンや、経年により高齢となった上記生物から抽出したエラスチンを用いることができる。 The denatured elastin used in the present invention can be elastin that has been artificially denatured by carrying out the above-mentioned operations, or elastin that has already been denatured without being denatured as described above can also be used as the denatured elastin. Examples of elastin that has already been denatured include elastin extracted from the above-mentioned organisms that have been exposed to ultraviolet rays for a long period of time, and elastin extracted from the above-mentioned organisms that have aged over time.
変性エラスチンの存在量を測定する方法としては、変性エラスチンを直接測定する方法、又は変性エラスチン中の変性部位を特異的に検出しこれを変性エラスチンの存在量として測定する方法が挙げられる。例えば、吸光度、蛍光強度、液体クロマトグラフィー（ＨＰＬＣ）、ガスクロマトグラフィー、質量分析、核磁気共鳴（ＮＭＲ）、免疫染色、電気泳動、ウエスタンブロッティング、放射免疫測定（Ｒａｄｉｏｉｍｍｕｎｏａｓｓａｙ）、Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ（ＥＬＩＳＡ）、ゲル浸透クロマトグラフィー、限外濾過、タンパク質定量、重量測定等により直接的に変性エラスチンの存在量を測定することができる。また変性エラスチン中に存在するニトロ基、糖化物、カルボニル基の存在量や、ミスフォールディングによる立体構造の変化、或いは他のエラスチンやタンパク質・分子との分子間架橋構造、またこれらを原因とする凝集物の形成を上述の方法や公知の手法により測定し、これらの存在量を変性エラスチンの存在量とすることも出来る。なお凝集物とは、エラスチン線維又はエラスチンタンパク質の分子若しくは複合体を含む重合集合体をさし、可視的な沈殿物が形成される程度にまで達したものも含まれる。エラスチンの重合集合体の重合形態は、分子間イオン結合、疎水相互作用、ファンデルワールス力等によるものを含む。凝集物の評価方法としては、電気泳動やゲル浸透クロマトグラフィー、限外濾過法、質量分析計等にてタンパク質の重合集合体の分子量を評価する方法、チオフラビンＴ等の凝集分子間に保持される凝集指示薬等を用いることで凝集の程度を評価する方法、或いは凝集にともなう溶液の屈折率の変化や凝集沈殿物の発生を光散乱法や吸光度法、又は凝集に伴う分子・線維形態の変化を顕微鏡等にて目視で直接評価することで凝集物の量を判断する方法が挙げられる。測定方法は、上記に限定されない。 Methods for measuring the amount of denatured elastin include a method for directly measuring denatured elastin, or a method for specifically detecting the denatured site in denatured elastin and measuring it as the amount of denatured elastin. For example, the amount of denatured elastin can be measured directly by absorbance, fluorescence intensity, liquid chromatography (HPLC), gas chromatography, mass spectrometry, nuclear magnetic resonance (NMR), immunostaining, electrophoresis, Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), gel permeation chromatography, ultrafiltration, protein quantification, weight measurement, etc. In addition, the amount of nitro groups, glycated products, and carbonyl groups present in denatured elastin, changes in three-dimensional structure due to misfolding, intermolecular crosslinked structures with other elastins, proteins, and molecules, and the formation of aggregates caused by these can also be measured by the above-mentioned methods or known techniques, and the amount of these can be used as the amount of denatured elastin. The term "aggregates" refers to polymerized assemblies containing elastin fibers or elastin protein molecules or complexes, and includes those that have reached the level of forming visible precipitates. Polymerization forms of polymerized elastin assemblies include those due to intermolecular ionic bonds, hydrophobic interactions, van der Waals forces, etc. Methods for evaluating aggregates include methods for evaluating the molecular weight of protein polymerized assemblies using electrophoresis, gel permeation chromatography, ultrafiltration, mass spectrometry, etc., methods for evaluating the degree of aggregation using an aggregation indicator held between aggregated molecules such as thioflavin T, and methods for determining the amount of aggregates by directly evaluating the change in the refractive index of the solution or the occurrence of aggregate precipitates due to aggregation using a light scattering method or absorbance method, or the change in molecular/fiber morphology due to aggregation visually using a microscope, etc. Measurement methods are not limited to those mentioned above.
本発明のスクリーニング方法は、変性エラスチン量を指標とすることで、エラスチンの異常沈着を抑制し、皮膚の硬化および皮膚弾力性の低下、それに伴うシワ、タルミの形成、ハリの低下といった光老化の兆候を予防又は改善することができる物質を評価、選択する方法である。本願の「抑制剤」は、「予防改善」を含む概念として用いる。 The screening method of the present invention uses the amount of denatured elastin as an indicator to evaluate and select substances that can inhibit abnormal deposition of elastin and prevent or improve signs of photoaging such as hardening of the skin, loss of skin elasticity, and the associated formation of wrinkles, sagging, and loss of firmness. In this application, the term "inhibitor" is used as a concept that includes "prevention and improvement."
本発明の変性エラスチン量を指標とするとは、任意の方法を用いて測定した変性エラスチンの存在量を効果判定の基準にするという趣旨である。例えば、被験物質の存在により、変性エラスチン量を無添加群より減少させることができる場合、効果ありと判定することができる。なおこの時の変性エラスチン量の減少は、既存の変性エラスチンの分解の促進による場合、又は変性エラスチンの新規生成の抑制による場合のどちらでもあり得る。 In the present invention, the amount of denatured elastin is used as an index, meaning that the amount of denatured elastin measured using any method is used as the standard for determining effectiveness. For example, if the presence of the test substance can reduce the amount of denatured elastin compared to a non-addition group, it can be determined that there is an effect. Note that the reduction in the amount of denatured elastin in this case can be due to either the promotion of the decomposition of existing denatured elastin or the inhibition of the new production of denatured elastin.
既存の変性エラスチンの分解の促進による変性エラスチン量の減少剤をスクリーニングする場合は、任意の方法を用いて測定した変性エラスチンの分解の程度を効果判定の基準にする。例えば、被験物質の存在により、変性エラスチンの分解を無添加群よりも増加させることができる場合、効果ありと判定することができる。変性エラスチンを用いてエラスチンの分解を評価する系において、被験物質の有無により系中で生じる変性エラスチンの分解を増加させる物質を選択すれば良く、例えば、被験物質の無添加に対して好ましくは３％以上、さらに好ましくは５％以上、変性エラスチンの分解を増加させる物質を選択すればよい。このときの変性エラスチンの分解は、変性エラスチンの直接的な分解又は細胞外基質分解酵素による分解のどちらでも良い。 When screening for an agent that reduces the amount of denatured elastin by promoting the degradation of existing denatured elastin, the degree of degradation of denatured elastin measured using any method is used as the criterion for determining effectiveness. For example, if the presence of the test substance can increase the degradation of denatured elastin compared to a non-addition group, it can be determined that the agent is effective. In a system in which denatured elastin is used to evaluate the degradation of elastin, a substance that increases the degradation of denatured elastin that occurs in the system depending on the presence or absence of the test substance can be selected. For example, a substance that increases the degradation of denatured elastin by preferably 3% or more, more preferably 5% or more compared to the absence of the test substance can be selected. The degradation of denatured elastin in this case can be either direct degradation of denatured elastin or degradation by extracellular matrix degrading enzymes.
変性エラスチンの直接的な分解とは、酵素などを介さず分解することを意味する。変性エラスチンの直接的な分解には、公知の方法を用いることができるが、例えば、任意の変性エラスチンと被験物質を混合し、一定時間後に変性エラスチンの存在量或いは変性エラスチンの分解産物量を測定することで確認することができる。変性エラスチンと被験物質を混合する順番やタイミングは問わず、両者が試験系中で共存するタイミングがあればよい。 Direct degradation of denatured elastin means degradation without the intervention of enzymes or the like. Publicly known methods can be used for direct degradation of denatured elastin, but for example, it can be confirmed by mixing any denatured elastin with a test substance and measuring the amount of denatured elastin present or the amount of decomposition products of denatured elastin after a certain period of time. The order and timing of mixing the denatured elastin and the test substance are not important, as long as the two are able to coexist in the test system.
細胞外基質分解酵素による変性エラスチンの分解には、公知の方法を用いることができる。例えば、好中球エラスターゼ、線維芽細胞エラスターゼ、膵エラスターゼのほか、マトリクスメタロプロテアーゼ、セリンプロテアーゼ、システインプロテアーゼ等の酵素を用いるほか、これらの酵素を産生する培養細胞や細胞破砕物或いは培養上清等を用いることができる。変性エラスチンと酵素を反応させる、又は変性エラスチンを培養細胞に添加或いは培養上清と混合して反応させたのちに変性エラスチンの存在量或いは変性エラスチンの分解産物量を測定することで、変性エラスチンの分解を確認することができる。なお変性エラスチンと細胞外基質分解酵素、及び被験物質を混合する順番やタイミングは問わず、両者が試験系中で共存するタイミングがあればよい。 For the degradation of denatured elastin by extracellular matrix degrading enzymes, known methods can be used. For example, enzymes such as neutrophil elastase, fibroblast elastase, pancreatic elastase, matrix metalloprotease, serine protease, cysteine protease, etc. can be used, and cultured cells, cell lysates, culture supernatant, etc. that produce these enzymes can also be used. Degradation of denatured elastin can be confirmed by reacting denatured elastin with an enzyme, or by adding denatured elastin to cultured cells or mixing it with culture supernatant and reacting it, and then measuring the amount of denatured elastin present or the amount of decomposition products of denatured elastin. The order and timing of mixing denatured elastin, extracellular matrix degrading enzyme, and test substance are not important, as long as they are both present at the same time in the test system.
変性エラスチンの分解の有無や程度は、特に限定されないが、公知の方法で測定することができる。例えば、変性エラスチンの存在量を測定する、或いは変性エラスチンの分解産物量を測定することで、変性エラスチンの分解を算出することができる。変性エラスチンの存在量を測定する方法としては、前述の方法を用いることができる。他方、変性エラスチンの分解の測定方法として、系中に存在する分解産物量を測定し算出することもできる。変性エラスチンの分解産物量の測定方法は、特に限定されないが、公知の方法で行うことができる。例えば、色素結合不溶性エラスチンに変性処理を行い不溶性の変性エラスチンを調製したのちに上述の方法を用いて分解反応を行い、上清中に遊離した分解産物の色素を吸光度或いは蛍光強度にて測定する方法が挙げられる。また、可溶性の変性エラスチンを用いて上述の方法で分解反応を行ったのち、電気泳動又はゲル浸透クロマトグラフィー等を用いて分子量を測定し、検出された可溶性の変性エラスチンよりも分子量が小さい物質を分解産物として定量することもできる。さらに、変性処理を行ったエラスチンペプチドに上述の方法にて分解反応を行ったのち、系中に存在するペプチド断片分解産物をＨＰＬＣにて測定する方法などがある。測定方法は、上記に限定されない。 The presence or absence and degree of decomposition of denatured elastin can be measured by known methods, without particular limitation. For example, the amount of denatured elastin present can be measured, or the amount of decomposition products of denatured elastin can be measured to calculate the decomposition of denatured elastin. The method for measuring the amount of denatured elastin present can be the above-mentioned method. On the other hand, the decomposition of denatured elastin can also be measured and calculated by measuring the amount of decomposition products present in the system. The method for measuring the amount of decomposition products of denatured elastin can be measured by known methods, without particular limitation. For example, a method can be used in which dye-bound insoluble elastin is denatured to prepare insoluble denatured elastin, and then a decomposition reaction is performed using the above-mentioned method, and the dye of the decomposition product released in the supernatant is measured by absorbance or fluorescence intensity. In addition, after performing a decomposition reaction using the above-mentioned method using soluble denatured elastin, the molecular weight can be measured using electrophoresis or gel permeation chromatography, etc., and substances with molecular weights smaller than the detected soluble denatured elastin can be quantified as decomposition products. In addition, a method can be used in which a decomposition reaction is performed using a denatured elastin peptide using the above-mentioned method, and then the peptide fragment decomposition products present in the system are measured by HPLC. The measurement method is not limited to the above.
変性エラスチンの新規生成の抑制による変性エラスチン量の減少剤をスクリーニングする場合は、変性エラスチンの新規生成の有無や程度を効果判定の基準にする。例えばエラスチンに前述の変性処理を行い変性エラスチンを生成させる系において、被験物質の存在により、変性エラスチンの存在量を無添加群よりも減少させることができる場合、効果ありと判定することができる。変性エラスチンの存在量を評価する系において、被験物質の有無により系中に存在する変性エラスチンの存在量を減少させる物質を選択すればよく、例えば、被験物質の無添加に対して被験物質の無添加に対して好ましくは３％以上、さらに好ましくは５％以上、変性エラスチンの存在量を減少させる物質を選択すればよい。エラスチンと被験物質を混合する順番やタイミングは問わず、両者が試験系中で共存したのちに変性処理を実施すればよい。 When screening for agents that reduce the amount of denatured elastin by inhibiting the new formation of denatured elastin, the presence or absence and the degree of new formation of denatured elastin are used as the criterion for determining effectiveness. For example, in a system in which elastin is subjected to the above-mentioned denaturation treatment to generate denatured elastin, if the presence of the test substance can reduce the amount of denatured elastin present in the system compared to the non-addition group, it can be determined that there is an effect. In a system for evaluating the amount of denatured elastin, a substance that reduces the amount of denatured elastin present in the system depending on the presence or absence of the test substance can be selected, for example, a substance that reduces the amount of denatured elastin present in the system by preferably 3% or more, more preferably 5% or more, compared to the absence of the test substance can be selected. The order and timing of mixing elastin and the test substance do not matter, and the denaturation treatment can be performed after the two coexist in the test system.
変性エラスチンの新規生成の有無や程度は、特に限定されないが、公知の方法で測定することができる。例えば、生成する変性エラスチンの存在量を測定することで、変性エラスチンの新規生成を評価することができる。変性エラスチンの存在量を測定する方法としては、前述の方法を用いることができる。 Whether or not new denatured elastin is produced and its degree can be measured by known methods, without particular limitation. For example, the amount of denatured elastin produced can be measured to evaluate the new production of denatured elastin. The method described above can be used to measure the amount of denatured elastin produced.
用いる被験物質には、特に制限はない。動植物由来エキス、菌類の培養物、又はこれらの酵素等処理物、化合物又はその誘導体等であっても被検物質として用いることが出来、液状の他、粉末状、ジェル状等であっても差し支えない。被験物質に応じて変性エラスチンの分解の分析方法、および変性エラスチンの生成抑制の分析方法を適宜選択すればよい。 There are no particular limitations on the test substance used. Animal and plant extracts, fungal cultures, or products treated with enzymes or the like, compounds or derivatives thereof, etc. can be used as test substances, and they can be in liquid, powder, gel, etc. form. The method of analysis of the decomposition of denatured elastin and the method of analysis of the inhibition of the production of denatured elastin can be appropriately selected depending on the test substance.
以下、本発明を実施例によりさらに具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.
＜実験１＞光老化部および自然老化部皮膚切片中のエラスチンおよびニトロチロシンの局在
以下の手順で、皮膚切片中のエラスチン、ニトロチロシンを検出した。
インフォームドコンセントを得た９０代女性から得た光老化部皮膚（露光部、頬部）、自然老化部皮膚（非露光部、臀部）のホルマリン固定パラフィン包埋切片を作成し、一次抗体に抗ニトロチロシン抗体（Ｓｔｒｅｓｓｍａｒｑ社）、エラスチン抗体（Ａｂｃａｍ社）、二次抗体にＡｌｅｘａＦｌｕｏｒ（R）４８８標識抗マウス抗体（Ａｂｃａｍ社）、ＡｌｅｘａＦｌｕｏｒ（R）５９４標識抗ウサギ抗体（Ａｂｃａｍ社）を用いて免疫染色し、蛍光顕微鏡（キーエンス社）にて蛍光像を観察した（倍率１０倍）。
<Experiment 1> Localization of elastin and nitrotyrosine in sections of photoaged and naturally aged skin Elastin and nitrotyrosine were detected in sections of skin by the following procedure.
Formalin-fixed, paraffin-embedded sections of photoaged skin (exposed area, cheek) and naturally aged skin (unexposed area, buttocks) were prepared from a woman in her 90s who gave informed consent, and immunostained using anti-nitrotyrosine antibody (Stressmarq) and elastin antibody (Abeam) as primary antibodies, and AlexaFluor(R) 488-labeled anti-mouse antibody (Abeam) and AlexaFluor(R) 594-labeled anti-rabbit antibody (Abeam) as secondary antibodies, and the fluorescent images were observed under a fluorescence microscope (Keyence) (magnification 10x).
図１に示すように、露光部皮膚の真皮では非露光部の真皮よりもニトロチロシンが多く検出され、光老化部皮膚では真皮の細胞外基質にニトロチロシンが存在することが示された。また、露光部皮膚のニトロチロシンはエラスチンとほぼ同じ局在を示しており、光老化部皮膚にはニトロ化エラスチンが存在することが示唆された。 As shown in Figure 1, more nitrotyrosine was detected in the dermis of sun-exposed skin than in the dermis of non-sun-exposed skin, indicating that nitrotyrosine is present in the extracellular matrix of the dermis in photoaged skin. Furthermore, nitrotyrosine in sun-exposed skin was localized in roughly the same location as elastin, suggesting the presence of nitrated elastin in photoaged skin.
＜実験２＞エラスターゼによるニトロ化エラスチンタンパク質の分解
以下の手順で、ニトロ化修飾処理したエラスチンのエラスターゼによる分解を測定した。
＜Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理エラスチン溶液の調製＞
オルセイン標識不溶性エラスチン（ＳＩＧＭＡ社）を２０ｍｇ／ｍＬの濃度になるようＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒ（０．２Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、２０ｍＭ ＣａＣｌ２）によく懸濁し、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ溶液を１００μＭ、３００μＭ、１ｍＭの濃度になるよう添加し、３７℃で１日間インキュベートし、これを３回繰り返した。
＜テトラニトロメタン処理エラスチン溶液の調製＞
オルセイン標識不溶性エラスチン（ＳＩＧＭＡ社）を４０ｍｇ／ｍＬの濃度になるようＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒ（０．２Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、２０ｍＭ ＣａＣｌ２）によく懸濁し、ＥｔＯＨに溶解した１０％テトラニトロメタン（ＴＮＭ）溶液を０．１％の濃度になるよう添加した。５分間混合後、尿素水溶液を終濃度２Ｍになるよう添加して５分間混合し反応を停止した。遠心分離後に上清を捨て、再度Ｒｅａｃｔｉｏｎ Ｂｕｆｆｅｒを添加し洗浄し、この操作を計６回繰り返した。最後の遠心分離後、沈査にＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒを添加し溶液を調製した。
＜エラスターゼによるエラスチン分解試験＞
Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理エラスチン溶液又はテトラニトロメタン処理エラスチン溶液に、好中球エラスターゼ（ＳＩＧＭＡ）を０．５ｕｎｉｔｓ／ｍＬになるよう添加、或いは培養線維芽細胞より回収した線維芽細胞エラスターゼ溶液を１／６量添加し、３７℃で６日間インキュベートした。遠心分離後に上清を９６ｗｅｌｌ－ｐｌａｔｅに回収し、エラスチン分解産物量を５９５ｎｍの吸光度で測定し、数式１に従って無変性のエラスチン分解を１としたときのニトロ化エラスチンの分解を算出した。
<Experiment 2> Decomposition of nitrated elastin protein by elastase The decomposition of nitrated elastin by elastase was measured by the following procedure.
<Preparation of Peroxynitrite-treated Elastin Solution>
Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in reaction buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl 2 ) to a concentration of 20 mg/mL, and peroxynitrite solution was added to concentrations of 100 μM, 300 μM, and 1 mM. The mixture was incubated at 37°C for 1 day, and this was repeated three times.
<Preparation of Tetranitromethane-treated Elastin Solution>
Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl 2 ) to a concentration of 40 mg/mL, and a 10% tetranitromethane (TNM) solution dissolved in EtOH was added to a concentration of 0.1%. After mixing for 5 minutes, an aqueous urea solution was added to a final concentration of 2 M and mixed for 5 minutes to stop the reaction. After centrifugation, the supernatant was discarded, and Reaction Buffer was added again to wash, and this operation was repeated a total of 6 times. After the final centrifugation, Reaction Buffer was added to the sediment to prepare a solution.
<Elastin Decomposition Test with Elastase>
Neutrophil elastase (SIGMA) was added to the peroxynitrite-treated elastin solution or tetranitromethane-treated elastin solution to a concentration of 0.5 units/mL, or 1/6 volume of a fibroblast elastase solution recovered from cultured fibroblasts was added, and the solution was incubated for 6 days at 37° C. After centrifugation, the supernatant was recovered in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm, and the degradation of nitrated elastin was calculated according to Equation 1, assuming that the degradation of unmodified elastin is 1.
表１に示すように、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理およびＴＮＭ処理は、無変性エラスチンに比べて好中球エラスターゼ又は線維芽細胞エラスターゼによるエラスチンの分解を低下させた。つまり、ニトロ化エラスチンではエラスターゼによる分解が抑制されることが確認された。 As shown in Table 1, peroxynitrite treatment and TNM treatment reduced the degradation of elastin by neutrophil elastase or fibroblast elastase compared to unmodified elastin. In other words, it was confirmed that degradation by elastase is suppressed in nitrated elastin.
＜実験２＞エラスターゼによる変性エラスチンタンパク質の分解
以下の手順で、各種の変性処理を行ったエラスチンのエラスターゼによる分解を測定した。
＜各種変性エラスチン溶液の調製＞
オルセイン標識不溶性エラスチン（ＳＩＧＭＡ社）を４０ｍｇ／ｍＬの濃度になるようＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒ（０．２Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、２０ｍＭ ＣａＣｌ２）によく懸濁し、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ溶液を１００μＭの濃度になるよう添加、或いはリボース水溶液を３００ｍＭの濃度になるよう添加、或いはアクロレイン溶液を１ｍＭの濃度になるようそれぞれ添加し、３７℃で３日間インキュベートした。遠心分離後に上清を捨て、再度Ｒｅａｃｔｉｏｎ Ｂｕｆｆｅｒを添加し洗浄し、この操作を計３回繰り返した。最後の遠心分離後、沈査にＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒを添加した。
＜エラスターゼによるエラスチン分解試験＞
各種変性エラスチン溶液に、好中球エラスターゼ（ＳＩＧＭＡ）を０．２ｕｎｉｔｓ／ｍＬになるよう添加し、３７℃で１日間インキュベートした。遠心分離後に上清を９６ｗｅｌｌ－ｐｌａｔｅに回収し、エラスチン分解産物量を５９５ｎｍの吸光度で測定し、数式２に従って無変性のエラスチン分解を１としたときの各種変性エラスチンの分解を算出した。
<Experiment 2> Decomposition of denatured elastin protein by elastase The decomposition by elastase of elastin that had been subjected to various denaturing treatments was measured according to the following procedure.
<Preparation of Various Modified Elastin Solutions>
Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in Reaction Buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl 2 ) to a concentration of 40 mg/mL, and Peroxynitrite solution was added to a concentration of 100 μM, or ribose aqueous solution was added to a concentration of 300 mM, or acrolein solution was added to a concentration of 1 mM, and the mixture was incubated at 37° C. for 3 days. After centrifugation, the supernatant was discarded, and Reaction Buffer was added again to wash the mixture, and this operation was repeated three times in total. After the final centrifugation, Reaction Buffer was added to the sediment.
<Elastin Decomposition Test with Elastase>
Neutrophil elastase (SIGMA) was added to each type of denatured elastin solution to a final concentration of 0.2 units/mL, and the solution was incubated for 1 day at 37° C. After centrifugation, the supernatant was collected in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm. The degradation of each type of denatured elastin was calculated according to Equation 2, assuming that the degradation of undenatured elastin is 1.
表２に示すように、ニトロ化（Ｐｅｒｏｘｙｎｉｔｒｉｔｅ）処理、糖化（リボース）処理、カルボニル化（アクロレイン）処理を行ったエラスチンは、すべて無変性エラスチンよりも好中球エラスターゼによるエラスチンの分解が低下した。つまり、変性エラスチンではエラスターゼによる分解が抑制されることが確認された。中でも、ニトロ化処理は最もエラスチンの分解を低下させた。 As shown in Table 2, elastin that had been treated with nitration (peroxynitrite), glycation (ribose), or carbonylation (acrolein) all showed reduced degradation of elastin by neutrophil elastase compared to unmodified elastin. In other words, it was confirmed that degradation by elastase was inhibited in modified elastin. Of these, nitration treatment reduced the degradation of elastin the most.
＜実験３＞ニトロ化処理によるエラスチンの凝集
以下の手順で、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理したエラスチン線維の形態変化を観察した。
エラスチンファイバーシート（細胞外基質研究所社）を０．２Ｍリン酸緩衝液（ｐＨ７．５）に浸し、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ（Ｄｏｊｉｎｄｏ社）を１００μＭになるよう添加した。３７℃で３日間インキュベートした後、蒸留水にてエラスチンファイバーシートを洗浄・乾燥し、Ａｕ蒸着後、走査型電子顕微鏡（ＪＥＯＬ社）にて線維形態を観察した。
<Experiment 3> Aggregation of elastin by nitration treatment The following procedure was used to observe the morphological changes of elastin fibers treated with Peroxynitrite.
An elastin fiber sheet (Extracellular Matrix Research Institute) was immersed in 0.2 M phosphate buffer (pH 7.5) and Peroxynitrite (Dojindo) was added to a concentration of 100 μM. After incubation at 37° C. for 3 days, the elastin fiber sheet was washed with distilled water, dried, and Au was evaporated, after which the fiber morphology was observed under a scanning electron microscope (JEOL).
図２に示すように、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理によりエラスチンファイバーは線維が束なり合一した様相を呈しており、ニトロ化処理はエラスチンの凝集を生じさせることが示された。 As shown in Figure 2, peroxynitrite treatment caused the elastin fibers to appear bundled and united, indicating that nitration treatment causes elastin aggregation.
＜実験４＞ニトロ化処理によるエラスチンゲルシートの硬度および弾力性の変化
以下の手順で、テトラニトロメタン処理したエラスチンゲルシートの硬度および弾力性を測定した。
エラスチンゲルシート（細胞外基質研究所社）を０．２Ｍリン酸緩衝液（ｐＨ７．４）に浸漬し、ＥｔＯＨに溶解した１０％テトラニトロメタン（ＴＮＭ）溶液を０．１％の濃度になるよう添加した。室温にて３時間インキュベートした後、溶液を捨て、シートをリン酸緩衝液にて洗浄した。ゲル硬度（ヤング率）は引張試験機（細胞外基質研究所社）を用いて測定した。総伸長および弾力性は、Ｃｕｔｏｍｅｔｅｒ（Ｃｏｕｒａｇｅ＋Ｋｈａｚａｋａ社）にて測定した。具体的には、得られたＵｆ値（減圧吸引後の総伸長）およびＵｒ値（減圧開放直後の収縮）を用い、弾力性は数式３に従って算出した。
<Experiment 4> Changes in hardness and elasticity of elastin gel sheet due to nitration treatment The hardness and elasticity of the elastin gel sheet treated with tetranitromethane were measured according to the following procedure.
An elastin gel sheet (Extracellular Matrix Research Institute, Inc.) was immersed in 0.2 M phosphate buffer (pH 7.4), and a 10% tetranitromethane (TNM) solution dissolved in EtOH was added to a concentration of 0.1%. After 3 hours of incubation at room temperature, the solution was discarded, and the sheet was washed with phosphate buffer. The gel hardness (Young's modulus) was measured using a tensile tester (Extracellular Matrix Research Institute, Inc.). The total elongation and elasticity were measured using a Cutometer (Courage+Khazaka Co., Ltd.). Specifically, the elasticity was calculated according to Formula 3 using the obtained Uf value (total elongation after vacuum suction) and Ur value (shrinkage immediately after vacuum release).
表３に示すように、テトラニトロメタン（ＴＮＭ）処理したエラスチンゲルシートでは硬度（ヤング率）が増加し、また表４に示すように、伸長率（Ｕｆ）および弾力性（Ｕｒ／Ｕｆ）が低下することが確認された。 As shown in Table 3, the hardness (Young's modulus) of the elastin gel sheet treated with tetranitromethane (TNM) was increased, and as shown in Table 4, the elongation rate (Uf) and elasticity (Ur/Uf) were decreased.
前述の結果より、光老化部皮膚ではニトロ化修飾された変性エラスチンが増していること、またニトロ化修飾はエラスターゼによるエラスチンの分解を抑制し、かつエラスチンの凝集を促進する特性があることを見出した。加えて、これらのエラスチンの性質変化の結果、エラスチンは硬化し弾力性が減少することも見出した。つまり、長年の紫外線暴露により生じたニトロ化修飾により生じた変性エラスチンは、エラスターゼによるエラスチンの分解を低下させ、光老化部皮膚のエラスチンの異常沈着に寄与することを突き止めた。つまり、変性エラスチン量を減少させることにより、皮膚の硬化および皮膚弾力性の低下、それに伴うシワ、タルミの形成、ハリの低下といった光老化の兆候を予防又は改善することができると考えられ、本発明の変性エラスチンの分解促進を評価及び／又は選択する方法、或いは変性エラスチンの生成抑制を評価及び／又は選択する方法は、上記知見に基づくものである。 The above results show that the amount of nitrated denatured elastin is increased in photoaged skin, and that nitration has the property of inhibiting the decomposition of elastin by elastase and promoting the aggregation of elastin. In addition, it was found that these changes in the properties of elastin result in hardening of elastin and loss of elasticity. In other words, it was found that denatured elastin caused by nitration modification resulting from long-term exposure to ultraviolet rays reduces the decomposition of elastin by elastase and contributes to the abnormal deposition of elastin in photoaged skin. In other words, it is believed that by reducing the amount of denatured elastin, it is possible to prevent or improve signs of photoaging such as hardening of the skin and loss of skin elasticity, as well as the associated formation of wrinkles, sagging, and loss of firmness. The method of the present invention for evaluating and/or selecting the promotion of decomposition of denatured elastin, or the method of evaluating and/or selecting the inhibition of the production of denatured elastin, is based on the above findings.
＜実施例１＞ニトロ化エラスチンの分解促進物質のスクリーニング方法
以下の手順で、エラスターゼによるニトロ化エラスチンの分解を測定し、光老化予防改善剤を選択した。
＜被験物質の調製＞
複数の乾燥植物原体にそれぞれ１０倍の重量の５０％（ｖ／ｖ）エタノール水溶液を加えて室温で１週間抽出した。抽出物の乾燥残分に対して、エタノール、水を重量比で１：５０：４９となるように加えて希釈したものを被験物質とした。対照物質としては５０％エタノール溶液を用いた。
＜ニトロ化エラスチンの分解試験＞
オルセイン標識不溶性エラスチン（ＳＩＧＭＡ社）を２０ｍｇ／ｍＬの濃度になるようＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒ（０．２Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、２０ｍＭ ＣａＣｌ２）によく懸濁し、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ溶液を０、１００μＭの濃度になるよう添加し、３７℃で１日間インキュベートしてニトロ化エラスチンを調製した。ここに被験物質又は対照物質を１００ｐｐｍの濃度になるよう添加した後、さらに好中球エラスターゼ（ＳＩＧＭＡ）を０．５ｕｎｉｔｓ／ｍＬになるよう添加し、３７℃で２日間インキュベートした。遠心分離後に上清を９６ｗｅｌｌ－ｐｌａｔｅに回収し、エラスチン分解産物量を５９５ｎｍの吸光度で測定した。数式４に従って対照物質添加を１００％としたときの各被験物質のニトロ化エラスチンの分解度を算出し、対照物質添加に対し分解が５％以上増加した被験物質を効果成分と判定した。
Example 1 Screening method for substances promoting the decomposition of nitrated elastin The decomposition of nitrated elastin by elastase was measured according to the following procedure, and a photoaging prevention and improvement agent was selected.
Preparation of test substances
A 50% (v/v) ethanol solution was added to each of a number of dried plant materials in an amount ten times the weight of the original material, and the mixture was extracted at room temperature for one week. The dried residue of the extract was diluted with ethanol and water in a weight ratio of 1:50:49 to prepare test substances. A 50% ethanol solution was used as a control substance.
<Decomposition test of nitrated elastin>
Orcein-labeled insoluble elastin (SIGMA) was thoroughly suspended in reaction buffer (0.2 M Tris-HCl (pH 7.5), 20 mM CaCl 2 ) to a concentration of 20 mg/mL, peroxynitrite solution was added to concentrations of 0 and 100 μM, and the mixture was incubated at 37° C. for 1 day to prepare nitrated elastin. The test substance or control substance was added to the suspension to a concentration of 100 ppm, and neutrophil elastase (SIGMA) was added to a concentration of 0.5 units/mL, followed by incubation at 37° C. for 2 days. After centrifugation, the supernatant was collected in a 96-well plate, and the amount of elastin degradation products was measured by absorbance at 595 nm. The degree of decomposition of nitrated elastin for each test substance was calculated according to formula 4, assuming the addition of the control substance to be 100%, and test substances that showed a 5% or greater increase in decomposition compared to the addition of the control substance were determined to be effective ingredients.
被験物質を添加したときのエラスターゼによるニトロ化エラスチンの分解が、対照物質を添加したときに対して５％以上増加していれば、被験物質にはニトロ化エラスチンの分解促進作用が十分あると判断でき、エラスチンの異常沈着の抑制、皮膚の硬化抑制、皮膚の弾力性低下抑制、シワ、タルミの形成、ハリ低下の予防改善等、光老化症状の予防改善が期待される。 If the decomposition of nitrated elastin by elastase when the test substance is added increases by 5% or more compared to when the control substance is added, it can be determined that the test substance has a sufficient effect of promoting the decomposition of nitrated elastin, and is expected to prevent and improve photoaging symptoms, such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging and loss of firmness.
＜実施例２＞熱変性エラスチンの分解促進物質のスクリーニング方法
以下の手順で、線維芽細胞由来分解酵素による熱変性エラスチンの分解を測定し、光老化予防改善剤を選択した。
＜被験物質の調製＞
実施例１と同様に調製した。
＜熱変性エラスチンの分解試験＞
ＦＩＴＣ標識不溶性エラスチン（ＡｎａＳｐｅｃ社）を１００ｍｇ／ｍＬの濃度になるようＲｅａｃｔｉｏｎ Ｂｕｆｆｅｒ（０．２Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ８．５）、２０ｍＭ ＣａＣｌ２）によく懸濁し、９５℃で１０分間加熱したのちに冷却し、熱変性エラスチンを調製した。２４ｗｅｌｌ－ｐｌａｔｅに、１０％ＦＢＳ含有Ｄ－ＭＥＭに懸濁した成人由来ヒト真皮線維芽細胞を５００μＬずつ播種、又は１０％ＦＢＳ含有Ｄ－ＭＥＭのみを５００μＬずつ分注し、３７℃、５％ＣＯ２／９５％空気の加湿条件で３日間培養した。培地を捨て、１０％ＦＢＳを加えたフェノールレッド不含有Ｄ－ＭＥＭを３００μＬずつ分注したのち、各ウェルに被験物質又は対照物質を１００ｐｐｍの濃度になるよう添加した後、調製した熱変性エラスチンをそれぞれ３０μＬずつ添加した。３７℃、５％ＣＯ２／９５％空気の加湿条件で３日間培養し、プレートの培養上清を回収した。培養上清を遠心分離した後、９６ｗｅｌｌ－ｐｌａｔｅに上清を分注し、分解産物量を、蛍光プレートリーダー（ＴＥＣＡＮ社）を用いて励起波長４９５ｎｍ、測定波長５１５ｎｍの蛍光で測定した。数式５に従って対照物質添加を１００％としたときの各被験物質の熱変性エラスチンの分解を算出し、分解が５％以上増加した被験物質を効果成分と判定した。
Example 2 Screening method for substances promoting the decomposition of thermally denatured elastin The decomposition of thermally denatured elastin by fibroblast-derived decomposing enzymes was measured according to the following procedure, and a photoaging prevention and improvement agent was selected.
Preparation of test substances
Prepared in the same manner as in Example 1.
<Decomposition test of thermally denatured elastin>
FITC-labeled insoluble elastin (AnaSpec) was thoroughly suspended in reaction buffer (0.2 M Tris-HCl (pH 8.5), 20 mM CaCl2) to a concentration of 100 mg/mL, heated at 95°C for 10 minutes, and then cooled to prepare thermally denatured elastin. 500 μL of adult human dermal fibroblasts suspended in 10% FBS-containing D-MEM were seeded into a 24-well plate, or 500 μL of 10% FBS-containing D-MEM alone was dispensed, and the plate was cultured at 37°C under humidified conditions of 5% CO2/95% air for 3 days. The medium was discarded, and 300 μL of phenol red-free D-MEM containing 10% FBS was dispensed into each well. Then, the test substance or control substance was added to each well to a concentration of 100 ppm, and 30 μL of the prepared thermally denatured elastin was added to each well. The wells were cultured at 37°C under humidified conditions of 5% CO2/95% air for 3 days, and the culture supernatant was collected from the plate. The culture supernatant was centrifuged, and the supernatant was dispensed into a 96-well plate, and the amount of decomposition product was measured using a fluorescent plate reader (TECAN) with an excitation wavelength of 495 nm and a measurement wavelength of 515 nm. The decomposition of thermally denatured elastin for each test substance was calculated according to formula 5 when the control substance was added as 100%, and the test substance that increased the decomposition by 5% or more was determined to be an effective ingredient.
被験物質を添加したときの熱変性エラスチンの分解が、対照物質を添加したときに対して５％以上増加していれば、被験物質には熱変性エラスチンの分解促進作用が十分あると判断でき、エラスチンの異常沈着の抑制、皮膚の硬化抑制、皮膚の弾力性低下抑制、シワ、タルミの形成、ハリ低下の予防改善等、光老化症状の予防改善が期待される。 If the decomposition of thermally denatured elastin increases by 5% or more when the test substance is added compared to when the control substance is added, it can be determined that the test substance has a sufficient effect of promoting the decomposition of thermally denatured elastin, and is expected to prevent and improve photoaging symptoms, such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging and loss of firmness.
＜実施例３＞糖化エラスチンの分解物質のスクリーニング方法
以下の手順で、糖化エラスチンの直接分解を測定し、光老化予防改善剤を選択した。
＜被験物質の調製＞
複数の乾燥植物原体にそれぞれ１０倍の重量の１，３－ブチレングリコール溶液を加えて５０℃、６時間加熱抽出したものを被験物質とした。対照物質としては１，３－ブチレングリコール溶液を用いた。
＜糖化エラスチン分解試験＞
５０ｍＭ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．５）にブタ由来可溶性エラスチン－Ａ（ＦＵＪＩＦＩＬＭ社）を１ｍｇ／ｍＬの濃度で溶解し、リボース（ＦＵＪＩＦＩＬＭ社）を終濃度が１００ｍＭの濃度になるよう添加し、６０℃で１５日間インキュベートして糖化エラスチンを調製した。糖化エラスチンに被験物質又は対照物質を１０００ｐｐｍの濃度になるよう添加したのち、蛍光プレートリーダーにて励起波長３７０ｎｍ、測定波長４４０ｎｍで反応前の糖化エラスチンの蛍光を測定した。３７℃で５日間インキュベートした後、反応溶液を９６ｗｅｌｌ－ｐｌａｔｅに２００μＬずつ分注し、反応後の糖化エラスチン量を蛍光プレートリーダーにて励起波長３７０ｎｍ、測定波長４４０ｎｍの蛍光で測定した。数式６に従って対照物質添加を１００％としたときの各被験物質の糖化エラスチンの分解を算出し、分解が５％以上増加した被験物質を効果成分と判定した。
Example 3 Screening method for decomposition substances of glycated elastin Direct decomposition of glycated elastin was measured according to the following procedure, and a photoaging prevention and improvement agent was selected.
Preparation of test substances
A 1,3-butylene glycol solution was added to each of a number of dried plant materials in an amount ten times by weight, and the mixture was heated at 50° C. for 6 hours for extraction to prepare test substances. A 1,3-butylene glycol solution was used as a control substance.
<Glycated elastin degradation test>
Soluble porcine elastin-A (FUJIFILM) was dissolved in 50 mM Tris-HCl buffer (pH 7.5) at a concentration of 1 mg/mL, ribose (FUJIFILM) was added to a final concentration of 100 mM, and incubated at 60°C for 15 days to prepare glycated elastin. After adding a test substance or a control substance to the glycated elastin at a concentration of 1000 ppm, the fluorescence of the glycated elastin before the reaction was measured using a fluorescent plate reader at an excitation wavelength of 370 nm and a measurement wavelength of 440 nm. After incubation at 37°C for 5 days, the reaction solution was dispensed in 200 μL portions into a 96-well plate, and the amount of glycated elastin after the reaction was measured using a fluorescent plate reader at an excitation wavelength of 370 nm and a measurement wavelength of 440 nm. The degradation of glycated elastin for each test substance was calculated according to formula 6, assuming the addition of the control substance to be 100%, and test substances that increased degradation by 5% or more were determined to be effective ingredients.
被験物質を添加したときの糖化エラスチンの分解が、対照物質を添加したときに対して５％以上増加していれば、被験物質には糖化エラスチンの分解促進作用が十分あると判断でき、エラスチンの異常沈着の抑制、皮膚の硬化抑制、皮膚の弾力性低下抑制、シワ、タルミの形成、ハリ低下の予防改善等、光老化症状の予防改善が期待される。 If the decomposition of glycated elastin increases by 5% or more when the test substance is added compared to when the control substance is added, it can be determined that the test substance has a sufficient effect of promoting the decomposition of glycated elastin, and is expected to prevent and improve photoaging symptoms, such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging and loss of firmness.
＜実施例４＞エラスチンの凝集物の生成抑制物質のスクリーニング方法
以下の手順で、紫外線照射により生じるエラスチンの凝集を測定し、光老化予防改善剤を選択した。
＜被験物質の調製＞
複数の乾燥植物原体にそれぞれ１００倍の重量の水を加えて８０℃、８時間加熱抽出したものを被験物質とした。対照物質としては水を用いた。
＜エラスチンの凝集抑制試験＞
０．２Ｍリン酸緩衝液（ｐＨ７．５）にトロポエラスチンタンパク質（ＳＩＧＭＡ社）を１ｍｇ／ｍＬの濃度で溶解し、被験物質又は対照物質を終濃度が１００ｐｐｍになるよう添加したのち、９６ｗｅｌｌ－ｐｌａｔｅに２００μLずつ分注した。ＵＶクロスリンカー（ＡｎａｌｙｔｉｋＪｅｎａ社）を用いてＵＶ－Ａ（３６５ｎｍ）をプレートに１００Ｊ／ｃｍ２照射した。３７℃で１日間インキュベート後、１０×Ｂｌｕｅ ｎａｔｉｖｅ ＰＡＧＥ Ｓａｍｐｌｅ Ｂｕｆｆｅｒ（５％（ｗ／ｖ）ＣＢＢ－Ｇ２５０、０．５Ｍ ６－ａｍｉｎｏｃａｐｒｏｉｃ ａｃｉｄ、０．１Ｍ Ｂｉｓ－Ｔｒｉｓ（ｐＨ７．０）、５０％ Ｇｌｙｃｅｒｏｌ）を１／１０量添加し、４℃で１０分間静置した。４－１６％ポリアクリルアミドグラジエントゲル（Ｉｎｖｉｔｒｏｇｅｎ社）に２０μＬずつアプライし、泳動槽の陽極側にＡｎｏｄｅ Ｂｕｆｆｅｒ（５０ｍＭＢｉｓ－Ｔｒｉｓ（ｐＨ７．０））、陰極側にＣａｔｈｏｄｅ Ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｃｉｎｅ（ｐＨ７．０）、１５ｍＭ Ｂｉｓ－Ｔｒｉｓ（ｐＨ７．０）、０．０２％ ＣＢＢ－Ｇ２５０）を満たし、泳動槽を冷却しながら１５０ｍＡで電気泳動した。途中、泳動を停止しＣａｔｈｏｄｅ ＢｕｆｆｅｒをＣＢＢ－Ｇ２５０を含まないものに置換し、再度電気泳動した。泳動終了後のゲルを固定液（５０％メタノール、１０％酢酸）に一晩浸漬して固定および脱色処理した後、銀染色キット（ＡＴＴＯ社）を用いて銀染色を行った。ゲルの写真を撮影後、ＩｍａｇｅＪ（Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ）にてバンド強度を定量した。トロポエラスチンタンパク質は単量体では分子量約７０ｋＤａのタンパク質であるため、この分子量の２倍以上大きい分子量を示すバンドの強度を総和したものを凝集物量とした。対照物質添加の凝集物量を１００％としたとき、凝集物量が５％以上減少した被験物質を効果成分と判定した。
Example 4 Screening method for elastin aggregate formation inhibitors Elastin aggregation caused by ultraviolet irradiation was measured and photoaging prevention and improvement agents were selected according to the following procedure.
Preparation of test substances
Test substances were prepared by adding 100 times the weight of water to each of the dried plant materials and extracting them at 80° C. for 8 hours. Water was used as a control substance.
<Elastin aggregation inhibition test>
Tropoelastin protein (SIGMA) was dissolved in 0.2 M phosphate buffer (pH 7.5) at a concentration of 1 mg/mL, and the test substance or control substance was added to a final concentration of 100 ppm, and then 200 μL of the solution was dispensed into a 96-well plate. The plate was irradiated with UV-A (365 nm) at 100 J/ cm2 using a UV crosslinker (Analytik Jena). After incubation at 37° C. for 1 day, 10× Blue native PAGE Sample Buffer (5% (w/v) CBB-G250, 0.5 M 6-aminocaproic acid, 0.1 M Bis-Tris (pH 7.0), 50% glycerol) was added in an amount of 1/10, and the mixture was allowed to stand at 4° C. for 10 minutes. 20 μL of each solution was applied to a 4-16% polyacrylamide gradient gel (Invitrogen), and the anode side of the electrophoresis tank was filled with anode buffer (50 mM Bis-Tris (pH 7.0)) and cathode buffer (50 mM Tricine (pH 7.0), 15 mM Bis-Tris (pH 7.0), 0.02% CBB-G250) on the cathode side. Electrophoresis was performed at 150 mA while cooling the electrophoresis tank. During the electrophoresis, the electrophoresis was stopped and the cathode buffer was replaced with one that did not contain CBB-G250, and electrophoresis was performed again. After the electrophoresis, the gel was immersed overnight in a fixing solution (50% methanol, 10% acetic acid) for fixation and decolorization, and then silver staining was performed using a silver staining kit (ATTO). After taking a photograph of the gel, the band intensity was quantified using ImageJ (National Institutes of Health). Since tropoelastin protein has a molecular weight of about 70 kDa in monomer form, the sum of the intensities of bands showing a molecular weight at least twice as large as this molecular weight was determined as the amount of aggregates. When the amount of aggregates in the presence of the control substance was taken as 100%, the test substance that reduced the amount of aggregates by 5% or more was determined to be an effective ingredient.
被験物質を添加したときのエラスチンの凝集物量が、対照物質を添加したときに対して５％以上減少していれば、被験物質にはエラスチンの凝集の生成抑制作用が十分あると判断でき、エラスチンの異常沈着の抑制、皮膚の硬化抑制、皮膚の弾力性低下抑制、シワ、タルミの形成、ハリ低下の予防改善等、光老化症状の予防改善が期待される。 If the amount of elastin aggregates when the test substance is added is reduced by 5% or more compared to when the control substance is added, it can be determined that the test substance has sufficient inhibitory effect on the formation of elastin aggregates, and is expected to prevent and improve photoaging symptoms such as inhibiting abnormal deposition of elastin, inhibiting skin hardening, inhibiting loss of skin elasticity, and preventing and improving the formation of wrinkles and sagging and loss of firmness.
本発明のスクリーニング方法を用いることで、エラスチンの異常沈着の抑制剤、および皮膚の硬化抑制剤、皮膚の弾力性低下抑制剤、シワ、タルミの形成、ハリ低下の予防改善剤等の、光老化を予防改善する物質を選択することが可能である。
By using the screening method of the present invention, it is possible to select substances that prevent and improve photoaging, such as inhibitors of abnormal elastin deposition, inhibitors of skin hardening, inhibitors of loss of skin elasticity, and agents for preventing and improving the formation of wrinkles and sagging and loss of firmness.
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| WO2020138023A1 (en) | 2018-12-27 | 2020-07-02 | サントリーホールディングス株式会社 | Agent for inhibiting reduction in decomposition of denatured elastin, agent for maintaining normal elastin fibers, agent for inhibiting formation of elastin-elafine composite, and screening method for substance having elastin-elafine composite formation inhibitory effect |
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| WO2020138023A1 (en) | 2018-12-27 | 2020-07-02 | サントリーホールディングス株式会社 | Agent for inhibiting reduction in decomposition of denatured elastin, agent for maintaining normal elastin fibers, agent for inhibiting formation of elastin-elafine composite, and screening method for substance having elastin-elafine composite formation inhibitory effect |
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| 芋川玄爾，光老化によるシワの形成機序とその予防，Skin Cancer，2003年，18(2)，pp.157-164 |
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