JP7423331B2 - Screening method for skin photoaging inhibitors/improvers - Google Patents
Screening method for skin photoaging inhibitors/improvers Download PDFInfo
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- JP7423331B2 JP7423331B2 JP2020016178A JP2020016178A JP7423331B2 JP 7423331 B2 JP7423331 B2 JP 7423331B2 JP 2020016178 A JP2020016178 A JP 2020016178A JP 2020016178 A JP2020016178 A JP 2020016178A JP 7423331 B2 JP7423331 B2 JP 7423331B2
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- Prior art keywords
- elastin
- nitrated
- amount
- protein
- skin
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Description
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、たるみ等の変化を生じる。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、顕著にシワやたるみ、シミが形成する。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。 Skin ages and loses its elasticity with age, resulting in changes such as a decrease in firmness, wrinkles, and sagging. Particularly in areas that are susceptible to chronic UV rays, such as the face, wrinkles, sagging, and age spots are most noticeable. Skin aging that occurs in areas exposed to ultraviolet rays is particularly called photoaging, and prevention and improvement of this is one of the biggest challenges for researchers in external skin preparations.
皮膚の支持組織である真皮は膠原線維（コラーゲン線維）、弾性線維（エラスチン線維）等の細胞外基質が主となり構成される。コラーゲン線維は皮膚の強度、エラスチン線維は皮膚の柔軟性、弾力性に寄与する。光老化部真皮では、コラーゲン線維の減少、エラスチン線維の異常沈着（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。光老化部位では皮膚の硬度が増加しており、また特に光老化により生じたシワ部位では皮膚の弾力性が低下していることが報告されていることから、光老化の進行には異常沈着したエラスチン線維が関与すると考えられる（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 (collagen fibers) and elastic fibers (elastin fibers). Collagen fibers contribute to the strength of the skin, and elastin fibers contribute to the flexibility and elasticity of the skin. It is known that in the dermis of photoaged areas, there is a decrease in collagen fibers and abnormal deposition of elastin fibers (fiber thickening, amorphous mass accumulation, etc.), and these fiber abnormalities are associated with loss of skin strength and flexibility. bring about. It has been reported that the hardness of the skin increases in areas of photoaging, and that the elasticity of the skin decreases, especially in areas with wrinkles caused by photoaging. It is thought that elastin fibers are involved (Non-patent Documents 1 and 2)
エラスチン線維は、前駆体であるトロポエラスチンタンパク質から切断され生成するエラスチンタンパク質がミクロフィブリル上に連なり、架橋されることで弾力性を持った線維として形成される。また形成されたエラスチン線維はエラスターゼ等の細胞外基質切断酵素により切断され、ターンオーバーが生じることも知られている。光老化部で観察されるエラスチン線維の肥厚・無定形塊蓄積は、長期間の紫外線への暴露によりエラスチン線維を構成するエラスチンタンパク質が変性し、その結果エラスターゼ等分解酵素による分解速度が低下し、変性したエラスチン線維が真皮で異常沈着することで生じると考えられる。 Elastin fibers are formed as elastic fibers by elastin protein, which is produced by being cleaved from the precursor tropoelastin protein, connected on microfibrils and crosslinked. It is also known that the formed elastin fibers are cleaved by extracellular matrix-cleaving enzymes such as elastase, resulting in turnover. The thickening and amorphous mass accumulation of elastin fibers observed in photoaged areas is due to the denaturation of the elastin protein that makes up elastin fibers due to long-term exposure to ultraviolet light, which results in a decrease in the rate of decomposition by degrading enzymes such as elastase. It is thought to be caused by abnormal deposition of degenerated elastin fibers in the dermis.
従来用いられてきたシワ、たるみ、ハリ等を改善する光老化改善剤は、コラーゲン線維、エラスチン線維を増加させるまたはそれらの分解を抑制することを目的としたものが多く用いられてきた。しかしながら、特にエラスチン線維を対象にしたものにおいては、エラスチン線維を増加またはその分解を抑制することにより、光老化部位で生じた弾力性の低下を補うためにエラスチン線維を増加させることを目的としたものである。そのため、光老化部位で生じるエラスチン線維の異常沈着を抑制することは出来ず、光老化の進行に対して根本的な改善をもたらすものではなかった。 Conventionally used photoaging-improving agents for improving wrinkles, sagging, firmness, etc. have often been used for the purpose of increasing collagen fibers and elastin fibers or suppressing their decomposition. However, in those that specifically target elastin fibers, the aim is to increase elastin fibers to compensate for the decrease in elasticity that occurs in photoaging areas by increasing elastin fibers or suppressing their degradation. It is something. Therefore, it has not been possible to suppress the abnormal deposition of elastin fibers that occurs in photoaging areas, and it has not brought about any fundamental improvement in the progress of photoaging.
一方でタンパク質のニトロ化は、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３）。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反応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４）。タンパク質のニトロ化が生じると、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５）。また、タンパク質中のニトロチロシンは加齢に伴う数々の疾患（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６）。ニトロチロシンは皮膚中に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１、特許文献２）。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, protein nitration is one of the post-translational modifications of proteins caused by active nitrogen species generated in vivo, and a nitro group is attached to the benzene ring in the residues of the aromatic amino acids tyrosine and tryptophan that make up proteins. It is what was done. The nitration reaction occurs when a benzene ring in an amino acid undergoes an electrophilic substitution reaction with nitronium ions (NO 2 + ), nitrogen dioxide radicals, etc. formed by active nitrogen species (Non-Patent Document 3). The content of tryptophan in many proteins existing in living organisms is much lower than that of tyrosine, and it is thought that protein nitration reactions occur mainly at tyrosine residues (Non-Patent Document 4). It is known that when protein nitration occurs, it causes a decrease in the function of enzymes and tyrosine kinase receptors, thereby affecting cell functions (Non-Patent Document 5). Additionally, nitrotyrosine in proteins is known to accumulate in a number of age-related diseases (arteriosclerosis, cerebral ischemic disease, etc.), and has been reported to be involved in these diseases (non-patent Reference 6). Nitrotyrosine is known to be present in the skin, and in particular, nitrotyrosine present in the stratum corneum is associated with skin color (Patent Document 1, Patent Document 2). However, the relationship between abnormal elastin deposition in photoaged skin was not known.
このような状況下、本発明者は光老化部皮膚でのエラスチンの異常沈着にかかわ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線維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。そこで鋭意検討の結果、エラスチンのニトロ化は紫外線照射により増加すること、またニトロ化はエラスチンの凝集を促進する特性があること、およびエラスターゼによる分解に対して抵抗性を付与することを見出した。つまり、紫外線照射によりエラスチン線維に生成したニトロ化物が光老化部でのエラスチン線維の異常沈着に寄与することを突き止めた。 Under these circumstances, the present inventor searched for new factors involved in the abnormal deposition of elastin in photoaged skin, and found that the constituent amino acids of elastin fibers abnormally deposited in photoaged skin were modified with nitration. We found that this was occurring. As a result of extensive research, we discovered that nitration of elastin increases with ultraviolet irradiation, that nitration promotes the aggregation of elastin, and that it confers resistance to degradation by elastase. In other words, we found that nitrations generated in elastin fibers by ultraviolet irradiation contribute to the abnormal deposition of elastin fibers in photoaged areas.
更に発明者は、さらにニトロ化して凝集したエラスチン線維は硬度が増加し、弾力性が低下すること、加えて真皮に存在する線維芽細胞の活性にも影響を及ぼすことを見出した。 Furthermore, the inventors have found that elastin fibers that have been further nitrated and aggregated have increased hardness and decreased elasticity, and have also affected the activity of fibroblasts present in the dermis.
以上の新知見から、ニトロ化物の生成を抑制する、またニトロ化物を分解することで皮膚真皮中のニトロ化エラスチンの存在量を制御することにより、光老化に伴うエラスチン線維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、たるみの形成、ハリの低下等を予防又は改善することができる物質を評価、選択することができると確信し、本発明に至った。 Based on the above new findings, it is possible to suppress the abnormal deposition of elastin fibers associated with photoaging by suppressing the production of nitrates and controlling the amount of nitrated elastin in the skin dermis by decomposing nitrates. We believe that it is possible to evaluate and select a substance that can prevent or improve the hardening of the skin, the decrease in skin elasticity, the formation of wrinkles, sagging, and the decrease in firmness associated with this, and have thus arrived at the present invention.
皮膚の硬化、皮膚弾力性の低下を抑制することにより、光老化に伴う皮膚のシワ、たるみ、ハリの低下を予防および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。 An object of the present invention is to provide a screening method for a substance that can prevent and improve skin wrinkles, sagging, and a decrease in firmness associated with photoaging by suppressing skin hardening and decrease in skin elasticity.
エラスチンタンパク質のアミノ酸残基に生じるニトロ化物量、およびニトロ化により惹起されるエラスチンタンパク質の凝集を指標とすることで上記課題を解決した。 The above problems were solved by using as indicators the amount of nitration produced in amino acid residues of elastin protein and the aggregation of elastin protein caused by nitration.
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〕ニトロ化物量を指標とした光老化改善剤のスクリーニング方法
〔２〕ニトロ化物量を指標としたシワ、ハリ、たるみ改善剤のスクリーニング方法
〔３〕ニトロ化物量を指標とした皮膚の弾力性低下改善剤のスクリーニング方法
〔４〕ニトロ化物量を指標とした皮膚の硬化抑制剤のスクリーニング方法
〔５〕ニトロ化物量を指標とした細胞外基質タンパク質の凝集改善剤のスクリーニング方法
〔６〕前記ニトロ化物量の指標が、ニトロ化物量の減少である〔１〕乃至〔５〕いずれかに記載のスクリーニング方法。
〔７〕前記ニトロ化物量の減少が、ニトロ化物の新規生成の抑制、または既存のニトロ化物の分解を作用機序とする〔１〕乃至〔６〕いずれかに記載のスクリーニング方法。
〔８〕前記ニトロ化物が芳香族アミノ酸のニトロ化物またはこれを構成アミノ酸とするニトロ化タンパク質である〔１〕乃至〔７〕いずれかに記載のスクリーニング方法。
〔９〕前記芳香族アミノ酸のニトロ化物がニトロチロシン、３，５－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、２，４－ジニトロフェニルアラニン、ニトロシステインから選択される少なくとも１種以上である〔１〕乃至〔８〕いずれかに記載のスクリーニング方法。
〔１０〕前記細胞外基質タンパク質がエラスチンである〔５〕記載のスクリーニング方法。
〔１１〕ニトロ化に起因するエラスチンの凝集を指標とした光老化改善剤のスクリーニング方法
〔１２〕ニトロ化に起因するエラスチンの凝集を指標としたシワ、ハリ、たるみ改善剤のスクリーニング方法
〔１３〕ニトロ化に起因するエラスチンの凝集を指標とした皮膚の弾力性低下改善剤のスクリーニング方法
〔１４〕ニトロ化に起因するエラスチンの凝集を指標とした皮膚の硬化抑制剤のスクリーニング方法
The present invention provides the following screening method.
[1] Screening method for photoaging improving agents using nitrated content as an indicator [2] Screening method for wrinkle, firmness, and sagging improving agents using nitrated content as an indicator [3] Skin elasticity using nitrated content as an indicator Screening method for an agent for improving sexual deterioration [4] Method for screening a skin hardening inhibitor using the amount of nitration as an index [5] Method for screening for an agent for improving aggregation of extracellular matrix proteins using the amount of nitration as an index [6] The above The screening method according to any one of [1] to [5], wherein the indicator for the amount of nitrated compounds is a decrease in the amount of nitrated compounds.
[7] The screening method according to any one of [1] to [6], wherein the reduction in the amount of nitrated compounds has an action mechanism that suppresses new generation of nitrated compounds or decomposes existing nitrated compounds.
[8] The screening method according to any one of [1] to [7], wherein the nitrated product is a nitrated product of an aromatic amino acid or a nitrated protein having this as a constituent amino acid.
[9] The nitrated product of the aromatic amino acid is at least one selected from nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, 2,4-dinitrophenylalanine, and nitrocysteine [1] to [8] The screening method according to any one of the above.
[10] The screening method according to [5], wherein the extracellular matrix protein is elastin.
[11] Screening method for photoaging improving agents using elastin aggregation caused by nitration as an indicator [12] Screening method for wrinkle, firmness, and sagging improving agents using elastin aggregation caused by nitration as an indicator [13] Screening method for an agent for improving skin elasticity using elastin aggregation caused by nitration as an indicator [14] Screening method for a skin hardening inhibitor using elastin aggregation caused by nitration as an indicator
本発明によれば、ニトロ化物量を指標に用いることにより、光老化に伴うエラスチン線維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、ニトロ化物量またはエラスチンの凝集を指標とすることで、皮膚の硬度の増加、皮膚弾力性の低下を抑制することができ、それに伴うシワ、たるみの形成、ハリの低下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。 According to the present invention, a method of screening for a new substance that suppresses abnormal deposition of elastin fibers associated with photoaging is provided by using the amount of nitration as an index. In addition, by using the amount of nitration or elastin aggregation as an indicator, it is possible to suppress an increase in skin hardness and a decrease in skin elasticity, and to improve the associated wrinkles, sagging formation, and decrease in firmness. A method for screening substances is provided.
本発明は、エラスチン線維の凝集を抑制し、皮膚の硬化および皮膚弾力性の低下、それに伴うシワ、たるみの形成、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。 The present invention evaluates substances that can suppress the aggregation of elastin fibers and prevent or improve signs of photoaging such as hardening of the skin, decrease in skin elasticity, and associated wrinkles, sagging formation, and decrease in firmness. This is the method of choice.
本発明で指標となるニトロ化物は、化合物としてのニトロ基含有化合物のほか、ニトロ化されたペプチド或いはタンパク質が挙げられる。ニトロ基含有化合物としてはニトロチロシンや３，５－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、２，４－ジニトロフェニルアラニン、Ｓ－ニトロシステイン等、ニトロ化タンパク質を構成するアミノ酸残基となりうる化合物が挙げられる。またニトロ化されたペプチドあるいはタンパク質としては、前述のアミノ酸残基となりうる化合物を構成物中に含むペプチドまたはタンパク質が挙げられる。 Nitrated compounds that serve as indicators in the present invention include nitro group-containing compounds as well as nitrated peptides or proteins. Examples of nitro group-containing compounds include compounds that can serve as amino acid residues constituting nitrated proteins, such as nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, 2,4-dinitrophenylalanine, and S-nitrocysteine. . Furthermore, examples of nitrated peptides or proteins include peptides or proteins that contain a compound that can serve as the above-mentioned amino acid residue in their composition.
本発明の「ニトロ化物量を指標とする」とは、任意の方法を用いてニトロ化物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、ニトロ化物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。なおこの時のニトロ化物量の減少は、ニトロ化物の新規生成の抑制による場合、または既存のニトロ化物の分解による場合のどちらでもあり得る。いずれの作用機序に起因するかは後述する方法により選別することができる。 In the present invention, "using the amount of nitrated compounds as an index" means that the amount of nitrated compounds is used as a standard for determining effectiveness using any method. For example, if the presence of the test substance can reduce the amount of nitrated substances compared to the non-additive group, it can be determined that the test substance is effective. Note that the reduction in the amount of nitrated compounds at this time may be due to either suppression of new generation of nitrated compounds or decomposition of existing nitrated compounds. Which mechanism of action is attributable to this can be determined by the method described below.
ニトロ化物の新規生成の抑制を作用機序とするニトロ化物量減少物質をスクリーニングする場合は、アミノ酸、ペプチド、タンパク質、またはこれらの含有物とニトロ化試薬とを混合し、ニトロ化物を生成させる系において、被験物質の存在の有無により系中に生成されるニトロ化物を減少させる物質を選択すればよい。この場合において、例えばアミノ酸としては、チロシン、トリプトファン、フェニルアラニン、システイン等を用いることができる。ペプチドとしては、前述のアミノ酸を含むアミノ酸が約２－５０個程度連続して結合した構造をとる物質を用いることができる。タンパク質としては、前述のアミノ酸を構成物中に含みペプチドよりも分子量が大きな物質であり、例えばエラスチンやコラーゲン等の細胞外基質タンパク質のほか、アルブミンやカゼイン、スキムミルク等のタンパク質試薬等を用いることができる。また、これらの含有物としては、例えばテープストリッピングや研磨材等により収集した角層や、真皮線維芽細胞等の培養細胞の細胞破砕物などの混合物を用いることができる。
ニトロ化試薬としては、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、また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。ニトロ化自体は公知の手法を用いることができる。また本発明では、化合物あるいはタンパク質含有物と被験物質、ニトロ化試薬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。
When screening for substances that reduce the amount of nitrates whose mechanism of action is to suppress the new generation of nitrates, use a system that generates nitrates by mixing amino acids, peptides, proteins, or substances containing these with a nitration reagent. In this case, a substance that reduces the amount of nitrate produced in the system depending on the presence or absence of the test substance may be selected. In this case, for example, tyrosine, tryptophan, phenylalanine, cysteine, etc. can be used as the amino acid. As the peptide, a substance having a structure in which about 2 to 50 amino acids, including the above-mentioned amino acids, are consecutively bonded can be used. Proteins are substances that contain the above-mentioned amino acids in their composition and have a larger molecular weight than peptides. For example, in addition to extracellular matrix proteins such as elastin and collagen, protein reagents such as albumin, casein, and skim milk can be used. can. Further, as these substances, a mixture such as stratum corneum collected by tape stripping or an abrasive material, crushed cells of cultured cells such as dermal fibroblasts, etc. can be used.
As the nitration reagent, active nitrogen species such as peroxynitrite may be used directly, or a mixture of compounds capable of generating active nitrogen species in the test system may be used. For example, by mixing nitric oxide and superoxide anion, or myeloperoxidase (MPO) and NaNO2 , hydrogen peroxide, etc. In addition, reagents such as tetranitromethane and nitric acid that generate nitronium ions can also be used. In addition, reagents such as nitroyl chloride, nitrosoperoxycarboxylate, sodium azide, and catalase can also be used. A known method can be used for the nitration itself. Furthermore, in the present invention, the order and timing of mixing the compound or protein-containing substance, the test substance, and the nitration reagent do not matter, as long as there is a timing at which both coexist in the test system.
既存のニトロ化物の分解を作用機序とするニトロ化物量減少物質をスクリーニングする場合は、任意のニトロ化物と被験物質を混合したとき、一定時間後にニトロ化物の存在量を減少させるものを選択すればよい。ニトロ化物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。
この場合に使用するニトロ化物は前述のニトロ化物をそのまま使用する他、アミノ酸、ペプチド、タンパク質、またはこれらの含有物とニトロ化試薬とを反応さてニトロ化物としたものを使用することができる。この場合は、両者を十分に反応させ、反応が終了したものを用いることが好ましい。
When screening for substances that reduce the amount of nitrates whose mechanism of action is the decomposition of existing nitrates, select a substance that reduces the amount of nitrates after a certain period of time when the test substance is mixed with any nitrate. Bye. The order and timing of mixing the nitrated compound and the test substance do not matter, as long as the timing allows both to coexist in the test system.
In this case, the nitrated product used may be the above-mentioned nitrated product as it is, or a nitrated product obtained by reacting an amino acid, a peptide, a protein, or a substance containing these with a nitration reagent. In this case, it is preferable to allow both to sufficiently react and use the product after the reaction has been completed.
ニトロ化物の測定方法は、公知の方法で行うことができる。例えば、吸光度法、免疫染色法、ウエスタンブロッティング、放射免疫測定（Ｒａｄｉｏｉｍｍｕｎｏａｓｓａｙ）、ＥＬＩＳＡ、液体クロマトグラフィー、ガスクロマトグラフィー、質量分析法、ＮＭＲ法等を用いて直接的に存在量を測定することができる。また、ニトロ化物としてニトロ化ペプチド或いはニトロ化タンパク質量を測定する場合は、例えばニトロ化ペプチド或いはニトロ化タンパク質を酵素処理等にて分解し、得られた単離ニトロ基や単離ニトロ化アミノ酸残基の量を測定したものをニトロ化ペプチド量あるいはニトロ化タンパク質量とし、これらをニトロ化物量として把握しても良い。
また、ニトロ化物の新規生成の抑制を作用機序とするニトロ化物量減少物質をスクリーニングする場合には、上記の直接的な測定方法に加え間接的にニトロ化物量を測定する方法も用いることができる。間接的なニトロ化物測定方法として、反応後に系中に残存するニトロ化試薬の存在量や、被験物質とニトロ化試薬が反応して生成した反応副産物の存在量を測定することで、系中に存在するニトロ化物量を間接的に推定することもできる。例えば、被験物質を未添加の試験系と比較して、被験物質が存在する系中において反応後系中にニトロ化試薬の存在量が多い場合、反応していないニトロ化試薬が多く反応生成物のニトロ化物の存在量が少ないことが推定される。また、被験物質を未添加の試験系と比較して、被験物質が存在する系中において、被験物質とニトロ化試薬の反応により生成する反応副産物の存在量が多い場合、ニトロ化物量の存在量が少ないことが推定される。このようにニトロ化物の新規生成の抑制を作用機序とするニトロ化物量減少物質をスクリーニングする場合のニトロ化物の測定方法には、直接的な測定に加えて間接的な測定によりニトロ化物量を把握してもよい。
Nitride can be measured by a known method. For example, the abundance can be directly measured using absorbance method, immunostaining method, Western blotting, radioimmunoassay, ELISA, liquid chromatography, gas chromatography, mass spectrometry, NMR method, etc. . In addition, when measuring the amount of nitrated peptide or nitrated protein as a nitrated product, for example, the nitrated peptide or nitrated protein is decomposed by enzymatic treatment, etc., and the resulting isolated nitro group or isolated nitrated amino acid residue is used. The amount of the group measured may be defined as the amount of nitrated peptide or the amount of nitrated protein, and these may be understood as the amount of nitrated product.
Furthermore, when screening for substances that reduce the amount of nitrates whose mechanism of action is to suppress the new generation of nitrates, in addition to the above-mentioned direct measurement method, it is also possible to use a method that indirectly measures the amount of nitrates. can. As an indirect method for measuring nitration, it is possible to measure the amount of nitration reagent remaining in the system after the reaction, or the amount of reaction byproducts generated by the reaction between the test substance and the nitration reagent. It is also possible to indirectly estimate the amount of nitrate present. For example, if there is a large amount of nitrating reagent present in the system after the reaction in a system where the test substance is present, compared to a test system without the test substance added, there will be a large amount of unreacted nitrating reagent and the reaction product will be It is estimated that the amount of nitrated compounds present is small. In addition, if the amount of reaction byproducts produced by the reaction between the test substance and the nitration reagent is higher in the system where the test substance is present than in the test system without the addition of the test substance, the amount of nitration It is estimated that there are few In this way, when screening for substances that reduce the amount of nitrates whose mechanism of action is to suppress the new generation of nitrates, methods for measuring nitrates include direct measurement as well as indirect measurement. You can understand it.
本発明における「ニトロ化に起因するエラスチンの凝集を指標」とした試験で用いるエラスチンは、特に限定されないが、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、成熟したエラスチン線維あるいはそれを構成するエラスチンタンパク質を用いることができる。エラスチンタンパク質としては成熟したエラスチン線維から抽出された可溶性エラスチンタンパク質または不溶性エラスチンタンパク質、またはエラスチン線維の前駆体タンパク質であるトロポエラスチンタンパク質、およびそれらの加水分解物等が用いられる。また、上記生物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、人工的に産生させたリコンビナントのトロポエラスチンタンパク質やエラスチン線維、あるいは大腸菌やコムギ胚芽、培養細胞など抽出液中の酵素を用いた無細胞系にて産生されたリコンビナントのトロポエラスチンタンパク質やエラスチン線維を用いることもできる。加えて、これらの方法にて作製されたトロポエラスチンタンパク質を用い、人工的に架橋し調製したエラスチン線維を用いてもよい。 In the present invention, the elastin used in the test using "the aggregation of elastin due to nitration as an indicator" is, but is not particularly limited to, mature elastin extracted from vertebrates including humans, rats, cows, rabbits, salmon, etc. Fibers or elastin proteins that constitute them can be used. As the elastin protein, soluble elastin protein or insoluble elastin protein extracted from mature elastin fibers, tropoelastin protein which is a precursor protein of elastin fibers, hydrolysates thereof, etc. are used. In addition, recombinant tropoelastin proteins and elastin fibers that are artificially produced by transfecting the tropoelastin genes of the above organisms into microbial cells such as E. coli and yeast, or extracts of E. coli, wheat germ, cultured cells, etc. It is also possible to use recombinant tropoelastin protein or elastin fiber produced in a cell-free system using the following enzyme. In addition, elastin fibers prepared by artificially crosslinking using tropoelastin proteins produced by these methods may also be used.
本発明における「ニトロ化に起因するエラスチンの凝集」とは、エラスチンがニトロ化されたことによって生じる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が形成された状態をさし、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まれる。 In the present invention, "aggregation of elastin due to nitration" refers to a state in which polymeric aggregates containing elastin fibers or elastin protein molecules or complexes are formed due to nitration of elastin, and are visible. This also includes those that have reached the point where a precipitate is formed. The polymerization form of the polymerized elastin aggregate includes those based on intermolecular ionic bonds, hydrophobic interactions, van der Waals forces, and the like.
本発明でいう「ニトロ化に起因するエラスチンの凝集を指標とする」とは、ニトロ化によって生じるエラスチンの凝集の有無、程度を効果判定の基準にするという趣旨である。例えば、被験物質の添加により、エラスチンタンパク質またはエラスチン線維にニトロ化処理を行った際の重合集合体の形成を抑制することができる場合、効果ありと判定することができる。凝集の評価方法としては、公知の方法を用いることができる。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において凝集の有無や程度を判断することができ、例えばニトロ化処理を行ったエラスチンタンパク質の分子量が、ニトロ化処理を行っていないエラスチンタンパク質のそれと比べて２倍以上大きい分子量の物質が増加した場合、重合集合体が増加・形成されたと判断することができる。また、チオフラビンＴ等、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有無や程度を判断することもできる。ある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、または凝集に伴う分子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の形成を判断することもできる。ニトロ化処理を行ったエラスチン線維については、走査型電子顕微鏡等の微細構造を観察できる顕微鏡を用いて分子同士が隣接し接触した凝集形態に変化したことを直接観察することで凝集の有無や程度を判断することができる。凝集の評価方法は、上記に限定されない。 In the present invention, "using elastin aggregation caused by nitration as an indicator" means that the presence or absence and extent of elastin aggregation caused by nitration are used as a criterion for determining effectiveness. For example, if the addition of the test substance can suppress the formation of polymer aggregates when elastin protein or elastin fibers are subjected to nitration treatment, it can be determined that the test substance is effective. As a method for evaluating aggregation, a known method can be used. The existence and degree of aggregation can be determined by methods that evaluate the molecular weight of polymerized protein aggregates using electrophoresis, gel permeation chromatography, ultrafiltration, mass spectrometry, etc. If the molecular weight of elastin protein increases by at least twice that of elastin protein that has not undergone nitration treatment, it can be determined that polymer aggregates have increased and been formed. Moreover, the presence or absence and degree of aggregation can also be determined by using an aggregation indicator, such as thioflavin T, that is held between aggregated molecules. Alternatively, the formation of aggregation can be determined by directly evaluating changes in the refractive index of the solution due to aggregation and the generation of agglomerated precipitates using a light scattering method or absorbance method, or by visually evaluating changes in molecular morphology due to aggregation using a microscope, etc. You can also do that. For elastin fibers that have undergone nitration treatment, the presence or absence of aggregation and the degree of aggregation can be determined by directly observing that the molecules have changed to an agglomerated form in which they are adjacent to each other using a microscope that can observe the fine structure, such as a scanning electron microscope. can be judged. The method for evaluating aggregation is not limited to the above.
被験物質は、特に制限はない。動物由来エキス、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。被験物質に応じてニトロ化量の分析方法、およびエラスチンの凝集の分析方法を適宜選択すればよい。 There are no particular restrictions on the test substance. Animal-derived extracts, fungal cultures, enzyme-treated products of these, compounds, or derivatives thereof can be used as test substances, and in addition to liquid form, powdered, gel, etc. are also acceptable. do not have. The method for analyzing the amount of nitration and the method for analyzing elastin aggregation may 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。 EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.
＜実験１＞非露光部および光老化部皮膚切片中のエラスチン線維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線維、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のちに採取した光老化部皮膚（前腕部、６０代男性由来）、正常皮膚（腹部、３０代男性由来）をホルマリン固定したのちパラフィンに包埋して切片を作成し、一次抗体に抗ニトロチロシン抗体（Ｍｉｌｌｉｐｏｒｅ社）、エラスチン抗体（ＳａｎｔａＣｒｕｚ社）、二次抗体にＡｌｅｘａＦｌｕｏｒ（登録商標）４８８標識抗マウス抗体（Ａｂｃａｍ社）、ＡｌｅｘａＦｌｕｏｒ（登録商標）５９４標識抗ウサギ抗体（Ｉｖｎｉｔｒｏｇｅｎ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。
図１に示すように、非露光部皮膚では、エラスチン線維は真皮全体に分布しており、ニトロチロシンとは共局在していない。一方、露光部皮膚では真皮上部にエラスチン線維が多数沈着しており、かつその一部にニトロチロシンが共局在していることが確認され、露光部皮膚にニトロ化エラスチンが存在することが示唆された。
<Experiment 1> Localization of elastin fibers and nitrotyrosine in skin sections of non-exposed and photoaged areas Elastin fibers and nitrotyrosine were detected in skin sections using the following procedure.
After obtaining informed consent, photoaged skin (forearm, from a man in his 60s) and normal skin (abdomen, from a man in his 30s) were fixed in formalin and embedded in paraffin to create sections. The primary antibody was an anti-nitrotyrosine antibody (Millipore), the elastin antibody (Santa Cruz), and the secondary antibody was an AlexaFluor (registered trademark) 488-labeled anti-mouse antibody (Abcam), an AlexaFluor (registered trademark) 594-labeled anti-rabbit antibody (Ivnitrogen). Immunostaining was carried out using a fluorescent microscope (Keyence Corporation), and the fluorescent image was observed using a fluorescence microscope (Keyence Corporation).
As shown in Figure 1, in unexposed skin, elastin fibers are distributed throughout the dermis and are not colocalized with nitrotyrosine. On the other hand, in the exposed skin, a large number of elastin fibers were deposited in the upper dermis, and nitrotyrosine was found to be colocalized in some of them, suggesting the presence of nitrated elastin in the exposed skin. It was done.
＜実験２＞ＵＶ照射によるニトロ化タンパク質量の増加
以下の手順で、ＵＶを照射した線維芽細胞で生じるニトロ化タンパク質を検出した。
ヒト真皮線維芽細胞を、牛胎児血清１０％を加えたダルベッコＭＥＭ（Ｄ－ＭＥＭ）培地に懸濁し、３０ｍｍ培養ディッシュに播種し、３７℃、５％ＣＯ２／９５％空気の加湿条件で５日間培養した。培地を捨て、Ｐｈｏｓｐｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅ（ＰＢＳ（－））を添加した後、ＵＶクロスリンカー（Ａｎａｌｙｔｉｋ Ｊｅｎａ社）を用いて２Ｊ／ｃｍ２の強度で細胞にＵＶ－Ａを照射した。ディッシュ中のＰＢＳ（－）を捨て、新しい血清含有Ｄ－ＭＥＭ培地を添加し、３７℃、５％ＣＯ２／９５％空気の加湿条件で３日間培養した。培養終了後、培地を捨て、ＰＢＳ（－）で細胞を洗浄した後、ＲＩＰＡ Ｂｕｆｆｅｒ（０．１％ドデシル硫酸ナトリウム（ＳＤＳ）、５０ｍＭ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、１％ ＴｒｉｔｏｎＸ－１００、０．５％デオキシコール酸、１ｍＭ Ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌ ｆｌｕｏｒｉｄｅ（ＰＭＳＦ）、１０μｇ／ｍＬアプロチニン）を添加して細胞を溶解し回収した後、９５℃で５分間加熱し、超音波破砕処理を行った。細胞破砕物中の総タンパク質量はＢＣＡ法、ニトロチロシン量はＮＷＬＳＳＴＭ Ｎｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ（Ｎｏｒｔｈｗｅｓｔ Ｌｉｆｅ Ｓｃｉｅｎｃｅ Ｓｐｅｃｉａｌｔｉｅｓ社）にて定量した。
図２に示すように、ＵＶ－Ａを照射したサンプルでは、無照射のコントロールのサンプルに対して線維芽細胞破砕物中のニトロチロシン量が増加していることが確認され、ＵＶ－Ａ照射によりニトロ化タンパク質が増加することが示された。
<Experiment 2> Increase in the amount of nitrated proteins by UV irradiation Nitrated proteins produced in UV irradiated fibroblasts were detected using the following procedure.
Human dermal fibroblasts were suspended in Dulbecco's MEM (D-MEM) medium supplemented with 10% fetal bovine serum, seeded in a 30 mm culture dish, and incubated at 37°C under humidified conditions of 5% CO 2 /95% air for 5 days. Cultured for 1 day. After discarding the medium and adding Phosphate Buffered Saline (PBS(-)), the cells were irradiated with UV-A at an intensity of 2 J/cm 2 using a UV crosslinker (Analytik Jena). The PBS (-) in the dish was discarded, fresh serum-containing D-MEM medium was added, and the dish was cultured for 3 days at 37° C. under humidified conditions of 5% CO 2 /95% air. After culturing, discard the medium and wash the cells with PBS(-), then add RIPA Buffer (0.1% sodium dodecyl sulfate (SDS), 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% TritonX- 100, 0.5% deoxycholic acid, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg/mL aprotinin) were added to lyse and collect the cells, followed by heating at 95° C. for 5 minutes and ultrasonic disruption. The total protein amount in the cell homogenate was determined by the BCA method, and the nitrotyrosine amount was determined by NWLSS ™ Nitrotyrosine ELISA (Northwest Life Science Specialties).
As shown in Figure 2, it was confirmed that in the samples irradiated with UV-A, the amount of nitrotyrosine in the fibroblast fragments increased compared to the control sample without irradiation, and the It was shown that nitrated proteins were increased.
＜実験３＞ＵＶ照射によるニトロ化エラスチンタンパク質量の増加
以下の手順で、ＵＶを照射した線維芽細胞で生じるニトロ化エラスチンを検出した。
ヒト真皮線維芽細胞を、牛胎児血清１０％を加えたダルベッコＭＥＭ（Ｄ－ＭＥＭ）培地に懸濁し、３０ｍｍ培養ディッシュに播種し、３７℃、５％ＣＯ２／９５％空気の加湿条件で５日間培養した。培地を捨て、ＰＢＳ（－）を添加した後、ＵＶクロスリンカー（Ａｎａｌｙｔｉｋ Ｊｅｎａ社）を用いて１－２Ｊ／ｃｍ２の強度で細胞にＵＶ－Ａを照射した。ディッシュ中のＰＢＳ（－）を捨て、新しい血清含有Ｄ－ＭＥＭ培地を添加し、３７℃、５％ＣＯ２／９５％空気の加湿条件で１－３日間培養した。これを５回繰り返しながら、計１７日間培養した。培養終了後、培地を捨て、ＰＢＳ（－）で細胞を洗浄した後、ＬｙｓｉｓＢｕｆｆｅｒ（０．５％ ＳＤＳ、２ｍＭ ＥＤＴＡ、１ｍＭ ＰＭＳＦ、１０μｇ／ｍＬアプロチニン）を添加して細胞を溶解し回収した後、９５℃で５分間加熱し、超音波破砕処理を行った。ここに５倍量のＩＰ Ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、１５０ｍＭ ＮａＣｌ、１％（ｖ／ｖ）ＴｒｉｔｏｎＸ－１００、１ｍＭ ＰＭＳＦ、１０μｇ／ｍＬアプロチニン）を添加し混合したものをＴｏｔａｌ Ｆｒａｃｔｉｏｎとした。Ｔｏｔａｌ Ｆｒａｃｔｉｏｎの細胞破砕物２００μｇとマグネチックビーズ標識抗ニトロチロシン抗体（Ｍｉｌｌｉｐｏｒｅ社）２０μＬを混合し、４℃で一晩転倒混和した。遠心分離にてビーズ標識抗体複合体を分離後、上清を破棄し沈査に２×Ｓａｍｐｌｅ Ｂｕｆｆｅｒ（０．１Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ６．８）、４％ ＳＤＳ、１０％ ２－メルカプトエタノール、２０％グリセロール、０．１％ブロモフェノールブルー）を２５μＬ添加し、９５℃で５分間加熱処理したものをＩＰ Ｆｒａｃｔｉｏｎとした。７．５％ポリアクリルアミドゲルにＴｏｔａｌ Ｆｒａｃｔｉｏｎ、ＩＰ Ｆｒａｃｔｉｏｎの細胞破砕物を等量ずつアプライし、２時間程度電気泳動した。ゲル中のタンパク質をＰＶＤＦ膜にトランスファー後、５％スキムミルクで３０分間ブロッキングし、抗エラスチン抗体（ＳａｎｔａＣｒｕｚ社）を添加して４℃で一晩インキュベートした。抗体を洗浄後、ＨＲＰ標識抗マウス抗体（ＧＥ Ｈｅａｌｔｈｃａｒｅ社）を添加して室温で２時間インキュベートした後、抗体を洗浄し、ＰＶＤＦ膜上に発光基質（Ｉｎｖｉｔｒｏｇｅｎ社）を滴下し、ｉＢｒｉｇｈｔ ＣＬ１５００（Ｉｎｖｉｔｒｏｇｅｎ社）にて化学発光を検出した。
図３に示すように、同サンプルからニトロチロシン抗体にて免疫沈降を行いニトロチロシンを単離回収したところ、免疫沈降を行う前のサンプル（Ｔｏｔａｌ Ｆｒａｃｔｉｏｎ）ではＵＶ－Ａ照射に関わらずサンプル中のトロポエラスチン量にはほとんど差がなかったが、免疫沈降サンプル（ＩＰ Ｆｒａｃｔｉｏｎ）では、無照射に対しＵＶ－Ａ照射サンプルでは線維芽細胞破砕物中のエラスチンタンパク質量が増加していることが示された。つまり、線維芽細胞へのＵＶ－Ａ照射により、エラスチンタンパク質にニトロ化が増加したことが確認された。
<Experiment 3> Increase in the amount of nitrated elastin protein by UV irradiation Nitrated elastin produced in UV irradiated fibroblasts was detected using the following procedure.
Human dermal fibroblasts were suspended in Dulbecco's MEM (D-MEM) medium supplemented with 10% fetal bovine serum, seeded in a 30 mm culture dish, and incubated at 37°C under humidified conditions of 5% CO 2 /95% air for 5 days. Cultured for 1 day. After discarding the medium and adding PBS (-), the cells were irradiated with UV-A at an intensity of 1-2 J/cm 2 using a UV cross-linker (Analytik Jena). The PBS (-) in the dish was discarded, fresh serum-containing D-MEM medium was added, and the dish was cultured for 1-3 days at 37° C. under humidified conditions of 5% CO 2 /95% air. This was repeated 5 times for a total of 17 days of culture. After culturing, discard the medium, wash the cells with PBS (-), add LysisBuffer (0.5% SDS, 2mM EDTA, 1mM PMSF, 10μg/mL aprotinin) to lyse and collect the cells. It was heated at 95° C. for 5 minutes and subjected to ultrasonic crushing treatment. Add and mix 5 times the amount of IP Buffer (50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% (v/v) TritonX-100, 1mM PMSF, 10μg/mL aprotinin) to Total Fraction. And so. 200 μg of Total Fraction cell fragments and 20 μL of magnetic bead-labeled anti-nitrotyrosine antibody (Millipore) were mixed and mixed overnight at 4° C. by inversion. After separating the bead-labeled antibody complex by centrifugation, discard the supernatant and add 2x Sample Buffer (0.1M Tris-HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, 20% IP Fraction was obtained by adding 25 μL of glycerol, 0.1% bromophenol blue) and heating at 95° C. for 5 minutes. Equal amounts of total fraction and IP fraction cell lysates were applied to a 7.5% polyacrylamide gel and electrophoresed for about 2 hours. After the protein in the gel was transferred to a PVDF membrane, it was blocked with 5% skim milk for 30 minutes, anti-elastin antibody (Santa Cruz) was added, and the membrane was incubated overnight at 4°C. After washing the antibody, HRP-labeled anti-mouse antibody (GE Healthcare) was added and incubated at room temperature for 2 hours. After washing the antibody, a luminescent substrate (Invitrogen) was dropped onto the PVDF membrane, and an iBright CL1500 (Invitrogen) was added. Chemiluminescence was detected at
As shown in Figure 3, when nitrotyrosine was isolated and recovered from the same sample by immunoprecipitation with a nitrotyrosine antibody, in the sample before immunoprecipitation (Total Fraction), regardless of UV-A irradiation, the amount of nitrotyrosine in the sample was Although there was almost no difference in the amount of tropoelastin, the immunoprecipitation samples (IP Fraction) showed that the amount of elastin protein in the fibroblast fragments increased in the UV-A irradiated samples compared to the non-irradiated samples. It was done. In other words, it was confirmed that UV-A irradiation of fibroblasts increased nitration of elastin protein.
＜実験４＞ニトロ化処理によるエラスチンの凝集
以下の手順で、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理したトロポエラスチンタンパク質の分子量の変化およびエラスチン線維の形態変化を検出した。
＜分子量変化の検出＞
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、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ（Ｄｏｊｉｎｄｏ社）を１００μＭになるよう添加した。３７℃で１日間インキュベートした後、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（５０ｍＭ Ｂｉｓ－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。
＜線維形態の観察＞
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。
図４に示すように、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理により約４８０ｋＤａ以下の領域のバンドが薄く、約４８０ｋＤａ以上の高分子量側の領域（Ａ）のバンドが濃くなった。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、複数の分子が高度に会合したトロポエラスチンタンパク質の重合集合体が増加していることが確認された。また、図５に示すように、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理によりエラスチンファイバーの線維が束なり合一した。以上の結果から、ニトロ化処理はエラスチンに凝集を生じさせることが示された。
<Experiment 4> Aggregation of elastin by nitration treatment Changes in the molecular weight of the peroxynitrite-treated tropoelastin protein and changes in the morphology of elastin fibers were detected using the following procedure.
<Detection of molecular weight change>
Tropoelastin protein (SIGMA) was dissolved in 0.2M phosphate buffer (pH 7.5) at a concentration of 1 mg/mL, and Peroxynitrite (Dojindo) was added at a concentration of 100 μM. After incubation at 37°C for 1 day, 10x Blue native PAGE Sample Buffer (5% (w/v) CBB-G250, 0.5M 6-aminocaproic acid, 0.1M 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. Apply 20 μL each to a 4-16% polyacrylamide gradient gel (Invitrogen), add Anode Buffer (50mM Bis-Tris (pH 7.0)) to the anode side of the electrophoresis tank, and Cathode Buffer (50mM Tricine (pH 7.0) to the cathode side). ), 15mM Bis-Tris (pH 7.0), 0.02% CBB-G250), and electrophoresis was performed at 150mA while cooling the electrophoresis tank. Halfway through, electrophoresis was stopped, the Cathode Buffer was replaced with one that did not contain CBB-G250, and electrophoresis was performed again. After electrophoresis, the gel was immersed overnight in a fixative solution (50% methanol, 10% acetic acid) for fixation and decolorization, and then silver staining was performed using a silver staining kit (ATTO).
<Observation of fiber morphology>
An elastin fiber sheet (Extracellular Matrix Research Institute) was soaked in 0.2M phosphate buffer (pH 7.5), and Peroxynitrite (Dojindo) was added to 100 μM. After incubating at 37° C. for 3 days, the elastin fiber sheet was washed and dried with distilled water, and after Au deposition, the fiber morphology was observed using a scanning electron microscope (JEOL).
As shown in FIG. 4, the peroxynitrite treatment made the band in the region of about 480 kDa or less thinner, and the band in the higher molecular weight region (A) of about 480 kDa or more became darker. Since tropoelastin protein is a protein with a molecular weight of about 70 kDa as a monomer, it was confirmed that polymerized aggregates of tropoelastin protein in which multiple molecules are highly associated are increasing. Furthermore, as shown in FIG. 5, the elastin fibers were bundled and unified by the peroxynitrite treatment. The above results showed that nitration treatment caused elastin to aggregate.
＜実験５＞エラスターゼによるエラスチンタンパク質の分解
以下の手順で、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ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。
オルセイン標識不溶性エラスチン（ＳＩＧＭＡ社）を１０ｍｇ／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、好中球エラスターゼ（ＳＩＧＭＡ）を０．５ｕｎｉｔｓ／ｍＬになるよう添加し、３７℃で一晩インキュベートした。遠心分離後に上清を９６ ｗｅｌｌ－ｐｌａｔｅに回収し、５９５ｎｍの吸光度からエラスチン分解率を算出した。
図６に示すように、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理はエラスターゼによるエラスチンの分解率を抑制することが確認された。
<Experiment 5> Decomposition of elastin protein by elastase The decomposition of elastin treated with Peroxynitrite by elastase was measured using the following procedure.
Orcein-labeled insoluble elastin (SIGMA) was well suspended in Reaction Buffer (0.2M Tris-HCl (pH 7.5), 20mM CaCl 2 ) to a concentration of 10 mg/mL, and neutrophil elastase (SIGMA) was suspended at 0. The solution was added at a concentration of .5 units/mL and incubated overnight at 37°C. After centrifugation, the supernatant was collected in a 96 well-plate, and the elastin degradation rate was calculated from the absorbance at 595 nm.
As shown in FIG. 6, it was confirmed that the Peroxynitrite treatment suppressed the rate of elastin degradation by elastase.
＜実験６＞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（ｐＨ７．５）に浸漬し、ＥｔＯＨに溶解した１０％テトラニトロメタン溶液を０．１％の濃度になるよう添加した。室温にて２０分インキュベートした後、尿素水溶液を終濃度４Ｍになるよう添加し、反応を停止した。溶液を捨て、シートを蒸留水にて洗浄後、ＰＢＳ（－）に浸漬した。ゲル硬度は引張試験機（細胞外基質研究所社）を用いてヤング率を測定した。弾力性は、Ｃｕｔｏｍｅｔｅｒ（Ｃｏｕｒａｇｅ＋Ｋｈａｚａｋａ社）にて測定した。
図７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、また図８に示すように、弾力性（回復率；Ｕｒ／Ｕｆ）が低下することが確認された。
<Experiment 6> 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) was immersed in 0.2M phosphate buffer (pH 7.5), and a 10% tetranitromethane solution dissolved in EtOH was added to a concentration of 0.1%. After incubating for 20 minutes at room temperature, an aqueous urea solution was added to a final concentration of 4M to stop the reaction. The solution was discarded, the sheet was washed with distilled water, and then immersed in PBS (-). Gel hardness was determined by measuring Young's modulus using a tensile tester (Extracellular Matrix Research Institute). Elasticity was measured using Cutometer (Courage+Khazaka).
As shown in Figure 7, the hardness (Young's modulus) of the elastin gel sheet treated with tetranitromethane (TNM) increased, and as shown in Figure 8, it was confirmed that the elasticity (recovery rate; Ur/Uf) decreased. It was done.
＜実験７＞ニトロ化エラスチン線維上で培養した線維芽細胞の遺伝子発現量の変化
以下の手順で、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ処理したエラスチン線維上で培養した線維芽細胞の遺伝子発現量を測定した。
３０φガラスシャーレ（アズワン社）にシリコン支持シート（細胞外基質研究所社）を敷き、その上にエラスチンファイバーシート（細胞外基質研究所社）を重ねた。０．２Ｍリン酸緩衝液（ｐＨ７．５）を添加し、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ（Ｄｏｊｉｎｄｏ社）を１００μＭになるよう滴下した。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した。ヒト真皮線維芽細胞を、牛胎児血清１０％を加えたダルベッコＭＥＭ（Ｄ－ＭＥＭ）培地に懸濁し、２４ ｗｅｌｌ－ｐｌａｔｅ内に播種し、３７℃、５％ＣＯ２／９５％空気の加湿条件で７日間培養した。Ｔｏｔａｌ ＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｊｅｎａ Ｂｉｏｓｃｉｅｎｃｅ社）を用いて、Ｔｏｔａｌ ＲＮＡを抽出した。その後、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔ（ＴａＫａＲａ社）を用いて逆転写を行い、ｃＤＮＡを合成した。得られたｃＤＮＡを鋳型として、Ｉ型コラーゲン、トロポエラスチン、フィブリリン－１、ファイブリン５、およびＧＡＰＤＨ（グリセルアルデヒド３－リン酸 デヒドロゲナー ゼ；ハウスキーピング遺伝子として使用）の発現量を遺伝子特異的プライマー及びＰｏｗｅｒ ＳＹＢＲ Ｇｒｅｅｎ Ｍａｓｔｅｒ Ｍｉｘ（アプライドバイオシステムズ社）を用いて、リアルタイムＰＣＲ（７５００ Ｒｅａｌ Ｔｉｍｅ ＰＣＲ Ｓｙｓｔｅｍ、アプライドバイオシステムズ社）にて測定し、遺伝子発現の解析は比較Ｃｔ法にて行った。つまり、被験物質添加による遺伝子発現量の変化は、コントロール群のＣＳＥのＣｔ値をＧＡＰＤＨのＣｔ値で補正した値を１とし、それに対する相対量として求めた。
図９に示すように、ニトロ化処理したエラスチンファイバー上で培養した線維芽細胞では、コントロールに対してコラーゲン（ＣＯＬ１Ａ１）、フィブリリン－１（Ｆｉｂｒｉｌｌｉｎ－１）、ファイブリン５（Ｆｉｂｕｌｉｎ－５）の遺伝子発現量にはほぼ変化がなかったが、トロポエラスチン（Ｔｒｏｐｏｅｌａｓｔｉｎ）の遺伝子発現量は増加することが確認された。
<Experiment 7> Change in gene expression levels of fibroblasts cultured on nitrated elastin fibers The gene expression levels of fibroblasts cultured on peroxynitrite-treated elastin fibers were measured using the following procedure.
A silicon support sheet (Extracellular Matrix Research Institute, Inc.) was placed on a 30φ glass petri dish (As One Corporation), and an elastin fiber sheet (Extracellular Matrix Research Institute, Inc.) was layered thereon. A 0.2M phosphate buffer (pH 7.5) was added, and Peroxynitrite (Dojindo) was added dropwise to a concentration of 100 μM. After incubating at 37°C for 3 days, the elastin fiber sheet was washed with distilled water. Human dermal fibroblasts were suspended in Dulbecco's MEM (D-MEM) medium supplemented with 10% fetal bovine serum and seeded in 24 well-plates at 37°C under humidified conditions of 5% CO 2 /95% air. The cells were cultured for 7 days. Total RNA was extracted using Total RNA Purification Kit (Jena Bioscience). Thereafter, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, we measured the expression levels of type I collagen, tropoelastin, fibrillin-1, fibulin-5, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) in a gene-specific manner. Measurement was performed using real-time PCR (7500 Real Time PCR System, Applied Biosystems) using primers and Power SYBR Green Master Mix (Applied Biosystems), and gene expression analysis was performed using the comparative Ct method. In other words, the change in gene expression level due to the addition of the test substance was determined as a relative amount with respect to the value obtained by correcting the CSE Ct value of the control group with the GAPDH Ct value as 1.
As shown in Figure 9, in fibroblasts cultured on nitrated elastin fibers, the genes of collagen (COL1A1), fibrillin-1 (Fibrillin-1), and fibulin-5 (Fibulin-5) Although there was almost no change in the expression level, it was confirmed that the gene expression level of tropoelastin increased.
上記の結果より、エラスチンのニトロ化は紫外線照射により増加すること、またニトロ化はエラスチンの凝集を促進する特性があること、およびエラスターゼによる分解に対して抵抗性を付与することを見出した。加えてこれらの物性変化の結果、エラスチン線維は硬化し、弾力性が減少することも見出した。つまり、紫外線照射によりエラスチン線維に生成したニトロ化物が光老化部でのエラスチン線維の異常沈着に寄与することを突き止めた。本発明のエラスチン線維の凝集を抑制し、皮膚の硬化および皮膚弾力性の低下、それに伴うシワ、たるみの形成、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及び／又は選択する方法は、上記知見に基づくものである。 From the above results, we found that nitration of elastin increases with ultraviolet irradiation, that nitration has the property of promoting elastin aggregation, and that it confers resistance to degradation by elastase. In addition, they found that as a result of these changes in physical properties, elastin fibers harden and their elasticity decreases. In other words, we found that nitrations generated in elastin fibers by ultraviolet irradiation contribute to the abnormal deposition of elastin fibers in photoaged areas. Evaluate and/or evaluate the substance of the present invention that can suppress the aggregation of elastin fibers and prevent or improve the signs of photoaging such as skin hardening, decrease in skin elasticity, and accompanying wrinkles, sagging formation, and decrease in firmness. Alternatively, the method of selection is based on the above findings.
＜実施例１＞ニトロチロシン生成抑制物質のスクリーニング方法
以下の手順で、ニトロ化物であるニトロチロシンの生成抑制活性を測定した。
＜被験物質の調製＞
原体（ウーロン茶、煎茶、プーアール茶、ほうじ茶、紅茶、碁石茶（登録商標）、酒かす（月桂冠社製））に１０倍の重量の５０％（ｖ／ｖ）エタノール水溶液を加えて６０℃、４時間加熱抽出した。抽出物の乾燥残分に対して、エタノール、水を重量比で１：３０：６９となるように加えて希釈したものを被験物質とした。バラ乳酸菌発酵物に関しては、バラ科バラ属に属するゴールデンハートの花弁に２倍の重量の１％グルコース水溶液を添加したものを高圧蒸気滅菌処理し、乳酸菌（Ｌａｃｔｏｂａｃｈｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）の菌液を１％重量添加して３０℃で２週間静置培養した。高圧蒸気滅菌処理を行った後、ろ過液の乾燥残分に対してエタノール、水を重量比で１：３０：６９となるように加えて希釈したものを被験物質とした。
＜ニトロチロシン生成抑制試験＞
０．１Ｍリン酸緩衝液（ｐＨ７．４）に溶解した１ｍｇ／ｍＬウシ血清アルブミン（ＢＳＡ）溶液に、被験物質または対照物質を１０ｐｐｍの濃度になるよう添加した後、Ｐｅｒｏｘｙｎｉｔｒｉｔｅ発生剤であるＳＩＮ－１（Ｄｏｊｉｎｄｏ社）を終濃度が３ｍＭになるよう添加した。３７℃で一晩インキュベートしたのち、反応溶液中のＢＳＡのアミノ酸残基に生じたニトロチロシン量をＮＷＬＳＳＴＭ Ｎｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ （Ｎｏｒｔｈｗｅｓｔ Ｌｉｆｅ Ｓｃｉｅｎｃｅ Ｓｐｅｃｉａｌｔｉｅｓ社）を用いて定量した。
図１０に示すように、ニトロ化剤であるＳＩＮ－１の添加により、ＳＩＮ－１未添加に比べてニトロチロシン量は大きく増加した。ＳＩＮ－１とともに番茶、サケカスエキスを添加した場合では被験物質の未添加と同程度のニトロチロシン量が生成しておりニトロチロシン生成抑制作用は確認されなかったが、ウーロン茶、煎茶、プーアール茶、ほうじ茶、紅茶、碁石茶、バラ乳酸菌発酵物エキスでは、ニトロチロシンの生成量が被験物質未添加に比べて抑制されており、ニトロチロシン生成抑制作用が確認された。以上の結果から、この方法を用いてニトロ化物の生成抑制剤を選択することができ、本スクリーニング方法を用いることで、タンパク質の凝集蓄積改善剤、皮膚の硬化抑制剤、皮膚の弾力性抑制剤、シワ、ハリ、タルミ改善剤等、光老化を改善する物質を選択することが可能である。
<Example 1> Screening method for nitrotyrosine production inhibitor The activity of inhibiting the production of nitrotyrosine, a nitrated compound, was measured according to the following procedure.
<Preparation of test substance>
Add 10 times the weight of 50% (v/v) ethanol aqueous solution to the raw material (oolong tea, sencha, pu-erh tea, roasted tea, black tea, Goishicha (registered trademark), sake lees (manufactured by Gekkeikan)) at 60°C. Extraction was carried out by heating for 4 hours. A test substance was prepared by diluting the dried residue of the extract by adding ethanol and water at a weight ratio of 1:30:69. Regarding the rose lactic acid bacteria fermentation product, the petals of Golden Heart, which belongs to the Rosaceae family, are added with twice the weight of a 1% aqueous glucose solution and then sterilized using high-pressure steam. The cells were added and statically cultured at 30°C for 2 weeks. After high-pressure steam sterilization, the dried residue of the filtrate was diluted by adding ethanol and water at a weight ratio of 1:30:69, and the resulting product was used as a test substance.
<Nitrotyrosine production inhibition test>
After adding the test substance or control substance to a concentration of 10 ppm to a 1 mg/mL bovine serum albumin (BSA) solution dissolved in 0.1 M phosphate buffer (pH 7.4), the peroxynitrite generating agent SIN- 1 (Dojindo) was added to a final concentration of 3 mM. After overnight incubation at 37°C, the amount of nitrotyrosine generated in the amino acid residues of BSA in the reaction solution was quantified using NWLSS ™ Nitrotyrosine ELISA (Northwest Life Science Specialties).
As shown in FIG. 10, the addition of SIN-1, a nitrating agent, greatly increased the amount of nitrotyrosine compared to when SIN-1 was not added. When bancha and salmon cass extract were added together with SIN-1, the same amount of nitrotyrosine was produced as when no test substance was added, and no inhibitory effect on nitrotyrosine production was confirmed; , black tea, Goishi tea, and rose lactic acid bacteria fermented product extracts suppressed the amount of nitrotyrosine produced compared to when no test substance was added, confirming the inhibitory effect on nitrotyrosine production. From the above results, it is possible to select a nitrate generation inhibitor using this method, and by using this screening method, it is possible to select an agent for improving protein aggregation accumulation, an agent for suppressing skin hardening, and an agent for suppressing skin elasticity. It is possible to select substances that improve photoaging, such as agents that improve wrinkles, firmness, and sagging.
＜実施例２＞ニトロ化物分解物質のスクリーニング方法
以下の手順で、ニトロ化物であるニトロチロシンの分解活性を測定した。
＜被験物質の調製＞
実施例１と同様に調製した。
＜ニトロチロシン分解活性試験＞
３－ニトロチロシンを０．２Ｍリン酸緩衝液（ｐＨ７．０）に溶解し、５μｇ／ｍＬの濃度の溶液を調製した。リン酸緩衝液または３－ニトロチロシン溶液を９６ ｗｅｌｌ－ｐｌａｔｅに分注し、被験物質または対照物質を各ｗｅｌｌに終濃度が１００ｐｐｍになるよう添加した。３７℃で７２時間インキュベートしたのち、プレートリーダーにて４５０ｎｍの吸光度を測定し、ニトロチロシン残存率を算出した。
図１１に示すように、無添加のコントロールではニトロチロシン残存率が１００％であった。また、ほうじ茶、煎茶もニトロチロシン残存率はほぼ１００％であり、ニトロチロシン分解活性はないことが確認された。他方、碁石茶、酒かす、バラ乳酸菌発酵物の添加ではニトロチロシン残存率は８０－９０％程度に減少しており、これらの被験物質にはニトロチロシン分解作用があることが確認された。以上の結果から、この方法を用いてニトロ化物分解剤を選択することができ、本スクリーニング方法を用いることで、タンパク質の凝集蓄積改善剤、皮膚の硬化抑制剤、皮膚の弾力性抑制剤、シワ、ハリ、タルミ改善剤等、光老化を改善する物質を選択することが可能である。
<Example 2> Screening method for nitrated decomposition substances The degrading activity of nitrotyrosine, a nitrated product, was measured according to the following procedure.
<Preparation of test substance>
Prepared in the same manner as in Example 1.
<Nitrotyrosine degrading activity test>
3-Nitrotyrosine was dissolved in 0.2M phosphate buffer (pH 7.0) to prepare a solution with a concentration of 5 μg/mL. The phosphate buffer or 3-nitrotyrosine solution was dispensed into 96 well-plates, and the test substance or control substance was added to each well at a final concentration of 100 ppm. After incubating at 37°C for 72 hours, the absorbance at 450 nm was measured using a plate reader, and the residual rate of nitrotyrosine was calculated.
As shown in FIG. 11, the residual rate of nitrotyrosine was 100% in the additive-free control. Furthermore, the residual rate of nitrotyrosine in hojicha and sencha was almost 100%, and it was confirmed that they did not have nitrotyrosine degrading activity. On the other hand, the residual rate of nitrotyrosine decreased to about 80-90% with the addition of Goishi tea, sake lees, and fermented material of rose lactic acid bacteria, and it was confirmed that these test substances have a nitrotyrosine-degrading effect. From the above results, it is possible to select a nitride decomposition agent using this method, and by using this screening method, it is possible to select an agent for improving protein aggregation accumulation, an agent for suppressing hardening of the skin, an agent for suppressing skin elasticity, and an agent for suppressing wrinkles. It is possible to select substances that improve photoaging, such as , firmness and sagging improving agents.
＜実施例３＞エラスチンの凝集抑制物質のスクリーニング方法
以下の手順で、エラスチンの凝集を評価した。
＜被験物質の調製＞
実施例１と同様に調製した。
＜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、被験物質または対照物質を終濃度が１００ｐｐｍになるよう添加したのちＰｅｒｏｘｙｎｉｔｒｉｔｅ（Ｄｏｊｉｎｄｏ社）を終濃度が１００μＭになるよう添加した。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。
トロポエラスチンタンパク質は単量体では分子量約７０ｋＤａのタンパク質であり、凝集処理により複数の分子が会合した高分子量の重合集合体が形成される。よって、被験物質の処理により７０ｋＤａ以上の分子量のバンドが減少している場合、被験物質にはエラスチンタンパク質の凝集抑制作用があると判断される。この方法を用いることでエラスチンの凝集抑制剤を選択することができ、本スクリーニング方法を用いることで、皮膚の硬化抑制剤、皮膚の弾力性抑制剤、シワ、ハリ、タルミ改善剤等、光老化を改善する物質を選択することが可能である。
<Example 3> Screening method for elastin aggregation inhibitory substance Aggregation of elastin was evaluated using the following procedure.
<Preparation of test substance>
Prepared in the same manner as in Example 1.
<Elastin aggregation inhibition test>
Tropoelastin protein (SIGMA) was dissolved in 0.2M 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. (Company) was added to a final concentration of 100 μM. After incubation at 37°C for 1 day, 10x Blue native PAGE Sample Buffer (5% (w/v) CBB-G250, 0.5M 6-aminocaproic acid, 0.1M Bis-Tris (pH 7.0), 50% Glycerol ) was added in an amount of 1/10 and allowed to stand at 4°C for 10 minutes. Apply 20 μL each to 4-16% polyacrylamide gradient gel (Invitrogen), add Anode Buffer (50 mM Bis-Tris (pH 7.0)) to the anode side of the electrophoresis tank, and Cathode Buffer (50 mM Tricine (pH 7.0) to the cathode side). , 15mM Bis-Tris (pH 7.0), 0.02% CBB-G250), and electrophoresis was performed at 150 mA while cooling the electrophoresis tank. Halfway through, electrophoresis was stopped, the Cathode Buffer was replaced with one that did not contain CBB-G250, and electrophoresis was performed again. After electrophoresis, the gel was immersed overnight in a fixative solution (50% methanol, 10% acetic acid) for fixation and decolorization, and then silver staining was performed using a silver staining kit (ATTO).
Tropoelastin protein is a protein with a molecular weight of about 70 kDa as a monomer, and a high molecular weight polymeric aggregate in which a plurality of molecules are associated is formed by aggregation treatment. Therefore, if the number of bands with a molecular weight of 70 kDa or more is reduced by treatment with the test substance, it is determined that the test substance has an effect of inhibiting elastin protein aggregation. By using this method, it is possible to select elastin aggregation inhibitors, and by using this screening method, it is possible to select agents that inhibit skin hardening, skin elasticity inhibitors, wrinkle, firmness, and sagging agents, etc. It is possible to select substances that improve
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| US20190290574A1 (en) | 2015-12-08 | 2019-09-26 | University Of Pretoria | Topical skin care compositions comprising myrsine africana extracts |
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| JP2002326922A (en) | 2001-03-01 | 2002-11-15 | Kose Corp | Skin external preparation |
| JP2003081835A (en) | 2001-09-10 | 2003-03-19 | Shiseido Co Ltd | Promotive agent for recovery from photoaging and skin care preparation for promoting recovery from photoaging |
| JP2009084164A (en) | 2007-09-27 | 2009-04-23 | Septem Soken:Kk | Antiaging agent |
| US20190290574A1 (en) | 2015-12-08 | 2019-09-26 | University Of Pretoria | Topical skin care compositions comprising myrsine africana extracts |
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