JP2004067553A - Angiotensin-converting enzyme-inhibiting peptide - Google Patents
Angiotensin-converting enzyme-inhibiting peptide Download PDFInfo
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、アンギオテンシン変換酵素阻害剤等として有用なペプチドに関する。
【０００２】
【従来の技術】
アンギオテンシン変換酵素（ＡＣＥ）は主にヒトの血管の内皮細胞、肺、腎臓及び脳に存在する。この酵素は、血管の収縮を引き起こし、血圧を上昇させるために、Ｃ末端から２つのアミノ酸（Ｈｉｓ−Ｌｅｕ）を切断することにより、不活性なアンギオテンシンＩを活性なアンギオテンシンＩＩに変換し得る。したがって、ＡＣＥは血圧を上昇させることができる。
【０００３】
アンギオテンシン変換酵素阻害剤（ＡＣＥＩ）のＡＣＥへの結合はアンギオテンシンＩＩの生成を減少し得る。ＡＣＥＩを食品に加えると、高血圧症状を減少させるのにプラスの効果を引き起こすであろう。現在のところ、多くのペプチドがＡＣＥの阻害についての効果を有することが知られている。それらは異なったアミノ酸配列と長さを有する。
【０００４】
【発明が解決しようとする課題】
従って、本発明は、豚肉由来の機能性成分として、高血圧予防効果が期待されるアンギオテンシン変換酵素（ＡＣＥ）阻害ペプチドを提供することを主な課題とする。
【０００５】
【課題を解決するための手段】
本発明は、豚肉由来の新規なアンギオテンシン変換酵素（ＡＣＥ）阻害ペプチドを見出した。
【０００６】
本発明は、以下の項１〜４の活性ペプチド、並びにアンギオテンシン変換酵素阻害剤およびその製造法に関する。
項１． 以下の２種の少なくとも１種からなるポリペプチド：
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓ；及び
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ。
項２． 以下の７種の少なくとも１種からなるアンギオテンシン変換酵素阻害剤：
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ；
Ｔｈｒ−Ｐｒｏ；及び
Ｔｈｒ−Ｌｙｓ。
項３． トロポニンをペプシンで加水分解することを特徴とするアンギオテンシン変換酵素阻害剤の製造法。
項４． アンギオテンシン変換酵素阻害剤がＡｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓである項３の製造法。
【０００７】
【発明の実施の形態】
本発明は、豚肉由来の機能性成分として、高血圧予防効果が期待されるアンギオテンシン変換酵素（ＡＣＥ）阻害ペプチドに関する。
【０００８】
本発明において、豚肉由来のタンパク質から抽出することができる粗精製トロポニンをペプシンで加水分解すると、ＡＣＥ阻害活性を有するペプチドＡｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓ（ＲＭＬＧＱＴＰＴＫ、以下９ｍｅｒと言う）が生成することが見出された。このペプチドは、骨格筋トロポニンＣ（Ｌｏｒｋｉｎ ＰＡ ＆ Ｌｅｈｍａｎｎ Ｈ， ＦＥＢＳ Ｌｅｔｔｅｒ， １５３： ８１−８７，１９８３）の４４−５２位由来である。
【０００９】
９ｍｅｒの部分構造ペプチドであるＡｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ（ＲＭＬＧＱＴＰ、以下７ｍｅｒと言う）、Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ（ＲＭＬＧＱＴ）、Ａｒｇ−Ｍｅｔ−Ｌｅｕ（ＲＭＬ）、Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ（ＲＭＬＧＱＴＰＴ）、Ｔｈｒ−Ｐｒｏ（ＴＰ）及びＴｈｒ−Ｌｙｓ（ＴＫ）もＡＣＥ阻害活性を有することが分かった。
【００１０】
本発明のペプチド９ｍｅｒは豚肉由来のタンパク質から抽出した粗精製トロポニンをペプシンで加水分解することによって得られる。また、ペプチド９ｍｅｒの部分構造ペプチドは、Ｆｍｏｃ固相合成などの公知の方法に従い合成できる。
【００１１】
本発明のペプチド９ｍｅｒは、粗精製トロポニンのペプシン加水分解生成物を、イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィー、逆相ＨＰＬＣなどを組み合わせて精製することにより得られる。
【００１２】
本発明のＡＣＥ阻害ペプチドは、ウサギ肺由来及びウシ肺由来のＡＣＥに対して阻害活性を有する。
【００１３】
下記実施例において、より具体的に本発明を説明する。
【００１４】
【実施例】
以下実施例において測定されたＡＣＥに対する５０％阻害活性（ＩＣ５０）は、ウシ肺由来のＡＣＥに対するもので、Ｃｕｓｈｍａｎ −Ｃｈｅｕｎｇの方法（Ｂｉｏｃｈｅｍｉｃａｌ Ｐｈａｒｍａｃｏｌｏｇｙ， ２０： １６３７−１６４８， １９７１）で測定した。
【００１５】
実施例１
国産の豚ロース肉から抽出した粗精製トロポニンをペプシンで加水分解した。この粗精製トロポニンのペプシン加水分解物のウシ肺由来のＡＣＥに対する５０％阻害活性（ＩＣ５０）は２２５ｇ／ｍｌであった。未加水分解トロポニンにはＡＣＥ阻害活性が認められなかったことから、活性の発現はペプシン分解で生じたペプチドに由来することが分かった。
【００１６】
該加水分解物をまず、ＤＥ５３カラム（Ｗｈａｔｍａｎ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｌｔｄ．， Ｋｅｎｔ， ＵＫ） （１６ ｘ １５０ ｍｍ）を用いる陰イオン交換クロマトグラフィーに供した。すなわち、２０ｍＭトリス（ヒドロキシメチル）アミノメタン（酢酸でｐＨ ７．５に調整したもの）で該加水分解物をアプライし、同バッファーを含むＮａＣｌ水溶液によるグラジェント（０−３００ ｍＭ）溶出を行った。流速は１．１３ ｍｌ／分とし、７分毎（７．９１ ｍｌ）に分画した。この画分をＳＥＰ−ＰＡＫ Ｐｌｕｓ Ｃ１８カートリッジ（Ｗａｔｅｒｓ Ｃｏ．， Ｍｉｌｆｏｒｄ， ＭＡ， ＵＳＡ）を用いて脱塩し、吸着画分を５０％アセトニトリルで溶出させて得られた画分を濃縮して、その活性を測定した。ここで得られた活性画分をＣｏｓｍｏｓｉｌ ５Ｃ１８ ＡＲ−ＩＩ （４．５ ｘ １５０ｍｍ） （Ｎａｃａｌａｉ Ｔｅｓｑｕｅ Ｉｎｃ．， Ｋｙｏｔｏ Ｊａｐａｎ）を用いる逆相（ＲＰ）−ＨＰＬＣに供し、０．１％トリフルオロ酢酸（ＴＦＡ）を含むアセトニトリルの１−８０％のグラジェント溶出（流速０．５ｍｌ／分）で分画した。活性画分はさらに、同じカラムで０．１％ＴＦＡを含む１４％アセトニトリルでイソクラティック溶出（流速０．５ｍｌ／分）した。ＲＰ−ＨＰＬＣで得られた活性画分をＴＳＫ−ｇｅｌ Ｇ２０００ＳＷＸＬ （７．８ ｘ ３００ｍｍ） （Ｔｏｓｏｈ Ｃｏ．， Ｔｏｋｙｏ， Ｊａｐａｎ） を用いるゲル濾過ＨＰＬＣに供して、２０ ｍＭリン酸Ｎａ緩衝液（ｐＨ ７．０）でイソクラティック溶出（流速０．５ｍｌ／分）した。最後に、活性画分をＣｏｓｍｏｓｉｌ ５ＰＥ−ＭＳ （４．６ ｘ ２５０ｍｍ） （Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用いるＲＰ−ＨＰＬＣに供し、０．１％ＴＦＡを含む１２％アセトニトリルでイソクラティック溶出（流速１ｍｌ／分）した。いずれの場合も、２１５ｎｍの吸光度をモニターした。
【００１７】
精製で得られた画分のアミノ酸配列を、プロテインシーケンサー（ＰＰＳＱ−１０， Ｓｈｉｍａｄｚｕ Ｃｏ．， Ｋｙｏｔｏ， Ｊａｐａｎ）でシーケンス解析した。得られたペプチドは骨格筋トロポニンＣの４４位から５２位にあたるＲＭＬＧＱＴＰＴＫ（９ｍｅｒ）であると認められた。これはこれまでに報告のない、新規なＡＣＥ阻害ペプチドであった。９ｍｅｒのＡＣＥ阻害活性は３４Ｍ（３５μｇ／ｍｌ）であった。
【００１８】
実施例２：９ｍｅｒの部分構造ペプチドの合成
９ｍｅｒの部分構造ペプチドであるＲＭＬＧＱＴＰＴ 、ＲＭＬＧＱＴＰ（７ｍｅｒ）、ＲＭＬＧＱＴ、ＲＭＬＧ、ＲＭＬ、ＲＭ、ＭＬ、ＬＧ、ＧＱ、ＱＴ、ＴＰ、ＰＴ、ＴＫ、ＰＴＫ及びＴＰＴＫをＦｍｏｃ固相合成法で合成した。これら合成ペプチドのＩＣ５０、分子量、及びＲＰ−ＨＰＬＣ（Ｃｏｓｍｏｓｉｌ ５Ｃ１８ ＡＲ−ＩＩ （４．５ ｘ １５０ｍｍ） （Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）で０．１％ ＴＦＡを含むアセトニトリルの１−３０％のグラジェント溶出（流速１ｍｌ／ｍｉｎ））の保持時間（ＲＴ）を表１に示す。また、９ｍｅｒのデータも表１に示す。
【００１９】
【表１】
実施例３：アンギオテンシン変換酵素との反応性
Ｆｕｊｉｔａら（Ｊｏｕｒｎａｌ ｏｆ Ｆｏｏｄ Ｓｃｉｅｎｃｅ， ６５： ５６４−５６９， ２０００）の方法を参考にして、ペプチド合成した９ｍｅｒおよび７ｍｅｒ（１ｍｇ／ｍｌ）をそれぞれ、ウシ肺由来ＡＣＥ（２０ ｍＵ／ｍｌ）と混合し、３７℃で２０時間まで反応させた。バッファー条件は、通常のＡＣＥ阻害活性測定と同様とした。反応終了後は、９５℃で２０分加熱してＡＣＥを失活させた。Ｃｏｓｍｏｓｉｌ ５Ｃ１８ ＡＲ−ＩＩ （４．５ ｘ １５０ｍｍ） （Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用いるＲＰ−ＨＰＬＣに供し、０．１％ ＴＦＡを含むアセトニトリルの１−３０％のグラジェント溶出（流速０．５ｍｌ／ｍｉｎ）で分画して分解程度を分析した。また、ＡＣＥによる分解産物の同定は、ＨＰＬＣ溶出時間を合成ペプチドと比較することで行った。ペプチド濃度はＨＰＬＣピーク面積で算出した。
【００２１】
その結果を図１に示す。９ｍｅｒは経時的にゆっくりとＡＣＥにより加水分解され、反応６０分後には６３％の基質が残存していることが示された。一方、７ｍｅｒはＡＣＥにより速やかに分解されており、６０分後には１９％の基質残存率であった。７ｍｅｒのＣ末端ジペプチドＴＰ、さらにその次のジペプチドＧＱは急速に遊離していることが認められた。
【００２２】
これらの結果から、９ｍｅｒではＣ末端ジペプチドＴＫが遊離しにくいために、その後のＴＰやＧＱの遊離速度が抑えられているものと考えられた。すなわち、７ｍｅｒはＡＣＥの非常に優れた基質である一方、９ｍｅｒはＡＣＥによる分解を比較的受けにくい基質であることが分かった。
【００２３】
実施例４：消化酵素耐性
経口で摂取したときの有効性を評価するために、消化酵素に対する耐性を検討した。
【００２４】
９ｍｅｒの合成品（１ ｍｇ／ｍｌ）を、それぞれａ−キモトリプシン、ペプシン、及びトリプシンと、基質：酵素比１００：１で反応させた。バッファーは１０ ｍＭ トリス−ＨＣｌ （ｐＨ７．５）を使用し、３７℃で６時間分解した。反応終了後は、９５℃、２０分間加熱して酵素を失活させた。Ｃｏｓｍｏｓｉｌ ５Ｃ１８ ＡＲ−ＩＩ （４．５ ｘ １５０ｍｍ） （Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用いるＲＰ−ＨＰＬＣに供し、０．１％ ＴＦＡを含む１４％アセトニトリルでイソクラティック溶出（流速０．５ｍｌ／ｍｉｎ）で分画して分解程度を分析した。ペプチド濃度はＨＰＬＣピーク面積で算出した。
【００２５】
その結果を表２に示す。３７℃、６時間の反応にも関わらず、いずれの酵素でも基質残存率が高く、ペプシンおよびキモトリプシンでは７５％、トリプシンでは８６％の基質が残存していた。
【００２６】
分解物全体のＡＣＥ阻害活性を測定したところ、未分解ペプチドの活性に近い３２−３８ｇ／ｍｌのＩＣ５０を示し、ＡＣＥ阻害活性に対する分解産物の寄与率が低いことが分かった（表２）。
【００２７】
【表２】
【発明の効果】
本発明により、豚肉タンパク質由来の新規なアンギオテンシン変換酵素（ＡＣＥ）阻害ペプチドを提供することができる。
【００２９】
９ｍｅｒは有原らが報告したミオシン由来ペプチド（Ｍｅａｔ Ｓｃｉｅｎｃｅ， ５７： ３１９−３２４，２００１）よりも強いＡＣＥ阻害活性を示し、７ｍｅｒは同等の活性だった。これまでに報告されている動物筋肉食品由来のＡＣＥ阻害ペプチドは、構造タンパク質であるミオシンやアクチンおよび水溶性タンパク質から見出されていた。本発明のペプチドは、調節タンパク質から見出された初めての例である。
【００３０】
ＡＣＥ阻害活性が比較的強い９ｍｅｒがＡＣＥによる加水分解を受けにくいということは、その活性を失いにくいことを意味しており、短期的に有効なＡＣＥ阻害剤としての利用が期待される。
【００３１】
９ｍｅｒのＡＣＥ阻害活性に対する分解産物の寄与率が低いことから、９ｍｅｒペプチドは消化酵素に対する耐性が比較的高く、経口投与した際に消化管中で消化されにくい可能性が示唆され、インヴィボでの有効性が期待される。
【００３２】
一般的にジペプチドもしくはトリペプチドなどの小さなペプチドのみが、腸管から吸収される（Ａｄｉｂｉ ｅｔ ａｌ． Ｃｌｉｎｉｃａｌ ｒｅｓｅａｒｃｈ， １７： ３７６， １９６９） とされているが、一方では、Ｐａｐｐｅｎｈｅｉｍｅｒ ｅｔ ａｌ． （Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙｏｆ Ｓｃｉｅｎｃｅｓ ｏｆ ｔｈｅ Ｕｎｉｔｅｄ Ｓｔａｔｅｓ ｏｆ Ａｍｅｒｉｃａ， ９１： １９４２−１９４５， １９９４） はオクタペプチドがｐａｒａｃｅｌｌｕｌａｒｌｙに腸管から吸収されることを示している。Ｓｈｉｍｉｚｕ ｅｔ ａｌ． （Ｐｅｐｔｉｄｅｓ， １８： ６８１−６８７， １９９７） は疎水性ノナペプチドが経細胞輸送により上皮細胞を通過することを明らかにしている。さらにＳａｔａｋｅ ｅｔ ａｌ． （Ｂｉｏｓｃｉｅｎｃｅ， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ， ６６： ３７８−３８４， ２００２）は消化酵素に対する耐性が高く腸管内に長時間滞留するペプチドの場合は、なんらかのトランスポート機能を利用して吸収される可能性を示唆している。これらのことから、本発明による消化耐性のある新規ＡＣＥ阻害ペプチド９ｍｅｒも腸管から吸収される可能性があると考えられる。すなわち、９ｍｅｒが腸管から吸収されて、ＡＣＥ阻害剤として有効に機能する可能性が期待される。
【図面の簡単な説明】
【図１】図１は、実施例３のＡＣＥとペプチド ９ｍｅｒ（ａ）及び７ｍｅｒ（ｂ）の反応における、反応時間とペプチド濃度の関係を表すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a peptide useful as an angiotensin converting enzyme inhibitor or the like.
[0002]
[Prior art]
Angiotensin converting enzyme (ACE) is present mainly in human vascular endothelial cells, lung, kidney and brain. This enzyme can convert inactive angiotensin I to active angiotensin II by cleaving two amino acids (His-Leu) from the C-terminus to cause vasoconstriction and increase blood pressure. Thus, ACE can raise blood pressure.
[0003]
Binding of an angiotensin converting enzyme inhibitor (ACEI) to ACE may reduce angiotensin II production. Adding ACEI to food will cause a positive effect in reducing hypertensive symptoms. At present, many peptides are known to have an effect on ACE inhibition. They have different amino acid sequences and lengths.
[0004]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide an angiotensin converting enzyme (ACE) inhibitory peptide, which is expected to have an effect of preventing hypertension, as a functional ingredient derived from pork.
[0005]
[Means for Solving the Problems]
The present invention has found a novel angiotensin converting enzyme (ACE) inhibitory peptide derived from pork.
[0006]
The present invention relates to the following active peptides of items 1 to 4, and an angiotensin converting enzyme inhibitor and a method for producing the same.
Item 1. A polypeptide consisting of at least one of the following two:
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys; and Arg-Met-Leu-Gly-Gln-Thr-Pro.
Item 2. Angiotensin converting enzyme inhibitors comprising at least one of the following seven:
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys;
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr;
Arg-Met-Leu-Gly-Gln-Thr-Pro;
Arg-Met-Leu-Gly-Gln-Thr;
Arg-Met-Leu;
Thr-Pro; and Thr-Lys.
Item 3. A method for producing an angiotensin converting enzyme inhibitor, which comprises hydrolyzing troponin with pepsin.
Item 4. Item 4. The method according to Item 3, wherein the angiotensin converting enzyme inhibitor is Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention relates to an angiotensin converting enzyme (ACE) inhibitory peptide, which is expected to have an effect of preventing hypertension as a functional ingredient derived from pork.
[0008]
In the present invention, when a partially purified troponin that can be extracted from a pork-derived protein is hydrolyzed with pepsin, a peptide having an ACE inhibitory activity, Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys (RMLGQTPTK, (Hereinafter referred to as 9mer). This peptide is from skeletal muscle troponin C (Lorkin PA & Lehmann H, FEBS Letter, 153: 81-87, 1983) at positions 44-52.
[0009]
Arg-Met-Leu-Gly-Gln-Thr-Pro (RMLGQTP, hereinafter referred to as 7mer), Arg-Met-Leu-Gly-Gln-Thr (RMLGQT), Arg-Met-Leu (9-mer partial structure peptide) RML), Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr (RMLGQTPT), Thr-Pro (TP) and Thr-Lys (TK) were also found to have ACE inhibitory activity.
[0010]
The peptide 9mer of the present invention can be obtained by hydrolyzing a crude troponin extracted from a pork-derived protein with pepsin. Further, the partial structural peptide of the peptide 9mer can be synthesized according to a known method such as Fmoc solid phase synthesis.
[0011]
The peptide 9mer of the present invention can be obtained by purifying a pepsin hydrolysis product of partially purified troponin using a combination of ion exchange chromatography, gel filtration chromatography, reverse phase HPLC, and the like.
[0012]
The ACE inhibitory peptide of the present invention has inhibitory activity on ACE derived from rabbit lung and bovine lung.
[0013]
The present invention will be described more specifically in the following examples.
[0014]
【Example】
The 50% inhibitory activity (IC 50 ) on ACE measured in the following examples was on ACE derived from bovine lung, and was measured by the method of Cushman-Cheng (Biochemical Pharmacology, 20: 1637-1648, 1971).
[0015]
Example 1
Crude troponin extracted from domestic pork loin was hydrolyzed with pepsin. The 50% inhibitory activity (IC 50 ) of this crude troponin hydrolyzate of pepsin against bovine lung-derived ACE was 225 g / ml. No ACE inhibitory activity was observed for unhydrolyzed troponin, indicating that the expression of the activity was derived from the peptide generated by pepsin degradation.
[0016]
The hydrolyzate was first subjected to anion exchange chromatography using a DE53 column (Whatman International Ltd., Kent, UK) (16 × 150 mm). That is, the hydrolyzate was applied with 20 mM tris (hydroxymethyl) aminomethane (adjusted to pH 7.5 with acetic acid), and a gradient (0-300 mM) elution was performed with an aqueous NaCl solution containing the same buffer. . The flow rate was 1.13 ml / min, and fractionation was performed every 7 minutes (7.91 ml). This fraction was desalted using a SEP-PAK Plus C18 cartridge (Waters Co., Milford, Mass., USA), and the fraction obtained by eluting the adsorbed fraction with 50% acetonitrile was concentrated. Its activity was measured. The active fraction obtained here was subjected to reverse phase (RP) -HPLC using Cosmosil 5C 18 AR-II (4.5 × 150 mm) (Nacalai Tesque Inc., Kyoto Japan) and 0.1% trifluoroacetic acid. Fractionation was performed by gradient elution of acetonitrile containing (TFA) with 1-80% (flow rate: 0.5 ml / min). The active fraction was further subjected to isocratic elution with the same column and 14% acetonitrile containing 0.1% TFA (flow rate: 0.5 ml / min). The active fraction obtained by RP-HPLC was subjected to gel filtration HPLC using TSK-gel G2000SW XL (7.8 × 300 mm) (Tosoh Co., Tokyo, Japan) to give a 20 mM Na phosphate buffer (pH 7.0), and was subjected to isocratic elution (flow rate: 0.5 ml / min). Finally, the active fraction was subjected to RP-HPLC using Cosmosil 5PE-MS (4.6 × 250 mm) (Nacalai Tesque), isocratic elution with 12% acetonitrile containing 0.1% TFA (flow rate 1 ml / min) )did. In each case, the absorbance at 215 nm was monitored.
[0017]
The amino acid sequence of the fraction obtained by the purification was subjected to sequence analysis using a protein sequencer (PPSQ-10, Shimadzu Co., Kyoto, Japan). The obtained peptide was identified as RMLGQTPTK (9 mer) corresponding to positions 44 to 52 of skeletal muscle troponin C. This was a novel ACE inhibitory peptide that had not been reported before. The ACE inhibitory activity of the 9mer was 34 M (35 μg / ml).
[0018]
Example 2 : Synthesis of 9mer partial structure peptide RMLGQTPT, RMLGQTP (7mer), RMLGQT, RMLG, RML, RM, ML, LG, GQ, QT, TP, PT, TK, PTK and TPTK which are 9mer partial structure peptides Was synthesized by the Fmoc solid phase synthesis method. IC 50 , molecular weight of these synthetic peptides and 1-30% gradient elution of acetonitrile containing 0.1% TFA by RP-HPLC (Cosmosil 5C 18 AR-II (4.5 × 150 mm) (Nacalai Tesque)) The retention time (RT) at a flow rate of 1 ml / min) is shown in Table 1. Table 1 also shows 9-mer data.
[0019]
[Table 1]
Example 3 Reactivity with Angiotensin-Converting Enzyme Referring to the method of Fujita et al. (Journal of Food Science, 65: 564-569, 2000), 9-mer and 7-mer (1 mg / ml) synthesized with a peptide were collected from bovine lung, respectively. It was mixed with derived ACE (20 mU / ml) and reacted at 37 ° C for up to 20 hours. The buffer conditions were the same as in the usual measurement of ACE inhibitory activity. After completion of the reaction, ACE was deactivated by heating at 95 ° C for 20 minutes. Cosmosil 5C 18 AR-II (4.5 x 150mm) (Nacalai Tesque) was subjected to RP-HPLC using a 1-30% of gradient elution acetonitrile containing 0.1% TFA (flow rate 0.5 ml / min) And the degree of decomposition was analyzed. The identification of the degradation product by ACE was performed by comparing the HPLC elution time with that of the synthetic peptide. The peptide concentration was calculated from the HPLC peak area.
[0021]
The result is shown in FIG. The 9mer was slowly hydrolyzed by ACE over time, indicating that 63% of the substrate remained after 60 minutes of reaction. On the other hand, 7mer was rapidly decomposed by ACE, and after 60 minutes, the residual ratio of the substrate was 19%. It was found that the 7-mer C-terminal dipeptide TP and the next dipeptide GQ were rapidly released.
[0022]
From these results, it was considered that the C-terminal dipeptide TK was hardly released with the 9-mer, and the subsequent release rate of TP and GQ was suppressed. That is, it was found that 7mer is a very excellent substrate of ACE, while 9mer is a substrate which is relatively hard to be degraded by ACE.
[0023]
Example 4 Digestive Enzyme Resistance To evaluate the efficacy when taken orally, the resistance to digestive enzymes was examined.
[0024]
A 9-mer synthetic product (1 mg / ml) was reacted with a-chymotrypsin, pepsin, and trypsin, respectively, at a substrate: enzyme ratio of 100: 1. As a buffer, 10 mM Tris-HCl (pH 7.5) was used and digested at 37 ° C. for 6 hours. After completion of the reaction, the enzyme was inactivated by heating at 95 ° C. for 20 minutes. Cosmosil 5C 18 subjected to AR-II (4.5 x 150mm) RP-HPLC using a (Nacalai Tesque), minute isocratic elution at 14% acetonitrile containing 0.1% TFA (flow rate 0.5 ml / min) And analyzed the degree of degradation. The peptide concentration was calculated from the HPLC peak area.
[0025]
Table 2 shows the results. Despite the reaction at 37 ° C. for 6 hours, the residual ratio of the substrate was high for all enzymes, and 75% of the substrate remained for pepsin and chymotrypsin and 86% for trypsin.
[0026]
When the ACE inhibitory activity of the whole degraded product was measured, it showed an IC 50 of 32-38 g / ml close to the activity of the undegraded peptide, indicating that the contribution of the degraded product to the ACE inhibitory activity was low (Table 2).
[0027]
[Table 2]
【The invention's effect】
According to the present invention, a novel angiotensin converting enzyme (ACE) inhibitory peptide derived from pork protein can be provided.
[0029]
9mer showed stronger ACE inhibitory activity than the myosin-derived peptide (Meat Science, 57: 319-324, 2001) reported by Arihara et al., And 7mer showed equivalent activity. The ACE inhibitory peptides derived from animal muscle foods which have been reported so far have been found from structural proteins such as myosin and actin and water-soluble proteins. The peptides of the present invention are the first examples found from regulatory proteins.
[0030]
The fact that a 9mer having a relatively strong ACE inhibitory activity is less susceptible to hydrolysis by ACE means that it is less likely to lose its activity, and is expected to be used as an effective ACE inhibitor in a short term.
[0031]
The low contribution of the degradation products to the 9-mer ACE inhibitory activity suggests that the 9-mer peptide has relatively high resistance to digestive enzymes and may not be easily digested in the gastrointestinal tract when administered orally. Sex is expected.
[0032]
Generally, only small peptides such as dipeptides or tripeptides are absorbed from the intestinal tract (Adibi et al. Clinical research, 17: 376, 1969), while Pappenheimer et al. (Proceedings of the National Academyof Sciences of the United States of America, 91: 1942-1945, 1994) shows that octapeptide is absorbed from the intestinal tract into paracellularly. Shimizu et al. (Peptides, 18: 681-687, 1997) show that hydrophobic nonapeptides pass through epithelial cells by transcellular transport. Further, Satake et al. (Bioscience, Biotechnology, and Biochemistry, 66: 378-384, 2002) suggest that peptides having high resistance to digestive enzymes and staying in the intestinal tract for a long time may be absorbed using some transport function. are doing. From these facts, it is considered that the novel ACE inhibitory peptide 9mer which is resistant to digestion according to the present invention may also be absorbed from the intestinal tract. That is, it is expected that 9-mer will be absorbed from the intestinal tract and function effectively as an ACE inhibitor.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the reaction time and the peptide concentration in the reaction of ACE of Example 3 with 9-mer (a) and 7-mer (b) peptides.
Claims (4)
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓ；及び
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ。A polypeptide consisting of at least one of the following two:
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys; and Arg-Met-Leu-Gly-Gln-Thr-Pro.
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ−Ｌｙｓ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ｔｈｒ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ｔｈｒ；
Ａｒｇ−Ｍｅｔ−Ｌｅｕ；
Ｔｈｒ−Ｐｒｏ；及び
Ｔｈｒ−Ｌｙｓ。Angiotensin converting enzyme inhibitors comprising at least one of the following seven:
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys;
Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr;
Arg-Met-Leu-Gly-Gln-Thr-Pro;
Arg-Met-Leu-Gly-Gln-Thr;
Arg-Met-Leu;
Thr-Pro; and Thr-Lys.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007230978A (en) * | 2006-03-03 | 2007-09-13 | Hokkaido Univ | Composition containing pork-original peptide having antifeeding activity |
| JP2016128435A (en) * | 2009-03-31 | 2016-07-14 | エボニック デグサ ゲーエムベーハーＥｖｏｎｉｋ Ｄｅｇｕｓｓａ ＧｍｂＨ | Dipeptides as feed additives |
-
2002
- 2002-08-05 JP JP2002226829A patent/JP4106539B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007230978A (en) * | 2006-03-03 | 2007-09-13 | Hokkaido Univ | Composition containing pork-original peptide having antifeeding activity |
| JP2016128435A (en) * | 2009-03-31 | 2016-07-14 | エボニック デグサ ゲーエムベーハーＥｖｏｎｉｋ Ｄｅｇｕｓｓａ ＧｍｂＨ | Dipeptides as feed additives |
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