JP7433337B2 - 血小板由来成長因子(pdgf)-bb産生亢進剤、及びそれを含む幹細胞安定化剤、並びにそれらを含む皮膚抗老化剤 - Google Patents
血小板由来成長因子(pdgf)-bb産生亢進剤、及びそれを含む幹細胞安定化剤、並びにそれらを含む皮膚抗老化剤 Download PDFInfo
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Description
[1]ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物の少なくともいずれかを有効成分として含んでなる血小板由来成長因子-BB(PDGF-BB)産生亢進剤。
[2][1]に記載のPDGF-BB産生亢進剤を含んでなる、幹細胞安定化剤。
[3][1]に記載のPDGF-BB産生亢進剤を含んでなる皮膚抗老化剤であって、皮膚における幹細胞を安定化することにより皮膚の老化を抑制する、皮膚抗老化剤。
ワサビ(Eutrema属)は、アブラナ科ワサビ属の多年草である。本発明に用いられるワサビの抽出物としては、本ワサビ(Eutrema japonicum (Miq.) Koidz.)が好ましいが、ユリワサビ(Eutrema tenue)や近縁種(Eutrema yunnanense)といった他の種を用いてもよい。また、ワサビの葉の抽出物が好ましいが、種、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ワサビの抽出物は、金印株式会社等から市販されており、このような市販品を用いることもできる。
カミツレ(学名:Matricaria chamomilla)は、キク科シカギク属に属する一年草である。本発明に用いられるカミツレの抽出物としては、カミツレの頭花の抽出物が好ましいが、カミツレの種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。カミツレの抽出物は、丸善製薬株式会社等から市販されており、このような市販品を用いることもできる。
ハイビスカス(Hibiscus属)は、アオイ目アオイ科フヨウ属に属する一年生または多年生の亜灌木である。本発明に用いられるハイビスカスの抽出物としては、ローゼル(Hibiscus sabdariffa)の抽出物が好ましい。ハイビスカスの花の抽出物が好ましいが、ハイビスカスの果実、種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ハイビスカスの抽出物は、日研フード株式会社等から市販されており、このような市販品を用いることもできる。
PDGF-BBの産生亢進作用の評価対象試料として以下を用いた。
1. 測定前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。
2. サンプル希釈液(10mL⇒50mL)とアッセイ希釈液(6mL⇒30mL)は、測定前に脱イオン水または蒸留水で5倍に希釈した。細胞ライセート緩衝液は、脱イオン水または蒸留水で2倍に希釈した。
3. 試料の希釈:HUVEC(ヒト臍帯静脈内皮細胞)と評価対象試料との反応液を1×サンプル希釈液で少なくとも5倍に希釈して測定試料を調製した(PDGF-BBのレベルは、サンプルによって異なるので、各サンプルの最適希釈係数は適宜決定した)。
4. 標準の調製:凍結乾燥した標準バイアルに280μLの1×サンプル希釈液を加えて50ng/mLの標準液を調製した。穏やかに混合して粉末を完全に溶解させた。400pg/mLのストック標準溶液を調製するために、496μLのサンプル希釈液を含むチューブに、再構成標準のバイアルから4μLのPDGF-BB標準液を加えた。各チューブに400μLの1×サンプル希釈液をピペットで注入した。下記のように原液標準液を使用して希釈系列を作成した。次の移送の前に各チューブを十分に混合した。1×サンプル希釈液をゼロ標準液(0pg/mL)とした。
6. 使用前に、100μLの1×アッセイ希釈液をバイアルに加えて、ビオチン化抗体濃縮物を調製した。ピペットで上下に軽く混ぜた。ビオチン化抗体濃縮物は、1×アッセイ希釈液で80倍に希釈し(180μL⇒14400μL)、以下に記載のアッセイ手順のステップ4で使用した。
7. ストレプトアビジン-HRP試薬は、1×アッセイ希釈液で800倍に希釈した。HRP-ストレプトアビジン濃縮液20μLを16mLの1×アッセイ希釈液の入ったチューブに加え、800倍希釈HRP-ストレプトアビジン溶液を調製した。
1. 使用前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。すべての標準とサンプルは、少なくとも2連で実行した。
2. 各標準液100μLを添加し(試薬調製手順のステップ3を参照)、適切なウェルにサンプルを分注した。ウェルを覆い、穏やかに振盪しながら室温で2.5時間インキュベートした。
3. 溶液を捨て、1×洗浄緩衝液で4回洗浄した。マルチチャンネルのピペットを使用して各ウェルに洗浄緩衝液(300μL)を満たして洗浄した。最後の洗浄の後、吸引またはデカントすることによって残りの洗浄緩衝液をすべて除去した。プレートを逆さまにし、きれいな紙タオルで水分を十分に除去した。
4. 100μLの1×調製ビオチン化抗体(試薬調製手順のステップ6を参照)を各ウェルに加えた。穏やかに振とうしながら室温で1時間インキュベートした。
5. 溶液を捨て、ステップ3の洗浄を再び行った。
6. 調製したストレプトアビジン-HRP溶液(試薬調製手順のステップ7を参照)100μLを各ウェルに加えた。穏やかに振とうしながら室温で45分間インキュベートした。
7. 溶液を捨て、ステップ3の洗浄を再び行った。
8. 各ウェルに100μLのTMB基質を加えた。穏やかに振とうしながら暗所・室温で30分間インキュベートした。
9. 50μLの停止液を各ウェルに加えた。
10. プレートは反応を停止してから30分以内に評価した。450nmおよび550nmに設定したELISAプレートリーダーで吸光度を測定した。550nm値を450nm値から差し引いて、マイクロプレートの光学的不完全性を補正した。
感度または検出の下限(LLD)は、ゼロおよび測定毎の標準曲線によって決定した。標準曲線から読み取ったゼロ+2標準偏差の値がLLDである(95%信頼度でゼロでない最小線量)。
陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量に対する割合を、図1及び下記表3に示す。図1、表3の結果から、52種類のサンプルのうち、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物にとりわけ高い効果がみられることが判明し、これらの成分はPDGF-BBの産生を亢進させる作用を有することが分かる。図中、試料A~Cは、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物以外の47種類の物質のうち3種の試料を示すが、他の物質も試料A~Cと同様に陰性対照(DMSO)より低いか又は有意な差が見られない等、基準を満たすものではなかった。
実験2において有意なPDGF-BB産生亢進作用が見られたワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物について、上記評価手順に従ってPDGF-BBタンパク量を測定し、PDGF-BB産生亢進作用を有することが知られている15μg/mLリンゴンベリー抽出物および2μg/mLアムラ抽出物と比較した。
対照(15μg/mLリンゴンベリー抽出物)について得られたタンパク量を100.0としてそれに対する割合を図2に示し、陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量を100.0としてそれに対する割合を図3に示す。図2,3より、陰性対照のみならず、従前に高いPDGF-BB産生亢進作用を有することが知られていたリンゴンベリーエキスやアムラエキス(特許文献2)と比較しても有意に高いPDGF-BB産生亢進能を奏することが分かった。
Claims (1)
- ハイビスカス抽出物を有効成分として含んでなる血小板由来成長因子-BB(PDGF-BB)産生亢進剤。
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CN109762782A (zh) | 2019-02-27 | 2019-05-17 | 嘉文丽(福建)化妆品有限公司 | 一种促间充质干细胞生长的培养基及其制备方法 |
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