JP2018537455A - 低分子rnaオニユリ抽出物を含む局所組成物および加齢に関する皮膚の徴候を低減するための美容ケア方法 - Google Patents
低分子rnaオニユリ抽出物を含む局所組成物および加齢に関する皮膚の徴候を低減するための美容ケア方法 Download PDFInfo
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Abstract
Description
低分子量(最大長150ヌクレオチド)の低分子RNAが濃縮された水性抽出物を、ユリ科(Liliaceae)のオニユリ球根(リリウムティグリヌム)から得る。
− 日焼け止め、紫外線および赤外線スクリーン、
− 抗フリーラジカル成分、
− DHEA(デヒドロエピアンドロステロン)、
− デヒドロ酢酸(DHA)、
− 天然または合成フィトステロール、
− αおよびβヒドロキシ酸、シラノール、
− 糖アミン、グルコサミン、D−グルコサミン、N−アセチルグルコサミン、N−アセチル−D−グルコサミン、マンノサミン、N−アセチルマンノサミン、ガラクトサミン、N−アセチルガラクトサミン、
− ポリフェノール、イソフラボン、フラボノイド、例えばブドウ抽出物、マツ抽出物、オリーブ抽出物、
− 脂質、例えばセラミドまたはリン脂質、
− 動物油、例えばスクアレンまたはスクアラン
− 植物油、例えばアーモンド油、ココナツ油、ヒマシ油、ホホバ油、オリーブ油、ナタネ油、落花生油、ヒマワリ油、コムギ胚芽油、トウモロコシ胚芽油、ダイズ油、綿実油、アルファルファ油、ケシ油、カボチャ種子油、月見草オイル、キビ油、オオムギ油、ライムギ油、ベニバナ油、パッションオイル、ヘーゼルナッツ油、パーム油、杏仁油、アボカド油、カレンデュラオイル、エトキシル化植物油、またはシアバター、
を含む。
<環境ストレス抵抗性(細胞生存率)に及ぼす低分子RNAオニユリ(リリウムティグリヌム)抽出物の評価>
本試験の目的は、環境ストレス後の細胞生存率に及ぼすオニユリ(リリウムティグリヌム)抽出物の効果を示すことである。環境ストレスは、汚染微粒子状物質(PM(<4μm);NIST2786)の適用によって誘発する。細胞生存率を、乳酸デヒドロゲナーゼ(LDH)活性の用量によって測定する。LDHは、ピルビン酸塩の乳酸塩への変換を触媒するオキシドレダクターゼ酵素である。その活性は、組織および細胞における病変および毒性の存在に関連している。
<UVストレス後のDNA損傷に及ぼす低分子RNAオニユリ(リリウムティグリヌム)抽出物の評価>
本試験の目的は、UVストレス後のDNA損傷に及ぼすオニユリ(リリウムティグリヌム)抽出物の好ましい効果を示すことである。DNA損傷を、個々の細胞における一本鎖および二本鎖DNA切断の検出を可能にする微小電気泳動技術である「単細胞ゲル電気泳動」(SCGE)としても知られる「コメットアッセイ」を使用して定量する。
<細胞外基質評価による加齢に関する、および老化に関する低分子RNAオニユリ(リリウムティグリヌム)抽出物の評価>
本試験の目的は、トロポエラスチン発現に関する細胞外基質(ECM)の評価に基づく、加齢に及ぼすオニユリ(リリウムティグリヌム)抽出物の効果、およびβ−ガラクトシダーゼ活性老化マーカーを使用することによる、老化に及ぼすオニユリ(リリウムティグリヌム)抽出物の効果を初めて示すことである。
トロポエラスチンの評価:
正常なヒト線維芽細胞をP15まで複製老化によって老化させた。
正常なヒト線維芽細胞をP15まで複製老化によって老化させた。
トロポエラスチンの評価(図3):
0.2%オニユリ(リリウムティグリヌム)溶液による48時間の処置は、老化していない(非老化)(P6)の線維芽細胞および老化(P15)の線維芽細胞においてトロポエラスチン発現の非常に有意な増加(Student’s t−検定)を示した。
予想されたように、β−ガラクトシダーゼ活性は、若い細胞(P8)と比較して複製老化線維芽細胞(P15)において増加した。0.2%オニユリ(リリウムティグリヌム)抽出物溶液による48時間の処置は、誘発された老化を有意に低減させた(Student’s t−検定)。
<光老化損傷に対する皮膚の維持に関する低分子RNAオニユリ(リリウムティグリヌム)抽出物の評価>
本試験の目的は、UVストレスによって誘発された光加齢損傷に対してオニユリ(リリウムティグリヌム)抽出物が皮膚に及ぼす好ましい効果を示すことである。ECM構造に関係するフィブリリンおよびトロポエラスチンを評価する。
抗フィブリリン抗体による免疫標識のために、組織を凍結した。凍結した皮膚生検を切断して、切片を冷アセトン中で固定した。特異的抗フィブリリン抗体(Abcam,ref.ab3090、マウスモノクローナル)を適用した後、蛍光色素に結合させた適切な二次抗体を適用した。特定の媒体中で標本を作製した後、スライドガラスを落射蛍光顕微鏡(Zeiss Axiovert 200M顕微鏡)によって観察した。
抗トロポエラスチン抗体による免疫標識に関して、組織を固定してパラフィンに包埋した。包埋した皮膚生検を切断し、切片のパラフィンを除去して再度水和した。次に、抗原賦活化プロトコールを実施した後、特異的抗トロポエラスチン抗体(Abcam,ref.ab3090、ウサギポリクローナル)を適用し、次いで蛍光色素に結合させた適切な二次抗体を適用した。特定の媒体中で標本を作製した後、スライドガラスを落射蛍光顕微鏡(Zeiss Axiovert 200M顕微鏡)によって観察した。
フィブリリンの評価(図5):
UVストレス後、図5に記載のようにフィブリリン線維の崩壊が観察された。ストレスと平行して実施した0.5%オニユリ(リリウムティグリヌム)抽出物溶液による処置は、線維の組織化に及ぼすUVの影響を可視的に低減させた。
UVストレス後、図6に記載のようにトロポエラスチン線維の崩壊が観察された。ストレスと平行して実施した0.2%および0.5%のオニユリ(リリウムティグリヌム)抽出物溶液による処置は、線維の組織化に及ぼすUVの影響を可視的に低減させた。
<低分子RNAリリウムティグリヌム抽出物によって処置した線維芽細胞におけるエラスチン量の評価>
プロトコール:プロトコールのスキームを図7に示す。19歳のドナーから得たP10および62歳のドナーから得たP8の正常なヒト皮膚線維芽細胞(NHDF)を、35mmガラス底培養皿に細胞11,000個/皿の濃度で播種して、24時間インキュベートした。オニユリ(リリウムティグリヌム)の20%保存溶液を、オニユリ(リリウムティグリヌム)1.3mLを培地(10%ウシ胎仔血清および1%ペニシリン/ストレプトマイシンを添加したDMEM)5.2mLで希釈した後、0.2μmのPVDFフィルターを通して濾過することによって調製した。オニユリ(リリウムティグリヌム)処置溶液は、保存溶液を培地で0.2%および2%となるように希釈することによって調製した。細胞をオニユリ(リリウムティグリヌム)によって24時間処置した。細胞をPBSで1回洗浄し、PBSの薄層(1mL)で覆った。Grobel照射チャンバー内で細胞に10J/cm2のUVA+40mJ/cm2のUVBを照射した。照射後、細胞を、オニユリ(リリウムティグリヌム)によって24時間処置した。
<低分子RNAリリウムティグリヌム抽出物によって処置した線維芽細胞におけるDNA断片化の評価>
プロトコール:
19歳ドナーから得たP10および62歳のドナーから得たP8のNHDFを、60mm培養皿に細胞80,000個/皿の濃度で播種して、24時間インキュベートした。細胞をPBSで1回洗浄し、PBSの薄層(2mL)で覆った。Grobel照射チャンバー内で細胞に10J/cm2のUVA+40mJ/cm2のUVBを照射した。照射後、細胞を、実施例5に記載のように調製した0.2%または2%オニユリ(リリウムティグリヌム)によって6時間処置した。
Claims (8)
- オニユリ(リリウムティグリヌム(Lilium tigrinum))の球根の抽出物、および生理的に許容される媒体を含む、局所適用のための化粧品組成物であって、前記オニユリの抽出物が、10〜12g/kgの乾燥重量、4〜8g/kgの糖濃度、0.5〜1.5g/kgのタンパク質断片濃度、50〜200mg/kgのフェノール化合物濃度、および15〜45mg/kg濃度の最大長150ヌクレオチドのRNAの含有量を有する、化粧品組成物。
- 前記オニユリ(リリウムティグリヌム)の球根の抽出物がDNAを全く含まない、請求項1に記載の化粧品組成物。
- 前記オニユリ(リリウムティグリヌム)の球根の抽出物が、本発明の前記組成物中に、前記組成物の総重量を基準として、0.1〜5%の間、好ましくは0.2〜2.5%の間の濃度で存在する、請求項1に記載の化粧品組成物。
- 皮膚における加齢および光加齢の徴候を低減するための、請求項1〜3のいずれか1項に記載の組成物の局所適用を含む美容ケアの方法。
- 細胞生存率を改善するための、請求項1〜3のいずれか1項に記載の組成物の局所適用を含む美容ケアの方法。
- 汚染に対する細胞保護および微粒子状物質に対する細胞保護を改善するための、請求項1〜3のいずれか1項に記載の組成物の局所適用を含む美容ケアの方法。
- UVにより誘発されるDNA損傷に対する細胞保護を改善するための、請求項1〜3のいずれか1項に記載の組成物の局所適用を含む美容ケアの方法。
- 細胞の老化を低減するための、請求項1〜3のいずれか1項に記載の組成物の局所適用を含む美容ケアの方法。
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HK1256880A1 (zh) | 2019-10-04 |
EP3377623B1 (fr) | 2021-10-20 |
WO2017087245A1 (en) | 2017-05-26 |
CN108291222A (zh) | 2018-07-17 |
CN108473980A (zh) | 2018-08-31 |
CA3005605A1 (en) | 2017-05-26 |
FR3043554B1 (fr) | 2019-07-12 |
CN108473980B (zh) | 2022-10-21 |
KR102268324B1 (ko) | 2021-06-24 |
JP2018533615A (ja) | 2018-11-15 |
US20180371000A1 (en) | 2018-12-27 |
KR20180080319A (ko) | 2018-07-11 |
EP3377624B1 (en) | 2021-07-21 |
FR3043695A1 (fr) | 2017-05-19 |
EP3377624A1 (en) | 2018-09-26 |
CN108291222B (zh) | 2022-10-21 |
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