JP2020510005A - 物質pを含むしわの改善または抗炎症化粧料組成物 - Google Patents
物質pを含むしわの改善または抗炎症化粧料組成物 Download PDFInfo
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- A61K2800/48—Thickener, Thickening system
Abstract
Description
本発明者らは物質Pを含む皮膚のしわまたは炎症改善用組成物の製造の前に、抗酸化剤及び界面活性剤の含量に応じた物質Pの安定性の評価実験を介して、前記化粧料組成物の適用に適合な抗酸化剤及び界面活性剤の最適含量を確認した。
物質Pに抗酸化剤としてチオ硫酸ナトリウム、界面活性剤としてポリソルベート80及び粘増剤としてヒドロキシエチルセルロースを追加した新規な剤形を製造した。物質Pは、ペプチド合成技術であるFmocケミストリー(Fmoc-chemistry)を用いた固相/液相の段階(Solid/ Solution phase)を介して合成し、高速液体クロマトグラフィーを介して精製して、最終的に純度85%以上のものを用いた。
物質Pを含有した組成物の安定性を皮膚繊維芽細胞の培養培地で確認した。前記表1のように製造した物質Pを含有した組成物(物質P、5μg/ml)または比較例としてリン酸緩衝溶液(PBS)に溶解した物質P(物質P、5μg/ml)を皮膚繊維芽細胞の培養培地(FGM、Lonza、USA)に0、3、6、12及び24時間まで適用した後、時間の経過に応じて培地内に残っている物質Pの含量を測定した。物質Pの含量は、Substance P ELISA Kit(Abcam、USA)を用いて、ELISA法で定量した。このとき、対照群としては0時間を適用したサンプルを用い、対照群を100%にして、時間の経過に応じて培地内に残っている物質Pの含量をパーセント(%)で計算した。
物質Pを含有した組成物のコラーゲン合成効果を皮膚繊維芽細胞で確認した。96個の穴がある平板培養プレート(96-well plate)に3×104 CFU/ウェルの人間の皮膚繊維芽細胞を入れて、37℃で24時間培養した。培地を(FGM、Lonza、USA)除去した後、新しい培地180μlを各ウェルに分注し、物質Pを含有した組成物またはリン酸緩衝溶液(PBS)に溶解した物質Pを20μlずつ入れた後、37℃で24時間培養した。このとき、添加された組成物及び物質Pの濃度は、全体の培地量を考慮して、最終濃度が所望の濃度になるように計算して添加した。24時間培養した後、上澄み液を集めてProcollagen Type1−Peptide(P1P)EIA Kit(TaKaRa、Japan)を用い、培地のコラーゲン量を測定した。
物質Pを含む組成物のコラーゲン分解酵素(Collagenase、MMP−1)の抑制効果を皮膚繊維芽細胞で確認した。96個の穴がある平板培養プレート(96-well plate)に3×104 CFU/ウェルの人間の皮膚繊維芽細胞を入れて、37℃で24時間培養した。培地を(FGM、Lonza、USA)除去した後、新しい培地180μlを各ウェルに分注し、物質Pを含有した組成物及びリン酸緩衝溶液(PBS)に溶解した物質Pを20μlずつ入れた後、37℃で24時間培養した。このとき、添加された組成物または物質Pの濃度は、全体培地量を考慮して、最終濃度が所望の濃度になるように計算して添加した。24時間培養した後、上澄み液を集めてHuman total MMP−1 Kit(R&D systems、USA)を用い、培地にあるコラーゲン分解酵素(MMP−1)の量を測定した。
物質Pを含有した組成物の炎症性サイトカインIL−6の減少効果をマウスのマクロファージであるRaw 264.7細胞で確認した。24個の穴がある平板培養プレート(24-well plate)に4×105 CFU/ウェルの人間の皮膚繊維芽細胞を入れて、37℃で24時間培養した。Raw 264.7細胞の成長用培地は、DMEM(Welgene、Korea)に200mMのL−グルタミン(Gibco、USA)を混ぜた培地を用いた。24時間培養した後、培地を除去した後に新しい培地1mlを各ウェルに分注した。細胞内の炎症反応の誘導のためにLPS(Sigma、USA)を100ng/ml処理すると同時に、物質Pを含有した組成物またはリン酸緩衝溶液(PBS)に溶解した物質Pを分注して、全体培地量が2ml/ウェルになるようにした後、37℃で24時間追加培養した。このとき、添加された組成物及び物質Pの濃度は、全体培地量を考慮して、最終濃度が所望の濃度になるように計算して添加した。24時間培養した後、上澄み液を集めてMouse IL−6 Immunoassay(R&D systems、USA)を用い、培地の炎症性サイトカインIL−6の量を測定した。
物質Pを含む組成物の炎症環境内の一酸化窒素(NO)の減少効果をマウスのマクロファージであるRaw 264.7細胞で確認した。24個の穴がある平板培養プレート(24-well plate)に4×105 CFU/ウェルの人間の皮膚繊維芽細胞を入れて、37℃で24時間培養した。Raw 264.7細胞の成長用培地は、DMEM(Welgene、Korea)に200mMのL−グルタミン(Gibco、USA)を混ぜた培地を用いた。24時間培養した後、培地を除去した後に新しい培地1mlを各ウェルに分注した。細胞内の炎症反応の誘導のためにLPS(Sigma、USA)を100ng/ml処理すると同時に、物質Pを含有した組成物またはリン酸緩衝溶液(PBS)に溶解した物質Pを分注して、全体培地量が2ml/ウェルになるようにした後、37℃で24時間追加培養した。このとき、添加された組成物及び物質Pの濃度は、全体培地量を考慮して、最終濃度が所望の濃度になるように計算して添加した。
Claims (11)
- 配列番号1のアミノ酸配列からなる物質P(substance P)、抗酸化剤、界面活性剤及び粘増剤を含む皮膚のしわまたは炎症改善用化粧料組成物。
- 前記物質Pの濃度が、1〜10μg/mlである、請求項1に記載の化粧料組成物。
- 前記物質Pの濃度が、5〜10μg/mlである、請求項1に記載の化粧料組成物。
- 前記抗酸化剤が、チオ硫酸ナトリウム(sodium thiosulfate)である、請求項1に記載の化粧料組成物。
- 前記抗酸化剤の含量が、全体組成物の総重量に対して0.01〜1重量%である、請求項1に記載の化粧料組成物。
- 前記界面活性剤が、ポリソルベート80(polysorbate 80)である、請求項1に記載の化粧料組成物。
- 前記界面活性剤の含量が、組成物の総重量に対して0.001〜0.1重量%である、請求項1に記載の化粧料組成物。
- 前記粘増剤が、ヒドロキシエチルセルロース(hydroxyethylcellulose)である、請求項1に記載の化粧料組成物。
- 前記粘増剤の含量が、組成物の総重量に対して1〜5重量%である、請求項1に記載の化粧料組成物。
- 請求項1〜9のいずれか一項に記載の化粧料組成物を個体の皮膚に塗布する段階を含む、皮膚のしわまたは炎症改善方法。
- 皮膚のしわまたは炎症改善のための化粧品を生産するための、請求項1〜9のいずれか一項に記載の化粧料組成物の用途。
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EP3574892A1 (en) | 2019-12-04 |
US20210137809A1 (en) | 2021-05-13 |
WO2018230751A1 (ko) | 2018-12-20 |
SG11201907845SA (en) | 2019-09-27 |
EP3574892A4 (en) | 2020-08-12 |
JP6901801B2 (ja) | 2021-07-14 |
CN111182887A (zh) | 2020-05-19 |
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