JP2020094047A - 皮膚常在細菌叢改善又は多様化用剤及びそれを含有する組成物 - Google Patents
皮膚常在細菌叢改善又は多様化用剤及びそれを含有する組成物 Download PDFInfo
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Landscapes
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Abstract
Description
〔1〕 ラクトフェリン又はその塩を有効成分とする皮膚常在細菌叢の改善又は多様化用剤;
〔2〕 前記〔1〕記載の皮膚常在細菌叢の改善又は多様化用剤を含む、組成物;
〔3〕 前記〔1〕記載の皮膚常在細菌叢の改善又は多様化用剤を含む、医薬組成物;
〔4〕 前記〔1〕記載の皮膚常在細菌叢の改善又は多様化用剤を含む、化粧品組成物;
〔5〕 ラクトフェリン又はその塩を0.001〜50%(W/W)の量で含有する皮膚外用剤である、前記〔3〕記載の医薬組成物;
〔6〕 ラクトフェリン又はその塩を0.00001〜50%(W/W)の量で含有する、前記〔4〕記載の化粧品組成物;
〔7〕 ラクトフェリン又はその塩を0.00001〜10%(W/W)の量で含有する入浴剤である、前記〔6〕記載の化粧品組成物
が、提供される。
結合剤としては、たとえば、澱粉、デキストリン、アラビアガム末、ゼラチン、ヒドロキシプロピルスターチ、カルボキシメチルセルロース・ナトリウム塩、メチルセルロース、結晶性セルロース、エチルセルロース、ポリビニルピロリドンが挙げられる。
液体培養法及び固体培養法(改変カップ法)を用いてラクトフェリンの黄色ブドウ球菌(Staphylococcus aureus)、表皮ブドウ球菌(Staphylococcus epidermidis)、アクネ菌キューティバクテリウム・アクネス(Cutibacterium acnes)に対する抗菌活性を調べた。
Staphylococcus aureus IFO12732(以下、SAと表記)及びStaphylococcus epidermidis NBRC 100911(以下、SEと表記)はミュラーヒントン培地(BD社)、Cutibacterium acnes NBRC 10765(以下、CAと表記)はGAM培地(ニッスイ)の希釈培地(1/2、1/5、1/10)を用いて37℃で静置培養(液体培養、固体培養)を行い、20時間後の生育状態を観察した。なお、CAは嫌気性菌であるため、嫌気ジャーとアネロパック(型番A−110、三菱ガス化学社製)を用い嫌気培養を行った。
SEは、1/10 MH培地を用い、37℃、120rpm、22時間振盪培養後、倍々希釈(4段階)して濁度(OD660)を測定した。それらを適宜希釈後、各3枚ずつ1/10 MH寒天培地に撒き、37℃で44時間静置培養後、コロニー数をカウントして検量線を作成した。
液体培養法により、主な皮膚常在菌に対するラクトフェリンの最小発育阻止濃度(MIC)を測定した。
ラクトフェリン(Tatura社製又は森永乳業製など、乳由来の精製ラクトフェリン粉末、他の例においても使用;以下、LFと表記)及びネガティブコントロールとして用いたバクトペプトン(BD社製;以下、BPと表記)は1/10培地で終濃度100mg/mlに、ポジティブコントロールとして用いたクロロテトラサイクリン塩酸塩(以下、CTCと表記)は1/10培地で終濃度10μg/mlに調製後、それぞれフィルター滅菌して使用した。
SA及びSEは、1/10 MH培地を用い37℃、120rpmで20時間振盪培養した菌液のOD660を測定し、先に求めた検量線より終濃度 1×104 cfu/mlとなるよう調製した。また、CAは1/10 GAM寒天培地を用い37℃、20時間静置嫌気培養後、シャーレよりかき取って1/10 GAM培地に懸濁した菌液のOD660を測定し、先に求めた検量線より終濃度 1×104 cfu/mlとなるよう調製した。
検証1の結果、LFの抗菌活性が想定以上に強く出たため、LFのみ定法で再アッセイを行った。すなわち、通常濃度の培地を用い、接種菌量を終濃度1×104cfu/mlに戻し、培養開始より22時間後(SA、SE)及び48時間後(SA、SE、CA)に生育を観察した。
LFは、各1/10培地で終濃度100mg/mlに調製後、フィルター滅菌し、倍々希釈(5段階)した。
表5に示す処方のラクトフェリン含有皮膚外用剤(クリーム剤)を製造した。
ラクトフェリン含有化粧水を、表6に示す処方で製造した。
ラクトフェリン含有乳液を、表7に示す処方で製造した。
ラクトフェリン含有美容液を、表8に示す成分で製造した。
ラクトフェリン含有ジェルを、表9に示す成分で製造した。
皮膚常在菌叢及び角質状態に対するラクトフェリン配合クリーム剤の有用性の検証を目的としてオープン試験を実施した。実施に当たって、株式会社NRLファーマの開催する倫理委員会にて承認を得て行った。
上記のように採取した皮膚常在菌採取用テープからDNAの抽出を行った。抽出から、菌叢の解析は、MySkin(登録商標)を採用し、TAK−Circulator社に依頼して実施した。なお、DNAの抽出は、森田らの方法(MicrobesEnviron. Vol22, No.3, 214-222, 2007)によって行うこともできる。
式 h’=−門相対発現量/全門発現総量×log2門相対発現量/全門発現総量
H’=h’の総和
角質チェッカーを染色液に浸漬させ、10分程度染色を行った。染色液は、ゲンチアナバイオレット(細胞質染色用)を1.0(w/v)%、ブリリアントグリーン(核染色用)を0.5(w/v)%となるように蒸留水に溶解した。角質チェッカーを10分間程度水に浸し、余分な染色液を洗い流した。この際に角質細胞が剥離しないように気を付けて洗浄した。
このことから、本発明の一態様のラクトフェリン含有皮膚外用剤の使用により、皮膚常在菌叢が多様化され、皮膚の状態を改善することが分かった。
被験者に対し、ビジュアルアナログスケール(VAS)(0〜10、0はすごく改善、10はすごく悪化、5は変化なし)を用いて、製造例1の皮膚外用剤の使用感(かさつき、くすみ、赤み、しみ・そばかす、にきび、吹き出物、額・眉間のしわ、目尻のしわ、目元のたるみ、口もとのたるみ、滑らかさ、はり、つや、きめ、化粧のり、化粧もち、肌の状態総合評価)についてアンケート調査し、本発明の一態様のラクトフェリン含有皮膚外用剤による改善度を求めた。
ラクトフェリン含有入浴剤を、表12に示す処方で製造した。
(使用時、150〜300Lの温水に対し、本入浴剤10〜60gを溶解する。)
Claims (7)
- ラクトフェリン又はその塩を有効成分とする皮膚常在細菌叢の改善又は多様化用剤。
- 請求項1記載の皮膚常在細菌叢の改善又は多様化用剤を含む、組成物。
- 請求項1記載の皮膚常在細菌叢の改善又は多様化用剤を含む、医薬組成物。
- 請求項1記載の皮膚常在細菌叢の改善又は多様化用剤を含む、化粧品組成物。
- ラクトフェリン又はその塩を0.001〜50%(W/W)の量で含有する皮膚外用剤である、請求項3記載の医薬組成物。
- ラクトフェリン又はその塩を0.00001〜50%(W/W)の量で含有する、請求項4記載の化粧品組成物。
- ラクトフェリン又はその塩を0.00001〜10%(W/W)の量で含有する入浴剤である、請求項6記載の化粧品組成物。
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