JP5443637B1 - コラーゲン合成促進剤 - Google Patents
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Abstract
【解決手段】シークヮーサー種子油を含有することを特徴とする皮膚外用剤。
【選択図】図1
Description
シークヮーサー種子油の調製:
シークヮーサーの果実から、キャタピラ式搾汁機を用いてパルプ、内皮等を除去し、種子を分離した後、40℃で48時間乾燥し、水分が3%以下となるように調整した。乾燥した種子を直圧式圧搾機KT100−200(サン精機社製)を用いて、コールドプレス製法(25℃、75MPa、15分)により圧搾した。得られた圧搾油を一夜静置し、上澄みを分取して活性炭濾過し、シークヮーサー種子油を得た。
コラーゲン合成促進試験:
製造例1で得られたシークヮーサー種子油を試験試料とし、線維芽細胞に対するI型コラーゲン合成促進作用を評価した。正常ヒト線維芽細胞を、0.5%仔牛血清含有ダルベッコ変法MEM(0.5%FBS-DMEM)を用いて2.0×104cells/wellの細胞密度にて96穴プレートに播種した。播種24時間後に試験可能な最高濃度から希釈した試験試料を含有する0.5%FBS-DMEMと交換した。なお、試験試料未処理細胞をコントロールとした。また陽性コントロールとして25μMアスコルビン酸リン酸マグネシウム塩(VC-PMg)を用いた。試験試料含有培地で24時間培養したのち、培養上清を回収してELISAに供した。細胞は0.1%Triton X-100溶液にて溶解したのち、BCA法にてタンパク量を定量した。より詳細には、培地および検量線用I型コラーゲン溶液を高吸着型ELISAプレートに入れ、4℃にて一昼夜コーティングしたのち、1%牛血清アルブミン(BSA)溶液を用いて37℃にて1時間ブロッキングした。一次抗体反応は、Anti-Human Collagen Type I antibody(Rabbit)を0.3%BSA溶液で希釈したものを添加し、37℃にて1時間反応させた。二次抗体反応は、ヒストファインMAX-PO(R)(Rabbit)をリン酸緩衝液で希釈したものを添加し、37℃にて1時間反応させた。次に、0.3mg/mLの2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammouniumsalt(ABTS)および0.03%の過酸化水素を含むリン酸-クエン酸緩衝液(0.1M,pH4.0)を添加して20分間反応させ、マイクロプレートリーダーにて405nmの吸光度を測定した。培地中のI型コラーゲン量は、市販のI型コラーゲンにて作成した検量線から算出した。全細胞のタンパク量で培地中のI型コラーゲン量を除することによって単位タンパク量あたりのI型コラーゲン産生量を算出した。それぞれのI型コラーゲン産生量はStudent t検定を用いて有意差検定を行い、コントロールとの差を評価した。結果を表1および図1に示す。
安全性試験:
実施例1のシークヮーサー種子油を試験試料とし、EpiSkin法による皮膚一次刺激性代替法を用い、皮膚一次刺激性を評価した。
試験は、OECD Guidelines for the Testing of Chemicals Test No. 439 およびEuropean Center for the Validation of Alternative Methods (ECVAM)にて公開されているプロトコールである-ECVAM Skin Irritation Validation Study- VALIDATION OF THE EpiSkinTMTEST METHOD15 min- 42 hours FOR THE PREDICTION OF ACUTE SKIN IRRITATION OF CHEMICALS.に準じて実施した。皮膚一次刺激性評価は、MTT法による皮膚モデルの細胞生存率 (MTT viability (%))を指標とした。EpiSkin-SMTM(0.38 cm2)を、維持培地にて24時間インキュベーションし、試験試料10μLを、表皮側から曝露した。なお陰性コントロールとしてPBS(+)、刺激性コントロールとして5%SDS(Sigma)を用いた。試験試料を15分間曝露した後、速やかに試験試料を洗浄操作により皮膚モデルから除去し、新鮮な維持培地にてさらに42時間培養した。培養後、皮膚モデルを0.3mg/mLの3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT、Sigma)を含有する試験培地(フェノールレッド未含有)に移し、3時間培養した。その後、トランスウェルより皮膚モデルをパンチで外し、さらに酸性イソプロパノールに72時間浸漬することによりブルーホルマザンを抽出し、抽出液の550nmにおける吸光度をマイクロプレートリーダー(Molecular devices, V max)にて測定した。MTT viability(%)は、陰性コントロール曝露皮膚モデルの吸光度を100としたときの百分率で表した。得られた結果を基にして、試験成立の判断基準および試験試料の皮膚一次刺激性(R38、Risk Phrase 38)判定基準を表2および3に示す。また試験試料のMTT試験による細胞生存率変化の結果および皮膚刺激性の判定結果を表4および5に示す。
下記組成の乳液を常法により製造した。
実施例1のシークヮーサー種子油 0.01g
ホホバオイル 4.0g
オリーブオイル 2.0g
スクワラン 2.0g
セタノール 2.0g
モノステアリン酸グリセリル 2.0g
ポリオキシエチレンセチルエーテル(20E.0) 2.5g
オレイン酸ポリオキシエチレンソルビタン(20E.0) 2.0g
グリチルレチン酸ステアリル 0.1g
1,3−ブチレングリコール 3.0g
ヒノキチオール 0.15g
香料0.05g
精製水 残量(全量100g)
下記組成の化粧水を常法により製造した。
実施例1のシークヮーサー種子油 2g
グリセリン 3g
1,3−ブチレングリコール 3g
オレイン酸ポリオキシエチレンソルビタン(20E.0) 0.5g
パラオキシ安息香酸メチル 0.15g
クエン酸 0.1g
クエン酸ソーダ 0.1g
香料 0.05g
精製水 残量(全量100g)
下記組成のクリームを常法により製造した。
実施例1のシークヮーサー種子油 0.05g
アロエエキス 0.1g
流動パラフィン 5.0g
サラシミツロウ 4.0g
スクワラン 10.0g
セタノール 3.0g
ラノリン 2.0g
ステアリン酸 1.0g
オレイン酸ポリオキシエチレンソルビタン(20E.0) 1.5g
モノステアリン酸グリセリル 3.0g
油溶性甘草エキス0.1g
1,3−ブチレングリコール6.0g
パラオキシ安息香酸メチル1.5g
香料0.1g
精製水 残量(全量100g)
下記組成のパックを常法により製造した。
実施例1のシークヮーサー種子油0.05g
ポリビニルアルコール15g
ポリエチレングリコール3g
プロピレングリコール7g
エタノール10g
パラオキシ安息香酸メチル0.05g
グリチルリチン酸ジカリウム0.1g
香料0.05g
精製水 残量(全量100g)
Claims (2)
- シークヮーサー種子の圧搾油を有効成分として含有することを特徴とするコラーゲン合成促進剤。
- コラーゲンがI型コラーゲンである請求項1記載のコラーゲン合成促進剤。
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