JP2012528147A - ヒノキ多糖体を含有する皮膚外用剤組成物 - Google Patents
ヒノキ多糖体を含有する皮膚外用剤組成物 Download PDFInfo
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- JP2012528147A JP2012528147A JP2012512973A JP2012512973A JP2012528147A JP 2012528147 A JP2012528147 A JP 2012528147A JP 2012512973 A JP2012512973 A JP 2012512973A JP 2012512973 A JP2012512973 A JP 2012512973A JP 2012528147 A JP2012528147 A JP 2012528147A
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Abstract
【選択図】図1
Description
乾燥したヒノキ葉1kgにエタノール10Lを添加し、常温で24時間撹拌した後、遠心分離を通じて溶媒を除去した。製造されたヒノキ葉の残渣を30℃で7時間熱水抽出し、得られた熱水抽出液は、フィルタープレス機器を利用して濾過(珪藻土濾過)及び回収した後、初期体積の1/10となるように50℃で減圧濃縮した。次いで、上記濃縮液を限外濾過(分子量CUT OFF:1,000ダルトン)し、低分子量の遊離タンパク質を除去し、最終限外濾過濃縮液の5倍体積のエタノールを100mL/minの速度で徐々に加えて、エタノール沈殿反応を進行した。沈殿されたヒノキ多糖体を45℃で真空乾燥し、パウダー形態のヒノキ多糖体(53g)を得た。
ヒト正常線維芽細胞を105個の濃度で、12孔平板培養器で培養した後、製造例1で得たヒノキ多糖体を1ppm、10ppm、50ppmの濃度で含む培地に交替した。陽性対照群としてTGF−β(Human Transforming Growth Factor-β1, Roche Co.)を使用した。培養3日目、細胞を収穫し、ELISA方法で生成されたI型プロコラーゲン(type I procollagen)の量を定量した。上記ヒノキ多糖体を含まない対照群のプロコラーゲンの量を0、陽性対照群を100に設定し、各測定値との比較値を算出した。その結果は、下記表1に示した。
ヒト正常線維芽細胞を105個の濃度で、12孔平板培養器で培養し、紫外線Bを40mJ/cm2で照射した後、製造例1で得たヒノキ多糖体を1ppm、10ppm、または50ppmで含む培地に交替した。培養2日目、細胞を収穫し、ELISA方法で生成されたMMP−1(matrix metalloproteinase I)の量を定量した。陽性対照群としては、レチノイン酸(Retinoic acid;RA、Sigma.米国)を使用した。試験物質を含まず、紫外線を照射しない対照群を100にして測定した値との比較値を算出し、その結果を下記表2に示した。
30〜50才の顔面にしわがある試験対象者40名に対して下記表3に記載した組成のヒノキ多糖体を含有する栄養クリーム(実施例1)及びヒノキ多糖体を含有しない栄養クリーム(比較例1)を利用して皮膚しわ改善効果を比較評価した。
上記実施例1及び比較例1の皮膚弾力改善効果を測定した。30〜50才の女性40名を対象にして2個グループに分けて、各グループに上記実施例1及び比較例1の栄養クリームを毎日2回ずつ12週間顔面に塗布した後、皮膚弾力測定器(Cutometer SEM 575, C+Kエレクトロニック社, ドイツ)を利用して皮膚弾力を測定した。
本発明のヒノキ多糖体のメラニン生成抑制効果を調べるために、ねずみの色素細胞を利用した。先に、C57BL/6マウス来由のねずみ色素細胞(Mel−Ab cell)(Dooley, T. P. et al, Skin pharmacol, 7, pp188-200)を、DMEM(Dulbeccos modified Eagles media)に、10%牛胎盤血清、100nMの2−O−テトラデカノイルホルボール(tetradecanoylphorbol)−13−アセテート、及び1nMのコレラ毒素を添加した培地で、37℃、5%CO2の条件で培養した。培養したMel−Ab細胞を0.25%トリプシン−EDTAで取り外し、24−孔プレートに105細胞/孔(cells/well)の濃度で細胞を培養した後、二日間から3日連続で10ppm、50ppm、または100ppmのヒノキ多糖体を加えて培養した。この際、アルブチン(Sigma, 米国)を陽性対照群として使用した。次に、培養液を除去し、PBSで洗浄した後、1N水酸化ナトリウムで細胞を溶かし、400nmで吸光度を測定した。測定した吸光度を利用して下記数式2によってメラニン生成抑制率(%)を計算し、その結果を下記表4に示した(Dooleyの方法)。
本発明のヒノキ多糖体の人体皮膚に対する美白効果を調べるために、下記のような実験を行った。まず、元気な12名の男性を対象にして被検者の上膊部位に直径1.5cmの孔が穿設された不透明テープを付着した後、各被検者の最小紅斑量(Minimal Erythema Dose)の1.5〜2倍程度の紫外線(UVB)を照射し、皮膚の黒化を誘導した。
角質形成細胞及びヒト皮膚細胞株(HaCaT)の分化時に生成されるCE(Cornified Envelop)の量を測定し、本発明によるヒノキ多糖体の細胞分化促進効果を調べた。
ヒト角質形成細胞を96孔細胞培養プレートの各孔に5x104個を入れ、24時間付着させた。付着させた皮膚細胞株に試験物質を処理した後、2日が経過してから培地を除去し、−20℃冷蔵庫に保管した。凍結−解凍(Freeze-thawing)を2回繰り返して、物質処理した細胞を破壊させた後、−20℃に保管したアセトン:エタノール(1:1、v/v)を処理し、4℃で30分間放置し、細胞を固定させた。その後、室温に放置し、有機溶媒が蒸発されるようにし、ブロッキング(1%牛血清アルブミン)、トランスグルタミナーゼ抗体(primary)、HRPアンチ−マウス抗体(secondary)を処理し、発色は、OPD(σ−phenyldiamine)を添加して行った。発現量は、490nmで吸光度を測定し、補正は、630nmでバックグラウンド(background)を測定して行った。低カルシウム(0.03mM)処理群と高カルシウム(1.2mM)処理群をそれぞれ陰性/陽性対照群とし、低カルシウム濃度に試験物質を添加して実施した結果を100とし、測定した値との比較値を計算し、下記表7に示した。
皮膚乾燥症を示す50〜60代の大人男女50名を2個グループに分けて、各グループに実施例1及び比較例1を毎日2回ずつ4週間顔面に塗布した。塗布開始前と、塗布後1週、2週、4週が経過した時点及び塗布を中止してから2週経過(全体で6週経過)後に、恒温、恒湿条件(24℃、相対湿度40%)でコルネオメーターで皮膚水分量を測定し、試験結果は、塗布開始前に測定したコルネオメーター値を基準にして一定期間処理後の測定値の増加分を百分率で表示した。その結果は、下記表8に示した。
ヒト線維芽細胞を105個の濃度で12孔平板培養器に培養した後、LPS処理をした後、ヒノキ多糖体を1ppm、10ppm及び100ppmを含む培地に交替した。対照群は、ヒノキ多糖体を処理せず、培養した未処理群である。培養2日目、細胞を収穫し、ウェスタンブロット(Westren blot)方法を使用して生成されたシクロオキシゲナーゼ−2(cyclooxygenase−2、COX−2)の量を定量した。対照群を100にして濃度計(densitometer)で測定した値との比較値を下記表9に示した。
テトラデカノイルホルボールアセテート(tetradecanoyl phorbol acetate、TPA)誘発性マウス耳炎症モデルは、炎症反応の作用機作と抑制物質の効能を試すために広く使用されている実験方法である(De Young LM et al., Agents and Actions, 26;335-341(1989))。
レチノイン酸(retinoic acid)の場合、皮膚角質層の分化促進、にきび治療、しわ改善効果などの様々な優れた皮膚効能によって化粧品と医薬品に広く利用されている(Fisher et al., J. Investig. Dermatol., 3:61-68(1998))。しかし、レチノイン酸を皮膚に局所的に適用する場合、初期に刺激感を誘発し、ひいては、皮膚紅斑とむくみを誘発する副作用があるものと報告されている(Varani et al., Arch. Dermatol. Res., 295:255-262(2003))。これより、レチノイン酸のこのような皮膚刺激を低減するための試みが多方面に進行されている(Kim et al., Toxicol.Letters., 146:65-73(2003))。一方、ニュージーランド白色兎の場合、様々な皮膚刺激物質に対する反応性に優れていて、個体差異が少なく、皮膚刺激実験に広く使用されている。したがって、本試験例では、レチノイン酸でニュージーランド白色兎に皮膚刺激を誘発させた後、試験物質による皮膚刺激減少程度を評価した。
ヒト角質形成細胞であるHaCaT細胞は、韓国細胞部銀行(Korean Cell Line Bank; Seoul, Korea)から分譲されて使用した。HaCaT細胞を10%(v/v)FBS、ペニシリン100U/mL及びストレプトマイシン100μg/mLを含むDMEM培地に注入し、37℃、5%CO2供給条件を備えた動物細胞培養器で培養した。孔(Well)当たり1.5×106濃度で用意したHaCaT細胞を24時間培養し、細胞単一層を形成させた後、HaCaT細胞単一層をp200ピペットチップで「スクラッチ損傷」を誘導した。「スクラッチ損傷」された細胞層を製造例1で得たヒノキ多糖体10ppm濃度で処理した培養培地で24時間培養し、陰性対照群は、上記ヒノキ多糖体をとかすのに使用したDMSO(Dimethyl sulfoxide)で同量処理し、スクラッチ傷が回復する程度をマイクロイメージビデオカメラ(Boyertown、PA)が装着された顕微鏡で確認した。その結果を図1に示した。
[剤形例1]柔軟化粧水(スキンローション)
Claims (10)
- ヒノキ多糖体を有効成分として含有する皮膚外用剤組成物。
- 上記ヒノキ多糖体が組成物全体重量に対して0.001〜10重量%で含有されることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が抗老化用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚しわ改善用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚弾力改善用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚美白用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚保湿用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚乾燥疾患の予防及び改善用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が抗炎症用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
- 上記組成物が皮膚再生用であることを特徴とする請求項1に記載の皮膚外用剤組成物。
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |