JP2022540460A - グラフェン量子ドットを有効成分として含む化粧料組成物 - Google Patents
グラフェン量子ドットを有効成分として含む化粧料組成物 Download PDFInfo
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Abstract
Description
製造例1:グラフェン量子ドットの製造
本実験に使用されたグラフェン量子ドット(Graphene Quantum Dots、GQD)は、2018年、Nature Nanotechnologyに発表された論文[Nat.Nanotech.3,812-818(2018)]を参照して製造した。硫酸と窒酸とが3:1の体積比率で混合された溶液にカーボンファイバー(Carbon fiber)を入れた後、80℃で24時間撹拌しながら加熱する。以後、酸除去と真空濾過により精製し、水分を除去して、パウダー状のグラフェン量子ドットを得る。このように製造したグラフェン量子ドットは20ナノメートル以下の多様な大きさを有することができ、本実験に使用されたグラフェン量子ドットは約3ナノメートル水準である。
実施例1のグラフェン量子ドットの細胞毒性評価のために、ヒトの線維芽細胞細胞株(Human dermal fibroblast、Hs68)における細胞数の変化を測定した。Epigallocatechin gallate(EGCG)を処理した細胞を陽性対照群として使用した。
細胞内で発生する活性酸素種(ROS)と反応して蛍光を呈するDCF-DAを用いて、ROSの生成量を確認することにより抗酸化力を測定した。線維芽細胞株Hs68を6well plateに3.5×105の数だけ分注した後、37℃、5%CO2条件の培養器で24時間培養した。以後、培地を除去し、DPBSを入れた後、Controlを除いた残りの細胞群にH2O2を添加した後、24時間追加培養した。以後、光を遮断した状態でDCF-DA(10μM)を入れて、37℃で1時間染色した。その後、培地を除去した後にDPBSで2回washingした後、wellごとに1mlのDPBSを入れて、cell scrapperで掻いてEP tubeに入れた。このように集めたcell溶液を200ulずつ96well black plateに3回繰り返し入れた後、Ex/Em=485/535nmとしてVictor3でplate readingした。下記数式1のように無処理群(None)に対する%を計算して、細胞内ROS(intracellular ROS)を測定した結果を図2に示した。
[数1]
細胞内ROS(%)=
{(UVおよび試料処理群OD-UV無処理群OD)/UV無処理群OD}×100
(ここで、OD(Optical Density)は吸光度単位を意味する。)
生きている細胞内のOHラジカルに選択的に結合して蛍光を呈するOH580 probeを添加して、グラフェン量子ドット(GQD)のOHラジカル消去能を評価した。線維芽細胞株HS68を96well plateに2×104ずつ分注した後、37℃、5%CO2条件の培養器で24時間培養した。以後、plateを800rpmで2分間遠心分離した後、培地を除去し、OH580 stain working solution(abcam、Massachusetts、USA)をwellごとに100ulずつ処理した後、ホイルに包んで37℃、5%CO2で1時間反応させる。以後、DPBSにH2O2(100uM)をはじめとするグラフェン量子ドットをそれぞれ10、100、1000ng/mlに合わせて処理後、2時間37℃、5%CO2で反応させる。以後、Solutionを除去し、DPBSで2~3回washingした後、100uLのAssay Bufferを各wellに入れて、蛍光顕微鏡で594nmの波長で観察する。
グラフェン量子ドット(GQD)の細胞再生促進効果を評価するために、実験例1、2と同一の細胞を用いて実験を進行させた。Ascorbic acid処理細胞を陽性対照群として使用した。
紫外線によって増加するコラゲナーゼ(MMP-1)に対するグラフェン量子ドット(GQD)の阻害能を比較するために、ヒト線維芽細胞株Hs68を用いてMMP-1の発現量測定を進行させた。線維芽細胞株Hs68を6well plateに3×105の数だけ分注した後、37℃、5%CO2条件の培養器で24時間培養した。以後、培地を除去し、DPBSを入れた後、UVB非照射群を除いた残りの細胞群に12mJ/cm2のUVBを照射した後、GQDを濃度別に添加して24時間追加的に培養した。その後、各サンプルを処理した細胞からトリゾール(RNA iso、DAKARA、日本)を用いてRNAを分離した後、nanodropを用いて260nmでRNAを定量した後、それぞれ2μgのRNAを用いて増幅器でcDNAを合成した(C1000 Thermal Cycler、Bio-Rad、米国)。合成されたcDNAにターゲットタンパク質であるMMP-1 primerとシアニン染料であるサイバーグリーン(SYBR Green supermis、Applied Biosystems、米国)を添加した混合物を用いて、real-time PCR機械でリアルタイム重合酵素連鎖反応を実施することにより、最終的にMMP-1遺伝子の発現程度を評価した。Primerの配列と反応条件は下記表1の通りであり、遺伝子の発現量はβ-actin遺伝子に対する補正により最終的に分析した。
前記図2と図3の結果から示された抗酸化効果がヒト皮膚においてシワに重要な役割を果たすコラーゲンの生成を増加させるかを、次の方法で測定した。ヒト線維芽細胞を6well細胞培養皿に4×105の密度で接種した後、37℃、5%CO2培養器で24時間培養した。UVBを12mJ/cm2照射した後、GQDを濃度別に処理し、24時間追加培養した。その後、各サンプルの細胞からトリゾール(RNA iso、DAKARA、日本)を用いてRNAを分離した後、nanodropを用いて260nmでRNAを定量し、それぞれ2μgのRNAを用いて増幅器でcDNAを合成した(C1000 Thermal Cycler、Bio-Rad、米国)。合成されたcDNAにターゲットタンパク質であるprocollagen primerとシアニン染料であるサイバーグリーンを添加した混合物を用いてreal-time PCR機械でリアルタイム重合酵素連鎖反応を実施することにより、最終的にtype1 procollagen遺伝子の発現程度を評価した。遺伝子の発現量はβ-actin遺伝子に対する補正により最終的に分析した。
グラフェン量子ドット(GQD)の保湿効果を確認するために、ヒト線維芽細胞で発生するヒアルロン酸合成酵素の発現程度をHyaluronic acid synthase-3(HAS-3)とAquaporin-3(AQP-3)遺伝子発現の測定により評価した。
製造例2:グラフェン量子ドットを含む化粧料組成物の製造
(1)トナー剤の製造
グラフェン量子ドットを含む化粧料組成物を、下記表2に示した成分および含有量によって、通常の方法によりトナー剤に製造した。
グラフェン量子ドットを含む化粧料組成物を、下記表3に示した成分および含有量によって、通常の方法によりローション剤に製造した。
グラフェン量子ドットを含む化粧料組成物を、下記表4に示した成分および含有量によって、通常の方法によりクリーム剤に製造した。
Claims (7)
- グラフェン量子ドットを有効成分として含む、皮膚再生、シワ改善、保湿または抗酸化用化粧料組成物。
- 前記グラフェン量子ドットは、化粧料組成物の総重量対比、1.0×10-7~30重量%含まれることを特徴とする、請求項1に記載の化粧料組成物。
- 前記グラフェン量子ドットは、
i)活性酸素消去、ii)コラゲナーゼ(MMP-1)の発現抑剤、iii)タイプ1プロコラーゲン(type1 procollagen)の合成促進、iv)フィブリリン(fibrillin)の発現増加、v)ヒアルロン酸合成酵素-3(HAS-3)の発現増加、またはvi)アクアポリン-3(AQP-3)の発現増加からなる群より選択される1つ以上の効果を提供することを特徴とする、請求項1に記載の化粧料組成物。 - 前記化粧料組成物は、粉体(powder)、脂肪物質、有機溶媒、溶解剤、濃縮剤、ゲル化剤、軟化剤、抗酸化剤、懸濁化剤、安定化剤、発泡剤(foaming agent)、芳香剤、界面活性剤、水、イオン型または非イオン型乳化剤、充填剤、金属イオン封鎖剤、キレート化剤、保存剤、ビタミン、遮断剤、湿潤化剤、必須オイル、染料、顔料、香料、親水性または親油性活性剤からなる群より選択される1種以上をさらに含むことを特徴とする、請求項1に記載の化粧料組成物。
- 前記化粧料組成物は、トナー剤、ローション剤、およびクリーム剤からなる群より選択される1つ以上の剤形であることを特徴とする、請求項1に記載の化粧料組成物。
- グラフェン量子ドットを有効成分として含む、皮膚再生、シワ改善、保湿または抗酸化用医薬部外品組成物。
- グラフェン量子ドットを有効成分として含む、皮膚再生、シワ改善、保湿または抗酸化用薬学組成物。
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