JP3822959B2 - Anti-aging skin external preparation - Google Patents
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Description
【０００１】
【発明の属する技術分野】
この発明は、真皮線維芽細胞の代謝活性を活性化し、さらに紫外線による線維芽細胞の損傷を防止することにより、加齢や紫外線などの種々のストレスによるシワ，シミの発生，皮膚の弾性の低下といった皮膚老化症状の防止或いは改善に有効で、抗炎症作用，創傷治癒促進作用をも有する老化防止用皮膚外用剤に関する。
【０００２】
【従来の技術】
加齢や紫外線等外来ストレスにより生じるしわ，シミの発生、皮膚弾性の低下といった皮膚の老化症状には、皮膚真皮の線維芽細胞の機能低下やマトリックス線維の減少又は分解が重要な要因となっている。従って、皮膚の老化防止，改善作用を有する老化防止用皮膚外用剤を得るため、線維芽細胞の賦活或いは増殖促進作用を有する成分の検索と配合が試みられている。
【０００３】
例えば、ビワ抽出物（特公平５−１７２０６号公報），α−ヒドロキシ酢酸（特開平５−１１２４２２号公報），α−ヒドロキシ酸のステロールエステル（特開平８−１０４６３２号公報），6-ベンジルアミノプリン（特開平７−２３３０３７号公報），特定のリボヌクレアーゼ（特開平７−３０９７７８号公報），L-リシル-L-グリシル-L-ヒスチジン（特開平７−３１６１９２号公報），乳汁由来線維芽細胞増殖因子（特開平８−１１９８６７号公報），酸化型コエンザイムＡ（特開平８−１７５９６１号公報）等が開示されている。
【０００４】
しかしながら、上記の真皮線維芽細胞賦活効果を有する成分等の中には、作用効果が不十分であったり、安定性が悪かったりして、皮膚外用剤基剤中に含有させた場合、有効な効果を得るにはかなりの量を含有させなければならないものも存在していた。また、好ましくない副作用や刺激性などを有していたり、製剤安定性に悪影響を及ぼすものや、臭いや色の点で外用剤に配合しにくいもの、一定の作用，品質を維持することの困難なものも多かった。
【０００５】
【発明が解決しようとする課題】
そこで本発明においては、真皮線維芽細胞の代謝活性を向上させる細胞賦活作用に優れる新規成分を探求し、それを皮膚外用剤に含有させることにより、紫外線などの外来ストレスにより生じる皮膚の傷害や老化を、有効に防止或いは改善する作用に優れる老化防止用皮膚外用剤を得ることを目的とした。
【０００６】
【課題を解決するための手段】
上記の課題を解決するため、本発明者らは真皮線維芽細胞の代謝活性促進効果を指標として、有効な活性化作用を有する物質のスクリーニングを行った。その結果、珪藻の抽出物が、高い真皮線維芽細胞の代謝促進効果、及び紫外線による線維芽細胞の傷害を防止する効果を有することを見いだした。この珪藻抽出物においては、皮膚刺激性，接触感作性といった皮膚への悪影響もなく、また老化防止用皮膚外用剤に配合したときも、真皮線維芽細胞賦活効果の不活化は起こらずに、品質も安定していた。
【０００７】
珪藻は、淡水や海水，土壌中に見られる黄褐色の藻類で、植物プランクトンの一種である。珪藻については、珪藻を海水中で培養した培養上清を乾燥させて得られる海水塩（フランス国特許２４１７９７９）、培養した珪藻に含まれているエイコサペンタエン酸（特開昭５９−４６２２５、特開平５−５８０５７２６）、珪藻などの海藻の親水溶媒抽出物のラジカル消去活性（フランス国特許２６５７０１２）、珪藻から単離したポリ-β-N-アセチルグルコサミン類（国際公開９５１５３４３，米国特許５６２３０６４）等が開示されている。今回本発明者は、珪藻の溶媒抽出物において、顕著な線維芽細胞賦活作用及び、紫外線による真皮線維芽細胞への傷害を防止する効果を新たに発見し、本発明の完成に至った。
【０００８】
【発明の実施の形態】
本発明の実施の形態を説明する。
【０００９】
本発明で用いられる珪藻は、特に種類は限定されないが、線維芽細胞賦活作用、及び紫外線による細胞傷害防御能から、キートセロス(Chaetoceraceae)科の珪藻特にキートセロス グラシリス(Chaetoceros gracilis)及び、パブローバ属（Pavlova）の珪藻の抽出物が特に好ましい。
【００１０】
これらの海藻の抽出物を得る抽出溶媒としては、水、エタノール，メタノール，イソプロパノール，イソブタノール，n-ヘキサノール，メチルアミルアルコール，2-エチルブタノール，n-オクチルアルコールなどのアルコール類、グリセリン，エチレングリコール，エチレングリコールモノメチルエーテル，エチレングリコールモノエチルエーテル，プロピレングリコール，プロピレングリコールモノメチルエーテル，プロピレングリコールモノエチルエーテル，トリエチレングリコール，1,3-ブチレングリコール，ヘキシレングリコール等の多価アルコール又はその誘導体、アセトン，メチルエチルケトン，メチルイソブチルケトン，メチル-n-プロピルケトンなどのケトン類、酢酸エチル，酢酸イソプロピルなどのエステル類、エチルエーテル，イソプロピルエーテル，n-ブチルエーテル等のエーテル類などの極性溶媒から選択される１種又は２種以上の混合溶媒が好適に使用できる。また、リン酸緩衝生理食塩水等の無機塩類を添加した極性溶媒、界面活性剤を添加した極性溶媒を用いることもでき、特に限定はされない。上記の抽出溶媒の中でも、エタノール，メタノール，1,3-ブチレングリコール，水から選択される１種の溶媒又は２種以上の混合溶媒、及びこれらの溶媒に無機塩，界面活性剤を添加した溶媒が好ましく用いられる。
【００１１】
さらに、抽出方法としては、室温，冷却又は加温した状態で含浸させて抽出する方法、水蒸気蒸留等の蒸留法を用いて抽出する方法、生の珪藻から圧搾して抽出物を得る圧搾法等が例示され、これらの方法を単独で又は２種以上を組み合わせて抽出を行う。
【００１２】
抽出の際の珪藻と溶媒との比率は特に限定されるものではないが、珪藻１に対して溶媒０．５〜１０００重量倍、特に抽出操作、効率の点で０．５〜１００重量倍が好ましい。また、抽出温度は、常圧下で室温から溶剤の沸点以下の範囲とするのが便利であり、抽出時間は抽出温度などによって異なるが、２時間〜２週間の範囲とするのが好ましい。
【００１３】
また、このようにして得られた珪藻抽出物は、抽出物をそのまま用いることもできるが、真皮線維芽細胞賦活作用、及び紫外線による細胞傷害防御作用を失わない範囲内で脱臭，脱色，濃縮等の精製操作を加えたり、さらにはカラムクロマトグラフィー等を用いて分画物として用いてもよい。これらの抽出物や精製物、分画物は、これらから溶媒を除去することによって乾燥物とすることもでき、さらにアルコールなどの溶媒に可溶化した形態、或いは乳剤の形態で老化防止用皮膚外用剤に添加することができる。
【００１４】
これらの珪藻抽出物の老化防止用皮膚外用剤への配合量は、その効果や添加した際の香り，色調の点から考え、０．００１〜２０重量％の濃度範囲とすることが望ましい。配合量が０．００１重量％未満であると、十分な真皮線維芽細胞賦活作用、及び紫外線による細胞傷害防御作用老化防止効果が得られないが、あまり多量に配合する必要もなく、２０重量％を超えると老化防止用皮膚外用剤の安定性等に影響を及ぼすこともある。
【００１５】
本発明においては、上記の珪藻抽出物を含有させた老化防止用皮膚外用剤を提供し得るが、老化防止用皮膚外用剤としては、ローション，乳剤，クリーム，軟膏等の形態をとることができる。またさらに、柔軟性化粧水，収れん性化粧水，洗浄用化粧水等の化粧水類、エモリエントクリーム，モイスチュアクリーム，マッサージクリーム，クレンジングクリーム，メイクアップクリーム等のクリーム類、エモリエント乳液，モイスチュア乳液，ナリシング乳液，クレンジング乳液等の乳液類、ゼリー状パック，ピールオフパック，洗い流しパック，粉末パック等のパック類、美容液、及び洗顔料といった種々の製剤形態の老化防止用化粧料としても提供することができる。
【００１６】
本発明においてはさらに、他の細胞賦活剤や美白成分，保湿剤，抗炎症剤，紫外線吸収剤等、他の有効成分を併用することもでき、日焼け止め化粧料、皮膚保護用化粧料、美白剤等の薬用化粧料或いは医薬部外品等として提供することもできる。
【００１７】
【実施例】
本発明の実施例に使用した珪藻抽出物の製造例を、まず示す。
【００１８】
［製造例１〜製造例４］
表１に示した珪藻及び抽出溶媒を用いて、珪藻抽出物を調製した。培養した珪藻を遠心分離（４０００ｒｐｍ，１０分間）して原体を得る。それに１０倍量の抽出溶媒を添加し、ブレンダー処理した後再度遠心分離を行い上清を回収して珪藻抽出物とした。なお抽出溶媒として用いたリン酸緩衝生理食塩水は、塩化ナトリウム８．０ｇ，塩化カリウム０．２ｇ，リン酸水素二ナトリウム１．１５ｇ，リン酸二水素カリウム０．２ｇ，塩化カルシウム０．１ｇ，塩化マグネシウム・六水和物０．１ｇを蒸留水に溶解して１０００ｍｌとし、ｐＨを７．４に調製して用いた。
【００１９】
【表１】
［真皮線維芽細胞代謝活性化作用］
ヒト由来真皮線維芽細胞を１ウェルあたり２．０×１０4個となるように９６穴マイクロプレートに播種し、２４時間後に製造例１〜４に示した珪藻抽出物を含有する１．０容量％牛胎仔血清添加ダルベッコ最小必須培地にて、３７℃で４８時間培養した。次いで2-(4,5-ジメチル-2-チアゾリル)-3,5-ジフェニルテトラゾリウムブロミド（ＭＴＴ）を０．４mｇ／ｍｌ含有する前記培地に交換して３７℃で２時間培養し、テトラゾリウム環の開環により生じるフォルマザンを、2-プロパノールにて抽出し５５０ｎｍにおける吸光度により測定した。なお、１．０容量％牛胎仔血清添加ダルベッコ最小必須培地のみで培養した系を対照とし、５．０容量％牛胎仔血清添加ダルベッコ最小必須培地で培養した系を陽性対照とした。結果は対照における吸光度を１００．０％として表した活性化指数により表２に示した。
【００２１】
【表２】
その結果、表２に示したとおり、珪藻抽出物を添加して真皮線維芽細胞を培養することにより、活性化指数の上昇が認められ、有意な線維芽細胞代謝活性化が認められていた。特に、抽出溶媒として５０％エタノールを用いた製造例１及び製造例３においては、０．０１重量％ときわめて低濃度で、有意な活性化指数の上昇が認められ、高い線維芽細胞賦活作用を有することが示された。
【００２３】
［紫外線による細胞傷害防御作用］
ヒト由来真皮線維芽細胞を１ウェルあたり２．０×１０4個となるように９６穴マイクロプレートに播種し、２４時間後に製造例１〜４に示した珪藻抽出物を含有する５．０容量％牛胎仔血清添加ダルベッコ最小必須培地に交換し、３７℃で２４時間培養した。次いで培地をＨａｎｋｓ培地に交換し、紫外線を０．５Ｊ／ｃｍ2量照射した。再度、５．０容量％牛胎仔血清添加ダルベッコ最小必須培地に交換し、３７℃で２４時間培養した後、培地をニュートラルレッドを２０μｇ／ｍｌ含有する前記培地に交換して３７℃で２時間培養し、培地中に含まれるニュートラルレッドをリン酸緩衝生理食塩水で洗浄除去した。細胞内に取り込まれたニュートラルレッドは、０．１Ｎ塩酸含有３０％エタノール水溶液で抽出し、抽出液の５５０nmの吸光度を測定する。ニュートラルレッドは、生細胞の細胞膜だけを透過し、リソゾームに沈着するので、生細胞だけを特異的に染色することができる。なお、珪藻抽出物を添加せず、５．０容量％牛胎仔血清添加ダルベッコ最少必須培地のみで培養した系を対照とし、紫外線を照射しない系を非照射対照とした。結果は式１に従い算出した防御指数により表３に示した。
【００２４】
【数１】
【表３】
その結果、表３に示したとおり、珪藻抽出物を添加して真皮線維芽細胞を培養することにより、有意な紫外線による真皮線維芽細胞の傷害を防御する作用が認められていた。特に、抽出溶媒として５０％エタノールを用いた製造例１及び製造例３においては、０．１重量％以下のきわめて低濃度で、有意な防御指数の上昇が認められ、高い紫外線による真皮線維芽細胞の傷害を防御する作用を有することが示された。
【００２７】
次に、先に示した製造例を用いて調製した実施例を示し、更に本発明について詳細に説明する。
【００２８】
［実施例１〜４］Ｏ／Ｗ乳化型美容液
表４に示した珪藻抽出物を用いて、下記の処方によりＯ／Ｗ乳化型美容液を調製した。
（処方）
(１)スクワラン ５．０(重量％)
(２)白色ワセリン ２．０
(３)ミツロウ ０．５
(４)ソルビタンセスキオレエート ０．８
(５)ポリオキシエチレンオレイルエーテル(20EO) １．２
(６)パラオキシ安息香酸メチル ０．１
(７)プロピレングリコール ５．０
(８)精製水 ５９．１
(９)カルボキシビニルポリマー1.0重量％水溶液 ２０．０
(10)水酸化カリウム ０．１
(11)エタノール ５．０
(12)珪藻抽出物 １．０
(13)香料 ０．２
製法：(１)〜(５)の油相成分を混合し７５℃に加熱して溶解，均一化する。一方(６)〜(８)の水相成分を混合，溶解して７５℃に加熱し、油相成分を添加して予備乳化する。(９)を添加した後ホモミキサーにて均一に乳化し、(10)を加えてｐＨを調整する。冷却後４０℃にて(11)〜(13)を添加，混合，均一化する。
【００２９】
【表４】
上記実施例１〜実施例４を用いて、紫外線によるしわの発生に対する防止効果を評価した。なお珪藻抽出物を精製水に代替したものを比較例１とした。しわ発生防止効果は、ヘアレスマウス５匹を１群とし、各群について実施例及び比較例をそれぞれ１日１回背部に塗布し、１Ｊ／ｃｍ2／週の長波長紫外線（ＵＶＡ）を５０週間照射し、ヘアレスマウスにおけるしわの発生状況を観察し、表５に示す判定基準に従って点数化して行った。この際、精製水のみを塗布した群を対照とした。結果は各群の平均値を算出し、ＵＶＡ照射日数との関係により表６に示した。
【００３１】
【表５】
【表６】
表６に示されるように、対照群においては、ＵＶＡ照射日数が４０週を越える頃には形成されたしわの深さが中程度にまで達し、５０週後には深いしわの発生が認められていた。これに対し、本発明の実施例塗布群では、いずれにおいても５０週後に微小ないし軽微なしわが認められた程度で、しわの発生は顕著に抑制されていた。一方比較例塗布群では、有意なしわの発生抑制或いは軽減は認められなかった。
【００３４】
続いて、本発明の実施例１〜実施例４及び比較例１について、抗炎症作用及び創傷治癒促進効果を評価した。人工的に炎症又は創傷を形成した１群５匹のマウスを用い、各群に実施例及び比較例をそれぞれ０．５ｇずつ１日２回７日間塗布し、７日目に炎症部位及び創傷部位の状態を観察した。抗炎症作用については「有効」，「やや有効」，「無効」、創傷治癒促進効果については「完全治癒」，「ほぼ治癒」，「治癒不完全」の３段階でそれぞれ評価し、各評価を得たマウスの数にて表７に示した。
【００３５】
【表７】
表７より明らかなように、抗炎症作用については、本発明の実施例塗布群ではいずれにおいても無効と評価されたマウスは見られず、３例以上のマウスにおいて有効な抗炎症作用が認められていた。また創傷治癒促進効果についても、本発明の実施例塗布群では創傷治癒の不完全なマウスはいずれにおいても認められておらず、３例以上のマウスで完全な治癒を認めていた。これに対し比較例１塗布群では、やや有効な抗炎症作用の認められたマウスが１例見られたが、残り４例では炎症の改善は全く認められなかった。また比較例１塗布群すべてにおいて、創傷治癒は不完全であった。
【００３７】
次に本発明の実施例１〜実施例４及び比較例１について、６ヶ月間の実使用試験を行った。パネラーとして、顕著なしわの発生等の皮膚症状を有する４０歳〜６０歳代の女性を用い、１群２０名とした。使用試験は、各群に実施例及び比較例のそれぞれをブラインドにて使用させて行った。使用試験前および使用試験終了後の皮膚の状態を観察し、しわの改善状況について、「改善」，「やや改善」，「変化なし」の３段階にて評価した。なお、しわの程度については写真撮影及びレプリカにより評価した。結果は、各評価を得たパネラー数にて表８に示した。
【００３８】
【表８】
表８に示されるように、しわの改善状況については、本発明の実施例使用群ではすべてにおいて改善傾向が認められていた。特に、実施例１及び実施例３使用群では、７５％以上のパネラーで明確な改善を認めていた。これに対し、比較例使用群では、明確な改善を認めたパネラーは見られず、７５％のパネラーで症状の改善を認めなかった。
【００４０】
なお、本発明の実施例１〜実施例４については、上記使用試験期間中に含有成分の析出，分離，凝集，変臭，変色といった状態変化は全く見られなかった。また、各実施例使用群において、皮膚刺激性反応や皮膚感作性反応を示したパネラーも存在しなかった。
【００４１】
続いて本発明の他の実施例の処方を示す。
【００４２】
［実施例５］皮膚用ローション
(１)エタノール １０．０(重量％)
(２)ヒドロキシエチルセルロース １．０
(３)珪藻抽出物(製造例１) ０．５
(４)パラオキシ安息香酸メチル ０．１
(５)精製水 ８８．４
製法：(１)〜(５)を混合し均一とする。
【００４３】
［実施例６］皮膚用乳剤
(１)ステアリン酸 ０．２(重量％)
(２)セタノール １．５
(３)ワセリン ３．０
(４)流動パラフィン ７．０
(５)ポリオキシエチレン(10EO)モノオレイン酸エステル １．５
(６)酢酸トコフェロール ０．５
(７)グリセリン ５．０
(８)パラオキシ安息香酸メチル ０．１
(９)トリエタノールアミン １．０
(10)精製水 ７９．２
(11)珪藻抽出物(製造例２) １．０
製法：(１)〜(６)の油相成分を混合，加熱して均一に溶解し、７０℃に保つ。一方、(７)〜(10)の水相成分を混合，加熱して均一とし、７０℃とする。この水相成分に前記油相成分を攪拌しながら徐々に添加して乳化し、冷却した後４０℃にて(11)を添加，混合する。
【００４４】
［実施例７］皮膚用ゲル剤
(１)精製水 ８７．８(重量％)
(２)カルボキシビニルポリマー ０．５
(３)ジプロピレングリコール １０．０
(４)パラオキシ安息香酸メチル ０．１
(５)水酸化カリウム ０．１
(６)珪藻抽出物(製造例３) １．５
製法：(１)に(２)を均一に溶解した後、(３)に(４)を溶解して添加し、次いで(５)を加えて増粘させ、(６)を添加する。
【００４５】
【００４６】
［実施例９］水中油型乳剤性軟膏
(１)白色ワセリン ２５．０(重量％)
(２)ステアリルアルコール ２５．０
(３)グリセリン １２．０
(４)ラウリル硫酸ナトリウム １．０
(５)パラオキシ安息香酸メチル ０．１
(６)精製水 ３５．４
(７)珪藻抽出物(製造例３) １．５
製法：(１)〜(４)の油相成分を混合，溶解して均一とし、７５℃に加熱する。一方、(５)を(６)に溶解して７５℃に加熱し、これに前記油相成分を添加して乳化し、冷却後４０℃にて(７)を添加，混合する。
【００４７】
［実施例１０］化粧水
(１)エタノール １０．０(重量％)
(２)1,3-ブチレングリコール ５．０
(３)珪藻抽出物(製造例１) ０．２
(４)香料 ０．１
(５)精製水 ８４．７
製法：(１)〜(４)を順次(５)に添加して均一に混合，溶解する。
【００４８】
［実施例１１］エモリエントクリーム(油中水型)
(１)流動パラフィン ３０．０(重量％)
(２)マイクロクリスタリンワックス ２．０
(３)ワセリン ５．０
(４)ジグリセリルオレイン酸エステル ５．０
(５)L-グルタミン酸ナトリウム １．６
(６)L-セリン ０．４
(７)プロピレングリコール ３．０
(８)パラオキシ安息香酸メチル ０．１
(９)精製水 ５２．３
(10)香料 ０．１
(11)珪藻抽出物(製造例２) ０．５
製法：(５)，(６)を(９)の一部に溶解して５０℃とし、５０℃に加熱した(４)に攪拌しながら徐々に添加する。これをあらかじめ混合し７０℃に加熱溶解した(１)〜(３)に均一に分散し、これに(７)，(８)を(９)の残部に溶解して７０℃に加熱したものを攪拌しながら添加し、ホモミキサーにて乳化する。冷却後、４０℃にて(10)，(11)を添加，混合する。
【００４９】
［実施例１２］メイクアップベースクリーム
(１)ステアリン酸 １２．０(重量％)
(２)セタノール ２．０
(３)グリセリルトリ2-エチルヘキサン酸エステル ２．５
(４)自己乳化型グリセリルモノステアリン酸エステル ２．０
(５)プロピレングリコール １０．０
(６)水酸化カリウム ０．３
(７)精製水 ６８．６
(８)酸化チタン １．０
(９)ベンガラ ０．１
(10)黄酸化鉄 ０．４
(11)香料 ０．１
(12)珪藻抽出物(製造例１) ０．５
(13)珪藻抽出物(製造例３) ０．５
製法：(１)〜(４)の油相成分を混合し、７５℃に加熱して均一とする。一方(５)〜(７)の水相成分を混合し、７５℃に加熱，溶解して均一とし、これに(８)〜(10)の顔料を添加し、ホモミキサーにて均一に分散させる。この水相成分に前記油相成分を添加し、ホモミキサーにて乳化した後冷却し、４０℃にて(11)〜(13)を添加，混合する。
【００５０】
［実施例１３］乳液状ファンデーション
(１)ステアリン酸 ２．０(重量％)
(２)スクワラン ５．０
(３)ミリスチン酸オクチルドデシル ５．０
(４)セタノール １．０
(５)デカグリセリルモノイソパルミチン酸エステル ９．０
(６)1,3-ブチレングリコール ６．０
(７)水酸化カリウム ０．１
(８)パラオキシ安息香酸メチル ０．１
(９)精製水 ５３．３
(10)酸化チタン ９．０
(11)タルク ７．４
(12)ベンガラ ０．５
(13)黄酸化鉄 １．１
(14)黒酸化鉄 ０．１
(15)香料 ０．１
(16)珪藻抽出物(製造例３) ０．３
製法：(１)〜(５)の油相成分を混合し、７５℃に加熱して均一とする。一方(６)〜(９)の水相成分を混合し、７５℃に加熱，溶解して均一とし、これに(10)〜(14)の顔料を添加しホモミキサーにて均一に分散させる。この水相成分に前記油相成分を添加し、ホモミキサーにて均一に乳化した後冷却し、４０℃にて(15)，(16)を添加，混合する。
【００５１】
【００５２】
【発明の効果】
以上詳述したように、珪藻の抽出物は、真皮線維芽細胞賦活作用及び紫外線による真皮線維芽細胞の傷害を防御する作用を有し、さらにこれを含有する本発明の老化防止用皮膚外用剤は、シワ，シミの発生、皮膚弾性の低下といった皮膚老化症状の防止或いは改善に有効で、抗炎症作用，創傷治癒促進作用をも有し、さらに安定性，安全性も良好である。[0001]
BACKGROUND OF THE INVENTION
The present invention activates the metabolic activity of dermal fibroblasts and further prevents damage to fibroblasts caused by ultraviolet rays, thereby causing wrinkles and spots due to various stresses such as aging and ultraviolet rays, and reducing skin elasticity. It is effective for prevention or improvement of skin aging symptoms such as the above, and an anti-aging skin external preparation having an anti-inflammatory action and a wound healing promoting action.
[0002]
[Prior art]
For skin aging symptoms such as wrinkles, spots, and skin elasticity, which are caused by external stress such as aging and ultraviolet rays, decreased function of fibroblasts in the dermis and decrease or degradation of matrix fibers are important factors. Yes. Therefore, in order to obtain an anti-aging external preparation for skin having an anti-aging / improving action on the skin, attempts have been made to search for and mix components having an action to promote or promote fibroblasts.
[0003]
For example, loquat extract (JP-B-5-17206), α-hydroxyacetic acid (JP-A-5-112422), sterol ester of α-hydroxyacid (JP-A-8-104632), 6-benzylamino Purine (JP-A-7-233037), specific ribonuclease (JP-A-7-309778), L-lysyl-L-glycyl-L-histidine (JP-A-7-316192), milk-derived fibroblast Growth factors (Japanese Patent Laid-Open No. 8-119867), oxidized coenzyme A (Japanese Patent Laid-Open No. 8-175961) and the like are disclosed.
[0004]
However, among the above-mentioned components having a dermal fibroblast activation effect, the action effect is insufficient or the stability is poor, and it is effective when contained in a skin external preparation base. Some have had to contain significant amounts to achieve an effect. In addition, it has undesirable side effects and irritation, adversely affects the stability of the preparation, is difficult to mix with external preparations in terms of odor and color, and is difficult to maintain a certain level of action and quality There were also many things.
[0005]
[Problems to be solved by the invention]
Therefore, in the present invention, by searching for a novel component having an excellent cell activating effect that improves the metabolic activity of dermal fibroblasts and including it in an external preparation for skin, skin damage or aging caused by external stress such as ultraviolet rays The purpose of the present invention is to obtain an anti-aging skin external preparation excellent in the effect of effectively preventing or improving the skin.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors screened a substance having an effective activating action using the metabolic activity promoting effect of dermal fibroblasts as an index. As a result, it has been found that the diatom extract has a high dermal fibroblast metabolism promoting effect and an effect of preventing fibroblast damage caused by ultraviolet rays. In this diatom extract, there is no adverse effect on the skin such as skin irritation and contact sensitization, and even when formulated in an anti-aging skin external preparation, the dermal fibroblast activation effect is not inactivated, The quality was stable.
[0007]
Diatoms are yellow-brown algae found in freshwater, seawater, and soil, and are a type of phytoplankton. As for diatoms, sea salt obtained by drying a culture supernatant obtained by cultivating diatoms in seawater (French Patent No. 2417979), eicosapentaenoic acid contained in the cultured diatoms (JP 59-46225, JP 5-5805726), radical scavenging activity of hydrophilic solvent extracts of seaweeds such as diatoms (French Patent 2657012), poly-β-N-acetylglucosamines isolated from diatoms (International Publication No. 9515343, US Pat. No. 5,562,064) and the like It is disclosed. The inventor of the present invention newly found a remarkable fibroblast activation action and an effect of preventing damage to dermal fibroblasts caused by ultraviolet rays in a diatom solvent extract, and the present invention has been completed.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described.
[0009]
The type of diatom used in the present invention is not particularly limited, but diatoms of the family Chaetoceraceae, particularly Chaetoceros gracilis and Pavlova (Pavlova), from the fibroblast activation action and the ability to protect against cytotoxicity by ultraviolet rays. The diatom extract of) is particularly preferred.
[0010]
Extraction solvents for obtaining these seaweed extracts include water, ethanol, methanol, isopropanol, isobutanol, n-hexanol, methyl amyl alcohol, 2-ethylbutanol, n-octyl alcohol and other alcohols, glycerin, ethylene glycol , Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, triethylene glycol, 1,3-butylene glycol, hexylene glycol and other polyhydric alcohols or derivatives thereof, acetone , Methyl ethyl ketone, methyl isobutyl ketone, ketones such as methyl-n-propyl ketone, esters such as ethyl acetate and isopropyl acetate, ethyl ether One or two or more mixed solvents selected from polar solvents such as ethers such as isopropyl ether and n-butyl ether can be suitably used. Moreover, the polar solvent which added inorganic salts, such as phosphate buffered saline, and the polar solvent which added surfactant can also be used, and it does not specifically limit. Among the above extraction solvents, one solvent selected from ethanol, methanol, 1,3-butylene glycol, water, or a mixed solvent of two or more, and a solvent obtained by adding an inorganic salt or a surfactant to these solvents Is preferably used.
[0011]
Furthermore, as an extraction method, a method of impregnating and extracting in a cooled or heated state at room temperature, a method of extracting using a distillation method such as steam distillation, a pressing method of obtaining an extract by squeezing from raw diatom, etc. And these methods are used alone or in combination of two or more.
[0012]
The ratio of the diatom and the solvent during the extraction is not particularly limited, but the solvent is 0.5 to 1000 times by weight, particularly 0.5 to 100 times by weight in terms of extraction operation and efficiency with respect to diatom 1. preferable. The extraction temperature is conveniently in the range from room temperature to the boiling point of the solvent under normal pressure, and the extraction time is preferably in the range of 2 hours to 2 weeks, although it varies depending on the extraction temperature.
[0013]
In addition, the diatom extract thus obtained can be used as it is, but deodorizing, decolorizing, concentrating, etc. within a range not losing the dermal fibroblast activation action and the cytotoxic protection action by ultraviolet rays. These may be used as a fraction by adding the purification procedure described above or using column chromatography or the like. These extracts, purified products, and fractions can also be dried by removing the solvent from them, and further used in the form of an anti-aging skin in a form solubilized in a solvent such as alcohol or in the form of an emulsion. It can be added to the agent.
[0014]
The blending amount of these diatom extracts into the anti-aging skin external preparation is preferably in a concentration range of 0.001 to 20% by weight in view of the effect, scent and color tone when added. If the blending amount is less than 0.001% by weight, sufficient dermal fibroblast activation action and anti-aging effect by ultraviolet rays can not be obtained, but it is not necessary to add too much, and 20% by weight. Exceeding this may affect the stability of the anti-aging external preparation for skin.
[0015]
In the present invention, an anti-aging skin external preparation containing the above diatom extract can be provided, but the anti-aging skin external preparation can take the form of a lotion, emulsion, cream, ointment and the like. . Furthermore, lotions such as flexible lotions, astringent lotions, and lotions for cleaning, emollient creams, moisture creams, massage creams, cleansing creams, makeup creams, emollient emulsions, moisture emulsions, nourishing It can also be provided as anti-aging cosmetics in various formulation forms such as emulsions such as emulsions, cleansing emulsions, jelly-like packs, peel-off packs, wash-out packs, packs such as powder packs, cosmetic liquids, and face wash. .
[0016]
In the present invention, other active ingredients such as other cell activators, whitening ingredients, moisturizers, anti-inflammatory agents, ultraviolet absorbers and the like can also be used in combination, sunscreen cosmetics, skin protection cosmetics, whitening. It can also be provided as a medicinal cosmetic such as an agent or a quasi-drug.
[0017]
【Example】
An example of producing the diatom extract used in the examples of the present invention will be shown first.
[0018]
[Production Example 1 to Production Example 4]
A diatom extract was prepared using the diatom and extraction solvent shown in Table 1. The cultured diatom is centrifuged (4000 rpm, 10 minutes) to obtain the drug substance. A 10-fold amount of extraction solvent was added thereto, blended, and then centrifuged again to recover the supernatant to obtain a diatom extract. The phosphate buffered saline used as the extraction solvent was 8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 0.1 g calcium chloride, Magnesium chloride hexahydrate 0.1 g was dissolved in distilled water to 1000 ml, and the pH was adjusted to 7.4.
[0019]
[Table 1]
[Activation of dermal fibroblast metabolism]
Human-derived dermal fibroblasts are seeded in a 96-well microplate so as to be 2.0 × 10 4 per well, and after 24 hours, 1.0 volume containing the diatom extract shown in Production Examples 1 to 4 The cells were cultured at 37 ° C. for 48 hours in Dulbecco's minimum essential medium supplemented with% fetal bovine serum. Subsequently, the medium containing 0.4 mg / ml of 2- (4,5-dimethyl-2-thiazolyl) -3,5-diphenyltetrazolium bromide (MTT) was exchanged and cultured at 37 ° C. for 2 hours. Formazan produced by ring opening was extracted with 2-propanol and measured by absorbance at 550 nm. In addition, the culture | cultivation only in the 1.0 volume% fetal bovine serum addition Dulbecco minimum essential medium was set as the control | contrast, and the system | cultivation in the 5.0 volume% fetal bovine serum addition Dulbecco minimum essential medium was made into the positive control. The results are shown in Table 2 by the activation index expressed as the absorbance in the control as 100.0%.
[0021]
[Table 2]
As a result, as shown in Table 2, when the dermal fibroblasts were cultured by adding the diatom extract, an increase in the activation index was observed, and significant fibroblast metabolic activation was observed. In particular, in Production Example 1 and Production Example 3 using 50% ethanol as the extraction solvent, a significant increase in the activation index was observed at a very low concentration of 0.01% by weight, and a high fibroblast activation effect was observed. It was shown to have.
[0023]
[Protection against UV damage by cells]
Human-derived dermal fibroblasts are seeded in a 96-well microplate so as to be 2.0 × 10 4 per well, and after 24 hours, 5.0 volumes containing the diatom extract shown in Production Examples 1 to 4 The medium was replaced with Dulbecco's minimum essential medium supplemented with 1% fetal calf serum and cultured at 37 ° C. for 24 hours. Subsequently, the medium was replaced with a Hanks medium, and ultraviolet rays were irradiated in an amount of 0.5 J / cm 2 . The medium was replaced with Dulbecco's minimum essential medium supplemented with 5.0% by volume fetal calf serum and cultured at 37 ° C. for 24 hours, and then the medium was replaced with the medium containing 20 μg / ml of neutral red and cultured at 37 ° C. for 2 hours. The neutral red contained in the medium was removed by washing with phosphate buffered saline. Neutral red taken up into cells is extracted with 30% ethanol aqueous solution containing 0.1N hydrochloric acid, and the absorbance at 550 nm of the extract is measured. Since neutral red permeates only the cell membrane of living cells and deposits on lysosomes, only living cells can be specifically stained. In addition, the system which did not add a diatom extract and culture | cultivated only with the 5.0 volume% fetal bovine serum addition Dulbecco's minimum essential medium was set as the control, and the system which did not irradiate with ultraviolet rays was set as the non-irradiated control. The results are shown in Table 3 based on the defense index calculated according to Equation 1.
[0024]
[Expression 1]
[Table 3]
As a result, as shown in Table 3, by adding diatom extract and culturing dermal fibroblasts, a significant effect of protecting dermal fibroblasts from being damaged by ultraviolet rays was observed. In particular, in Production Example 1 and Production Example 3 using 50% ethanol as an extraction solvent, a significant increase in the defense index was observed at an extremely low concentration of 0.1% by weight or less, and dermal fibroblasts due to high ultraviolet rays. It was shown to have an action to protect against injury.
[0027]
Next, examples prepared using the production examples shown above will be shown, and the present invention will be described in detail.
[0028]
[Examples 1 to 4] O / W emulsified essence liquid Using the diatom extract shown in Table 4, O / W emulsified essence liquid was prepared according to the following formulation.
(Prescription)
(1) Squalane 5.0 (% by weight)
(2) White petrolatum 2.0
(3) Beeswax 0.5
(4) Sorbitan sesquioleate 0.8
(5) Polyoxyethylene oleyl ether (20EO) 1.2
(6) Methyl paraoxybenzoate 0.1
(7) Propylene glycol 5.0
(8) Purified water 59.1
(9) Carboxyvinyl polymer 1.0 wt% aqueous solution 20.0
(10) Potassium hydroxide 0.1
(11) Ethanol 5.0
(12) Diatom extract 1.0
(13) Fragrance 0.2
Production method: The oil phase components (1) to (5) are mixed and heated to 75 ° C. to dissolve and homogenize. On the other hand, the water phase components (6) to (8) are mixed and dissolved and heated to 75 ° C., and the oil phase component is added and pre-emulsified. After (9) is added, the mixture is uniformly emulsified with a homomixer, and (10) is added to adjust the pH. After cooling, add (11) to (13) at 40 ° C, mix and homogenize.
[0029]
[Table 4]
Using Examples 1 to 4 described above, the effect of preventing wrinkles due to ultraviolet rays was evaluated. In addition, the thing which replaced the diatom extract with the purified water was made into the comparative example 1. The wrinkle-preventing effect consists of 5 hairless mice in one group, and each example and comparative example is applied to the back of each group once a day, and 1 J / cm 2 / week of long wavelength ultraviolet rays (UVA) is applied for 50 weeks. Irradiation was performed, and the occurrence of wrinkles in the hairless mouse was observed and scored according to the criteria shown in Table 5. At this time, a group to which only purified water was applied was used as a control. As a result, the average value of each group was calculated and shown in Table 6 according to the relationship with the number of UVA irradiation days.
[0031]
[Table 5]
[Table 6]
As shown in Table 6, in the control group, when the number of UVA irradiation days exceeded 40 weeks, the depth of wrinkles formed reached a medium level, and after 50 weeks, deep wrinkles were observed. It was. On the other hand, in the example application group of the present invention, the occurrence of wrinkles was remarkably suppressed to the extent that fine to slight wrinkles were observed after 50 weeks in all cases. On the other hand, no significant suppression or reduction of wrinkles was observed in the comparative application group.
[0034]
Subsequently, with respect to Examples 1 to 4 and Comparative Example 1 of the present invention, the anti-inflammatory action and the wound healing promoting effect were evaluated. Using 5 mice per group artificially inflamed or wounded, 0.5 g each of Examples and Comparative Examples was applied to each group twice a day for 7 days. The state of was observed. The anti-inflammatory action is evaluated as “effective”, “slightly effective”, “ineffective”, and the wound healing promotion effect is evaluated in three stages, “complete healing”, “almost healing”, and “incomplete healing”. The number of obtained mice is shown in Table 7.
[0035]
[Table 7]
As is clear from Table 7, regarding the anti-inflammatory effect, no mice evaluated as invalid in any of the application groups of the examples of the present invention were found, and an effective anti-inflammatory effect was observed in 3 or more mice. It was. In addition, regarding the wound healing promoting effect, none of the mice with incomplete wound healing was observed in any of the application groups of Examples of the present invention, and complete healing was observed in three or more mice. On the other hand, in the Comparative Example 1 application group, one mouse was found to have a slightly effective anti-inflammatory action, but no improvement in inflammation was observed in the remaining four cases. Moreover, wound healing was incomplete in all the application groups of Comparative Example 1.
[0037]
Next, for Examples 1 to 4 and Comparative Example 1 of the present invention, an actual use test for 6 months was conducted. As panelists, women in their 40s to 60s having skin symptoms such as the occurrence of significant wrinkles were used, and 20 people per group were used. The use test was conducted by allowing each group to use each of the examples and comparative examples blindly. The condition of the skin before and after the use test was observed, and the improvement state of wrinkles was evaluated in three stages: “Improved”, “Slightly improved”, and “No change”. Note that the degree of wrinkles was evaluated by photography and replica. The results are shown in Table 8 in terms of the number of panelists that obtained each evaluation.
[0038]
[Table 8]
As shown in Table 8, with respect to the wrinkle improvement situation, an improvement tendency was recognized in all of the examples using groups of the present invention. In particular, in the use group of Example 1 and Example 3, a clear improvement was recognized with 75% or more of panelists. On the other hand, in the group using the comparative example, no panelists who showed a clear improvement were found, and 75% of the panelists showed no improvement in symptoms.
[0040]
In Examples 1 to 4 of the present invention, no state changes such as precipitation, separation, aggregation, odor change, and discoloration of the components were observed during the above-described use test period. In addition, there was no panel exhibiting a skin irritation reaction or a skin sensitization reaction in the groups used in each example.
[0041]
Subsequently, the formulation of another embodiment of the present invention will be shown.
[0042]
[Example 5] Skin lotion
(1) Ethanol 10.0 (wt%)
(2) Hydroxyethyl cellulose 1.0
(3) Diatom extract (Production Example 1) 0.5
(4) Methyl paraoxybenzoate 0.1
(5) Purified water 88.4
Production method: (1) to (5) are mixed to make uniform.
[0043]
[Example 6] Emulsion for skin
(1) Stearic acid 0.2 (% by weight)
(2) Cetanol 1.5
(3) Vaseline 3.0
(4) Liquid paraffin 7.0
(5) Polyoxyethylene (10EO) monooleate 1.5
(6) Tocopherol acetate 0.5
(7) Glycerin 5.0
(8) Methyl paraoxybenzoate 0.1
(9) Triethanolamine 1.0
(10) Purified water 79.2
(11) Diatom extract (Production Example 2) 1.0
Production method: The oil phase components (1) to (6) are mixed, heated and uniformly dissolved, and kept at 70 ° C. On the other hand, the aqueous phase components (7) to (10) are mixed and heated to be uniform, and set to 70 ° C. The oil phase component is gradually added to the aqueous phase component while stirring to emulsify, and after cooling, (11) is added and mixed at 40 ° C.
[0044]
[Example 7] Gel for skin
(1) Purified water 87.8 (% by weight)
(2) Carboxyvinyl polymer 0.5
(3) Dipropylene glycol 10.0
(4) Methyl paraoxybenzoate 0.1
(5) Potassium hydroxide 0.1
(6) Diatom extract (Production Example 3) 1.5
Production method: (2) is uniformly dissolved in (1), (4) is dissolved and added to (3), then (5) is added to increase the viscosity, and (6) is added.
[0045]
[0046]
[Example 9] Oil-in-water emulsion ointment
(1) White petrolatum 25.0 (wt%)
(2) Stearyl alcohol 25.0
(3) Glycerin 12.0
(4) Sodium lauryl sulfate 1.0
(5) Methyl paraoxybenzoate 0.1
(6) Purified water 35.4
(7) Diatom extract (Production Example 3) 1.5
Production method: The oil phase components (1) to (4) are mixed, dissolved and made uniform, and heated to 75 ° C. On the other hand, (5) is dissolved in (6) and heated to 75 ° C., the oil phase component is added thereto to emulsify, and after cooling, (7) is added and mixed at 40 ° C.
[0047]
[Example 10] Lotion
(1) Ethanol 10.0 (wt%)
(2) 1,3-butylene glycol 5.0
(3) Diatom extract (Production Example 1) 0.2
(4) Fragrance 0.1
(5) Purified water 84.7
Production method: (1) to (4) are sequentially added to (5) and mixed and dissolved uniformly.
[0048]
[Example 11] Emollient cream (water-in-oil type)
(1) Liquid paraffin 30.0 (wt%)
(2) Microcrystalline wax 2.0
(3) Vaseline 5.0
(4) Diglyceryl oleate 5.0
(5) Sodium L-glutamate 1.6
(6) L-serine 0.4
(7) Propylene glycol 3.0
(8) Methyl paraoxybenzoate 0.1
(9) Purified water 52.3
(10) Fragrance 0.1
(11) Diatom extract (Production Example 2) 0.5
Production method: (5) and (6) are dissolved in a part of (9) to 50 ° C. and gradually added to (4) heated to 50 ° C. with stirring. This was mixed in advance and uniformly dispersed in (1) to (3) heated and dissolved at 70 ° C. Then, (7) and (8) were dissolved in the remainder of (9) and heated to 70 ° C. Add with stirring and emulsify with homomixer. After cooling, add and mix (10) and (11) at 40 ° C.
[0049]
[Example 12] Makeup base cream
(1) Stearic acid 12.0 (wt%)
(2) Cetanol 2.0
(3) Glyceryl tri-2-ethylhexanoate 2.5
(4) Self-emulsifying glyceryl monostearate 2.0
(5) Propylene glycol 10.0
(6) Potassium hydroxide 0.3
(7) Purified water 68.6
(8) Titanium oxide 1.0
(9) Bengala 0.1
(10) Yellow iron oxide 0.4
(11) Fragrance 0.1
(12) Diatom extract (Production Example 1) 0.5
(13) Diatom extract (Production Example 3) 0.5
Production method: The oil phase components (1) to (4) are mixed and heated to 75 ° C. to be uniform. On the other hand, the water phase components (5) to (7) are mixed, heated and dissolved at 75 ° C. to make uniform, and the pigments (8) to (10) are added to this and uniformly dispersed with a homomixer. . The oil phase component is added to the water phase component, emulsified with a homomixer, cooled, and (11) to (13) are added and mixed at 40 ° C.
[0050]
[Example 13] Emulsion foundation
(1) Stearic acid 2.0 (wt%)
(2) Squalane 5.0
(3) Octyldodecyl myristate 5.0
(4) Cetanol 1.0
(5) Decaglyceryl monoisopalmitate 9.0
(6) 1,3-butylene glycol 6.0
(7) Potassium hydroxide 0.1
(8) Methyl paraoxybenzoate 0.1
(9) Purified water 53.3
(10) Titanium oxide 9.0
(11) Talc 7.4
(12) Bengala 0.5
(13) Yellow iron oxide 1.1
(14) Black iron oxide 0.1
(15) Fragrance 0.1
(16) Diatom extract (Production Example 3) 0.3
Production method: The oil phase components (1) to (5) are mixed and heated to 75 ° C. to be uniform. On the other hand, the aqueous phase components (6) to (9) are mixed, heated and dissolved at 75 ° C. to make uniform, and the pigments (10) to (14) are added thereto and uniformly dispersed with a homomixer. The oil phase component is added to the water phase component, and the mixture is uniformly emulsified with a homomixer, cooled, and (15) and (16) are added and mixed at 40 ° C.
[0051]
[0052]
【The invention's effect】
As described above in detail, the diatom extract has an action of activating dermal fibroblasts and an effect of protecting dermal fibroblasts from being damaged by ultraviolet rays, and further contains an anti-aging skin external preparation according to the present invention. Is effective in preventing or ameliorating skin aging symptoms such as wrinkles, spots, and skin elasticity, and has an anti-inflammatory action and a wound healing promoting action, and also has good stability and safety.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22716097A JP3822959B2 (en) | 1997-08-07 | 1997-08-07 | Anti-aging skin external preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22716097A JP3822959B2 (en) | 1997-08-07 | 1997-08-07 | Anti-aging skin external preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1149695A JPH1149695A (en) | 1999-02-23 |
| JP3822959B2 true JP3822959B2 (en) | 2006-09-20 |
Family
ID=16856443
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22716097A Expired - Fee Related JP3822959B2 (en) | 1997-08-07 | 1997-08-07 | Anti-aging skin external preparation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3822959B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012052356A2 (en) | 2010-10-19 | 2012-04-26 | Cutech S.R.L. | Extracts of microalgae and their application |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3690771B2 (en) * | 1997-10-13 | 2005-08-31 | 株式会社ノエビア | Topical skin preparation |
| FR2822701B1 (en) | 2001-04-03 | 2005-03-18 | Lvmh Rech | USE OF ALGAE PHAEODACTYLUM EXTRACT AS A COSMETIC AGENT PROMOTING PROTEASOME ACTIVITY OF SKIN CELLS AND COSMETIC COMPOSITION CONTAINING SAME |
| JP6573400B2 (en) * | 2014-09-08 | 2019-09-11 | 公立大学法人兵庫県立大学 | A novel diatom transformation vector and a novel promoter sequence contained therein |
| CN104523563B (en) * | 2015-01-09 | 2017-11-21 | 青岛泉佳美硅藻泥科技有限公司 | A kind of diatom facial mask and preparation method thereof |
-
1997
- 1997-08-07 JP JP22716097A patent/JP3822959B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012052356A2 (en) | 2010-10-19 | 2012-04-26 | Cutech S.R.L. | Extracts of microalgae and their application |
| EP3795216A1 (en) | 2010-10-19 | 2021-03-24 | Symrise AG | Extracts of thalassiosira sp. and their application |
| EP3804693A1 (en) | 2010-10-19 | 2021-04-14 | Symrise AG | Extracts of chaetoceros sp. and their application |
| EP3808334A1 (en) | 2010-10-19 | 2021-04-21 | Symrise AG | Extracts of chlorococcum sp. and their application |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH1149695A (en) | 1999-02-23 |
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