JP7406782B2 - 皮膚外用剤 - Google Patents
皮膚外用剤 Download PDFInfo
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- JP7406782B2 JP7406782B2 JP2018230471A JP2018230471A JP7406782B2 JP 7406782 B2 JP7406782 B2 JP 7406782B2 JP 2018230471 A JP2018230471 A JP 2018230471A JP 2018230471 A JP2018230471 A JP 2018230471A JP 7406782 B2 JP7406782 B2 JP 7406782B2
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- extract
- acid
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- derived
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Description
滅菌したGP液体培地2700gに、予め同培地で培養しておいたササユリ由来の酵母(サッカロマイセス・セレビシエ)の前培養液300gを添加し、30℃で通気攪拌しながら20時間培養した。加熱殺菌した後、水酸化ナトリウム水溶液でpH8.5に調整し、3時間、攪拌しながら90℃で加熱処理した。この液をpH調整した後、濾過し1604gの酵母培養液抽出物を得た(固形分濃度1.12%)。
製造例1で示した方法で作製した酵母培養液抽出物を濃縮し、酵母培養液抽出物粉末を17g得た。これに水を少量加え溶解した後、少量のエタノール、及びD-マンニット833gを加え、撹拌しながら真空乾燥し、D-マンニット賦形化酵母培養液抽出物粉末850gを得た。
滅菌したGP液体培地2700gに、予め同培地で培養しておいたササユリ由来の酵母(サッカロマイセス・セレビシエ)の前培養液300g添加し、30℃で通気攪拌しながら20時間培養した。加熱殺菌した後、塩酸水溶液でpH2.5に調整し、3時間、攪拌しながら90℃で加熱処理した。この液をpH調整した後、濾過し1219gの酵母培養液抽出物を得た(固形分濃度1.08%)。
滅菌したGP液体培地2700gに、予め同培地で培養しておいたササユリ由来の酵母(サッカロマイセス・セレビシエ)の前培養液300g添加し、30℃で通気攪拌しながら20時間培養した。加熱殺菌した後、3時間、90℃で加熱処理した。この液をpH調整した後、濾過し1536gの酵母培養液抽出物を得た(固形分濃度0.60%)。
ヒト真皮由来線維芽細胞NB1RGBを、0.5%NCS含有イーグル最少必須培地を入れた96穴マイクロプレートに1×104個/穴播種し、37℃,5.0%CO2の条件下に1日間プレ培養した後、製造例1を1.0%、2.0%の濃度(溶液として)となるように培地に添加し、同条件でさらに5日間培養した。次に、培地を除去し、冷メタノール、冷エタノールで細胞を固定した後、0.1%シリウスレッド含有飽和ピクリン酸水溶液で染色を行った。精製水で洗浄後、0.1%NaOH:メタノール=1:1溶液にて抽出を行い、マイクロプレートリーダー(Model 680、バイオラッド社製)を用いて波長540nmでコラーゲン量を測定した。製造例1に代えてPBS(-)を添加した試料無添加の場合(コントロール)についても上記と同様の操作を行い、ここに得られたコラーゲン量に対する各試料添加時のコラーゲン量の相対値を求め、線維芽細胞コラーゲン合成率(%)とした。また、試験系が正常に機能しているかを確認するために、製造例1の代わりに陽性対照として1mMのアスコルビン酸リン酸マグネシウム塩を添加した場合についても、同様の試験を行った。
[表1]
ヒト真皮由来線維芽細胞NB1RGBを、0.5%NCS含有イーグル最少必須培地を入れた96穴マイクロプレートに1×104個/穴播種し、37℃,5.0%CO2の条件下に1日間プレ培養した後、製造例1を1.0%、2.0%の濃度(溶液として)となるように培地に添加し、同条件でさらに5日間培養した。その後、それぞれのウェルから上清を回収し、ヒアルロン酸測定キット(Quantikine(登録商標) ELISA Hyaluronan Immunoassay、R&D SYSTEMS、USA)を用いて上清中のヒアルロン酸量を定量した。製造例1の代わりにPBS(-)を添加した区をコントロールとし、コントロール区のヒアルロン酸合成量に対する各試料添加区のヒアルロン酸合成量の相対値を算出し、ヒアルロン酸合成促進効果(%)を求めた。試験が正常に行われたことを確認するために、ポジティブコントロールとしてNアセチルDグルコサミン10mM添加する区も設定した
[表2]
ヒト真皮由来線維芽細胞(NB1RGB)を10%NCS含有イーグル最少必須培地にて2×105個/mLに調製し、96穴マイクロプレートに100μLを播種して、5%炭酸ガス、飽和水蒸気下、37℃で培養した。24時間後、終濃度が0.5%、1%または2%となるように製造例1を含んだ培養液を追添加して培養した。また、終濃度が2%となるようにPBS(-)を含んだ培養液を追添加した試験区をcontrolとして設定し比較区とした。48時間後、試験区の上清を除去し、2’,7’-dichlorodihydrofluorescein diacetate(DCFH-2DA)を細胞に取り込ませた後、蛍光プレートリーダー(フルオロスキャンアセント、Thermo Labsystems社製)を用いて蛍光強度(励起波長485nm、吸収波長538nm)を測定し、これを酸化ダメージ量とした。測定後、細胞溶解液中のタンパク質量を測定し、タンパク質量当たりの酸化ダメージ量を算出した。
[表3]
ヒト真皮由来線維芽細胞NB1RGBを、10%NCS含有イーグル最少必須培地を入れた24穴マイクロプレートに9×104個/穴播種し、37℃,5.0%CO2の条件下に1日間プレ培養した後、製造例1を1.0%の濃度(溶液として)となるように培地に添加しさらに培養した。翌日、紫外線ランプ(アズワン社 型番SLUV-6)を用いて5Jの紫外線A波を照射し、照射終了24時間後の培養上清をケモカイン誘導溶液とした。同時に、比較として紫外線未照射区を設定し、回収した培養上清をケモカイン未誘導溶液とした。回収した培養液をトランズウェル(コーニング社製 型番3415)のレセプター側に500μL添加した。1.25%DMSO及び10%FBS含有RPMI培地で5日間培養することで好中球様細胞に分化誘導させたヒト白血病細胞株HL60細胞を1×106個/mLに調整し、トランズウェルのセルカルチャーインサート側に300μL播種した。分化誘導させたHL60細胞を播種した2時間後、レセプター側に遊走した細胞を得た。また、播種とは別ウェルに0.5mM EDTA/PBS(-)溶液を200μL添加し、遊走終了後のセルカルチャーインサートを移動させ、30分静置する事で、セルカルチャーインサートの底面に残った細胞を得た。遊走した細胞を合わせ2’,7’-dichlorodihydrofluorescein diacetateを取り込ませた後、蛍光プレートリーダー(フルオロスキャンアセント、Thermo Labsystems社製)を用いて蛍光強度(励起波長485nm、吸収波長538nm)を測定した。得られた蛍光強度を、ケモカイン未誘導コントロールを100とした時の相対値で示した。
[表4]
ヒト真皮由来線維芽細胞NB1RGBを、10%NCS含有イーグル最少必須培地を入れた24穴マイクロプレートに9×104個/穴播種し、37℃,5.0%CO2の条件下に1日間プレ培養した後、製造例1を1.0%の濃度(溶液として)となるように培地に添加しさらに培養した。翌日、紫外線ランプ(アズワン社 型番SLUV-6)を用いて5Jの紫外線A波を照射し、照射終了24時間後の培養上清をIL-8誘導溶液とした。同時に、比較として紫外線未照射区を設定し、回収した培養上清をIL-8未誘導溶液とした。回収した培養液のIL-8量をIL-8測定キット(Boster Biological Technology社製)を用いて測定した。得られたIL-8量を、未誘導コントロールを100とした時の相対値で示した。
[表5]
培養B16マウスメラノーマ細胞B16-F10を、24穴マイクロプレートに2.4×104個/穴播種し、10%FBS含有RPMI1640培地中、37℃、5.0%CO2の条件下に1日間プレ培養した後、10%FBS含有RPMI1640培地で、製造例1を2.0%の終濃度(溶液として)となるように希釈した溶液と終濃度1mMになるように調整したテオフィリン含有培地を添加し、同条件で2日間培養した。次に培養液を除去し、1N NaOH/10%ジメチルスルフォキシド溶液を1穴あたり200μL添加し、シールして50℃、2時間インキュベートして細胞を溶解させた。この溶液100μLを別の96穴マイクロプレートに移し、マイクロプレートリーダー(Model 680、バイオラッド社製)を用い、波長490nmでメラニン値を測定した。一方同じ細胞を溶解させた溶液を5μL別の96穴マイクロプレートに移し、さらに精製水で5倍希釈したDye Reagent Concentrate(バイオラッド社)溶液を200μL添加し、 マイクロプレートリーダー(Model 680、バイオラッド社製)を用い、波長570nmの吸光度を測定した。別で既知の量の牛血清アルブミン(Sigma社製)を段階希釈し、同様に操作して得られた検量線から、アルブミン当量のタンパク質量を計測した。得られた吸光度をタンパク質量で徐して、タンパク質あたりのメラニン量を求めた。製造例1に代えてPBS(-)を添加した試料無添加の場合(対照)についても上記と同様の操作を行い、ここに得られたタンパク質あたりのメラニン量に対する各試料添加時のタンパク質あたりのメラニン量の相対値を求め、メラニン合成率(%)とした。なお、比較のため、製造例1の代わりに、2mMのコウジ酸を添加した場合(陽性対照)についても同様の試験を行った。
[表6]
[成分] 部
ホホバ油 1.0
ポリオキシエチレン(5.5)セチルアルコール 5.0
メチルパラベン 0.1
製造例1の酵母抽出物 2.0
グリセリン 5.0
1,3-ブチレングリコール 5.0
クエン酸ナトリウム 0.2
精製水 全量が100部となる量
処方例1に含まれる製造例1の酵母抽出物に代えて、製造例3の酵母抽出物2.0部を用いるほかは、処方例1と同様にして化粧水を得た。
処方例1に含まれる製造例1の酵母抽出物に代えて、製造例4の酵母抽出物2.0部を用いるほかは、処方例1と同様にして化粧水を得た。
[成分] 部
スクワラン 5.0
ヘキサラン 3.0
ホホバ油 1.0
ツバキ油 1.5
ポリオキシエチレン(20)ソルビタンモノステアレート 1.0
親油型ステアリン酸グリセリル 1.0
水添大豆レシチン 1.5
製造例1の酵母抽出物 3.0
L-アスコルビン酸-2-グルコシド 2.0
水酸化カリウム 0.5
グリセリン 3.0
1,3-ブチレングリコール 2.0
カルボキシメチルセルロース 0.3
キサンタンガム 0.2
ヒアルロン酸ナトリウム 0.01
精製水 全量が100部となる量
処方例4の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてアルブチン3.0部を用いるほかは処方例4と同様にして乳液を得た。
処方例4の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてニコチン酸アミド5.0部を用いるほかは処方例4と同様にして乳液を得た。
処方例4の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてトラネキサム酸2.0部を用いるほかは処方例5と同様にして乳液を得た。
処方例4の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてトラネキサム酸セチルエステル塩酸塩2.0部を用いるほかは処方例4と同様にして乳液を得た。
[成分] 部
スクワラン 3.0
セチルアルコール 1.0
モノステアリン酸ポリエチレングリコール 0.2
ジプロピレングリコール 1.0
イソオクタン酸セチル 1.0
ジメチルポリシクロキサン 1.0
製造例1の酵母抽出物 2.0
乳酸菌発酵米 2.0
海藻抽出物 1.0
ラフィノース 2.0
1,3-ブチレングリコール 2.0
L-アスコルビン酸-2-グルコシド 2.0
水酸化カリウム 0.5
アセチル化ヒアルロン酸ナトリウム 0.2
グァーガム 3.0
セラミド 1.0
精製水 全量が100部となる量
処方例9の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてアルブチン3.0部を用いるほかは処方例9と同様にして乳液を得た。
処方例9の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてニコチン酸アミド5.0部を用いるほかは処方例9と同様にして乳液を得た。
処方例9の成分中、L-アスコルビン酸-2-グルコシド2.0部及び水酸化カリウム0.5部に代えてトラネキサム酸2.0部を用いるほかは処方例10と同様にして乳液を得た。
[成分] 部
オリーブ油 5.0
ホホバ油 5.0
スクワラン 5.0
ベヘニルアルコール 2.0
パルミチン酸 2.5
製造例2の酵母抽出物粉末 2.0
乳酸菌発酵米 2.0
水素添加レシチン 0.5
カルボキシビニルポリマー 0.3
アルギン酸ナトリウム 0.2
海藻抽出物 2.0
メチルパラベン 0.15
エチルパラベン 0.03
精製水 全量が100部となる量
[成分] 部
ポリオキシエチレンソルビタンモノステアレート 1.5
セチルアルコール 3.0
モノステアリン酸グリセリル 1.0
スクワラン 5.0
オリーブ油 5.0
ジプロピレングリコール 3.0
製造例3の酵母抽出物 2.0
ゲンチアナ抽出物 1.0
甘草抽出物 2.0
クエン酸 0.2
メチルパラベン 0.2
精製水 全量が100部となる量
[成分] 部
ステアリン酸 2.4
モノステアリン酸プロピレングリコール 2.0
セトステアリルアルコール 0.2
液状ラノリン 2.0
流動パラフィン 3.0
ミリスチン酸イソプロピル 8.5
プロピルパラベン 0.05
製造例2の酵母抽出物粉末 5.0
カルボキシメチルセルロースナトリウム 0.2
ベントナイト 0.5
プロピレングリコール 4.0
トリエタノールアミン 1.1
メチルパラベン 0.1
酸化チタン 8.0
タルク 4.0
着色顔料 適量
精製水 全量が100部となる量
[成分] 部
N-ラウロイルメチルアラニンナトリウム 25.0
ヤシ油脂肪酸カリウム液(40%) 26.0
ヤシ油脂肪酸ジエタノールアミド 3.0
メチルパラベン 0.1
製造例2の酵母抽出物粉末 5.0
1,3-ブチレングリコール 2.0
精製水 全量が100部となる量
[成分] 部
N-ヤシ油脂肪酸メチルタウリンナトリウム 10.0
ポリオキシエチレン(3)アルキルエーテル硫酸ナトリウム 20.0
ラウリルジメチルアミノ酢酸ベタイン 10.0
ヤシ油脂肪酸ジエタノールアミド 4.0
メチルパラベン 0.1
クエン酸 0.1
製造例1の酵母抽出物 2.0
1,3-ブチレングリコール 2.0
精製水 全量が100部となる量
[成分] 部
ポリオキシエチレン(10)硬化ヒマシ油 1.0
塩化ジステアリルジメチルアンモニウム 1.5
塩化ステアリルトリメチルアンモニウム 2.0
2-エチルヘキサン酸グリセリル 1.0
セタノール 3.0
ステアリルアルコール 1.0
メチルパラベン 0.1
製造例2の酵母抽出物粉末 2.0
1,3-ブチレングリコール 5.0
精製水 全量が100部となる量
Claims (3)
- ユリ科(Liliaceae)ユリ属(Lilium)のササユリ(Lilium japonicum)由来の酵母(特許微生物寄託センター受託番号NITE AP-01947)抽出物、或いはその濃縮物又は乾燥粉末を有効成分とする肌荒れ改善用皮膚外用剤。
- ユリ科(Liliaceae)ユリ属(Lilium)のササユリ(Lilium japonicum)由来の酵母(特許微生物寄託センター受託番号NITE AP-01947)の抽出物、或いはその濃縮物又は乾燥粉末を有効成分とする老化防止用皮膚外用剤。
- ユリ科(Liliaceae)ユリ属(Lilium)のササユリ(Lilium japonicum)由来の酵母(特許微生物寄託センター受託番号NITE AP-01947)の抽出物、或いはその濃縮物又は乾燥粉末を有効成分とする美白用皮膚外用剤。
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JP2000109408A (ja) | 1998-10-02 | 2000-04-18 | Kanebo Ltd | 美白化粧料 |
JP2004502712A (ja) | 2000-07-08 | 2004-01-29 | コグニス・フランス・ソシエテ・アノニム | 皮膚を老化から保護する方法 |
JP2004137212A (ja) | 2002-10-18 | 2004-05-13 | Noevir Co Ltd | 水性スティック状化粧料 |
JP2009179642A (ja) | 2009-05-21 | 2009-08-13 | Tsujido Chemical Corp | 皮膚外用剤 |
JP2018193341A (ja) | 2017-05-19 | 2018-12-06 | 共栄化学工業株式会社 | 植物発酵物 |
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JP2000109408A (ja) | 1998-10-02 | 2000-04-18 | Kanebo Ltd | 美白化粧料 |
JP2004502712A (ja) | 2000-07-08 | 2004-01-29 | コグニス・フランス・ソシエテ・アノニム | 皮膚を老化から保護する方法 |
US20040028697A1 (en) | 2000-07-08 | 2004-02-12 | Gilles Pauly | Method for protecting the skin from aging |
JP2004137212A (ja) | 2002-10-18 | 2004-05-13 | Noevir Co Ltd | 水性スティック状化粧料 |
JP2009179642A (ja) | 2009-05-21 | 2009-08-13 | Tsujido Chemical Corp | 皮膚外用剤 |
JP2018193341A (ja) | 2017-05-19 | 2018-12-06 | 共栄化学工業株式会社 | 植物発酵物 |
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