JP6803113B2 - 評価方法及び皮膚外用剤 - Google Patents
評価方法及び皮膚外用剤 Download PDFInfo
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- JP6803113B2 JP6803113B2 JP2016131908A JP2016131908A JP6803113B2 JP 6803113 B2 JP6803113 B2 JP 6803113B2 JP 2016131908 A JP2016131908 A JP 2016131908A JP 2016131908 A JP2016131908 A JP 2016131908A JP 6803113 B2 JP6803113 B2 JP 6803113B2
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Description
また、本発明において、ケラトヒアリン顆粒の合成を測定する工程は、さらに、被験物質を投与した細胞におけるフィラグリン生成量を測定する工程と、被験物質を投与した細胞内の顆粒状構造を確認する工程とを含むことを特徴とする。
また、本発明は、ケラトヒアリン顆粒合成を促進する剤を有効成分とする皮膚外用剤である。
また、ケラトヒアリン顆粒合成を促進し、皮膚の保湿機能及び透明感を向上させる剤を提供することができる。
本発明は、皮膚の状態(保湿機能、透明感等)を改善する物質をケラトヒアリン顆粒の合成能を指標として評価する方法、及びケラトヒアリン顆粒の合成を促進する剤を有効成分として含む皮膚外用剤である。
(i)被験物質の調製
被験物質の調製例1.アッケシソウ抽出物の調製(1)
アッケシソウ(Salicornia herbacea)の全草の乾燥細切物20gに精製水200gを加え、40℃で1時間抽出した。得られた抽出液をろ過して、褐色透明の抽出物溶液(固形分含量:2.0%)156gを得た。
アッケシソウ(Salicornia herbacea)の全草の乾燥細切物20gに50%1,3−ブチレングリコール水溶液200gを加え、40℃で5時間抽出した。得られた抽出液をろ過して、淡褐色透明の抽出物溶液(固形分含量:2.1%)163gを得た。
アッケシソウ(Salicornia herbacea)の全草の乾燥細切物20gに30%1,3−プロパンジオール水溶液200gを加え、40℃で5時間抽出した。得られた抽出液をろ過して、淡褐色透明の抽出物溶液(固形分含量:1.95%)157gを得た。
ケラトヒアリン顆粒には、プロフィラグリンが多量に含まれることから、フィラグリン抗体による免疫染色により得られた蛍光強度と、蛍光顕微鏡観察による表細細胞の顆粒状構造の観察結果から総合的に評価した。正常ヒト皮膚由来表皮細胞(NHEK)をHuMedia KG2培地(クラボウ社製)を入れた96穴マイクロプレートに4×103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1〜3の抽出物を試料溶液として含むHuMedia KB2培地を添加し、同条件でさらに3日間培養した。ここで、試料溶液の濃度は、追添加する培地全量に対する溶液としての終濃度が2.0%となるように調整した。次に、培養後の細胞に対してフィラグリン抗体を用いた免疫的検出を行った。すなわち、PBS(-)洗浄後、15%中性緩衝ホルマリン液を用いて細胞を30分間処理して固定し、0.5%Triton X-100溶液で1時間浸透処理、5倍希釈ブロッキングワンP(ナカライテスク社)溶液で2時間処理によるブロッキングを行った後、フィラグリン抗体を添加し、室温で2時間静置した。その後、PBS(-)を用いて洗浄し、蛍光ラベルした二次抗体を添加してさらに暗所で一定時間静置した。その後、PBS(-)で洗浄し、蛍光強度の測定を行った。すなわち、蛍光マイクロプレートリーダー(フルオロスキャンアセント、Thermo Fisher Scientific社製)を用いて二次抗体の蛍光ラベル(Alexa Fluor546)をEx=544nm、Em=590nmで測定し、その後、Hoechst33342によるDNA染色を行い、Ex=355nm、Em=460nmの測定を行った。それぞれの試験区のAlexa Fluor546の蛍光強度をHoechst33342の蛍光強度で割ることで、フィラグリンの生成度合いを求めた。試料溶液に代えて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、ここに得られたフィラグリン生成度合いに対する各試料添加時のフィラグリン生成度合いの相対値を求め、フィラグリン合成量(%)とした。さらに蛍光顕微鏡観察を行いて、細胞内の顆粒状構造の形成を確認した。そして、上記蛍光強度測定によるフィラグリン生成量の増大と、顆粒状構造の形成という2つの条件を満たす被験物質をケラトヒアリン顆粒合成促進効果を有するものとして評価した。また、試験系が正常に機能しているかを確認するために、試料溶液の代わりに陽性対照として塩化カルシウム1.8mMを添加した場合についても、同様の試験を行った。
[成分] 部
オリーブ油 1.0
ポリオキシエチレン(5.5)セチルアルコール 5.0
ブチルパラベン 0.1
製造例1の抽出物 5.0
エタノール 5.0
グリセリン 5.0
1,3−ブチレングリコール 5.0
水酸化カリウム 適量
香料 適量
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の抽出物に代えて、製造例2の抽出物5.0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の抽出物に代えて、製造例3の抽出物5.0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
流動パラフィン 6.0
ヘキサラン 4.0
ホホバ油 1.0
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
大豆レシチン 1.5
製造例1の抽出物 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
ヒアルロン酸ナトリウム 0.01
香料 適量
精製水 全量が100部となる量
処方例4の成分中、製造例1の抽出物剤3.0に代えて、製造例2の抽出物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、製造例1の抽出物3.0に代えて、製造例3の抽出物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部に代えてトラネキサム酸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。
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。
[成分] 部
スクワラン 3.0
ベヘニルアルコール 3.0
ヘキサラン 4.0
ホホバ油 1.0
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
グリセリン脂肪酸エステル 1.0
大豆レシチン 1.5
製造例1の抽出物 5.0
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
グリチルリチン酸ジカリウム 0.1
グリセリン 3.0
1,3−ブチレングリコール 2.0
水溶性コラーゲン 0.1
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10の成分中、グリチルリチン酸ジカリウム1.0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
エタノール 10.0
グリセリン 3.0
1、3−ブチレングリコール 2.0
メチルパラベン 0.2
クエン酸 0.1
クエン酸ナトリウム 0.3
カルボキシビニルポリマー 0.1
キサンタンガム 0.1
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
製造例1の抽出物 10.0
米抽出物の加水分解物 2.0
米糠抽出物の加水分解物 1.0
香料 適量
精製水 全量が100部となる量
[成分] 部
エタノール 2.0
グリセリン 5.0
1、3−ブチレングリコール 5.0
メチルパラベン 0.1
ヒアルロン酸 0.1
製造例2の抽出物 5.0
クエン酸 0.3
クエン酸ナトリウム 0.6
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3の抽出物 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1の抽出物 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
グリチルリチン酸ジカリウム 0.1
モノニトログアヤコールナトリウム 0.02
塩酸ピリドキシン 0.03
l−メントール 0.8
タマサキツヅラフジ根エキス 0.3
褐藻エキス 0.3
オタネニンジンエキス 0.3
ゲンチアナエキス 2.0
製造例1の抽出物 3.5
トリメチルグリシン 0.5
乳酸 0.2
1、3−ブチレングリコール 10.0
フェノキシエタノール 0.2
ポリオキシエチレン硬化ヒマシ油 0.4
L−アルギニン 適量
エタノール 20.0
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 3.2
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の抽出物 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 (1)
- 皮膚の状態を改善する物質の評価方法であって、細胞に被験物質を投与する工程と、被験物質を投与した細胞におけるケラトヒアリン顆粒の合成を測定する工程とを含み、ケラトヒアリン顆粒の合成を測定する工程は、被験物質を投与した細胞におけるフィラグリン生成量を測定する工程と、被験物質を投与した細胞内の顆粒状構造を確認する工程とを含むことを特徴とする評価方法。
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JP6437297B2 (ja) * | 2013-12-19 | 2018-12-12 | 日本メナード化粧品株式会社 | 特定の波長域を有する光を照射して栽培したセージの抽出物を含有する皮膚外用剤や内用剤 |
JP6362138B2 (ja) * | 2014-09-22 | 2018-07-25 | 御木本製薬株式会社 | 皮膚外用剤、皮膚角化促進剤、タイトジャンクション強化剤、皮膚バリア機能強化剤、トランスグルタミナーゼ遺伝子発現促進剤、ロリクリン遺伝子発現促進剤、フィラグリン遺伝子発現促進剤、インボルクリン遺伝子発現促進剤、ケラチン遺伝子発現促進剤、クローディン遺伝子発現促進剤、オクルディン遺伝子発現促進剤。 |
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