JP6324792B2 - Wnt発現抑制剤 - Google Patents
Wnt発現抑制剤 Download PDFInfo
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- JP6324792B2 JP6324792B2 JP2014080926A JP2014080926A JP6324792B2 JP 6324792 B2 JP6324792 B2 JP 6324792B2 JP 2014080926 A JP2014080926 A JP 2014080926A JP 2014080926 A JP2014080926 A JP 2014080926A JP 6324792 B2 JP6324792 B2 JP 6324792B2
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- wnt
- phalaenopsis
- extract
- expression
- seedling
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Description
(1)胡蝶蘭の抽出物を有効成分として含有するWnt発現抑制剤。
(2)Wntが、Wnt/β−カテニンシグナルに関与するWntである、(1)に記載のWnt発現抑制剤。
(3)胡蝶蘭の抽出物を有効成分として含有するメラニン合成抑制剤。
(4)胡蝶蘭が、胡蝶蘭の幼苗である、(1)〜(3)のいずれかに記載の剤。
(5)(1)〜(4)のいずれかに記載の剤を含む化粧品。
(6)(1)〜(4)のいずれかに記載の剤を含む医薬品または医薬部外品。
(7)皮膚外用製剤である、(6)に記載の医薬品または医薬部外品。
(8)(1)〜(4)のいずれかに記載の剤を含む飲食品。
本発明のWnt発現抑制剤は、胡蝶蘭の抽出物を有効成分として含有する。本発明でいう胡蝶蘭には、ファレノプシス属(Phalaenopsis)の各種、及びそれらと交配可能な近縁種であるドリティス属(Doritis)、ドリティノプシス属(Doritaenopsis)等の原種、その種間雑種あるいは属間雑種等が挙げられ、市販されている株を利用することができる。市販されている胡蝶蘭園芸品種の交配親の代表的品種としては、Phalaenopsis amabilis、Phalaenopsis aphrodite、Phalaenopsis schilleriana、Phalaenopsis lueddemanniana、Phalaenopsis equestris、Phalaenopsis intermedia等が挙げられる。また、ファレノプシス属以外の交配親の代表品種としては、ドリティス・プルケリマ(Doritis pulcherima)等が挙げられる。また、ファレノプシス属とドリティス属の交配種を、ドリティノプシス属と称し交配親として用いている。
胡蝶蘭の抽出物を以下のとおり製造した。製造例1〜6において抽出材料として用いた胡蝶蘭幼苗(フラスコ苗)は、葉の長径が3〜8cmの範囲であって、幼苗分化から花芽形成前のメリクロン苗をいう。
胡蝶蘭のフラスコ苗の葉の乾燥物10gを200mLの50%(V/V)エタノール水溶液に室温で4日間浸漬した。得られた抽出液を濾過した後エバポレーターで濃縮乾固して胡蝶蘭の50%エタノール抽出物を1.6g得た。
胡蝶蘭のフラスコ苗の根の乾燥物10gを200mLの50%(V/V)エタノール水溶液に室温で4日間浸漬した。得られた抽出液を濾過した後エバポレーターで濃縮乾固して胡蝶蘭の50%エタノール抽出物を1.7g得た。
胡蝶蘭のフラスコ苗全草の乾燥物10gを200mLの50%(V/V)エタノール水溶液に室温で4日間浸漬した。得られた抽出液を濾過した後エバポレーターで濃縮乾固して胡蝶蘭の50%エタノール抽出物を1.5g得た。
胡蝶蘭のフラスコ苗全草の乾燥物10gに200mLの水を加え、95〜100℃で2時間抽出した。得られた抽出液を濾過し、そのろ液を濃縮し、凍結乾燥して胡蝶蘭の熱水抽出物を2.3g得た。
胡蝶蘭のフラスコ苗全草の乾燥物10gを200mLの50%(V/V)1,3−BG水溶液に室温で5日間浸漬した。得られた抽出液を濾過して胡蝶蘭の1,3−BG抽出物を190g得た。
胡蝶蘭の成体全草の乾燥物10gを200mLの50%(V/V)エタノール水溶液に室温で4日間浸漬した。得られた抽出液を濾過した後エバポレーターで濃縮乾固して胡蝶蘭の50%エタノール抽出物を1.8g得た。
胡蝶蘭の抽出物の効果の評価実験を次のとおり行った。
20% ウシ胎児血清(FBS、ニチレイバイオ社製)を含有するDulbecco's Modified Eagle's Medium(DMEM、Sigma-Aldrich社製)を用いて培養したヒト扁平上皮がん細胞HSC-1(Wnt1発現細胞、JCRB細胞バンク製)を、2%FBS含有DMEMに懸濁し、8ウェルカルチャースライドに1x103個ずつ播種した。24時間培養後、胡蝶蘭抽出物(製造例1〜6)を最終濃度が0.01%になるように添加した2%FBS含有DMEMに交換し、さらに72時間培養した。細胞をPBS(-)にて2回洗浄し、4%パラホルムアルデヒドを加え、室温で10分間インキュベーションして細胞を固定した。
(1) セルカルチャーインサート内でのHSC-1の単独培養
24ウェルプレートの各ウェルに0.7mLの2%FBS含有DMEMを添加し、ポアサイズ0.4 μmのセルカルチャーインサートをその上からセットした。ヒト扁平上皮がん細胞HSC-1を、2%FBS含有DMEMに懸濁し、セルカルチャーインサートに4x104個ずつ播種した。このとき、対照としてHSC-1を含まない2%FBS含有DMEMを添加したものも用意した。24時間後、培地を胡蝶蘭の抽出物(製造例1〜6)を最終濃度が0.01%になるように添加した2%FBS含有DMEMに交換し、さらに72時間培養した。
Medium254に1%となるようにhuman melanocyte growth supplement(Life Technologies社製)を添加した培地(Medium254+)にて培養した継代数1のヒト正常メラノサイト(NHEM、TOYOBO社製)を、Medium254+に懸濁し、24ウェルプレートに4x104個ずつ播種し、72時間培養した。
(2)で用意したNHEMの培地を除き、新しいMedium254を各ウェルに0.7mLずつ添加した。(1)で用意したセルカルチャーインサート内の培地を除き、NHEMを培養した24ウェルプレートにセットし、新しい2%FBS含有DMEMを各セルカルチャーインサートに0.2mLずつ添加し、NHEMとHSC-1を72時間共培養した。
NHEMとHSC-1の共培養後、セルカルチャーインサートと培地を除き、NHEMをPBS(-)にて2回洗浄した後、Trizol Reagent(Invitrogen社製)によって細胞からRNAを抽出した。2-STEPリアルタイムPCRキット(Applied Biosystems社製)を用いて、抽出したRNAをcDNAに逆転写した後、ABI7300(Applied Biosystems社製)により、下記のプライマーセットを用いてリアルタイムPCR(95℃:15秒間、60℃:30秒間、40cycles)を実施し、メラニン合成関連遺伝子(Mitf、Dct、Tyr、Tyrp1)の発現を確認した。その他の操作は定められた方法に従って実施した。
5'-GCAGTGGTTTGGGCTT-3' (配列番号1)
5'-AATTCTGCACCCGGGAATC-3' (配列番号2)
Dct用プライマーセット:
5'-GGAATGCTTTGGAAGGGTTTG-3' (配列番号3)
5'-AAAGCGTTTGTCCCGTTCAG-3' (配列番号4)
Tyr用プライマーセット:
5'-TGCGGTGGGAACAAGAAATC-3' (配列番号5)
5'-GAAGAATGATGCTGGGCTGAGT-3' (配列番号6)
Tyrp1用プライマーセット:
5'-CGAAACACAGTGGAAGGTTACAGT-3' (配列番号7)
5'-CTCCTCAGCCATTCATCAAAGACT-3' (配列番号8)
18srRNA(内部標準)用プライマーセット:
5'-CCGAGCCGCCTGGATAC-3' (配列番号9)
5'-CAGTTCCGAAAACCAACAAAATAGA-3'(配列番号10)
製造例1〜6で製造した胡蝶蘭の抽出物を配合した製品の処方例を以下に示す。以下の処方例において「胡蝶蘭の抽出物」は、製造例1〜6で製造した胡蝶蘭の抽出物のいずれかを意味する。
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.1,3−ブチレングリコール 8.0
3.グリセリン 2.0
4.キサンタンガム 0.02
5.クエン酸 0.01
6.クエン酸ナトリウム 0.1
7.エタノール 5.0
8.パラオキシ安息香酸メチル 0.1
9.ポリオキシエチレン硬化ヒマシ油(40E.O.) 0.1
10.香料 0.1
11.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.スクワラン 5.5
3.オリーブ油 3.0
4.ステアリン酸 2.0
5.ミツロウ 2.0
6.ミリスチン酸オクチルドデシル 3.5
7.ポリオキシエチレンセチルエーテル(20E.O.) 3.0
8.ベヘニルアルコール 1.5
9.モノステアリン酸グリセリン 2.5
10.香料 0.1
11.パラオキシ安息香酸メチル 0.2
12.パラオキシ安息香酸エチル 0.05
13.1,3−ブチレングリコール 8.5
14.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.スクワラン 5.0
3.オリーブ油 5.0
4.ホホバ油 5.0
5.セタノール 1.5
6.モノステアリン酸グリセリン 2.0
7.ポリオキシエチレンセチルエーテル(20E.O.) 3.0
8.ポリオキシエチレンソルビタン
モノオレエート(20E.O.) 2.0
9.香料 0.1
10.プロピレングリコール 1.0
11.グリセリン 2.0
12.パラオキシ安息香酸メチル 0.2
13.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.エタノール 5.0
3.パラオキシ安息香酸メチル 0.1
4.ポリオキシエチレン硬化ヒマシ油 0.1
5.香料 適量
6.1,3−ブチレングリコール 5.0
7.グリセリン 5.0
8.キサンタンガム 0.1
9.カルボキシビニルポリマー 0.2
10.水酸化カリウム 0.2
11.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 2.0
2.ポリオキシエチレンセチルエーテル(30E.O.) 2.0
3.モノステアリン酸グリセリン 10.0
4.流動パラフィン 5.0
5.セタノール 6.0
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 10.0
8.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.ポリビニルアルコール 12.0
3.エタノール 5.0
4.1,3−ブチレングリコール 8.0
5.パラオキシ安息香酸メチル 0.2
6.パラオキシエチレン硬化ヒマシ油(20E.O.) 0.5
7.クエン酸 0.1
8.クエン酸ナトリウム 0.3
9.香料 適量
10.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 1.0
2.ステアリン酸 2.4
3.ポリオキシエチレンソルビタンモノステアレート(20E.O.) 1.0
4.ポリオキシエチレンセチルエーテル(20E.O.) 2.0
5.セタノール 1.0
6.液状ラノリン 2.0
7.流動パラフィン 3.0
8.ミリスチン酸イソプロピル 6.5
9.パラオキシ安息香酸ブチル 0.1
10.カルボキシメチルセルロースナトリウム 0.1
11.ベントナイト 0.5
12.プロピレングリコール 4.0
13.トリエタノールアミン 1.1
14.パラオキシ安息香酸メチル 0.2
15.二酸化チタン 8.0
16.タルク 4.0
17.ベンガラ 1.0
18.黄酸化鉄 2.0
19.香料 適量
20.精製水 残量
処方 配合量(重量%)
1.胡蝶蘭の抽出物 5.0
2.乾燥コーンスターチ 25.0
3.カルボキシメチルセルロースカルシウム 20.0
4.微結晶セルロース 40.0
5.ポリビニルピロリドン 7.0
6.タルク 3.0
処方 配合量(重量%)
1.胡蝶蘭の抽出物 0.1
2.ステビア 0.05
3.リンゴ酸 5.0
4.香料 0.1
5.精製水 残量
Claims (7)
- 胡蝶蘭のメリクロン苗の抽出物を有効成分として含有するWnt発現抑制剤。
- Wntが、Wnt/β−カテニンシグナルに関与するWntである、請求項1に記載のWnt発現抑制剤。
- 胡蝶蘭のメリクロン苗の抽出物を有効成分として含有するメラニン合成抑制剤。
- 胡蝶蘭のメリクロン苗の抽出物を含むWnt発現抑制用化粧品。
- 胡蝶蘭のメリクロン苗の抽出物を含むWnt発現抑制用医薬品または医薬部外品。
- 皮膚外用製剤である、請求項5に記載のWnt発現抑制用医薬品または医薬部外品。
- 胡蝶蘭のメリクロン苗の抽出物を含むWnt発現抑制用飲食品。
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