JP6393772B2 - 発毛及び育毛促進剤並びにそれらの利用 - Google Patents
発毛及び育毛促進剤並びにそれらの利用 Download PDFInfo
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- JP6393772B2 JP6393772B2 JP2016556224A JP2016556224A JP6393772B2 JP 6393772 B2 JP6393772 B2 JP 6393772B2 JP 2016556224 A JP2016556224 A JP 2016556224A JP 2016556224 A JP2016556224 A JP 2016556224A JP 6393772 B2 JP6393772 B2 JP 6393772B2
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Description
例えば、特定の配列を有する大豆タンパク由来のペプチドを有効成分として含む抗脱毛剤が知られている(特許文献1)。
〔1〕卵黄タンパク質加水分解物を有効成分として含有することを特徴とする発毛促進剤。
〔2〕毛乳頭細胞の増殖因子産生を促進させる作用を有することを特徴とする〔1〕に記載の剤。
〔3〕増殖因子が血管内皮細胞増殖因子(VEGF)、線維芽細胞増殖因子−7(FGF−7)及びインスリン様成長因子−1(IGF−1)からなる群より選択される1以上であることを特徴とする〔2〕に記載の剤。
〔4〕経口剤であることを特徴とする〔1〕〜〔3〕のいずれかに記載の剤。
〔5〕発毛を促進するための医薬であって、〔1〕〜〔3〕のいずれかに記載の剤を含む医薬。
〔6〕発毛を促進するためのサプリメントであって、〔1〕〜〔3〕のいずれかに記載の剤を含むサプリメント。
〔7〕発毛を促進するための食品添加剤であって、〔1〕〜〔3〕のいずれかに記載の剤を含む食品添加剤。
〔8〕発毛促進剤を製造するための卵黄タンパク質加水分解物の使用。
〔9〕卵黄タンパク質加水分解物を、発毛を促進することが必要なヒトに経口的に摂取させることを特徴とする非治療的な発毛促進方法。
〔10〕〔1〕〜〔3〕のいずれかに記載の剤の有効量を、発毛を促進することが必要な動物に投与することを特徴とする発毛促進方法。
〔11〕発毛促進のための〔1〕〜〔3〕のいずれかに記載の剤の使用。
〔12〕発毛促進のために使用する〔1〕〜〔3〕のいずれかに記載の剤。
〔13〕発毛促進をコンセプトとする旨が表示されている〔1〕〜〔3〕のいずれかに記載の剤を含む特定保健用食品、サプリメント、栄養補助食品または機能性食品。
卵黄タンパク質加水分解物は、卵黄タンパク質を加水分解して得られるものであればよい。卵黄タンパク質加水分解物の原料として用いる卵黄は、鶏、アヒル、うずら等の卵黄が挙げられるが、生産性の点から鶏の卵黄が好ましく用いられる。卵黄は、卵黄液、卵黄粉末、脱脂卵黄粉末等を用いることができ、中でも卵黄粉末、脱脂卵黄粉末が好ましく用いられる。また、資源の有効利用やコストの点から、卵黄から卵黄油や卵黄レシチンを製造する際に副産物として生じる脱脂卵黄を用いることが好ましい。卵黄の脱脂処理は、卵黄に食品加工に使用可能な有機溶媒(例えば、エタノール、イソプロパノール、ヘキサンなどの少なくとも一種)を作用させて実施するのが好ましい。通常は、卵黄にこれらの溶媒を添加して、攪拌して生成する固形物を採取することにより行われる。この操作を複数回繰り返してもよい。簡便性と安全性の点からエタノールが好ましく用いられる。
得られた卵黄タンパク質加水分解物は、適宜脱塩してそのまま用いることができる。また、限外ろ過膜、ゲルろ過や各種カラムクロマトグラフィー、メンブレンフィルター、等電点を利用した方法などで精製や分画して用いてもよい。精製、分画後の卵黄タンパク質加水分解物が発毛促進作用を有することは、例えば、実施例2、3に記載の方法等で確認することができる。
また、より好ましい卵黄タンパク質加水分解物としては、分子量1,000の限外濾過膜を用いた分画により得られた卵黄タンパク質加水分解物であって、ゲルろ過クロマトグラフィーによる分子量分布分析において、タンパク質・ペプチド・アミノ酸の合計の面積比に対して、分子量約500以上約20,000以下の部分の面積比が約85%以上、好ましくは約90%以上を占める卵黄タンパク質加水分解物が挙げられる。
本発明は、発毛促進のための本発明の発毛促進剤の使用を含む。
本発明は、発毛促進のために使用する本発明の発毛促進剤を含む。
(1)脱脂卵黄の調製
卵黄粉末1kgにエタノール5Lを加え、ブレンダーで30分間攪拌したのち固形物を回収した。この操作を3回繰り返して卵黄から脱脂を行い、風乾し、568gの脱脂卵黄粉末を得た。
(2)卵黄タンパク質加水分解物の調製
(1)で得られた脱脂卵黄粉末500gに水2.5kg及びノボザイムズ社製のアルカラーゼ(商品名、Bacillus licheniformis 由来のプロテアーゼ)25gを加え、pHを7にあわせて、55℃で3時間酵素反応させ、その後、80℃で15分間の熱処理で酵素を失活させ、3000×gで20分間遠心分離し、不溶物を除去し、ろ過後、ろ液をスプレー乾燥して、約140gの卵黄タンパク質加水分解物を得た。
カラム:Diol 60(6.0×300mm)(商品名、ワイエムシイ社製)
溶出液:0.2Mリン酸カリウム緩衝液、0.2M NaCl(pH6.9)/アセトニトリル(70:30)
流速:0.7ml/分
検出波長:280nm
分子量分析の結果を図1に示した。図1から、実施例1の卵黄タンパク質加水分解物はタンパク質・ペプチド・アミノ酸の合計を示す全面積に対して、分子量100以上20,000以下の部分の面積比が約90%を占めることが明らかとなった。
マウスは、日本エスエルシー株式会社が取り扱うSPF C3H/HeN Slc(雄性)を使用した。各試験群の平均体重がなるべく均等になるよう無作為に個体を振り分けた。マウスの飼育条件は1ケージあたり5匹、室温25℃、明暗12時間サイクルとした。
6週齢のマウスを購入した後、1週間の予備飼育を行い、試験環境に馴化させた。7週齢となったマウスの背部被毛を安全カミソリで剃毛した。
試験開始前において、飼料は固型飼料CRF−1(オリエンタル酵母工業株式会社)を、飲料水は公共水道水をそれぞれ自由に摂取させた。
最終観察日(17日目)には全試験群のマウスを麻酔後に安楽死させて撮影台に固定し、剃毛部を撮影した。撮影した画像から画像解析ソフトを用いて被毛再生部の面積を定量化し、発毛促進効果の有無を判定した。
(1)血管内皮細胞増殖因子(VEGF)産生量の測定
対数増殖期にあるヒト頭髪毛乳頭細胞(HFDPC、Cell Applications, Inc.)を毛乳頭細胞増殖培地(PCGM、Cell Applications, Inc.)で3×104cells/mlに調製し、1mlを24−well plate(collagen−coated)に植え継いで前培養を行った。
顕微鏡観察によって細胞の生育状況を確認した後、培養液全量を交換した。
培養液中に各サンプルを添加してインキュベートを開始した。この際、卵黄タンパク質加水分解物をPBS(−)に100mg/ml又は500mg/mlとなるように溶解し、それぞれ培養液全量に対して1%となるように添加した(最終濃度1mg/ml又は5mg/ml)。また、VEGFを産生促進するモデル薬剤として、ミノキシジル(Sigma-Aldrich Co. LLC.)をエタノールに3mMとなるように溶解し、培養液全量に対して1%となるように添加した(最終濃度30μM)。
2日間のインキュベート後、Human VEGF ELISAキット(R&D SYSTEMS)を用いて培養液中のVEGF濃度を測定した。
対数増殖期にあるヒト頭髪毛乳頭細胞をDMEM培地(10%血清)で3×104cells/mlに調製し、1mlを24−well plate(collagen−coated)に植え継いで前培養を行った。
培養液全量を除いて無血清DMEM培地に置換した。その後、速やかに各サンプルを培養液中に添加し、CO2インキュベーター内で2時間インキュベートした。この際、卵黄タンパク質加水分解物をPBS(−)に100mg/mlとなるように溶解し、培養液全量に対して1%となるように添加した(最終濃度1mg/ml)。また、FGF−7の発現誘導剤として、アデノシン(和光純薬工業株式会社製)をDMSOに10mMとなるように溶解し、培養液全量に対して1%となるように添加した(最終濃度100μM)。
培養液全量を除き、速やかに細胞を1mlのISOGEN2(ニッポンジーン)に溶解し、製造会社が推奨するプロトコルに従ってtotal RNAを精製した。これを鋳型とし、PrimeScript RT reagent kit (Takara)を用いて、製造会社が推奨するプロトコルに従ってcDNAを合成した。この反応液を鋳型とし、SYBR Premix EX Taqに(Takara)及びLightCycler 480(Roche)を用いて、それぞれ製造会社が推奨するプロトコルに従ってreal−time PCRを行った。この際使用したプライマーの塩基配列を表2に示した。
対数増殖期にあるヒト頭髪毛乳頭細胞をDMEM培地(10%血清)で3×104 cells/mlに調製し、1mlを24−well plate(collagen−coated)に植え継いで前培養を行った。
培養液全量を除いて無血清DMEM培地に置換した。その後、速やかにサンプルを培養液中に添加し、CO2インキュベーター内で4時間インキュベートした。この際、卵黄タンパク質加水分解物をPBS(−)に100mg/mlとなるように溶解し、培養液全量に対して1%となるように添加した(最終濃度1mg/ml)。また、IGF−1の発現誘導剤として、AdenosineをDMSOに10mMとなるように溶解し、培養液全量に対して1%となるように添加した(最終濃度100μM)。
培養液全量を除き、速やかに細胞を1mlのISOGEN2(ニッポンジーン)に溶解し、製造会社が推奨するプロトコルに従ってtotal RNAを精製した。これを鋳型とし、PrimeScript RT reagent kit (Takara)を用いて、製造会社が推奨するプロトコルに従ってcDNAを合成した。この反応液を鋳型とし、SYBR Premix EX Taqに(Takara)及びLightCycler 480(Roche)を用いて、それぞれ製造会社が推奨するプロトコルに従ってreal−time PCRを行った。この際使用したプライマーの塩基配列を表3に示した。
マウスは、日本チャールス・リバー株式会社が取り扱うC57BL/6(雌性)を使用した。各試験群の平均体重がなるべく均等になるよう無作為に個体を振り分けた。マウスの飼育条件は1ケージあたり5匹、室温25℃、明暗12時間サイクルとした。
6週齢のマウスを購入した後、1週間の予備飼育を行い、試験環境に馴化させた。7週齢となったマウスの背部被毛を市販の除毛クリームで脱毛した。
試験開始前において、飼料は固型飼料CRF−1(オリエンタル酵母工業株式会社)を、飲料水は公共水道水をそれぞれ自由に摂取させた。
最終観察日(投与9日目)には全試験群のマウスを麻酔後に安楽死させ、背部皮膚を電気シェーバーで剃毛し、正中線の両側の皮膚(耳の付け根より下側)を摘出した。摘出した皮膚を10%中性緩衝ホルマリン液に固定し、体軸に平行な4μmのパラフィン切片を作製してHE染色を行った。この際、各群について500個の毛包を観察し、成熟した成長期(Anagen VI)にある毛包の頻度を計測した。
マウスは、日本チャールス・リバー株式会社が取り扱うC57BL/6(雌性)を使用した。各試験群の平均体重がなるべく均等になるよう無作為に個体を振り分けた。マウスの飼育条件は1ケージあたり3匹、室温25℃、明暗12時間サイクルとした。
6週齢のマウスを購入した後、1週間の予備飼育を行い、試験環境に馴化させた。7週齢となったマウスの背部被毛を市販の除毛クリームで脱毛した。
試験開始前において、飼料は固型飼料CRF−1(オリエンタル酵母工業株式会社)を、飲料水は公共水道水をそれぞれ自由に摂取させた。
Claims (10)
- 卵黄タンパク質加水分解物を有効成分として含有することを特徴とする発毛促進剤。
- 毛乳頭細胞の増殖因子産生を促進させる作用を有することを特徴とする請求項1に記載の剤。
- 増殖因子が血管内皮細胞増殖因子(VEGF)、線維芽細胞増殖因子−7(FGF−7)及びインスリン様成長因子−1(IGF−1)からなる群より選択される1以上であることを特徴とする請求項2に記載の剤。
- 経口剤であることを特徴とする請求項1〜3のいずれかに記載の剤。
- 発毛を促進するための医薬であって、請求項1〜3のいずれかに記載の剤を含む医薬。
- 発毛を促進するためのサプリメントであって、請求項1〜3のいずれかに記載の剤を含むサプリメント。
- 発毛を促進するための食品添加剤であって、請求項1〜3のいずれかに記載の剤を含む食品添加剤。
- 発毛促進剤を製造するための卵黄タンパク質加水分解物の使用。
- 発毛促進のために使用する請求項1〜3のいずれかに記載の剤。
- 発毛促進をコンセプトとする旨が表示されている請求項1〜3のいずれかに記載の剤を含む発毛促進用特定保健用食品、発毛促進用サプリメント、発毛促進用栄養補助食品または発毛促進用機能性食品。
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