JP2007514695A - フラバノン類を含んでいる皮膚、毛髪及び外皮の健康を改善するための組成物 - Google Patents
フラバノン類を含んでいる皮膚、毛髪及び外皮の健康を改善するための組成物 Download PDFInfo
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- JP2007514695A JP2007514695A JP2006544359A JP2006544359A JP2007514695A JP 2007514695 A JP2007514695 A JP 2007514695A JP 2006544359 A JP2006544359 A JP 2006544359A JP 2006544359 A JP2006544359 A JP 2006544359A JP 2007514695 A JP2007514695 A JP 2007514695A
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- skin
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- hair
- flavanone
- milk
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Abstract
Description
生き物における最も重要な上皮組織は皮膚で、皮膚は生物の中で最も大きな組織であるといえる。表皮、真皮及び角質層を含む皮膚外皮の系は内部器官の系と相関関係を有するとともに外界と相互に作用している。環境及び生物の間の接触面でありながら、当該皮膚は外部因子群とともに、生物内部系の種々なパラメーター群に非常に影響を受ける。当該皮膚の調節機構はそれ故に、皮膚外皮の形態及び働きに関する通常の病的事象を保守するために必要な全身的変化を誘導するために常に活動的である必要がある。
第1の態様では、本発明はヒト又はペットのための栄養、美容又は医薬組成物に関し、それらは少なくとも活性化合物として、1種のフラバノン化合物又はその誘導体群或いはそれらの混合物を、皮膚、毛髪及び/又は外皮の不調を防止又は治療及び皮膚、毛髪及び外皮の健康を改善するのに十分な量を含有する。
第1の目的によれば、当該発明はヒト又はペットに経口投与するための栄養、美容又は医薬組成物を提供することで、これは活性化合物、少なくとも1種のフラバノン化合物又はその誘導体或いはそれらの混合物を皮膚、毛髪及び/又は外皮の不調或いは損傷を軽減する効果量を含有しており、これにより皮膚、毛髪及び外皮の健康を改善する。
平均消費量が1日当たり1リットルと推定して、ミネラルウォーターにヘスペリジンをリットル当たり0.01mgから200mgを加えて調製した。
トリ肉、ブタ肺及びウシ肝臓(挽肉)を73%、小麦粉16%、水7%、染料2%、香味、ビタミン類及び無機塩類から混合物を調製する。この混合物を12℃で乳化し、腸詰の形態に押出して、これをその後90℃で調理する。これを30℃に冷却し、厚切りにする。厚切り45%を、水98%、染料1%及びグアガム1%で調製したソース55%と混合する。ブリキ缶に充填し、125°で40分間滅菌する。
栄養補助食品をフラクトオリゴサッカライドとイヌリンにつき、重量で約70%のフラクトオリゴサッカライド及び約30%のイヌリンの比率とし、ヘスペレチン当量で500mgを加えて混合するか混和して調製した。得られたプレバイオティック混合物はいずれかの適切な担体、例えば発酵乳、ヨーグルト、生チーズ、レンネット処理乳、菓子バー、朝食用シリアルフレーク類又はバー類、飲料、粉乳、大豆を基にした製品、非乳発酵製品或いは臨床栄養用の栄養補助食品に混合又は混和することができる。
細胞毒評価
ヒト不死化ケラチノサイト(HaCaT)を10μMヘスペレチン、10μMヘスペレチン‐7‐O‐グルクロナイド或いは陰性対照としてDMSOの等量とともに16時間、誘発の1時間前まで培養した。細胞群はその後、細胞内で反応性酸素種を生成する生物異物である100μMのメナジオンで処理した。非メナジオン処理細胞群を陽性対照として使用した。5時間後当該上澄液につき細胞死に関する尺度として乳酸デヒドロゲナーゼ(LDH)活性をCytoTox96非放射性細胞毒性評価法(Promega,米国)を用いて分析した。
ラットの皮膚生検をヘスペリジンでの成長試行(図2)から採取した。真皮を切り取り、一部分を4%PFAで固定してパラフィン包埋し、一部分は凍結保存し、他の部分を直ちに液体窒素中で凍結した。
パラフィン切片
ラット皮膚を切断し、4%のパラアルデヒドPBS液(pH7.4)中に4日間固定し、Leica Microsysteme包埋装置を用いてパラフィン中に包埋した。当該組織をPBS及び生理食塩水(0.9%NaCl)で洗浄し、増加するエタノール濃度を伴う生理食塩水:30%、50%、70%、90%、99%、100%各々に30分間及び100%に更に1時間通過させて脱水した。組織試料をキシレン中で2回30分温置し、次いで2〜3時間及び3時間60℃でパラフィン蝋中に温置した。Leica Microtomeを用いて6μm厚さの切片に切断した。切片群をキシレン中で5分間脱蝋し、増加するエタノール濃度:100%、96%、90%、80%、70%及び50%エタノール各々に1分間通過させて脱水した。最終的に、切片群は蒸留水中に移し、染色した。
再水和した切片をメイヤーのヘマトキシリン溶液中で45秒間染色し、以下の一連の溶液:蒸留水、水道水、蒸留水及び70%エタノールで各々1分間洗浄した。エオシン溶液(90%エタノール中で1%(容量/容量))中で10秒間染色した後、切片群を90%及び100%エタノール中で洗浄した。キシレン中で2回10分間温置した後、Eukitとともにカバーガラスを載せ、室温で2時間空気乾燥した。
RNAを扱う実験に関する一般的方法
RNAに関する実験では、滅菌プラスチック又は熱処理ガラス容器(180℃で少なくとも8時間)を用いた。ピペットマンを含む全ての表面は使用前にRNase ZAPで清浄化し、Aerosol resistant tipsのみを使用した。
ABI PRISM(登録商標)7000HT Sequence Detection System,Applied Biosystems,USA
ABI PRISM(登録商標)7000RT‐PCR software,Applied Biosystems,USA
PCRサイクラー(Cycler),例えばPTC‐100 Programmable Thermal Controller,MJ Research Inc.,USA
Agilent 2100 bioanalyzer,Agilent Technologies,USA
蛍光プレートリーダー(Fluorescence Plate Reader),例えばSpectra Flour Plus F129005,Tecan,USA
Multifuge 3S,Heraeus with special buckets for MFC centrifugation,Kendro Laboratory Products,Switzerland
冷却遠心分離機(Cooling Centrifuge),例えばCentrifuge 5417R,Eppendorf,Germany
Totally RNA Kit(Art.No.1910),Ambion,USA
Lysing Matrix D(Art.No.6913‐100)、Q BIOgene,France
RNA 6000 Nano Assay(Art.No.5065‐4475 and 5065‐4476)、Agilent Technologies,USA
Assays‐on‐demand(20x stock,Applied Biosystems,USA)
RNase ZAP(Art.No.9780),Ambion,USA
ヌクレアーゼフリー水(Nuclease‐free water)(ddH2O,Art.No.9939),Ambion,USA
Milli‐Qろ過水(filtered water)(0.22μM,ddH2O)
エタノール,分析用(Art.No.02860),Fluka
ダルベッコリン酸緩衝生理食塩水(PBS,Art.No.D8537),Sigma
β‐メルカプトエタノール(Art.No.M7522),Sigma,USA
RiboGreen RNA Quantitation Kit(Art.No.R‐11490),Molecular Probes,USA
SUPERase‐In RNase阻害剤(Inhibitor)(20U/μl,Art.No.2694),Ambion,USA
SuperScript II RNase H−逆転写酵素(reverse transcriptase)(200U/μl,Art.No.18064−014),Invitrogen,USA
First‐strand緩衝液(buffer)(5x):250mM NaCl,0.1mM EDTA,1mM DTT,0.1%(v/v)NP‐40,50%(v/v)グリセロール,SuperScriptII RNaseH−逆転移酵素を含む
ジチオスレイトール(DTT,1mM),SuperScriptII RNase H−逆転移酵素を含む
2’‐デオキシアデノシン‐5’‐トリホスフェート(dATP,100mM,Art,No.272050)、Amersham Biosciences,England
2’‐デオキシシチジン‐5’‐トリホスフェート(dCTP,100mM,Art,No.272060)、Amersham Biosciences,England
2’‐デオキシグアノシン‐5’‐トリホスフェート(dGTP,100mM,Art,No.272070)、Amersham Biosciences,England
2’‐デオキシチミジン‐5’‐トリホスフェート(dTTP,100mM,Art,No.272080)、Amersham Biosciences,England
pd(N)6ランダム6量体(Random hexamer)(Art.No.27‐2166‐01),Amersham Biosciences,England
TaqMan Universal PCR Master Mix(Art.No.PN4304437),Applied Biosystems,USA
皮膚検体をLysing Matrix Dにてホモジナイズし、製造業者の使用説明書に従って全RNAをTotally RNA Kitを用いて抽出した。RNAはヌクレアーゼフリーの水40μlで溶出した。
当該定量は96穴プレートにてRibogreen(登録商標)RNA定量キット及び手動による蛍光マイクロプレートリーダーを用いて行った。当該検体群は最終容量100μl 1xTE緩衝液中に1:680又は1:3400のいずれかに希釈した。1、0.5、0.1、0.02、0μg/mlの濃度のリボソームRNA希釈液を標準として使用した。RNA 6000 Nano Assayを用いて1μlのRNAの完全性を調整した。
全ての操作は氷上で行った。2μlのpd(N)6ランダム6量体及び1μlのdNTP(10mM)を最終容量12μlのヌクレアーゼフリー水中のRNA2μgに添加した。65℃で5分間温置した後、検体を氷上に置いて、素早く遠心分離した。その後、4μlの5Xファーストストランドバッファー、2μlのジチオスレイトール、1μlのRNase阻害剤及び1μlの逆転写酵素のSuperScript II RNase H−を加えた(最終容量20μl)。当該逆転写反応は以下の温度プログラムを用いてPCRサイクラー中で実施した:当該酵素の阻害:25℃で10分間;逆転写反応:42℃で60分間;当該酵素の阻害:70℃で20分間。その後当該検体は20℃の冷蔵庫中にて使用するまで保存した。
当該実時間PCRをTaqMan(登録商標)法により96穴プレート(96WP)においてAssay‐on‐demandプライヤー及びプローブを用いて実施した。分析は43.7μlのTaqMan(登録商標)2x Universal PCRマスターミックス、4.4μlのAssay‐on‐demandプライヤー及びプローブ、21.9μlのヌクレアーゼフリー水及び17.5μlのcDNA(87.5ng=複製当たり25ng)を含有するマスターミックス(3.5x)を用いて3回行った。3通りの25μlのマスターミックスを96穴のABI Prism反応プレートに負荷し、透明なOptical adhesive coverで蓋をして3回2000rpmで1分間遠心分離或いは全ての空気泡が除かれるまで遠心分離した。当該PCR反応はその後以下の温度プログラムを用いてABI Prism(登録商標)7000 Sequence Detection Systemにおいて実施した:当該酵素の活性化を:50℃で2分間;変性を:95℃で10分間、及び40循環の標的増幅を:95℃で15分間のアニーリングと60℃で1分間延長。当該増幅プロットの分析はABI PRISM(登録商標)ソフトウェアーを用いて行った。ベースラインの調整は個々に行ったが(Il6:15〜25、Cd1d1:10〜20、Pcna:15〜25;Gapd:6〜15)、閾値は手動で全てのプライマーにつき0.2とした。当該得られたCtは更に分析するためにMicrosoft Excelに移した。
データはANOVAで分析した。
固定化ケラチノサイト(HaCat)を用いた生体外実験では、ヘスペレチン(hp)及びヘスペレチン‐7‐O‐グルクロニド(hp‐7‐O‐gluc)による処理は通常の培養条件下で細胞死を低減させる。hp及びhp‐7‐O‐glucの保護効果は反応性酸素種(ROS)の細胞内レベルを増加させる生体異物であるメナジオンで攻撃された細胞にてより顕著であった。更に、血液中でヘスペリジンの主な代謝物であるhp‐7‐O‐gluはそのアグリコンであるhpと比べてより強力である(図1)。
Claims (16)
- 活性成分として、少なくとも1種のフラバノン化合物又はその誘導体類或いはそれらの混合物を皮膚、毛髪及び/又は外皮の傷害を防止、緩和又は治療するのに十分な量を含有する、経口投与用の栄養、美容又は医薬組成物。
- 当該フラバノン化合物群がイソサクラネチン、ナリンジン、ヘスペリジン、エリオジクチオール、ポンシリン、ネオエリオシトリンである、請求項1記載の組成物。
- 当該誘導体群は当該アグリコン、カルコン、当該フラバノンのグルコシル化型又はメチル化型及び血液中におけるそれらの代謝生成物であるサルフェート化型及びグルクロン酸抱合型である、請求項1記載の組成物。
- 当該誘導体は酵素処理で得られるか又は合成的に作られる、請求項1記載の組成物。
- 当該フラバノン化合物はオレンジ、レモン、ダイダイ又はグレープフルーツなどの柑橘類果実の果肉又は果皮から抽出されるものである、請求項1から4のいずれかに記載の組成物。
- 更にビタミンC、ビタミンE、カロテノイド類、ユビキノン類、カテキン類、ポリフェノール類及び/又はジテルペン類を含有しているコーヒーエキス類、チコリーの抽出物類、イチョウ葉エキス類、プロアントシアニジン類に富むブドウ又はブドウ種子エキス類、スパイスエキス類、大豆エキス類、抗酸化活性を持つフラボノイド類の他の源、脂肪酸類、プレバイオティック繊維、プロバイオティック微生物、タウリン、レスベラトロール、アミノ酸類、セレン又はグルタチオンの前駆体のような他の活性化合物群を含有する、請求項1から5のいずれかに記載の組成物。
- 乳、ヨーグルト、カード、チーズ、発酵乳、乳に基づく発酵製品、アイスクリーム類、乳に基づく粉末類、特別調整粉乳、シリアル製品群、発酵シリアルに基づく製品群、ミネラルウォーター、チョコレート又はペットフード、栄養補助食品、錠剤或いは美容用製品である、請求項1から5のいずれかに記載の組成物。
- 皮膚、毛髪及び/又は外皮の障害或いは損傷を防止、緩和又は治療することを意図する組成物の調製用の活性成分として、少なくとも1種のフラバノン化合物又はその誘導体或いはそれらの混合物の使用。
- ヒト又はペット動物に投与して皮膚、毛髪及び/又は外皮の状態の改善を意図する組成物の調製用の活性成分として、少なくとも1種のフラバノン化合物又はその誘導体或いはそれらの混合物の使用。
- 当該フラバノン化合物群が、イソサクラネチン、ナリンジン、ヘスペリジン、エリオジクチオール、ポンシリン、ネオエリオシトリンであるか、或いはそれらのアグリコン、カルコン、グリコシレート化若しくはメチル化型、又はそれらの血液中での代謝生成物であるサルフェート化及びグルクロン酸抱合型から選択される誘導体である、請求項8又は9記載の使用。
- 当該フラバノン化合物は、オレンジ、レモン、ダイダイ又はグレープフルーツのような柑橘類果実の果肉又は果皮から抽出したものである、請求項8から10のいずれかに記載の使用。
- 当該組成物が美容、栄養又は医薬組成物である、請求項8から11のいずれかに記載の使用。
- 当該フラバノン化合物類、その誘導体類又はそれらの混合物が当該フラバノン化合物のアグリコン当量で0.01mgから1g、好ましくは0.1mgから800mgが存在する、請求項8から12のいずれかに記載の使用。
- 皮膚の障害又は損傷が、加齢又は化学的、生物的又は物理的ストレスのようなストレス状態、オキシダント又は発癌物質、細菌類、ウイルス類、真菌類、周辺細胞及び/又は微生物由来の脂質への曝露、又はUV照射への曝露により生じるものである、請求項8から13のいずれか記載の使用。
- 当該組成物は皮膚の光防護、水和、乾燥、硬さ、弾性、脂性、厚さ、正常な色素沈着、バリヤー機能、皮膚弾性を改善し、酸化状態、癌の危険性、炎症を防ぎ、或いは皮脂産生又は皮脂組成を調整し、及び/又は加齢の兆候を軽減することを意図するものである、請求項8から14のいずれかに記載の使用。
- 当該組成物は毛髪及び外皮の光沢、毛髪の密度、色、脂性を改善し、毛髪繊維の直径、皮脂産生を改良し、そして毛髪及び外皮の損失を防ぐことを意図する、請求項8から14のいずれかに記載の使用。
Applications Claiming Priority (2)
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EP03029183 | 2003-12-18 | ||
PCT/EP2004/014416 WO2005058255A1 (en) | 2003-12-18 | 2004-12-17 | Composition for improving skin, hair and coat health containing flavanones |
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JP2007514695A true JP2007514695A (ja) | 2007-06-07 |
JP2007514695A5 JP2007514695A5 (ja) | 2011-07-21 |
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JP2006544359A Pending JP2007514695A (ja) | 2003-12-18 | 2004-12-17 | フラバノン類を含んでいる皮膚、毛髪及び外皮の健康を改善するための組成物 |
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US (1) | US9717671B2 (ja) |
EP (1) | EP1750651B1 (ja) |
JP (1) | JP2007514695A (ja) |
KR (1) | KR20060123336A (ja) |
CN (1) | CN100553632C (ja) |
AR (1) | AR047854A1 (ja) |
AU (1) | AU2004298356A1 (ja) |
BR (1) | BRPI0417723A (ja) |
CA (1) | CA2549509C (ja) |
ES (1) | ES2658344T3 (ja) |
MX (1) | MXPA06006773A (ja) |
NO (1) | NO20063200L (ja) |
PL (1) | PL380446A1 (ja) |
PT (1) | PT1750651T (ja) |
RU (1) | RU2355378C2 (ja) |
TW (1) | TW200528134A (ja) |
WO (1) | WO2005058255A1 (ja) |
ZA (1) | ZA200605888B (ja) |
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MXPA06006773A (es) | 2006-09-04 |
PL380446A1 (pl) | 2007-02-05 |
CN1893909A (zh) | 2007-01-10 |
EP1750651A1 (en) | 2007-02-14 |
TW200528134A (en) | 2005-09-01 |
WO2005058255A1 (en) | 2005-06-30 |
ES2658344T3 (es) | 2018-03-09 |
US20070129428A1 (en) | 2007-06-07 |
AR047854A1 (es) | 2006-03-01 |
RU2355378C2 (ru) | 2009-05-20 |
KR20060123336A (ko) | 2006-12-01 |
CN100553632C (zh) | 2009-10-28 |
EP1750651B1 (en) | 2017-11-29 |
AU2004298356A1 (en) | 2005-06-30 |
PT1750651T (pt) | 2018-02-16 |
CA2549509C (en) | 2014-07-22 |
CA2549509A1 (en) | 2005-06-30 |
RU2006125735A (ru) | 2008-01-27 |
BRPI0417723A (pt) | 2007-04-03 |
ZA200605888B (en) | 2008-04-30 |
US9717671B2 (en) | 2017-08-01 |
NO20063200L (no) | 2006-09-12 |
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