JP4084726B2 - コラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックampホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤、並びに化粧料及び飲食品。 - Google Patents
コラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックampホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤、並びに化粧料及び飲食品。 Download PDFInfo
- Publication number
- JP4084726B2 JP4084726B2 JP2003339171A JP2003339171A JP4084726B2 JP 4084726 B2 JP4084726 B2 JP 4084726B2 JP 2003339171 A JP2003339171 A JP 2003339171A JP 2003339171 A JP2003339171 A JP 2003339171A JP 4084726 B2 JP4084726 B2 JP 4084726B2
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- JP
- Japan
- Prior art keywords
- extract
- inhibitor
- platelet aggregation
- cyclic amp
- tyrosinase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Description
しかし、紫外線の照射、空気の乾燥、過度の皮膚洗浄等の外的要因や加齢により、線維芽細胞の増殖が遅くなり、皮膚の保湿性や弾力性等が低下する。その結果、皮膚は張りや艶を失い、肌荒れ、シワ等の老化症状を呈するようになる。
そこで、コラーゲンの合成を促進することや、線維芽細胞の増殖を促進することが前記老化症状への効果的な方法であると考えられ、例えば、コラーゲン合成促進作用や線維芽細胞増殖促進作用を有する物質を天然物から抽出することが試みられている(特許文献1、特許文献2、非特許文献1参照)。
そこで、この酵素チロシナーゼの作用を阻害することが、シミやそばかす等の防止に有効であると考えられ、チロシナーゼ阻害作用を有する物質を天然物から抽出することが試みられており、例えば、藤茶抽出物(特許文献3参照)、Saussurea属植物の抽出物(特許文献4参照)などが報告されている。
そこで、血小板凝集抑制作用を有する物質を天然物から抽出することが試みられており、例えば、藤茶枝葉部抽出物(特許文献5参照)などが提案されている。
そこで、サイクリックAMPホスホジエステラーゼ阻害作用を有する物質を天然物から抽出することが試みられており、例えば、藤茶抽出物(特許文献6参照)、カエデ属植物の抽出物(特許文献7参照)、などが報告されている。
<1> ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物を含むコラーゲン合成促進剤である。
<2> ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物を含む線維芽細胞増殖促進剤である。
<3> ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物を含むサイクリックAMPホスホジエステラーゼ阻害剤である。
<4> ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物を含むチロシナーゼ阻害剤である。
<5> ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物を含む血小板凝集抑制剤である。
<6> 前記<1>から<5>のいずれかに記載のマンゴージンジャーの抽出物を有効成分として含有する化粧料である。
<7> 前記<1>から<5>のいずれかに記載のマンゴージンジャーの抽出物を有効成分として含有する飲食品
本発明の各種製剤、即ち、コラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックAMPホスホジエステラーゼ作用抑制剤、チロシナーゼ阻害剤及び血小板凝集抑制剤は、マンゴージンジャーの抽出物を含み、更に必要に応じてその他の成分を含有してなる。
ここで、前記マンゴージンジャーの根茎部は、古くから胆汁症、皮膚病、気管支炎、損傷による炎症に効果があるとされ、インドでは調味料として利用されており、その他の効果としては、解熱作用、健胃作用、駆風作用等が確認されている。しかし、前記マンゴージンジャーの抽出物が、高いコラーゲン合成促進作用、線維芽細胞増殖促進作用、サイクリックAMPホスホジエステラーゼ阻害作用、チロシナーゼ阻害作用、及び血小板凝集抑制作用を有し、肌荒れ、シワ等の老化、肥満、シミ、そばかすの発生動脈硬化症などを防止し得る製剤として有用であることについては全く知られておらず、これらのことは本発明者らの鋭意研究に基づく新知見である。
前記水に施す処理としては、例えば、精製、加熱、殺菌、滅菌、ろ過、イオン交換、浸透圧の調整、緩衝化等が挙げられる。従って、本発明において抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。
なお、水と親水性有機溶媒との混合溶媒を使用する場合には、前記親水性有機溶媒として低級アルコールを用いる場合には、前記水と前記低級脂肪族アルコールとの質量比が70:30〜20:80になるように混合することが好ましい。前記親水性有機溶媒として低級脂肪族ケトンを用いる場合には、前記水と前記低級脂肪族ケトンとの質量比が10:1〜10:40になるように混合することが好ましい。前記親水性有機溶媒として多価アルコールを用いる場合には、前記水と前記多価アルコールの質量比が10:1〜10:90が好ましい。
具体的には、前記マンゴージンジャーの抽出方法としては、例えば、抽出溶媒を満たした処理槽に、前記抽出原料を投入し、必要に応じて適宜攪拌しながら、可溶性成分を溶出した後、濾過し、抽出残渣を除くことによって、抽出液を得ることができる。この際、抽出条件は、前記抽出原料等に応じて適宜調整し得るが、前記抽出溶媒量は、前記抽出原料としてのマンゴージンジャーに対して通常5〜15倍量(質量比)であり、抽出時間は通常1〜3時間であり、抽出温度は通常、常温〜95℃であることが好ましい。
また、得られた前記抽出液は、そのままでもコラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックAMPホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤のいずれかとして使用することができるが、前記濃縮液又はその乾燥物としたものの方が、利用しやすい点で好ましい。乾燥物を得るに当たって、吸湿性を改善するためにデキストリン、シクロデキストリン等のキャリアーを加えても良い。
このようにして得られるマンゴージンジャーの抽出物は、各種製剤、例えばコラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックAMPホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤として配合するのに好適である。
また、本発明の各種製剤は、高いコラーゲン合成促進作用、線維芽細胞増殖促進作用、サイクリックAMPホスホジエステラーゼ阻害作用、チロシナーゼ阻害作用、血小板凝集抑制作用を有すると共に、高い安全性を有し、以下の本発明の化粧料又は飲食品に好適に使用することができる。
本発明の化粧料は、本発明のコラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックAMPホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤のいずれかを配合してなり、更に必要に応じてその他の成分を配合してなる。
前記化粧料としては、例えば、軟膏、クリーム、乳液、ローション、パック、リップ、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディシャンプー等が挙げられ、特に、皮膚に対して使用するものが好ましい。本発明の各種製剤を前記化粧料に配合して、皮膚、頭皮等に吸収させることにより、各種作用、即ち、コラーゲン合成促進作用、線維芽細胞増殖促進作用、サイクリックAMPホスホジエステラーゼ阻害作用、チロシナーゼ阻害作用及び血小板凝集抑制作用を高めるのに役立たせることができる。
前記化粧料は、必要に応じて本発明の効果を損なわない範囲で、その化粧料の製造に通常使用される各種主剤及び助剤、その他成分を使用することができる。
本発明の飲食品は、本発明のコラーゲン合成促進剤、線維芽細胞増殖促進剤、サイクリックAMPホスホジエステラーゼ阻害剤、チロシナーゼ阻害剤、及び血小板凝集抑制剤の少なくともいずれかを配合してなり、更に必要に応じてその他の成分を配合してなる。
また、本発明の飲食品は飲料、ハードカプセル、ソフトカプセル、顆粒などの形状に形態に加工することにより簡便に飲食でき、広範囲に利用することが可能である。
前記原料又は添加物としては、特に制限はなく、目的に応じて適宜選定することができるが、例えば、ブドウ糖、果糖、ショ糖、マルトース、ソルビトール、ステビオサイド、ルブソサイド、コーンシロップ、乳糖、クエン酸、酒石酸、リンゴ酸、コハク酸、乳酸、L−アスコルビン酸、dl−α−トコフェロール、エリソルビン酸ナトリウム、グリセリン、プロピレングリコール、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、ショ糖脂肪酸エステル、ソルビタン脂肪酸エステル、アラビアガム、カラギーナン、カゼイン、ゼラチン、ペクチン、寒天、ビタミンB類、ニコチン酸アミド、パントテン酸カカルシウム、アミノ酸類、カルシウム塩類、色素、香料、保存剤、などが挙げられる。
−マンゴージンジャー50質量%エタノール抽出物の製造−
マンゴージンジャー(インド産)の根茎部及び塊根部の乾燥粉末5kgを50質量%エタノール35リットルに加え、還流抽出器で95℃にて1時間加熱抽出し、熱時濾過した。残渣についてさらに同様の抽出処理を行った。得られた抽出液を合わせて減圧下で濃縮し、ペースト状のマンゴージンジャー抽出物を得た。抽出物の収率は表1に示すとおりであった。
−マンゴージンジャーエタノール抽出物の製造−
マンゴージンジャー(インド産)の根茎部及び塊根部の乾燥粉末5kgをエタノール35リットルに加え、還流抽出器で95℃にて1時間加熱抽出し、熱時濾過した。残渣についてさらに同様の抽出処理を行った。得られた抽出液を合わせて減圧下で濃縮し、ペースト状のマンゴージンジャー抽出物を得た。抽出物の収率は表1に示すとおりであった。
−マンゴージンジャーヘキサン抽出物の製造−
マンゴージンジャー(インド産)の根茎部及び塊根部の乾燥粉末5kgをヘキサン35リットルに加え、還流抽出器で60℃にて1時間加熱抽出し、熱時濾過した。残渣についてさらに同様の抽出処理を行った。得られた抽出液を合わせて減圧下で濃縮し、ペースト状のマンゴージンジャー抽出物を得た。抽出物の収率は表1に示すとおりであった。
−コラーゲン合成促進作用試験−
製造例1から3で得られたマンゴージンジャー抽出物について、以下のようにしてコラーゲン合成促進作用を試験した。
Websterらの方法(Anal.Biochem.,Vol.96,220,1979)に準拠して試験を行った。
ヒトの線維芽細胞を24穴プレートに播種し、37℃、5%CO2−95%airの下にて、製造例1〜3で得られた各試料(試料濃度:25ppm及び100ppm)を溶解した添加培地で数日間培養した後、β−アミノプロピオニトリルと、[3H]−プロリンとを添加し、24時間培養した。該培養液全体にペプシン/酢酸溶液を加えて4℃以下で16時間消化し、この消化液にキャリアーを加えて0.7mol/L食塩水溶液で沈殿させ、更に中性条件下で再溶解させて、4.2mol/L食塩水溶液で再沈殿させた。
得られた沈殿物を20質量%エタノールで洗浄した後、その沈殿物の放射活性を測定した。そして、試料無添加時の放射活性を100%として、コラーゲン合成促進率(%)を算出した。結果を表2に示す。
−線維芽細胞増殖作用試験−
製造例1から3で得られたマンゴージンジャー抽出物について、以下のようにして線維芽細胞増殖作用を試験した。
線維芽細胞増殖作用は、MTT法(J.Immunol.Method,93,157,1986)に準拠して行った。
25cm2の培養フラスコに入れた10v/v%FBS含有培地(α−MEM培地:GIBCO BLR社製,pH7.2)にヒト正常新生児皮膚繊維芽細胞(NBIRGB)1×106個を播種し、37℃、5%CO2−95%airの下で、4日間培養した。次いで、トリプシン処理をし、遠心分離して細胞を集めた。
沈殿として得られた細胞を5v/v%FBS含有培地(α−MEM培地:GIBCO BLR社製、pH7.2)に懸濁し、96穴プレートの1穴につき7×103個ずつ分注した。24時間培養後、製造例1〜3で得られた各試料(試料濃度:200ppm,100ppm)を溶解した5v/v%FBS含有培地を1穴につき100μLずつ加え、37℃、5%CO2−95%airの下で、3日間培養した。培養後、培地を1穴につき100μLずつ除去し、MTT試薬(3−(4,5−dimethy−2−thiazolyl)−2,5−diphenyl−2Htetrazolium bromide,5mg/mL PBS(−)溶液)20μLを添加し、4.5時間インキュベーションした(増殖した細胞中のミトコンドリア由来の活性酸素がMTT試薬と反応し、黄色であった試薬の色が570nmに吸収のピークを有する青黄色に変わる。)。その後、各穴に10質量%ドデシル硫酸ナトリウム−0.02mol/L硫酸溶液を100μLずつ分注し、18時間インキュベーションした。インキュベーション終了後、マイクロプレートリーダーを用いて570nmの吸光度を測定した。また、別途、製造例1〜3の試料だけでもブランクをとり、同様の操作を行い、吸光度の測定を行った。
また、各吸光度測定値は、同時に測定した650nmの吸光度を差し引いて、増殖した細胞による濁度の影響を補正した。そして、補正後の各吸光度により、下記の計算式に基づいて線維芽細胞増殖促進率(%)を求めた。結果を表3に示す。
線維芽細胞増殖促進率(%)=〔(A−C)−(B−D)〕/(B−D)×100
但し、前記数式中、Aは、試料添加時の吸光度を意味する。Bは、試料無添加時の吸光度を意味する。Cは、試料添加,細胞無添加時の吸光度を意味する。Dは、試料無添加,細胞無添加時の吸光度を意味する。
−サイクリックAMPホスホジエステラーゼ阻害作用試験−
製造例1〜3で得られたマンゴージンジャー抽出物について、以下のようにしてサイクリックAMPホスホジエステラーゼ阻害作用を試験した。
5mLの塩化マグネシウムを含有するトリス塩酸緩衝液(pH7.5)0.2mLに胎児血清アルブミン溶液0.1mL及びサイクリックAMPホスホジエステラーゼ溶液0.1mLを加え、更に製造例1〜3で得られた各試料溶液0.05mLを加え、37℃で5分間インキュベーションした。
次いで、サイクリックAMPホスホジエステラーゼ溶液0.05mLを加え、37℃で60分間インキュベーションした。沸騰浴中で3分間煮沸して反応を停止させ、4℃、3500rpmで遠心分離し、上清中の反応基質である5’−AMPを高速液体クロマトグラフィーにより定量した。
また、試料溶液を添加せずに同様の酵素反応と反応基質の分析を行い、試料無添加時の反応基質量に対する試料添加時の反応基質量の比率より、試料のサイクリックAMPホスホジエステラーゼ阻害率(%)を求めた。
−チロシナーゼ阻害作用試験−
製造例1〜3で得られたマンゴージンジャー抽出物について、以下のようにしてチロシナーゼ阻害作用を試験した。
試験管にマックルパイン緩衝液(pH6.8)1mL、チロシン溶液(0.3mg/mL)0.3mL、製造例1〜3で得られた各試料溶液5mgを50v/v%DMSO溶液5mLに溶解して得た試料溶液0.9mLを加え、37℃で10分間プレインキュベーションした。
その後、チロシナーゼ溶液(シグマ製、50,000unit、マッシュルーム由来、1mg/mL)0.1mLを加え、37℃で15分間インキュベーションを行った。
反応終了後、波長475nmにおける吸光度を測定した。また、同様の操作及び吸光度測定を、酵素を添加せずにマックルパイン緩衝液(pH6.8)を用いて行った。更に、試料溶液を添加せずに試料を溶かす溶媒に同様の測定を行い、下記の計算式によりチロシナーゼ活性阻害率(%)を求めた。
チロシナーゼ活性阻害率(%)=〔1−(A−B)/(C−D)〕×100
但し、前記数式中、Aは、酵素溶液添加,試料溶液添加時の吸光度を意味する。Bは、酵素溶液無添加,試料溶液添加時の吸光度を意味する。Cは、酵素溶液添加,試料溶液無添加時の吸光度を意味する。Dは、酵素溶液無添加,試料溶液無添加時の吸光度を意味する。
−血小板凝集抑制作用試験−
製造例1〜3で得られたマンゴージンジャー抽出物について、以下のようにして血小板凝集抑制作用を試験した。
日本種白色家兎の血液に77mM EDTAを血液量の1/10容量添加し、1000rpmで10分間遠心分離して沈殿物を除いた。上清を2100rpmで10分間遠心分離し、沈殿した血小板を採取した。得られた血小板を血小板洗浄液に浮遊させ、2100rpmで10分間遠心分離した。沈殿した血小板を採取し、血小板数が30万個/μLになるように血小板浮遊液に浮遊させた。
次に、調製した洗浄血小板浮遊液223μLに塩化カルシウム溶液1μLを加え、37℃にて1分間保持した。そこに製造例1〜3の各試料溶液1μLを加えて、更に2分間同温度に保持した後、1分間撹拌した。
次いで、凝集惹起剤として10ppmコラーゲン溶液25μLを添加し、37℃にて10分間インキュベーションした後、血小板凝集測定装置 PAM12CL(メバニクス株式会社製)を用いて血小板凝集率Aを測定した。別に、試料溶液の代わりに試料溶液の溶媒を添加しない以外は、上記と同様に操作して血小板凝集率Bを測定し、これらの結果から、次式により血小板凝集抑制率(%)を求めた。
血小板凝集抑制率(%)=〔(B−A)/B〕×100
ただし、前記数式中、Aは、凝集惹起剤添加、試料溶液添加時の血小板凝集率を意味する。Bは、凝集惹起剤添加、試料溶液無添加時の血小板凝集率を意味する。
−錠剤(健康食品)−
下記の原料を均一に混合し、常法により顆粒状にした後、打錠し、錠剤を製造した。
マンゴージンジャーヘキサン抽出物(製造例3) 32g
糖類 50g
結晶セルロース 10g
ショ糖脂肪酸エステル 8g
計 100g
−清涼飲料−
下記組成の清涼飲料を常法により製造した。なお、マンゴージンジャー抽出物(製造例2)のサイクロデキストリン包接物は、サイクロデキストリン水飴セルデックスSL−20(日本食品加工製)0.998gにマンゴージンジャー抽出物(製造例2)0.002gを加え、混合して製造したものである。
マンゴージンジャーエタノール抽出物(製造例2)の
サイクロデキストリン包接物 3g
液糖 10g
水 87g
計 100g
−カプセル剤−
下記の原料を混合し、打ち抜き法によりソフトカプセル状の健康食品を製造した。
マンゴージンジャー50質量%エタノール抽出物(製造例1) 90g
ウコンオレンジオレジンターメリック 25g
植物油 113g
グリセリン脂肪酸エステル 12g
ミツロウ 60g
計 300g
−乳液−
下記組成の乳液を常法により製造した。
ホホバオイル 4.0g
プラセンタエキス 0.1g
アスコルビン酸リン酸マグネシウム 0.1g
プルーン抽出物 1.0g
トウニン抽出物 1.0g
キョウニン抽出物 1.0g
タイオウ抽出物 0.5g
クジン抽出物 0.5g
オリーブオイル 2.0g
スクワラン 2.0g
セタノール 2.0g
モノステアリン酸グリセリル 2.0g
ポリオキシエチレンセチルエーテル(20E.O) 2.5g
オレイン酸ポリオキシエチレンソルビタン(20E.O) 2.0g
1,3−ブチレングリコール 3.0g
ヒノキチオール 0.15g
香料 0.15g
マンゴージンジャーヘキサン抽出物(製造例3) 1.0g
精製水 残部
計 100g
−クリーム−
下記組成のクリームを常法により製造した。
流動パラフィン 5.0g
サラシミツロウ 4.0g
セタノール 3.0g
スクワラン 10.0g
ラノリン 2.0g
アルブチン 0.1g
ルシノール 0.1g
黄杞エキス 0.1g
イチョウ葉エキス 0.1g
コンキオリン 0.1g
オウバクエキス 0.1g
ステアリン酸 1.0g
オレイン酸ポリオキシエチレンソルビタン(20E.O) 1.5g
モノステアリン酸グリセリル 3.0g
1,3−ブチレングリコール 6.0g
パラオキシ安息香酸メチル 0.05g
香料 0.1g
マンゴージンジャー50質量%エタノール抽出物(製造例1) 0.5g
精製水 残部
計 100g
−パック−
下記組成のパックを常法により製造した。
ポリビニルアルコール 15g
ポリエチレングリコール 3g
プロピレングリコール 7g
エタノール 10g
カミツレ抽出物 10g
エラグ酸 0.1g
油溶性甘草エキス 0.1g
海藻エキス 0.1g
酵母抽出液 0.1g
シソ抽出液 0.1g
チユ抽出液 0.1g
シナノキ抽出液 0.1g
パラオキシ安息香酸エチル 0.05g
香料 0.05g
セイロンベンケイエタノール抽出物 0.1g
マンゴージンジャー50質量%エタノール抽出物(製造例1) 0.05g
精製水 残部
計 100g
Claims (7)
- ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物(ただし、クルクミン類を含有する抽出物を除く)を含むコラーゲン合成促進剤。
- ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物(ただし、クルクミン類を含有する抽出物を除く)を含む線維芽細胞増殖促進剤。
- ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物(ただし、クルクミン類を含有する抽出物を除く)を含むサイクリックAMPホスホジエステラーゼ阻害剤。
- ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物(ただし、クルクミン類を含有する抽出物を除く)を含むチロシナーゼ阻害剤。
- ショウガ科マンゴージンジャー(Curcuma amada ROXB.)の抽出物(ただし、クルクミン類を含有する抽出物を除く)を含む血小板凝集抑制剤。
- 請求項1に記載のコラーゲン合成促進剤、請求項2に記載の線維芽細胞増殖促進剤、請求項3に記載のサイクリックAMPホスホジエステラーゼ阻害剤、請求項4に記載のチロシナーゼ阻害剤、及び、請求項5に記載の血小板凝集抑制剤の少なくともいずれかを含有する化粧料。
- 請求項1に記載のコラーゲン合成促進剤、請求項2に記載の線維芽細胞増殖促進剤、請求項3に記載のサイクリックAMPホスホジエステラーゼ阻害剤、請求項4に記載のチロシナーゼ阻害剤、及び、請求項5に記載の血小板凝集抑制剤の少なくともいずれかを含有する飲食品。
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