JP5070040B2 - 血管不全改善剤 - Google Patents
血管不全改善剤 Download PDFInfo
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- JP5070040B2 JP5070040B2 JP2007511216A JP2007511216A JP5070040B2 JP 5070040 B2 JP5070040 B2 JP 5070040B2 JP 2007511216 A JP2007511216 A JP 2007511216A JP 2007511216 A JP2007511216 A JP 2007511216A JP 5070040 B2 JP5070040 B2 JP 5070040B2
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- astaxanthin
- acid
- vascular
- added
- fluorescence intensity
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- 239000002478 γ-tocopherol Substances 0.000 description 1
- QUEDXNHFTDJVIY-DQCZWYHMSA-N γ-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 QUEDXNHFTDJVIY-DQCZWYHMSA-N 0.000 description 1
- OTXNTMVVOOBZCV-WAZJVIJMSA-N γ-tocotrienol Chemical compound OC1=C(C)C(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1 OTXNTMVVOOBZCV-WAZJVIJMSA-N 0.000 description 1
- 235000019150 γ-tocotrienol Nutrition 0.000 description 1
- 239000011722 γ-tocotrienol Substances 0.000 description 1
- 239000002446 δ-tocopherol Substances 0.000 description 1
- ODADKLYLWWCHNB-LDYBVBFYSA-N δ-tocotrienol Chemical compound OC1=CC(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1 ODADKLYLWWCHNB-LDYBVBFYSA-N 0.000 description 1
- 235000019144 δ-tocotrienol Nutrition 0.000 description 1
- 239000011729 δ-tocotrienol Substances 0.000 description 1
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Description
(2)有効成分としてアスタキサンチンとトコトリエノールを含有することを特徴とする血管内皮細胞保護剤であり、
(3)有効成分としてアスタキサンチンとトコトリエノールを含有することを特徴とする血管内皮機能改善剤であり、
(4)アスタキサンチンの投与量が1日あたり1〜20mgである(1)〜(3)のうちのいずれか記載の薬剤である。
Eagle(日水製薬製)粉末の最小培地(EMEM)9.4gを水1000mlに室温で撹拌しながら溶解し、オートクレーブ殺菌(121℃、15分)したものに、重炭酸ナトリウム溶液(大塚製薬メイロン製)、グルタミン(日水製薬)、必須アミノ酸溶液(ギブコ製)、非必須アミノ酸溶液(ギブコ製)、ビタミン混合液(ギブコ社製)、抗生物質混合液(ギブコ製)を添加した。この培地に牛胎児血清(ギブコ製)を10%あるいは0.4%加えたものを用意し、以降それぞれ「10%FBS加EMEM」および「0.4%FBS加EMEM」と称する。
10%FBS加EMEMで継代した血管内皮細胞(GM07373A)をトリプシン−EDTA溶液で剥離し、10%FBS加EMEMに浮遊させた細胞液の1.5mlに9mlの10%FBS加EMEMを加えて、9cmシャーレに入れ5%二酸化炭素雰囲気下37℃で3〜4日間前培養した。トリプシン−EDTA溶液で剥離し、10%FBS加EMEMに浮遊させた細胞浮遊液(細胞数0.7〜0.9×105cell/ml)の1mlを24穴プレートの各wellに移し、5%二酸化炭素雰囲気下37℃で2日間本培養した。培地を取り除いた後、0.4%FBS加EMEMの500μl/well加えて細胞を飢餓状態にして、5%二酸化炭素雰囲気下37℃で1晩培養した。試料の溶液とグルコース水溶液(200mg/ml)10μlを加えて、5%二酸化炭素雰囲気下37℃で2時間培養した。培地を取り除いた後、ハンクス氏液(日水製薬製)の1ml/wellを加えて2回洗浄し、溶液を取り除いた後、過酸化水素入り(あるいは過酸化水素無添加)染色液300μl/wellを加え、5%二酸化炭素雰囲気下37℃で30分間培養した。染色液を除去し、細胞の蛍光強度を測定した。
ライカ実体蛍光顕微鏡 MZ FL III(ライカマクロ蛍光装置GFP付)(ライカ製)に、フィルターセット(名称):GFP Plant 蛍光(GFP2)(励起フィルター:480/40nm、吸収フィルター:510nm)を用いて、100倍下で任意三箇所の蛍光強度を測定し、画像をCCDカメラで取り込み画像解析ソフト(Qwin:ライカ製)を用いて、グレー強度%に換算して、蛍光強度を測定した。1wellあたり3ヶ所蛍光強度を測定して、その平均値を求めた。また、1種類につき3〜4well作成した。
アスタキサンチンを添加した系は、過酸化水素とグルコースを添加したものにアスタキサンチンを加えたものである。前培養期間は5日間、細胞数は0.84×105Cell/Wellであった。
トコトリエノールまたはα−トコフェロールを添加した系は、過酸化水素とグルコースを
添加したものにトコトリエノールまたはα−トコフェロールを加えたものである。前培養
期間は3日間、細胞数は0.89×105Cell/Wellであった。
方法は実施例1と基本的には同一であるが、96wellプレートを使用し、マルチピペットで段階希釈し、蛍光プレートリーダーを用いた。すなわち、T25フラスコで植え継いだ血管内皮細胞をトリプシン−EDTA溶液を用いて剥離させ、10%FBS加EMEM培地で希釈してT75フラスコで前培養を4日間行った。コンフルエントになった細胞を再びトリプシン−EDTA溶液で細胞を剥離させ、10%FBS加EMEM培地で希釈して95×104cell/mlの細胞浮遊液とし、96wellプレートに100μl/well撒き2日間培養した。培地を吸引除去して0.4%FBS加EMEM培地50μlを加え、一晩飢餓培養を行った。培地を吸引除去し、グルコース(最終濃度400mg/dl)を含んだ0.4%FBS加EMEM培地を用いて各試料の希釈列(50μl/well、2倍×8段)をマルチピペットで作り、各試料溶液をそれぞれのWellに添加した。試料溶液は、実施例1と同様に各試薬をメタノールに適宜溶解して原液を調製し、0.4%FBS加EMEMで希釈して用いた。試料添加後、2時間5%炭酸ガス培養槽で37℃で刺激培養を行った。培地を吸引除去し、ハンクス氏液100μl/wellで2度洗浄後、実施例1と同様に、DMSOに分散させた蛍光色素(C−6827)と過酸化水素(最終0.003%)溶液加えたハンクス液30μl/well加え、5%炭酸ガス培養槽で25分培養し、蛍光色素を細胞に取り込ませた。色素液を吸引除去後、遮光下で室温に一晩放置した。Fluostarプレートリーダーで測定した。なお、1列は過酸化水素の希釈列(最終濃度100μM)を加えこれを標準とした。
蛍光強度の測定は、FluoStar(BMG Labtech社製)を用いた。各well毎の蛍光強度を自動的に測定する装置である。光源はキセノンランプで、Excitationは485nm、Emissionは538nmの条件で測定した。485nmの光を10回フラッシュしたときの蛍光強度を電圧として表示し、ゲインは最も蛍光強度が強い過酸化水素100μMのwellを8000(無単位)に調整した。
過酸化水素及びグルコース添加の蛍光強度は7655、過酸化水素及びグルコース添加なし蛍光強度は1338であった。24穴プレートを用い、直接測定法で蛍光強度を測定した。
過酸化水素及びグルコース添加の蛍光強度は7605、過酸化水素及びグルコース添加なし蛍光強度は1210であった。24穴プレートを用い、直接測定法で蛍光強度を測定した。
過酸化水素及びグルコース添加の蛍光強度は5250、過酸化水素及びグルコース添加なし蛍光強度は6689であった。96穴プレートを用い、直接測定法で蛍光強度を測定した
下記成分を下記組成比(重量%)で均一に混合し、1粒200mgの錠剤とした。
アスタリールパウダー 10重量部
ブルベリー末 2重量部
Vプレミックス 3重量部
乳糖 50重量部
バレイショデンプン 32重量部
ポリビニルアルコール 2重量部
ステアリン酸マグネシウム 1重量部
アスタリールパウダー(富士化学工業(株)製)はフリー体換算で1重量%のアスタキサンチンを含むヘマトコッカス藻抽出オイルから製造した粉末である。
下記成分からなるソフトカプセル剤皮の中にヘマトコッカス藻抽出オイル(アスタキサンチンを5重量%含有)を常法により充填し、1粒200mgのソフトカプセルを得た。
ゼラチン 70重量部
グリセリン 23重量部
パラオキシ安息香酸プロピル 0.5重量部
水 6.5重量部
下記の成分を配合し、常法に従ってドリンク剤を調製した。
ヘマトコッカス藻抽出オイル 5重量部
トコトリエノールオイル 5重量部
DL−酒石酸ナトリウム 0.1重量部
液糖 800重量部
クエン酸 12重量部
ビタミンC 10重量部
ビタミンE 30重量部
香料 1.5重量部
塩化カリウム 0.1重量部
硫酸マグネシウム 0.5重量部
水 10000重量部
ヘマトコッカス藻抽出オイルはアスタキサンチンフリー体換算で5重量%含有し、トコトリエノールオイルはトコトリエノールを40重量%含有したものである。
Claims (4)
- 有効成分として(A)アスタキサンチンと(B)トコトリエノールを含有することを特徴とする血管不全改善剤。
- 有効成分として(A)アスタキサンチンと(B)トコトリエノールを含有することを特徴とする血管内皮細胞保護剤。
- 有効成分として(A)アスタキサンチンと(B)トコトリエノールを含有することを特徴とする血管内皮機能改善剤。
- アスタキサンチンの投与量が1日あたり1〜20mgである請求項1〜3のうちのいずれか記載の薬剤。
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JPH10155459A (ja) * | 1996-11-27 | 1998-06-16 | Suntory Ltd | アスタキサンチン含有飲食物 |
JP2003064360A (ja) * | 2001-08-28 | 2003-03-05 | Nissui Pharm Co Ltd | 抗酸化剤 |
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JP2002226368A (ja) * | 2001-02-02 | 2002-08-14 | Fuji Chem Ind Co Ltd | 赤血球の酸化的損傷抑制剤 |
US7064101B2 (en) * | 2001-03-22 | 2006-06-20 | Fuji Chemical Industry Co., Ltd. | Stable astaxanthin-containing powdery compositions and process for producing the same |
US7090862B2 (en) * | 2001-03-30 | 2006-08-15 | Abbott Laboratories | Method of improving the antioxidant status of an infant |
JP2003055188A (ja) * | 2001-08-20 | 2003-02-26 | Fuji Chem Ind Co Ltd | シワ形成防止用皮膚外用剤、化粧料及び食品 |
JP2003335668A (ja) * | 2002-05-21 | 2003-11-25 | Fuji Chem Ind Co Ltd | 経口用美肌用剤 |
ES2587652T3 (es) * | 2004-02-04 | 2016-10-26 | Fuji Chemical Industry Co., Ltd. | Astaxantina para mejorar la atrofia muscular |
JP2006014730A (ja) * | 2004-05-31 | 2006-01-19 | Toyo Shinyaku:Kk | 食品 |
JP2006063021A (ja) * | 2004-08-27 | 2006-03-09 | Fuji Chem Ind Co Ltd | 高血圧降下剤および高血圧降下作用を有する食品 |
JP2007238441A (ja) * | 2004-12-03 | 2007-09-20 | Fuji Chem Ind Co Ltd | アスタキサンチンを有効成分とする体脂肪減少用組成物 |
-
2006
- 2006-03-31 JP JP2007511216A patent/JP5070040B2/ja not_active Expired - Fee Related
- 2006-03-31 EP EP06730917A patent/EP1864658A4/en not_active Withdrawn
- 2006-03-31 WO PCT/JP2006/306969 patent/WO2006106986A1/ja active Application Filing
- 2006-03-31 US US11/887,329 patent/US20090099255A1/en not_active Abandoned
-
2011
- 2011-08-18 US US13/199,056 patent/US20120004297A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JPH10155459A (ja) * | 1996-11-27 | 1998-06-16 | Suntory Ltd | アスタキサンチン含有飲食物 |
JP2003064360A (ja) * | 2001-08-28 | 2003-03-05 | Nissui Pharm Co Ltd | 抗酸化剤 |
Also Published As
Publication number | Publication date |
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WO2006106986A1 (ja) | 2006-10-12 |
EP1864658A4 (en) | 2008-04-23 |
JPWO2006106986A1 (ja) | 2008-09-25 |
US20120004297A1 (en) | 2012-01-05 |
EP1864658A1 (en) | 2007-12-12 |
US20090099255A1 (en) | 2009-04-16 |
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