JP6261059B2 - 有色タマネギの水抽出物を含有する生活習慣病改善剤 - Google Patents
有色タマネギの水抽出物を含有する生活習慣病改善剤 Download PDFInfo
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Description
(ピオグリタゾン、トログリタゾンなど)やフィブレート製剤(フェノフィブレートやベザフィブレートなど)があり、これらはPPAR(Peroxisome Proliferator Activated Receptor:ペルオキシソーム増殖薬活性化受容体)のアゴニストとして作用することが明らかにされている。前者は主に脂肪組織に分布するPPARγを、後者は肝臓、腎臓、心臓、消化管に存在するPPARαをターゲットとして作用する。
[1]可食部生重量100gあたりケルセチン配糖体を70mg以上含有する有色タマネギの水抽出物を有効成分とするPPARγ活性化剤。
[2]有色タマネギが長日系品種である前記[1]に記載のPPARγ活性化剤。
[3]有色タマネギが黄タマネギである前記[1]または[2]に記載のPPARγ活性化剤。
[4]可食部生重量100gあたりケルセチン配糖体を70mg以上含有する赤タマネギの水抽出物を有効成分とするPPARγ活性化剤。
[5]水抽出物が、タマネギ植物体の凍結乾燥物を水で抽出したものである前記[1]〜[4]のいずれかに記載のPPARγ活性化剤。
[6]ERβ活性化能を有することを特徴とする前記[1]〜[5]のいずれかに記載のPPARγ活性化剤。
[7]生活習慣病の予防、治療または改善用である前記[1]〜[6]のいずれかに記載のPPARγ活性化剤。
[8]生活習慣病が、糖尿病、脂質異常症、肥満または高血圧である前記[7]に記載のPPARγ活性化剤。
[9]メタボリックシンドロームの予防、治療または改善用である前記[1]〜[6]のいずれかに記載のPPARγ活性化剤。
[10]更年期障害の予防、治療または改善用である前記[6]に記載のPPARγ活性化剤。
[11]骨粗鬆症の予防、治療または改善用である前記[6]に記載のPPARγ活性化剤。
[12]前記[1]〜[11]のいずれかに記載のPPARγ活性化剤を含有する医薬。
[13]前記[1]〜[11]のいずれかに記載のPPARγ活性化剤を含有する飲食品。
また、本発明のPPARγ活性化剤の原料として用いられる有色タマネギは、以下の(a)〜(d)の特徴のうち、少なくとも1つ以上を有することが好ましい。
(a) Brix値が約9.0〜12.0%
(b) 栽培適応緯度が北緯約38〜45度または南緯約38〜45度
(c) 乾物率が約9〜12%
(d) 貯蔵可能期間が6か月程度
本発明のPPARγ活性化剤の原料となる可食部生重量100gあたりケルセチン配糖体を70mg以上含有する黄タマネギとしては、例えば、ヨーロッパラインズバーガータイプの黄タマネギとアメリカ東部タイプの黄タマネギとのF1などが挙げられる。以下、可食部生重量100gあたりケルセチン配糖体を70mg以上含有する赤タマネギおよび黄タマネギを合わせて「ケルセチン配糖体高含有有色タマネギ」という。
(1)腹囲(へそ周り)が、男性の場合は85cm以上、女性の場合は90cm以上
(2)中性脂肪が150mg/dL以上、HDLコレステロールが40mg/dL未満、のいずれか、または両方
(3)最高(収縮期)血圧が130mmHg以上、最低(拡張期)血圧が85mmHg以上、のいずれか、または両方
(4)空腹時血糖値が110mg/dL以上
本発明のPPARγ活性化剤をメタボリックシンドロームの診断基準を満たすヒトに適用すれば、治療対象を外れることが期待できる。
本発明の医薬および飲食品の投与量または摂取量は、患者または摂取者の年齢および体重、症状、投与時間、剤形、投与方法、薬剤の組み合わせ等に依存して決定できる。例えば、本発明の医薬を経口投与する場合、成人1人当たり0.5〜100mg/kg体重、好ましくは1〜50mg/kg体重の範囲で、また、非経口的に投与する場合は0.05〜50mg/kg体重、好ましくは0.5〜50mg/kg体重の範囲で一日1〜3回に分けて投与することができる。また、食品として摂取する場合には、成人1人1日当たり100〜6000mgの範囲、好ましくは200〜3000mgの範囲の摂取量となるように配合することができる。
1−1 試験方法
(1)タマネギ
タマネギには、ケルセチン配糖体高含有赤タマネギ「さらさらレッド(登録商標)」および一般のタマネギ「北もみじ(商品名)」を使用した。
(2)サンプル調製
2日間凍結乾燥後破砕したタマネギ可食部(鱗茎)を、100mg/mlとなるように超純水に浮遊し、4℃で24時間振盪した。孔径0.2μmのメンブレンフィルターでろ過したろ液をサンプルとした。
アフリカミドリザル腎由細胞株CV−1を2×105/wellとなるよう6穴プレートに播種し、DMEM(10%FBS)中で1日培養した。Gal4のDNA結合ドメイン(Gal4DBD)とPPARγのリガンド結合ドメイン(PPARγLBD)とのキメラタンパク質発現プラスミド(pGal4DBD/PPARγLBD)、およびGal4DNA応答配列とホタルルシフェラーゼ遺伝子を含むレポータープラスミド(pGal4−Luc)を、同時に遺伝子導入試薬(FuGENE HD、Roche社製)を用いて細胞に導入した。遺伝子導入細胞をトリプシンにより分散し、96穴プレートに1.6×104/wellとなるよう再度播種した。この際、培養液を各濃度のサンプルを含むDMEM培地(フェノールレッド無添加、10%活性炭処理血清)に交換した。サンプルの培地中の最終濃度は0.5、2、10%(v/v)とした。最高濃度は、事前に実施した細胞毒性試験において細胞毒性を示さない最高濃度であった10%(v/v)を採用した。陽性コントロールとして10μM pioglitazone(溶媒:DMSO)、陰性コントロールとして0.5%DMSOを用いた。48時間培養後、リン酸緩衝生理食塩水にて細胞を洗浄し、デュアルルシフェラーゼアッセイシステム(Promega社製)を用いて細胞を溶解した。さらにルシフェリンを含む基質溶液を加え、プレートリーダー(ARVO MX、Perkin Elmer社製)にてホタルルシフェラーゼ活性を測定した。PPARγ活性は、各サンプルのルシフェラーゼ活性を陰性コントロールのルシフェラーゼ活性で除した値で示した。実験は3回行い、平均値をデータとして採用した。
結果を図1に示した。図1から明らかなように、さらさらレッドは北もみじと比較して顕著に高いPPARγ活性を示した。
2−1 試験方法
(1)タマネギ
タマネギには、ケルセチン配糖体高含有赤タマネギ「さらさらレッド(登録商標)」の可食部(鱗茎)を使用した。
(2)タマネギサンプルの調製
実施例1と同様の方法で、タマネギ可食部の水抽出物を調製した。
(3)ケルセチン誘導体
ケルセチン(n)水和物(東京化成工業)、ケルセチン二水和物(Sigma−Aldrich)、ケルセチン4’−グルコシド(和光純薬工業)、ケルセチン3,4D−グルコシド(フナコシ(株)常盤植物科学研究所)の4種を使用した。これらの化合物はDMSOに溶解し、100mg/mlのワーキング溶液を調製した。
(4)レポーターアッセイによるPPARγ活性化試験
実施例1と同様の方法で実施した。なお、ケルセチン誘導体の濃度(培地中の最終濃度)は、事前に実施した細胞毒性試験により、細胞毒性を示さない最高濃度に基づいて設定した。
結果を図2に示した。図2から明らかなように、用いたケルセチン誘導体においては、ケルセチン4’−グルコシドの0.004mg/mlで僅かにPPARγ活性の上昇が認められたが、他では陰性コントロールより高い活性は認められなかった。一方、さらさらレッドの水抽出物は、顕著に高いPPARγ活性を示した。この結果から、ケルセチン配糖体高含有有色タマネギの水抽出物のPPARγ活性化能は、ケルセチン誘導体のPPARγ活性化能のみに起因するものではなく、ケルセチン配糖体高含有有色タマネギの水抽出物に含有される種々の成分(未知成分を含む)の組み合わせに基づくものであることが示唆された。
3−1 試験方法
(1)タマネギ
タマネギには、ケルセチン配糖体高含有赤タマネギ「さらさらレッド(登録商標)」の可食部(鱗茎)を使用した。
(2)サンプル調製
実施例1と同様の方法で、タマネギ可食部の水抽出物を調製した。
5種類の核内受容体(PPARα、PPARγ、ERα、ERβおよびPXR)について、実施例1と同様の方法で実施した。Gal4のDNA結合ドメイン(Gal4DBD)と核内受容体のリガンド結合ドメイン(核内受容体LBD)とのキメラタンパク質発現プラスミドとして、実施例1で用いたpGal4DBD/PPARγLBD以外に、pGal4DBD/PPARαLBD、pGal4DBD/ERαLBD、pGal4DBD/ERβLBD、およびpGal4DBD/PXRLBDを使用した。陽性コントロールとして、実施例1で用いた10μM pioglitazone(PPARγ)以外に、100μM WY14643(PPARα)、1μM β−estradiol(ERα、ERβ)、25μM Rifampicin(PXR)を、いずれもDMSOに溶解して使用し、陰性コントロールとして0.5%DMSOを使用した。
結果を図3に示した。図3から明らかなように、さらさらレッドの水抽出物は、PPARγ以外にERβを顕著に活性化した。この結果から、ケルセチン配糖体高含有有色タマネギの水抽出物は、PPARγおよびERβ活性化能を有することが明らかとなった。
4−1 試験方法
(1)タマネギ
表1に示す9種類の北海道産有色長日系タマネギを使用した。
(2)ケルセチン配糖体含量の測定
使用したタマネギのケルセチン配糖体含量を、文献(津志田藤二郎、鈴木雅博 1995 タマネギに存在するフラボノイド配糖体の分析および化学合成による同定 日本食品工業学会誌、42:100−108)の記載に準じてHPLC法で測定し、表1に示した。
5日間凍結乾燥後破砕したタマネギ可食部(鱗茎)を、100mg/mlとなるように超純水に浮遊し、4℃で24時間振盪した。孔径0.2μmのメンブレンフィルターでろ過したろ液をサンプルとした。
(4)レポーターアッセイによるPPARγ活性化試験
PPARγおよびERβについて、実施例1と同様の方法で実施した。陽性コントロールとして、実施例1で用いた10μM pioglitazone(PPARγ)以外に、1μM β−estradiol(ERβ)をDMSOに溶解して用いた。陰性コントロールには超純水を用いた。
(1)検体
可食部生重量100gあたりケルセチン配糖体を70mg以上含有する有色タマネギの水抽出物を検体とし、飼料に混餌して投与する。
(2)試験方法
Sprague Dawley雌ラットの卵巣を摘出して脂肪蓄積モデルラットを作製する。予備飼育(馴化)期間は標準飼料を給餌する。検体投与群、基礎飼料給餌群および無処置対照群(非卵巣摘出)の3群を設ける。検体投与群には検体混餌飼料を、他の2群には基礎飼料を給餌する。投与期間は4週間とする。1回/週の頻度で体重および摂餌量の測定を行う。投与期間終了後、全身麻酔下で腹大動脈から採血し、血清分離した後、放血して安楽死させる。血清中の中性脂肪、コレステロールおよびリン脂質を測定する。採血終了後、肝臓および白色脂肪(腸間膜、腎周囲および子宮周囲脂肪)を摘出し、それぞれの湿重量を測定する。子宮周囲脂肪をホルマリン系の固定液に浸漬して固定し、パラフィン包埋して薄切した標本をヘマトキシリン・エオジン染色する。脂肪細胞の大きさを画像解析装置により分析し、群間で比較する。
(1)検体
可食部生重量100gあたりケルセチン配糖体を70mg以上含有する有色タマネギの水抽出物を検体とし、飼料に混餌して投与する。
(2)試験方法
糖尿病モデルマウスとしてKK−Ay雄マウスを用いる。予備飼育(馴化)は標準飼料を給餌する。検体投与群および基礎飼料給餌群の2群を設ける。投与期間は4週間とする。1回/週の頻度で体重、摂餌量、尿糖および血糖の測定を行う。投与最終週に、3時間絶食させた後、糖負荷試験(投与前、30、60、90および120分に血糖値測定)を行う。投与期間終了後、全身麻酔下で放血して安楽死させる。肝臓および白色脂肪(腸間膜、腎周囲および子宮周囲脂肪)を摘出し、それぞれの湿重量を測定する。
Claims (6)
- 可食部生重量100gあたりケルセチン配糖体を70mg以上含有する有色タマネギの非加熱の可食部を凍結乾燥する工程、得られた凍結乾燥物を水に浸漬する工程、該凍結乾燥物を取り除きタマネギ水抽出物を取得する工程を含むPPARγ活性化剤の製造方法。
- さらに、得られたタマネギ水抽出物を有効成分として製剤化する工程を含む請求項1に記載の製造方法。
- 有色タマネギが長日系品種である請求項1または2に記載の製造方法。
- 有色タマネギが赤タマネギまたは黄タマネギである請求項1〜3のいずれかに記載の製造方法。
- タマネギ水抽出物が、ERβ活性化能を有することを特徴とする請求項1〜4のいずれかに記載の製造方法。
- PPARγ活性化剤が、更年期障害の予防、治療または改善に用いられる請求項1〜5のいずれかに記載の製造方法。
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