JP6928744B1 - 消化リパーゼ活性阻害剤、血中トリグリセリド濃度上昇抑制剤、及び脂肪吸収抑制剤 - Google Patents
消化リパーゼ活性阻害剤、血中トリグリセリド濃度上昇抑制剤、及び脂肪吸収抑制剤 Download PDFInfo
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- JP6928744B1 JP6928744B1 JP2020131574A JP2020131574A JP6928744B1 JP 6928744 B1 JP6928744 B1 JP 6928744B1 JP 2020131574 A JP2020131574 A JP 2020131574A JP 2020131574 A JP2020131574 A JP 2020131574A JP 6928744 B1 JP6928744 B1 JP 6928744B1
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- inhibitor
- lipase activity
- linoleic acid
- oleic acid
- digestive
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Abstract
Description
リノール酸及びオレイン酸について、消化リパーゼ阻害活性を下記の方法により評価した。なお、リノール酸及びオレイン酸としては、富士フイルム和光純薬社製のものを用いた。
ラット腸管アセトンパウダー(Sigma社製)30mgに0.1Mクエン酸緩衝液(pH6.0)30mLを加え、氷中で1時間振とう後、4℃、10000rpmで45分間遠心分離し、消化リパーゼ液を得た。当該消化リパーゼ液は、膵リパーゼ、腸リパーゼ等の混合物である。
1.5mL容チューブに、リノール酸0.19mg/mL又はオレイン酸0.16mg/mLのDMSO溶液(濃度は反応時の終濃度)、又は、比較対象であるセサミン0.044mg/mLのジメチルスルホキシド(DMSO)溶液(濃度は反応時の終濃度)を10μLと、発色液237μLと、上述の消化リパーゼ液9μLと、エステラーゼ阻害剤4μLと水90μLを添加し、30℃、5分間インキュベートした。その後、基質液25μLを添加し、遮光下30℃で30分間反応を行った。その後、反応停止液700μLを加え、この液について412nmの吸光度を測定した。発色液、基質液、エステラーゼ阻害剤、反応停止液としては、リパーゼキットS(SBバイオサイエンス社製)付属のものを用いた。
上記のサンプル及び試薬自身が有する吸光度を測定する目的で、新たな1.5mL容チューブにコントロールを作成した。コントロールとして、リノール酸0.19mg/mL又はオレイン酸0.16mg/mLのDMSO溶液、又は、セサミン0.044mg/mLのDMSO溶液を10μLと、発色液237μLと、上述した消化リパーゼ液9μLと、エステラーゼ阻害剤4μLと、水90μLとを添加し、遮光下30℃で35分間インキュベートした。その後、反応停止液700μL、及び基質液25μLを加え、412nmの吸光度を測定した。また、各サンプルのDMSO溶液の代わりにDMSOのみを添加した以外は活性評価と同様に調整したものをブランクaとし、DMSOのみを添加してコントロールと同様に調製したものをブランクb(コントロールのブランク)とした。ブランクa及びブランクbについても412nmの吸光度を測定した。
以下の計算式により、各サンプルを添加した際の消化リパーゼ活性を算出した。
消化リパーゼ活性(%)=100×(各サンプルの吸光度−コントロールの吸光度)/(ブランクaの吸光度−ブランクbの吸光度)
各サンプルを添加した際の消化リパーゼ活性の算出結果を図1に示す。セサミンを添加した際の消化リパーゼ活性が110.9%であったのに対して、リノール酸を添加した場合は8.9%、オレイン酸を添加した場合は7.1%であり、リノール酸又はオレイン酸の添加により消化リパーゼ活性が低下することが分かった。
リノール酸及びオレイン酸の混合物について、上記試験1と同様の方法で消化リパーゼ活性阻害効果を検討した。リノール酸及びオレイン酸としては上述したものを用いた。サンプルとして、0.009mg/mLのリノール酸水溶液、0.008mg/mLのオレイン酸水溶液、及び、0.009mg/mLのリノール酸水溶液と0.008mg/mLのオレイン酸水溶液との混合物をそれぞれ用いた(各サンプルにおける濃度は反応測定時の終濃度)。
まず、下記に示す計算式により、各サンプルの消化リパーゼ活性阻害率を算出した。
消化リパーゼ活性阻害率(%)=100−{100×(各サンプルの吸光度−コントロールの吸光度)/(ブランクaの吸光度−ブランクbの吸光度)}
また、混合物により相乗効果が得られるかどうかを評価するため、コルビーの式を用いた。以下のコルビーの式によって算出された理論値よりも実測値が大きい場合、相乗効果があると判断できる。
コルビーの式:理論値E=A+B−(A×B)/100
A:リノール酸の消化リパーゼ活性阻害率(%)
B:オレイン酸の消化リパーゼ活性阻害率(%)
結果を図2に示す。リノール酸、オレイン酸、及びこれらの混合物を添加した場合の消化リパーゼ活性阻害率は、それぞれ9.6%、12.8%、30.6%であった。コルビーの式により得られる、混合物による消化リパーゼ活性阻害率の理論値は21.1%であったため、リノール酸及びオレイン酸の混合物において、消化リパーゼ活性阻害作用の相乗効果があると判断できた。
リノール酸及びオレイン酸の混合物が、ラットに対して血中トリグリセリド濃度上昇抑制効果を有するか否かを、下記の試験により評価した。リノール酸及びオレイン酸としては上述したものを用いた。
ラットには、Wister Rat(雄性、6週齢)を用いた。ラットは、環境温度:23℃、設定湿度:55%、明暗各12時間(照明:午前8時〜午後8時)に維持された飼育室で飼育した。水道水を飲料水として自動給水装置を用いて自由に摂取させた。ラットは個別飼育し、搬入後5日間以上馴化させた。ケージ及び給餌器の交換は週に1回以上行った。
コーン油30mLを、コール酸400mg、コレステロールオリエート10g及び純水30mLと混合し、この混合物を10分間超音波処理することにより乳化して、脂質負荷食の乳化コーン油とした。
ラットの体重を測定し、リノール酸0.46mg/kg(ラット体重)及びオレイン酸0.72m/kg(ラット体重)を投与できるように投与用量を計算した。リノール酸及びオレイン酸を20%(v/v)エタノール溶液中で撹拌させ、試験液とした。ラットをリノール酸・オレイン酸投与群とコントロール群の2群(各群7匹)に分け、リノール酸・オレイン酸投与群には試験液400μLを経口投与した。コントロール群には20%(v/v)エタノール溶液400μLを経口投与した。いずれの群についても、経口投与から10分後、脂質負荷食である乳化コーン油1mLを経口投与した。脂質負荷食の投与から7.5時間経過後まで1.5時間毎にラットの採血を行った。採取した血液を30分間静置した後、遠心分離(3000g×15分)を行った。遠心分離後に血清を取り出し、血中トリグリセリド濃度(mg/dL)を測定した。血中トリグリセリド濃度の測定は、ラボアッセイTMトリグリセライド(富士フィルム和光純薬社製)の測定法に則って行った。
リノール酸・オレイン酸投与群、及びコントロール群における血中トリグリセリド濃度の変化を図3に示す。なお、コントロール群の被検体のうち1匹が試験続行不可能の状態になったため、コントロール群から除外した。リノール酸・オレイン酸投与群の血中トリグリセリド濃度は、脂質負荷食投与後、6時間後にコントロール群よりも有意に低値(P=0.04)であり、4.5時間後に抑制傾向(P=0.08)を示した。これにより、リノール酸及びオレイン酸の混合物によって血中トリグリセリド濃度の上昇が抑制されたことが分かる。すなわち、リノール酸及びオレイン酸により、脂肪の吸収が抑制され得ることが確認できた。
Claims (4)
- 前記リノール酸及び前記オレイン酸の両方を有効成分とする、請求項1〜3のいずれか一項に記載の剤。
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