JP5424677B2 - 血圧降下作用を有するローヤルゼリーの製造方法 - Google Patents
血圧降下作用を有するローヤルゼリーの製造方法 Download PDFInfo
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- JP5424677B2 JP5424677B2 JP2009063990A JP2009063990A JP5424677B2 JP 5424677 B2 JP5424677 B2 JP 5424677B2 JP 2009063990 A JP2009063990 A JP 2009063990A JP 2009063990 A JP2009063990 A JP 2009063990A JP 5424677 B2 JP5424677 B2 JP 5424677B2
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- royal jelly
- blood pressure
- proteolytic enzyme
- ace
- bacillus stearothermophilus
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Landscapes
- Jellies, Jams, And Syrups (AREA)
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Description
ACE活性を抑制させることにより血圧を降下させるACE阻害物質として、例えば、カプトプリルが開発され医薬品として利用されている。
また、ローヤルゼリーにバチルス ステアロサーモフィルス(Bacillus stearothermophilus)由来のタンパク質分解酵素を至適温度の70℃で作用させることによりACE阻害物質が産生されたことが記載されている(特許文献4)。しかしながら、この方法では反応時間を長くするとACE阻害活性が急激に低下し、実用的ではない。
上記のように種々の食品素材に対してそれぞれ異なったタンパク質分解酵素が用いられている。これは食品素材に含まれるタンパク質がそれぞれ異なっているため、それぞれのタンパク質に最適なタンパク質分解酵素が選択されているためだと考えられる。
1. ローヤルゼリーにバチルス ステアロサーモフィルス(Bacillus stearothermophilus)由来のタンパク質分解酵素を作用させる工程を含むことを特徴とする、血圧降下作用を有するローヤルゼリーの製造方法、
2. 血圧降下作用がアンジオテンシン変換酵素阻害作用である前記ローヤルゼリーの製造方法、
3. ローヤルゼリーが実質的に完全消化されたローヤルゼリーである前記いずれかのローヤルゼリーの製造方法、
4. ローヤルゼリーにバチルス ステアロサーモフィルス由来のタンパク質分解酵素を作用させる際の温度が40〜60℃である前記いずれかのローヤルゼリーの製造方法、
5. 前記いずれかの製造方法によって得られるローヤルゼリーを含有するローヤルゼリー組成物、および
6. 前記いずれかの製造方法によって得られるローヤルゼリーを含有する血圧降下剤、
からなる。
<ホウ酸緩衝液の調製>
50mM Na2B4O7 450mLと220mM H3BO4 550mLを混合し、pH8.3のホウ酸緩衝液を調製した。
<サンプルの調製>
上清をホウ酸緩衝液(pH8.3)で10倍希釈しサンプルとした。
<基質溶液の調製>
約30mLのホウ酸緩衝液にBz-Gly-His-Leu・H2O(分子量447.49 ペプチド研究所社製)170mg、NaCl 1.78gを溶解後、1N NaOHでpH8.3に調整し、ホウ酸緩衝液で50mLにメスアップして、基質溶液とした。
<ACE溶液の調製>
アンジオテンシン変換酵素(ウサギ肺由来、シグマ社製)をホウ酸緩衝液で溶解して60mU/mLの溶液をACE溶液とした。
<ACE阻害活性の測定方法>
(1)阻害活性の測定
サンプル30μLと基質溶液250μLを混合し、37℃で5分間放置した。5分間放置した後、ACE溶液を100μL添加し、37℃で30分間反応後、1N HClを250μL添加して反応を停止させた。反応停止後1.5mLの酢酸エチルを添加し、激しく1分間攪拌した。次に3,000rpmで10分間遠心分離を行い上層の酢酸エチル層を0.5mL分取し、遠心エバポレーター(40℃、1.5時間)で蒸発乾固させた。蒸発乾固させたものに2mLの精製水を加え、溶解後、228nmにおける吸光度を測定した。このときの吸光度をAとする。
(2)ブランクの測定
サンプル30μLおよび基質溶液250μLを混合し、37℃で35分間放置した。35分間放置した後、1N HCl 250μLを添加し、その後ACE溶液を100μL添加した。さらに、1.5mLの酢酸エチルを添加し、激しく1分間攪拌した。次に3,000rpmで10分間遠心分離を行い上層の酢酸エチル層を0.5mL分取し、遠心エバポレーター(40℃、1.5時間)で蒸発乾固させた。蒸発乾固させたものに2mLの精製水を加え、溶解後、228nmにおける吸光度を測定した。このときの吸光度をBとする。
(3)コントロールの測定
前記(1)阻害活性の測定でサンプルの代わりにホウ酸緩衝液を用いて同様に操作を行い、228nmにおける吸光度を測定した。このときの吸光度をCとする。さらに、前記(2)ブランクの測定でサンプルの代わりにホウ酸緩衝液を用いて同様に操作を行い、228nmにおける吸光度を測定した。このときの吸光度をDとする。
(4)計算
下式によりACE阻害活性を求めた。
ACE残存活性(%)=(A−B)×100/(C−D)
ACE阻害活性(%)=100−ACE残存活性
(1)基質溶液の調製
基質液Aの調製:75mLの100mM 乳酸緩衝液(pH3.0)に0.6gのカゼイン(ハマルステイン処方)を懸濁させ、沸騰水中で加熱溶解した。放冷後、pHを3.0に調整し、100mLにメスアップして、基質液Aとした。
基質液Bの調製:75mLの100mM ビス−トリスプロパン緩衝液(pH7.0)に0.6gのカゼイン(ハマルステイン処方)を懸濁させ、沸騰水中で加熱溶解した。放冷後、pHを7.0に調整し、100mLにメスアップして、基質液Bとした。
なお、表1に示す(2)、(3)および(5)のタンパク質分解酵素活性測定には基質液Aを使用し、表1に示す(1)および(4)のタンパク質分解酵素活性測定には基質液Bを使用した。さらに、タンパク質分解酵素活性が高く希釈が必要な場合は表1に示す(2)、(3)および(5)のタンパク質分解酵素活性測定には100mM 乳酸緩衝液(pH3.0)を使用し、表1に示す(1)および(4)のタンパク質分解酵素活性測定には100mM ビス−トリスプロパン緩衝液(pH7.0)を使用し希釈した。
(2)タンパク質分解酵素活性の測定
2mLの基質溶液を40℃で5分間放置した後、サンプル液を0.4mL添加し、40℃で30分間反応後、0.44M トリクロロ酢酸溶液を2mL添加し、反応を停止させた。反応停止後10分間室温に放置して、No2のろ紙(アドバンテック東洋)でろ過を行った。ろ液0.5mLに0.55M Na2CO3溶液1.25mLおよび精製水で3倍希釈したフェノール試薬0.25mLを添加し、37℃で30分間放置後660nmにおける吸光度を測定した。このときの吸光度をAとする。
(3)ブランクの測定
0.44M トリクロロ酢酸溶液を2mL添加後にサンプル液0.4mLを加えた以外は上記(2)タンパク質分解酵素活性の測定と同様に行った。このときの660nmにおける吸光度をBとする。
(4)計算
下式によりΔA660を求めた。下式中、Dは希釈率を意味する。
ΔA660=(A−B)×D
Claims (3)
- ローヤルゼリーにバチルス ステアロサーモフィルス(Bacillus stearothermophilus)由来のタンパク質分解酵素を作用させる工程を含み、ここで該工程が30〜40℃の温度で行われることを特徴とする、血圧降下作用を有するローヤルゼリーの製造方法。
- 請求項1に記載の血圧降下作用を有するローヤルゼリーの製造方法であって、前記工程が40℃の温度で行われることを特徴とする、血圧降下作用を有するローヤルゼリーの製造方法。
- 血圧降下作用がアンジオテンシン変換酵素阻害作用である請求項1または請求項2に記載のローヤルゼリーの製造方法。
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