JP6022738B2 - 記憶学習機能及び/又は認知機能を増強するための組成物 - Google Patents
記憶学習機能及び/又は認知機能を増強するための組成物 Download PDFInfo
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Description
ホエイペプチドエキスは以下のようにして調製した。5%(w/v)のホエイ(第一化成社製)水溶液に0.125%(w/v)の食品添加プロテアーゼ、又はペプチダーゼを混合し、50℃で4時間反応を行った。得られた酵素反応液を分画分子量(Molecular Weight Cut-off)10KDaの限外濾過膜を用い濾過し、濾液(以下、ホエイペプチドエキスともいう。)を回収し、実験に供した。
図1(A)に記憶学習機能・認知機能の評価方法の概要を示す。6週齢雄のCD−1マウスに、ペプチドエキスを用いた場合には、1mg/kg、合成ペプチドの場合には0.1〜3μmol/kgの量で胃内へ強制経口投与を行い、40分後に記憶障害を誘発するために0.80mg/kgのスコポラミン塩酸塩を腹腔内投与した。スコポラミンの腹腔内投与20分後に自発的交替行動を評価するY字迷路試験を実施した。
自発的交替行動変動率(%)=自発的交替行動数/(総進入数−2)×100
・・・・式(1)
自発的交替行動変動率が高いほど、短期記憶が保持されていることを示す。なお、一群10匹で実験を行い、平均、標準誤差を求めた。
乳タンパク質が豊富なホエイ中に脳機能を増強させるペプチドが含まれているか解析するため、ホエイをアスペルギルス(Asperugillus)属菌、バチルス(Bacillus)属菌、植物由来の酵素剤で処理し、Y字迷路試験を行い、脳機能を増強させる効果があるか検討した。
脳機能改善効果の高い、アマノP、又はサモアーゼC100で処理したホエイペプチドエキスは、C18固相抽出カラムに供した後、水、メタノールを各20、40、60、80%含む水溶液で段階的に溶出を行った。
ホエイをAspergillus melleus由来のタンパク質分解酵素プロテアーゼP「アマノ」3SDで処理し、記憶学習機能・認知機能の増強活性のある分画中に含まれるペプチド配列を解析した結果、GTWY(配列番号7)、GYGGVSLP(配列番号3)、KPTPEGDLEI(配列番号2)の3つのペプチドの配列情報が得られた(下記表2配列表参照)。
上記解析から、脳機能の増強効果があることが明らかとなった7つのペプチドのうち4つのペプチド、VAGTWYS(配列番号4)、GTWY(配列番号7)、TWYS(配列番号8)、TWY(配列番号11)にトリプトファン-チロシン(WY)の配列が含まれることが明らかとなった。WY配列が含まれる種々のペプチドで記憶学習機能・認知機能に対する効果が見られたことから、ジペプチドWYはコア配列として作用するものと考えられる。実際にWYを含む8アミノ酸からなるペプチドに記憶学習機能・認知機能を増強する効果が見られることを確認している。したがって、少なくともWYをコア配列として含む2から8アミノ酸の長さのペプチド、また、本発明で効果が得られている最長のペプチドが12アミノ酸からなるペプチドであることを鑑みると、WY配列を含む12残基程度までの長さのペプチドであれば、記憶学習機能・認知機能の増強効果があるものと考えられる。
ホエイペプチドエキス中で各種ペプチドがどの程度効果発揮に寄与しているか以下の方法で確認した。
乳に含まれるホエイ以外のタンパク質として、カゼインが含まれることが良く知られている。そこで、カゼイン(和光純薬工業株式会社製)を、上記表1に示した酵素剤のうち、アスペルギルス属菌由来の酵素剤であるプロテアーゼM「アマノ」SD(アマノMと略記することもある。)で消化し、記憶学習機能・認知機能増強活性を有するペプチドの解析を行った。ホエイの場合と同様にして、カゼインをアマノMで分解したペプチドエキスを、HPLCで分画し、活性のある分画のアミノ酸配列を決定した。その結果、記憶学習機能・認知機能増強活性のあるペプチドとして、KEMPFPKYPVEP(配列番号1)、AVPYP(配列番号6)、SLPQ(配列番号9)、YPE(配列番号12)、WY(配列番号13)、WM(配列番号14)の6つの配列情報を得ることができた。
以下の方法でミクログリアの貪食活性および抗炎症活性を増強するカゼインペプチドを単離同定した。
カゼインナトリウム(和光純薬工業社製)を食品添加プロテアーゼ、ペプチダーゼを用いて処理した。プロテアーゼ剤としては、ニューラーゼF3、プロテアーゼM「アマノ」SD、プロテアーゼA「アマノ」SD、プロテアーゼP「アマノ」3SD、プロチンSD−NY10,サモアーゼPC10F,プロチンSD−AY10、パパインW−40、ブロメラインF、ペプチダーゼ剤としては、プロテアックス、ペプチダーゼR(天野エンザイム株式会社)を用いた。
マウスより摘出した脳をパパイン処理することにより、脳組織分散液を得る。酵素反応を停止後、ミクログリアはマイクロビーズによって磁性標識された汎ミクログリアマーカーであるCD11b抗体(Miltenyi Biotec社製)と反応させ、MACS法によって単離した。
単離したミクログリアを種々のペプチドを添加した培地で12時間培養する。その後、蛍光色素フルオレセイン(FAM)で標識したアミロイドβ1−42(AnaSpec社製)を培地に500nMの濃度で添加し、5時間培養後に上清を除き、トリパンブルーで細胞外の蛍光を消光させ、マイクロプレートリーダーでミクログリア内に取り込まれたアミロイドβの量を蛍光量として測定し、貪食活性の増強を評価した。
単離したミクログリアを種々のペプチドを添加した培地で12時間培養する。その後、5ng/mlリポポリサッカライド(LPS、SIGMA-ALDRICH社)及び0.5ng/mlインターフェロンγ(IFN-γ、R&D systems社)を添加して12時間培養し、培養上清中に含まれるTNF−αをELISAキット(eBiosceince社)により定量した。
上記方法によって、ミクログリアの貪食活性の増強作用、抗炎症活性の増強作用を有する7種のペプチドを得た。すべての精製ペプチドについて、N末端アミノ酸配列分析を行い、アミノ酸配列を取得した。表4に取得したペプチドのアミノ酸配列を1文字表記で示す。
次に、取得したアミノ酸配列に基づいてペプチドを合成し、ミクログリアの貪食活性を評価した。
取得したアミノ酸配列に基づいてペプチドを合成し、抗炎症作用について評価した。単離したミクログリアを、0、25、50、100μMの合成ペプチド(KEMPFPKYPVEP、SMKEGIHAQQ、AVPYP、SLPQ、YPE、WM、WY)を添加した培地で12時間前処理する。その後は上記と同様に、LPS及びIFN-γを培地に添加して、12時間培養し、培養上清中に含まれるTNF−αをELISAにより測定した。結果を図11に示す。なお、図11では「CM1」がKEMPFPKYPVEPの結果を示し、「CM2」がSMKEGIHAQQの結果を示し、「CM3」がAVPYPの結果を示し、「CM4」がSLPQの結果を示し、「CM5」がYPEの結果を示し、「CM6」がWMの結果を示し、「CM7」がWYの結果を示す。
配列番号12〜16のペプチド配列が各種食品または食品素材に含まれるタンパク質のアミノ酸配列中に存在するか調べた。具体的には、下記表5に示す34種類のタンパク質のアミノ酸配列中にそれらペプチドの配列が現れるかを調べた。
上記同様に調製したホエイペプチドエキスを用いてコーヒー飲料および果汁飲料を製造した。各飲料組成および製法は表9及び表10に示す。
Claims (11)
- 記憶学習機能及び/又は認知機能を増強するための組成物であって、配列番号1〜16のいずれか一つのアミノ酸配列に示されるペプチド、又はこれらの薬学上許容される塩もしくは溶媒和物を含有することを特徴とする組成物。
- 食品組成物である、請求項1記載の組成物。
- 医薬組成物である、請求項1記載の組成物。
- 健康食品、機能性食品、栄養補助食品、サプリメント、保健機能食品、特定保健用食品、栄養機能性食品、機能性表示食品、又は病者用食品である、請求項2記載の組成物。
- 食品又は食品素材中に含まれるタンパク質の加水分解によって得られた配列番号1〜16のいずれか一つのアミノ酸配列に示されるペプチドを、記憶学習機能及び/又は認知機能を増強するための組成物に含有せしめることを特徴とする該組成物の製造方法。
- 前記ペプチドが、乳タンパク質又はそれを含有する食品もしくは食品素材を微生物由来の酵素剤で酵素処理することによって得られたものである、請求項5記載の組成物の製造方法。
- 前記乳タンパク質又はそれを含有する食品もしくは食品素材が、ホエイ又はカゼインである請求項6記載の組成物の製造方法。
- 前記酵素剤が、アスペルギルス属菌由来の酵素、バチルス属菌由来の酵素、及びリゾプス属菌由来の酵素からなる群から選ばれた1種又は2種以上の酵素を含むものである請求項6又は7記載の組成物の製造方法。
- ミクログリアの貪食活性及び/又は抗炎症活性を増強するための組成物であって、配列番号1、6、9、12、13、14、17のいずれか一つのアミノ酸配列に示されるペプチド、又はこれらの薬学上許容される塩もしくは溶媒和物を含有することを特徴とする組成物。
- 配列番号1〜16のいずれか一つのアミノ酸配列に示されるペプチドを有効成分とする記憶学習機能及び/又は認知機能の増強剤。
- 配列番号1、6、9、12、13、14、17のいずれか一つのアミノ酸配列に示されるペプチドを有効成分とするミクログリアの貪食活性及び/又は抗炎症活性の増強剤。
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JP2020097537A (ja) * | 2018-12-18 | 2020-06-25 | キリンホールディングス株式会社 | 脳血流を増加させるための組成物 |
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