JP6638161B2 - 脂肪蓄積抑制用飲食品組成物、脂肪肝の予防又は治療用飲食品組成物及び脂肪酸合成酵素抑制用飲食品組成物 - Google Patents
脂肪蓄積抑制用飲食品組成物、脂肪肝の予防又は治療用飲食品組成物及び脂肪酸合成酵素抑制用飲食品組成物 Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Description
シマアザミを有効成分として含有する。
シマアザミを有効成分として含有する。
シマアザミを有効成分として含有する。
シマアザミ葉からシマアザミ抽出物を調製し、培養細胞実験を行った。
シマアザミの葉を水で洗った後、次亜塩素酸で殺菌した。その後、水切りを十分行い、予備凍結させた。その後、真空乾燥装置(株式会社マルイ)を用いてシマアザミの葉を凍結乾燥させた。凍結乾燥させた葉を粉砕機で粉砕し、ふるいにかけることで非粉末物を除去して、シマアザミの葉の凍結乾燥粉末を得た。この凍結乾燥粉末1gに10mLのヘキサンを加え、37℃で2時間浸漬させることで、抽出処理を行った。抽出後、減圧濾過により抽出液を分離し、残渣を10mLのヘキサンで2回洗浄した。この洗浄により生じた液を、先に得た抽出液と合わせ、ヘキサン抽出物とした。
10%のウシ血清を加えたダルベッコ・フォークト変法イーグル最小必須培地(DMEM)にて、マウス線維芽細胞(3T3―L1細胞)を、5×103細胞/mL/ウェルとなるように24ウェルプレートに播種した。インキュベーター(37℃、5%CO2)内で、1日おきに培地交換を行いながら、コンフルエントに達するまで培養した。コンフルエントに達した後、さらに2日間の培養を行い、その後、分化誘導を開始させた。
3T3―L1細胞の脂肪細胞への分化誘導を、10%牛胎児血清(FBS)、50nMイソブチルメチルキサンチン(IBMX)、1μMデキサメタソン(DEX)及び10μg/mLインスリンを混合したDMEMにて、2日間培養することで行った。分化誘導開始後2日目に、培地を10%FBS及び10μg/mLインスリンを混合したDMEMに交換し、その後2日目に同培地の交換を行い、その後4日目まで培養を行った。なお、前述の通り得られたシマアザミの各抽出物を、終濃度50μg/mLとなるように、3T3―L1細胞の分化誘導開始時から実験終了時まで培地に添加した。
培養終了後の、脂肪細胞に分化誘導された3T3−L1細胞(以下、“培養脂肪細胞”という)内に形成される脂肪滴の数及び細胞内中性脂肪(トリグリセリド:TG)濃度を評価することによって、シマアザミ葉の各抽出物の脂質蓄積抑制効果を評価した。
オイルレッドO染色の結果を図1(a)に、細胞内中性脂肪(トリグリセリド:TG)濃度測定の結果を図1(b)に示す。図1(a)及び(b)において、「ND」は分化誘導処理を行なっていない3T3−L1細胞(Non−Diffenrentiation、ND)を示し、「Control」は分化誘導処理を行ってジメチルスルホキシドを添加して培養した細胞を示す。シマアザミ葉のヘキサン抽出物を添加して培養した細胞では、クロロホルム抽出物、エタノール抽出物及び水抽出物のそれに比して、形成された脂肪滴の数が明らかに少ないことが示された(図1(a))。また、シマアザミ葉のヘキサン抽出物を添加して培養した細胞では、コントロールに比して有意に細胞内中性脂肪(トリグリセリド:TG)濃度が低いことが示された(図1(b))。
シマアザミ葉のヘキサン抽出物を用いて、培養脂肪細胞の脂肪分解促進関連遺伝子及び脂肪合成関連遺伝子(表1)の発現に及ぼす影響について検討した。
結果を図2に示す。図2において、「ND」及び「Control」については図1と同様である。シマアザミ葉のヘキサン抽出物を添加して培養した細胞では、コントロールに比して有意に脂肪酸合成酵素(FAS)の発現が低下していた。
シマアザミ葉の凍結乾燥粉末を用いて、動物実験を行った。
・中性脂肪(図5):トリグリセライド E−テストワコー(和光純薬工業株式会社)
・総コレステロール(図5):コレステロールE−テストワコー(和光純薬工業株式会社)
・遊離脂肪酸(図5):NEFA C−テストワコー(和光純薬工業株式会社)
・インスリン(図5):マウスインスリン測定キット(株式会社森永生科学研究所)
・肝障害マーカー(ALT及びAST)(図6):トランスアミナーゼCII−テストワコー(和光純薬工業株式会社)
図3に体重、肝臓重量、脾臓重量及び腎臓重量の測定結果を示す。体重、脾臓重量及び腎臓重量には変化が見られなかった一方で、肝臓重量は、飼料中のシマアザミ葉の含量が多くなるほど有意に低下していた。
シマアザミ葉の摂取が、高脂肪食摂取マウスの脂肪細胞及び肝臓における脂肪分解促進関連遺伝子及び脂肪合成関連遺伝子(表3)の発現にどのような影響を及ぼすかについて検討した。
図7に皮下白色脂肪組織及び腎臓周辺白色脂肪組織での結果を、図8に肝臓での結果を示す。なお、図3−6と同様に、図7及び図8の「Control」は、シマアザミ葉の凍結乾燥粉末が含まれていない飼料を与えた群(つまり、表2の「0%」の群)を表す。シマアザミ葉を摂取した高脂肪食摂取マウスでは、皮下白色脂肪組織及び腎臓周辺白色脂肪組織の脂肪酸合成酵素(FAS)発現が用量依存的に低下しており(図7)、また、肝臓においても、FAS発現が用量依存的に低下していた(図8)。
シマアザミ葉から得られたヘキサン抽出物及びクロロホルム抽出物の脂肪蓄積抑制効果について、詳細に検証した。
実施例1と同様にシマアザミの葉の凍結乾燥粉末を得た。この凍結乾燥粉末に10倍量のヘキサンを加え、37℃で2時間振盪浸漬させることで、抽出処理を行った。抽出後、減圧濾過により抽出液を分離し、残渣を凍結乾燥粉末の10倍量のヘキサンで2回洗浄した。この洗浄により生じた液を、先に得た抽出液と合わせ、ヘキサン抽出物とした(図9)。
先のヘキサンによる洗浄後の残渣を減圧下に置くことで、残存するヘキサンを除去した。ヘキサン除去後の残渣に凍結乾燥粉末の10倍量のクロロホルムを加え、37℃で2時間振盪浸漬させることで、抽出処理を行った。抽出後、減圧濾過により抽出液を分離し、残渣を凍結乾燥粉末の10倍量のクロロホルムで2回洗浄した。この洗浄により生じた液を、先に得た抽出液と合わせ、クロロホルム抽出物とした(図9)。
以上の調製方法により得られたヘキサン抽出物を減圧乾固し、ヘキサンに溶解して、10mg/mLのヘキサン抽出物溶解液を得た(図10)。
細胞内中性脂肪濃度測定の結果を図11に示す。図11において、「NC」は分化誘導処理を行なっていない3T3−L1細胞を示し、「PC」は分化誘導処理を行ってジメチルスルホキシドを添加して培養した細胞を示す。HC(50/50)画分を添加して培養した細胞では、「PC」に比して有意に細胞内中性脂肪濃度が低いことが示された(図11)。
上記の調製方法により得られたクロロホルム抽出物を減圧乾固し、クロロホルムに溶解して、100mg/mLのクロロホルム抽出物溶解液を得た(図12)。
FAS−sense:TCGTGGGCTACAGCATGGT(配列番号33)
FAS−anti−sense:GCCCTCTGAAGTCGAAGAAG(配列番号34)
ACTB−sense:TCACCGAGCGCGGCT(配列番号35)
ACTB−anti−sense:TAATGTCACGCACGATTTCCC(配列番号36)
・0:00 Start(CHCl3:MeOH=100:0)
・60:00 (CHCl3:MeOH=75:15)
・70:00(CHCl3:MeOH=50:50)
・90:00 stop(CHCl3:MeOH=50:0)
本実施形態の第1の観点に係る脂肪蓄積抑制剤は、
アザミ属植物を有効成分として含有する。
本実施形態の第1の観点に係る脂肪蓄積抑制剤を有効成分として含有する。
本実施形態の第1の観点に係る脂肪蓄積抑制剤を有効成分として含有する。
本実施形態の第1の観点に係る脂肪蓄積抑制剤を含有する。
アザミ属植物に対して溶媒を用いて抽出操作を行うことで抽出液を得る工程を含む、
ことを特徴とする。
本実施形態の第5の観点に係る脂肪蓄積抑制剤の製造方法により得られる、
ことを特徴とする。
Claims (3)
- シマアザミを有効成分として含有する脂肪蓄積抑制用飲食品組成物。
- シマアザミを有効成分として含有する脂肪肝の予防又は治療用飲食品組成物。
- シマアザミを有効成分として含有する脂肪酸合成酵素抑制用飲食品組成物。
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