JP4594489B2

JP4594489B2 - Specific cancer killing agent and composition comprising the same

Info

Publication number: JP4594489B2
Application number: JP2000136449A
Authority: JP
Inventors: 陽夫宮澤; 美樹五十嵐; 尚彦羽田; 良計井上
Original assignee: Bizen Chemical Co Ltd
Current assignee: Bizen Chemical Co Ltd
Priority date: 2000-03-31
Filing date: 2000-03-31
Publication date: 2010-12-08
Anticipated expiration: 2020-03-31
Also published as: JP2001288079A

Description

【０００１】
【発明の属する技術分野】
本発明は、特定の共役不飽和脂肪酸類を有効成分としてなる殺癌細胞剤及びこれを配合してなる組成物に係るものである。より詳しくは、炭素−炭素間二重結合（以下、単に二重結合という）を３〜６個有する共役多価不飽和脂肪酸類であって、二重結合の全部もしくは一部が共役関係にある前記共役多価不飽和脂肪酸類を有効成分としてなる、ヒト由来の正常細胞には悪影響を与えず癌細胞のみを効率的に死滅させ得る特異的殺癌細胞剤、及び、これを配合してなる医薬用組成物又は食用組成物に関するものである。
【０００２】
【従来の技術】
近年、脂質の生理活性、薬理作用に関する研究が急速に進展し、とりわけ炭素数１８以上の不飽和脂肪酸と各種疾病との関係が解明されつつある。また、高齢化社会の本格的な到来を背景に、生活習慣や食事パターンと疾病との関連性が注目され、各種疾病の治療はもとより疾病予防の観点からも生活様式の見直しや各種食材に含まれる成分の機能評価が行われている。
【０００３】
前記不飽和脂肪酸のうち二重結合を３個以上もつ多価不飽和脂肪酸としては、シソ、エゴマ、アマ等の種子油を構成する脂肪酸であるα−リノレン酸（Ｃ１８：３ｎ−３系、以下α−ＬＮＡという。）、母乳や月見草の油脂の構成脂肪酸であるγ−リノレン酸（Ｃ１８：３ｎ−６系、以下γ−ＬＮＡという。）、動物臓器組織の油脂を構成するアラキドン酸（Ｃ２０：４ｎ−６系、以下ＡＡという。）、カツオやマグロ等の魚油や鶏卵黄油の構成脂肪酸であるエイコサペンタエン酸（Ｃ２０：５ｎ−３系、以下ＥＰＡという。）、ドコサヘキサエン酸（Ｃ２２：６ｎ−３系、以下ＤＨＡという。）等が知られている。これらは主に食品原材料に含れる脂質の構成成分であるが、微細藻類や微生物の組織中にも油分の構成脂肪酸として存在する。
【０００４】
また、これら多価不飽和脂肪酸の生理機能として、α−ＬＮＡの記憶学習能の向上作用（特開平１−１５３６２９号公報）、抗アレルギー作用、血清脂質改善作用等、ＡＡの血小板凝集作用等、ＥＰＡのコレステロール低減効果、血小板粘着能の低下や赤血球変形能の増加（田村泰ら、「食の科学」、第１６１巻、第３３−３９頁、１９９１年）等、ＤＨＡの血中脂質低下（今泉勝巳、「臨床栄養」、第８３巻、第４号、第４４０頁、１９９３年等）、血小板凝集能抑制（氷川祐三ら、「血液と脈管」、第１５巻、第２号、第１３８−１４１頁、１９８４年）、記憶・学習能の改善（Ａ．ｈｕｃａｓｅｔａｌ．，ＴｈｅＬａｎｃｅｔ，３３９，２６１，１９９２など）、抗痴呆（Ｍ．Ｓｏｄｅｒｂｅｒｇｅｔａｌ．，Ｌｉｐｉｄｓ，２６（６），４２１，１９９１）、抗腫瘍（成沢富雄ら、「医学のあゆみ」、第１４５巻、第９１１頁、１９８８年）、抗アレルギー（Ｍ．Ｓｈｉｋａｎｏｅｔａｌ．，Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，１５０，３５２５，１９９３）等の作用が見出され、治療薬や食品への利用をはじめ様々な用途への応用が提案されている。
【０００５】
ところで、多価不飽和脂肪酸の一種として共役多価不飽和脂肪酸がある。一般に、共役不飽和脂肪酸はそれに対応する非共役不飽和脂肪酸に比べて化学的反応性に富み、酸化されやすく、即乾性の被膜を形成するため、この特性を利用したものにキリ油あるいはキリ油分解脂肪酸であるエレオステアリン酸（シス−９，トランス−１１，トランス−１３−オクタデカトリエン酸）の塗料、ペンキ分野への応用が従来から行われてきた。また、バターやチーズ等の乳製品や牛肉にはリノール酸の共役異性体である共役リノール酸（シス−９，トランス−１１−オクタデカジエン酸を主体とする二重結合の位置異性体および幾何異性体の混合物）が微量ではあるが存在することが知られており、最近ではリノール酸を共役化処理した共役リノール酸が食品素材として上市され、体脂肪の低減作用（Ｌｉｐｉｄｓ，３１，８５３（１９９７））、血中コレステロール低減作用、抗腫瘍活性（Ｍ．Ａ．Ｂｅｌｕｒｙ，Ｎｕｔｒ．Ｒｅｓ．，５３，８３（１９９５））等の生理機能も見出されている。
【０００６】
【発明が解決しようとする課題】
前述のように、脂質を構成する多価不飽和脂肪酸の生理作用を解明し、これを各種疾病の、治療や予防に応用する試みが行われているが、共役不飽和脂肪酸の生理機能に関する研究はごく最近になって詳細に検討されはじめた段階であり、とりわけ二重結合を３個以上有する共役多価不飽和脂肪酸類についての知見はほとんど見あたらない。かかる現状に鑑み、本発明では、前記共役多価不飽和脂肪酸類の生理機能とりわけ癌細胞及び正常細胞に対する影響を解明し、癌細胞に対して特異的かつ効果的に増殖抑制作用ないしは殺細胞作用を有し、ひいては癌疾患の予防あるいは治療に応用し得る素材、及びこれを配合してなる医薬用組成物あるいは食用組成物を提供することを解決すべき課題とした。
【０００７】
【課題を解決するための手段】
本発明者らは、前記課題を解決するために、種々の形態の共役多価不飽和脂肪酸類について鋭意検討した結果、二重結合を３〜６個有する共役多価不飽和脂肪酸類が顕著な殺癌細胞効果を奏し、かつ正常細胞に対しては悪影響を及ぼさないことを見出し、本発明を完成するに至った。以下において、共役多価不飽和脂肪酸類とは、共役多価不飽和脂肪酸の単一物もしくは混合組成物や共役多価不飽和脂肪酸を構成脂肪酸とする各種誘導体を包含する意味で用いる。
【０００８】
すなわち、本発明において解決すべき課題である特異的殺癌細胞剤は、二重結合を３〜６個有する共役多価不飽和脂肪酸類を有効成分としてなる殺癌細胞剤によって達成される。ここで、共役多価不飽和脂肪酸類の構成脂肪酸は共役トリエン脂肪酸に富むものであることがより好ましく、さらには、α−ＬＮＡ、ＥＰＡ、ドコサペンタエン酸（Ｃ２２：５ｎ−３系、以下ＤＰＡという）及びＤＨＡからなる群から選ばれる１種又は２種以上の非共役多価不飽和脂肪酸を共役化処理して得られるものが望ましい。また、共役多価不飽和脂肪酸類のより望ましい態様は、二重結合を３〜６個有する共役多価不飽和脂肪酸を構成脂肪酸とするカルボン酸、エステル及び塩からなる群から選択される１種又は２種以上のものである。ここで、エステルとしては低級１価アルコールのエステルであることが望ましく、さらにはアシルグリセリド及び／又はグリセロリン脂質であることが望ましい。
【０００９】
なお、本発明において用いる前記共役多価不飽和脂肪酸類は、これに対応する非共役多価不飽和脂肪酸類を共役化処理して調製することができるが、該共役化処理は二重結合を３〜６個有する非共役多価不飽和脂肪酸類１重量部に対して１０〜３０重量％のアルカリ金属水酸化物を含むジオール溶液５０〜１００重量部を加え、１７０〜１９０℃にて５〜３０分間、共役化反応させることを特徴とする共役トリエン脂肪酸に富む共役多価不飽和脂肪酸類の製造方法によるものであることが望ましい。なお、本発明の特異的殺癌細胞剤が対象とする癌細胞は、ヒト由来のものであって、大腸癌細胞、肝臓癌細胞、肺癌細胞、乳癌細胞又は胃癌細胞であること、また、正常細胞はヒト由来の肺線維芽細胞、皮膚線維芽細胞又は胚線維芽細胞であることがより好ましい。
【００１０】
また、本発明の医薬用組成物及び食用組成物は、前記特異的殺癌細胞剤を配合した組成物となすことによって達成される。
【００１１】
【発明の実施の形態】
本発明の特異的殺癌細胞剤及びこれを配合してなる医薬用組成物又は食用組成物について以下にさらに詳述する。本発明によれば、まず、二重結合を３〜６個有する共役多価不飽和脂肪酸類を有効成分としてなる特異的殺癌細胞剤が提供される。本発明でいう共役多価不飽和脂肪酸類における共役多価不飽和脂肪酸とは、該脂肪酸１分子中に二重結合を３〜６個有する多価不飽和脂肪酸であって、二重結合の一部又は全部が共役の位置関係にある異性体を包含する。また、個々の二重結合の幾何異性体はシス型又はトランス型のいずれであってもよい。
【００１２】
本発明の共役多価不飽和脂肪酸類の構成脂肪酸は共役ジエン脂肪酸（以下、共役ジエン体ということがある。）、共役トリエン脂肪酸（以下、共役トリエン体ということがある。）、共役テトラエン脂肪酸（以下、共役テトラエン体ということがある。）、共役ペンタエン脂肪酸（以下、共役ペンタエン体ということがある。）及び共役ヘキサエン脂肪酸（以下、共役ヘキサエン体ということがある。）にタイプ分けできる。共役ジエン脂肪酸の例としてシス−９，トランス−１１，シス−１５−オクタデカトリエン酸、シス−５，トランス−７，シス−１１，シス−１４−エイコサテトラエン酸、シス−５，トランス−７，シス−１１，シス−１４，シス−１７−エイコサペンタエン酸、シス−４，トランス−６，シス−１０，シス−１３，シス−１６，シス−１９−ドコサヘキサエン酸等をあげることができる。また、共役トリエン脂肪酸としてシス−９，トランス−１１，トランス−１３−オクタデカトリエン酸（エレオステアリン酸）、シス−６，トランス−８，トランス−１０−オクタデカトリエン酸、シス−８，トランス−１０，シス−１２−エイコサトリエン酸、シス−５，トランス−７，トランス−９，シス−１４，シス−１７−エイコサペンタエン酸、シス−４，シス−６，トランス−８，シス−１３，シス−１６，シス−１９−ドコサヘキサエン酸等を例示できる。共役テトラエン脂肪酸としてシス−９，トランス−１１，トランス−１３，トランス−１５−オクタデカテトラエン酸（パリナリン酸）、シス−５，トランス−７，シス−９，シス−１１−エイコサテトラエン酸、シス−５，トランス−７，トランス−９，シス−１１，シス−１７−エイコサペンタエン酸、シス−４，シス−６，トランス−８，トランス−１０，シス−１６，シス−１９−ドコサヘキサエン酸等を例示できる。同様に、シス−５，トランス−７，トランス−９，トランス−１１，トランス−１３−エイコサペンタエン酸、シス−４，トランス−６，シス−８，トランス−１０，トランス−１２，シス−１９−ドコサヘキサエン酸等の共役テトラエン酸、シス−４，トランス−６，トランス−８、トランス−１０，トランス−１２，トランス−１４−ドコサヘキサエン酸等の共役ヘキサエン酸も本発明の共役多価不飽和脂肪酸類の範ちゅうに含めることができる。
【００１３】
本発明に係る共役多価不飽和脂肪酸類は、前記のように共役異性体の混合物であるが、二重結合の数が３〜６個であることが重要である。二重結合数が２個の共役リノール酸の場合は本発明の所望の効果である殺癌細胞作用が小さく、一方、７個以上の共役多価不飽和脂肪酸類はそのもの及びその原料の入手が困難であり、実用的ではない。また、前記の共役多価不飽和脂肪酸類の構成脂肪酸は共役トリエン脂肪酸に富むものがより好ましく、このような組成の共役多価不飽和脂肪酸類においては所望の効果がより一層顕著に発現するようになる。共役トリエン脂肪酸に富むものとは、共役多価不飽和脂肪酸類を構成する脂肪酸の総重量に対して共役トリエン脂肪酸を少なくとも５％（重量基準。以下とくにことわらないかぎり同様。）以上含有し、より望ましくは１０％以上含むものである。なお、かかる共役多価不飽和脂肪酸類はこれを構成する共役多価不飽和脂肪酸の炭素数の多少にかかわらず、また二重結合の位置異性体や幾何異性体の種類は任意の割合の組成であるものでもさしつかえない。
【００１４】
本発明においては、共役多価不飽和脂肪酸類の態様として、二重結合を３〜６個有する共役多価不飽和脂肪酸の単一物もしくは混合物を構成脂肪酸とするカルボン酸、エステル及び塩からなる群から選択される１種又は２種以上のものを採用することができる。ここに、塩としてはカリウム、ナトリウム、カルシウム、マグネシウム等のアルカリ金属塩又はアルカリ土類金属塩がよい。エステルは任意のものでよいが、メタノール、エタノール、ｎ−プロパノール、イソプロパノール、ｎ−ブタノール、イソブタノール等の炭素数が１〜５程度のもののエステルが望ましく、さらに、モノグリセリド、ジグリセリド、トリグリセリド等のアシルグリセリド、モノアシル型あるいはジアシル型のホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルイノシトール、ホスファチジルセリン等のアシルグリセロリン脂質がより望ましい。かかるアシルグリセリドとアシルグリセロリン脂質は単独で又は２種以上の混合物として用いることができ、これらにさらに前記のカルボン酸や塩や低級１価アルコールエステルを適宜併用してもさしつかえない。
【００１５】
本発明の共役多価不飽和脂肪酸類を得るにはさまざまな方法を採用でき、例えば、非共役多価不飽和脂肪酸類を原料としてこれを共役化処理する方法、動植物、微生物、藻類等の組織あるいはそれらの産生物から分離、精製処理して採取する方法等がある。
【００１６】
前者の方法では、二重結合を３〜６個有する非共役多価不飽和脂肪酸類、より好ましくは生理機能の点から望ましいｎ−３系脂肪酸であるα−ＬＮＡ、ＥＰＡ、ＤＰＡ及びＤＨＡからなる群から選ばれる１種又は２種以上もしくはこれらを構成脂肪酸とする前述のエステルや塩を原料とし、あるいはｎ−６系脂肪酸であるγ−ＬＮＡ、ジホモ−γ−リノレン酸（Ｃ２０：３）及びＡＡ等からなる群から選択される単独又は混合脂肪酸もしくはそれらのエステルや塩を原料とし、あるいは前記ｎ−３系脂肪酸およびｎ−６系脂肪酸の混合物もしくはこれらを構成脂肪酸とするエステルや塩を原料とする。
【００１７】
ついで、これを水酸化カリウム、水酸化ナトリウム等のアルカリ金属水酸化物、ヨウ素、ヨウ化カリウム、二酸化硫黄やチオール類等の硫黄化合物等を触媒とし、又は無触媒下で紫外線を照射しながら、窒素ガス等の不活性ガス雰囲気中で、１５０〜２５０℃にて５分〜１時間、共役化反応を行わしめ、必要に応じて触媒除去等の精製処理を施して目的とする共役多価不飽和脂肪酸類を製造することができる。
【００１８】
なお、共役化処理にあたっては、前記原料１重量部に対して、１０〜３０重量％、より好ましくは１５〜２５重量％のアルカリ金属水酸化物（前記同様）を含むジオール、より好ましくは水溶性のエチレングリコール、プロピレングリコール等の溶液５０〜１００重量部を加え、１７０〜１９０℃にて５〜３０分間、より好ましくは５〜１５分間、前記同様の不活性ガス雰囲気中で共役化反応を行わせることによって、特異的な殺癌細胞活性の高い共役トリエン脂肪酸に富む共役多価不飽和脂肪酸類を製造することが可能となる。
【００１９】
後者の方法では、動植物、微生物、藻類等の組織を原料とし、これから共役多価不飽和脂肪酸を構成脂肪酸とする油脂、例えばキリ油、サクランボやホウセンカの種子油を常法により搾油し、これをそのまま又は適宜にエステル交換、水素添加、ケン化分解、精製等の各処理を施すことによって目的とする共役多価不飽和脂肪酸類を採取することができる。なお、キリ油の構成脂肪酸はエレオステアリン酸であり、サクランボやホウセンカの種子油からパリナリン酸が得られる。これらの共役多価不飽和脂肪酸類は単独で又は混合して本発明の特異的殺癌細胞剤の有効成分として使用することが可能である。
【００２０】
本発明の特異的殺癌細胞剤は前述の共役多価不飽和脂肪酸類そのものを単一成分として又は適宜に組み合わせて混合成分として用いることによって調製することができる。また、該共役多価不飽和脂肪酸類を有効成分として、これに他の成分、すなわち食品及び／又は医薬品を製造する場合にはこれに許容され、かつ本発明の所望の効果の発現を阻害しない公知の添加物、安定化剤、賦活剤等を適宜に併用して本発明の特異的殺癌細胞剤を調製することも可能である。この併用成分あるいは原材料の例としてアミノ酸、ペプチド、蛋白質、各種糖質、澱粉およびこの分解物、油脂、アスコルビン酸、ビタミンＥ、トコフェロール、フィトステロール、ミネラル、カテキン等のポリフェノール類等およびこれらの誘導体をあげることができるが、本発明はこれらに限定されるものではない。本発明の特異的殺癌細胞剤に含まれる前記有効成分の量は、殺癌細胞剤の形態、使用目的、用途、操作および作業性等の条件により一律に規定しがたいが、概ね２０重量％以上、より好ましくは３０重量％以上である。２０重量％未満では後述する用途組成物を調製する際に多量の殺癌細胞剤を配合せざるを得ない等の使用上の利便性を欠くことがある。
【００２１】
なお、本発明の特異的殺癌細胞剤の形態は、本発明に係る共役多価不飽和脂肪酸類のみを用いるときは通常油状の液体であるが、前記併用物質を用いるときはその物性に適応させてさまざまな形態を採用できる。すなわち、併用物質がアスコルビン酸パルミテート、フィトステロール、ビタミンＥ等のように油溶性の場合は、本発明に係る共役多価不飽和脂肪酸類と混合して均一状態となし、また、アスコルビン酸、アミノ酸、ミネラル、蛋白質等のように水溶性ないしは水分散性の場合は、例えばその乾燥粉末を本発明に係る共役多価不飽和脂肪酸類と混練して分散状態にするか、水及び適宜に界面活性剤を共存させて乳化状態となすこともできる。
【００２２】
本発明の特異的殺癌細胞剤は、ヒト組織由来の癌細胞とりわけ大腸癌細胞、肝臓癌細胞、肺癌細胞、乳癌細胞又は胃癌細胞に対して顕著な死滅化効果を発揮する。この効果は二重結合を２個有する場合の共役ジエン脂肪酸のみから構成される共役リノール酸及びその誘導体に比べて著しく大きいものである。
【００２３】
本発明の特異的殺癌細胞剤は、ヒト組織由来の正常細胞とりわけ肺線維芽細胞、皮膚線維芽細胞、胚線維芽細胞に対しては該細胞の本来の機能の発現を阻害したり死滅させたりする作用は認められず、正常細胞に悪影響を及ぼさない。
【００２４】
本発明では、前述のように、特定の共役多価不飽和脂肪酸類を有効成分としてなる特異的殺癌細胞剤が提供されるが、さらにこれを配合してなる組成物も提供される。この組成物の態様としては医薬用組成物および食用組成物が好適である。
【００２５】
本発明の医薬用組成物は、前記の特異的殺癌細胞剤すなわち二重結合を３〜６個有する共役多価不飽和脂肪酸類を有効成分とし、これに本発明の趣旨に反しない公知の賦型剤や添加剤を必要に応じて加え、常法により加工して錠剤、カプセル剤、顆粒剤、散剤、注射剤等の製剤となしたものである。経口摂取、経管投与または注射投与して、癌疾患の予防あるいは治療のために適用する。前記製剤中に配合する本発明の特異的殺癌細胞剤は、適宜に溶剤分別や吸着剤処理等の分画精製処理を施して共役トリエン脂肪酸の含有量をできるだけ高めたものを用いるのが望ましい。その配合量は製剤の種類、形態や用法、用量等により一律に規定し難いが、概ね０．０１〜５０重量％である。経口摂取する場合の摂取量はとくに限定されないが、有効成分である前記共役多価不飽和脂肪酸として、成人（体重５０ｋｇ）１日あたり０．０１〜１０ｇ、より好ましくは０．１〜５ｇである。この範囲を外れて少ないと本発明の所望の効果を奏することが難しくなり、逆に多すぎてもさらに顕著な効果は認められない。
【００２６】
本発明の他の組成物の好適な態様は食用組成物である。すなわち、前述のようにして得られる二重結合を３〜６個有する共役多価不飽和脂肪酸類を有効成分として含む特異的殺癌細胞剤は、これをそのまま液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、ドレッシング、ゼリー、ヨーグルト、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキー等に添加したり、必要に応じてデキストリン、乳糖、澱粉等の賦型剤や香料、色素等とともにペレット、錠剤、顆粒等に加工したり、またゼラチン等で被覆してカプセルに成形加工して健康食品や栄養補助食品等として利用できる。これらの食品類あるいは食用組成物における本発明の殺癌細胞剤の配合量は、当該食品や組成物の種類や状態等により一律に規定しがたいが、約０．０１〜５０重量％、より好ましくは０．１〜３０重量％である。配合量が０．０１重量％未満では経口摂取による所望の効果が小さく、５０重量％を超えると食品の種類によっては風味を損なったり当該食品を調製できなくなる場合がある。なお、本発明の特異的殺癌細胞剤は、これをそのまま食用に供してもさしつかえない。
【００２７】
【実施例】
実施例１
反応容器に精製亜麻仁油（粗シグマ社製試薬。ＧＬＣによる脂肪酸組成分析によれば、α−ＬＮＡ：６７％）１ｇ、２１重量％水酸化カリウムを含むエチレングリコール溶液１００ｍｌを加え、容器内空気を窒素ガスで置換した後、密閉して１８０℃で１０分間共役化反応を行わせた。ついで、該反応物をエタノールで繰返し洗浄して、共役α−ＬＮＡを構成脂肪酸とするトリグリセリド（試料１）を調製した。共役多価不飽和脂肪酸の含量は、分光光度計（島津製作所（株）製、ＶＵＶ２４００ＰＣ）を用いて２２０〜３６０ｎｍの吸収スペクトルを測定し、ＡＯＡＣＯｆｆｉｃｉａｌＭｅｔｈｏｄｓｏｆＡｎａｌｙｓｉｓ（１９９０年、９５７．１３）に記載の方法に準じて求めた（以下、とくにことわらないかぎり同様）。試料１に含まれる共役多価不飽和脂肪酸の共役ジエン体は１９．５％であり、共役トリエン体は６３．０％であった。試料１をそのまま本発明の特異的殺癌細胞剤とした。
【００２８】
実施例２
実施例１の方法で別に調製した試料１の１ｇを常法により塩酸加水分解反応を行わしめ精製処理し、共役α−ＬＮＡを含む混合脂肪酸（試料２）を調製した。共役ジエン体：１８．０％、共役トリエン体：６２．０％であった。試料２を本発明の特異的殺癌細胞剤とした。
【００２９】
実施例３
反応容器に魚油由来のＥＰＡ（備前化成（株）製、ＧＣＬ純度：９８％）１ｇ、１５重量％水酸化ナトリウムを含むプロピレングリコール溶液１００ｍｌを加え、実施例１と同様にして１７０℃で２０分間共役化反応を行わせ、精製して共役ＥＰＡ（試料３）を調製した。これに含まれる共役多価不飽和脂肪酸の共役ジエン体：５８．０％、共役トリエン体：２３．７％、共役テトラエン体：１５．０％、共役ペンタエン体：２．５％であった。試料３を本発明の特異的殺癌細胞剤とした。
【００３０】
実施例４
反応容器に魚油由来のＤＨＡ（備前化成（株）製、ＧＬＣ純度：９９％）１ｇ、２５重量％水酸化カリウムを含むエチレングリコール溶液１００ｍｌを加え、実施例１と同様にして１８０℃で１５分間共役化反応を行わせ、精製して共役ＤＨＡ（試料４）を調製した。これに含まれる共役多価不飽和脂肪酸の共役ジエン体：５４．８％、共役トリエン体：２２．０％、共役テトラエン体：１４．７％、共役ペンタエン体：５．２％であった。試料４を本発明の特異的殺癌細胞剤とした。
【００３１】
実施例５
実施例３および４の方法で別にそれぞれ調製した試料３および試料４の各１ｇ混合物を冷却器つきエステル化用フラスコに入れ、硫酸−エタノール溶液を６０ｍｌ加えて１時間沸騰させ、室温まで冷却した。その後、水１００ｍｌおよび石油エーテル５０ｍｌを用いて抽出し、該抽出液を水洗し、洗浄水がメチルオレンジ指示薬で酸性を示さなくなるまで水洗を繰返した。石油エーテル溶液を無水硫酸ナトリウムで脱水後、減圧下に脱溶剤処理して共役ＥＰＡおよび共役ＤＨＡを構成脂肪酸とするエチルエステル（試料５）を調製した。これに含まれる共役多価不飽和脂肪酸の共役ジエン体：５６．０％、共役トリエン体：１５．０％、共役テトラエン体：１８．０％、共役ペンタエン体：３．５％であった。試料５を本発明の特異的殺癌細胞剤とした。
【００３２】
実施例６
実施例３および４の方法でそれぞれ別に調製した試料３および試料４の各１ｇ混合物を冷却器つきケン化用フラスコに入れ、１モル／リットル水酸化カリウム−エタノール溶液を５０ｍｌ加えて１時間加熱還流させ室温まで冷却した。その後、水５０ｍｌおよび石油エーテル１００ｍｌを用いて抽出、精製処理して共役ＥＰＡおよび共役ＤＨＡを構成脂肪酸とするカリウム塩を調製した。これに含まれる共役多価不飽和脂肪酸の共役ジエン体：５９．５％、共役トリエン体：２０．７％、共役テトラエン体：１２．０％、共役ペンタエン体：１．５％であった。
【００３３】
実施例７
脂肪酸組成がＥＰＡ：２７．５重量％およびＤＨＡ：１８．３重量％を含む精製分別魚油１０ｇと、１５重量％水酸化カリウム含有エチレングリコール溶液４００ｍｌとを用い、実施例１に記載の方法に準じて１８０℃で２５分間共役化反応を行わせ、精製して共役ＥＰＡおよび共役ＤＨＡを構成脂肪酸とするトリグリセリド（試料７）を調製した。これに含まれる共役多価不飽和脂肪酸の共役ジエン体：４７．０％、共役トリエン体：３３．０％、共役テトラエン体：１０．５％、共役ペンタエン酸：３．５％であった。試料７にビタミンＥ及びデキストリンを混合して本発明の特異的殺癌細胞剤とした。
【００３４】
比較例１
反応容器にリノール酸（米国シグマ社製試薬、ＧＬＣ純度：９９％）１ｇ、６重量％水酸化カリウムを含むエチレングリコール溶液１００ｍｌを加え、実施例１と同様にして１８０℃で２５分間共役化反応を行わせ、精製して共役リノール酸を調製した。このものは共役ジエン体：９０．６％であった。ついで、この共役リノール酸を実施例５に記載の方法に準じてエステル化処理し、精製処理して共役リノール酸を構成脂肪酸とするエチルエステル（比較試料１）を調製した。
【００３５】
試験例１
前述の各種共役多価不飽和脂肪酸類の殺癌細胞作用を以下に述べる方法で評価した。すなわち、東北大学加齢医学研究所付属癌細胞保存施設より分譲され、入手したヒト由来の５種類の癌細胞である大腸癌細胞（細胞番号（以下同様）：ＴＫＧ０３７９、以下ＤＬＤ−１という。）、肝臓癌細胞（ＴＫＧ０２０５、以下ＨｅｐＧ２という。）、肺癌細胞（ＴＫＧ０１８４、以下Ａ５４９という。）、乳癌細胞（ＴＫＧ０４７９、以下ＭＣＦ７という。）及び胃癌細胞（ＴＫＧ０２２８、以下ＭＫＮ−７という。）を用い、各細胞を９６穴マイクロプレートに播種し、８０％コンフレント（細胞充満度）に達した時点で、１５μＭの共役多価不飽和脂肪酸類を含む０．５％牛血清アルブミンを加え、５％二酸化炭素雰囲気下３７℃で２４時間インキュベートした後に生細胞数をＭＴＴ（３−（４，５−ジメチルアゾール−２−イル）−２，５−ジフェニル−２Ｈ−テトラゾリウムブロマイド）法で調べた。なお、ＭＴＴ法は生細胞内酵素活性を指標としているためほとんどの細胞に適用でき、この結果も比較的安定しているので殺細胞作用の評価法として採用した。この結果を表１に示す。また、非共役ＥＰＡおよび非共役ＤＨＡを構成脂肪酸とする魚油（比較試料２）について同様に試験した結果を併記した。
【００３６】
【表１】

【００３７】
表１において、数値は試験試料を添加しないときのＭＴＴ活性（細胞生存率、％）を１００とし、試験試料の添加濃度（１５μＭ）における相対値を平均値±標準偏差（ｎ＝６）で示す（以下同様）。各試験試料の数値は比較試料１および２の場合の値と比較して有意差があった（Ｐ＜０．０５）。このデータから、本発明に係る共役多価不飽和脂肪酸類は、魚油に比べて、大腸癌細胞に対して殺細胞効果が認められた。また、肝臓癌細胞、肺癌細胞及び胃癌細胞に対しても同様の殺細胞効果が認められた。
【００３８】
試験例２
前述の各種共役多価不飽和脂肪酸類の正常細胞に対する影響を試験例１に記載の方法と同様にして評価した。ヒト由来の正常細胞は、ヒューマンサイエンス研究資源バンク（大阪）から入手した肺線維芽細胞（以下ＭＲＣ−５という）、皮膚線維芽細胞（以下ＴＩＧ−１０３という）、胚線維芽細胞（以下ＫＭＳ−６という）を用いた。この結果を共役リノール酸エチルエステルおよび魚油（未変性）についての結果とともに表２に示す。同表中の数値、記号の意味は表１と同じである。これらのデータから、ｎ−３系脂肪酸の一種であるα−ＬＮＡ、ＥＰＡ、ＤＨＡ等の共役多価不飽和脂肪酸類はヒト正常細胞の生育（分裂、増殖等）に対してほとんど影響を及ぼさず、とりわけ共役トリエン体を多く含むものでは当該作用が強いことが明らかになった。
【００３９】
【表２】

【００４０】
実施例８
試料４又は試料７の共役多価不飽和脂肪酸類２５０ｍｇ、精製大豆油３０ｍｇ、ミツロウ１０ｍｇ及びビタミンＥ１０ｍｇを窒素ガス雰囲気下で約４０℃に加温し、十分に混合して均質な液状物とした。これをカプセル充填機に供給して１粒内容量が３００ｍｇのゼラチン被覆カプセル製剤を試作した。これらの製剤は医薬用組成物又は食用組成物として利用できるものである。
【００４１】
実施例９
市販の牛乳１リットルに対して試料７の共役多価不飽和脂肪酸類５ｇ及びビタミンＥ０．２ｇを添加し、十分に混合して均等な飲料物を試作した。このものは通常の牛乳と比較して風味、色あい、食感等の食品適性に遜色なく、癌予防用あるいは当該疾患用の食品として好適に利用され得るものである。
【００４２】
【発明の効果】
本発明によれば、二重結合を３〜６個有する共役多価不飽和脂肪酸類を有効成分としてなる特異的殺癌細胞剤が提供される。この殺癌細胞剤はヒト由来の大腸癌、肝臓癌、肺癌、乳癌あるいは胃癌等の癌細胞を死滅させる効果を奏し、かつ正常細胞の生育には悪影響を与えない。かかる効果は共役多価不飽和脂肪酸類を構成する脂肪酸が共役トリエン脂肪酸を多量に含む場合により一層顕著なものとなる。ちなみに、二重結合を２個しかもたない共役リノール酸等の共役ジエン脂肪酸では前記殺癌細胞作用が認められない。
また、本発明によれば、α−リノレン酸、エイコサペンタエン酸、ドコサヘキサエン酸等のｎ−３系非共役多価不飽和脂肪酸を構成脂肪酸とするものを原料として、これを共役トリエン脂肪酸に富む共役多価不飽和脂肪酸類に効率的に共役異性化できる共役化方法が提供される。
さらに、本発明によれば、前記特異的殺癌細胞剤を配合してなる医薬用組成物又は食用組成物が提供される。該組成物は癌疾患の予防あるいは治療用途に利用され得るものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cancer cell killing agent comprising a specific conjugated unsaturated fatty acid as an active ingredient and a composition comprising the same. More specifically, conjugated polyunsaturated fatty acids having 3 to 6 carbon-carbon double bonds (hereinafter simply referred to as double bonds), all or part of the double bonds being in a conjugated relationship. A specific cancer-killing cell agent comprising the conjugated polyunsaturated fatty acids as an active ingredient, and capable of efficiently killing only cancer cells without adversely affecting normal human-derived cells, and a combination thereof The present invention relates to a pharmaceutical composition or an edible composition.
[0002]
[Prior art]
In recent years, research on physiological activity and pharmacological action of lipids has progressed rapidly, and in particular, the relationship between unsaturated fatty acids having 18 or more carbon atoms and various diseases is being elucidated. In addition, due to the full-scale arrival of an aging society, the relationship between lifestyle habits and dietary patterns and diseases has attracted attention. In addition to the treatment of various diseases, lifestyles have been reviewed and included in various food ingredients. Functional evaluation of ingredients
[0003]
Among the unsaturated fatty acids, polyunsaturated fatty acids having 3 or more double bonds include α-linolenic acid (C18: 3 n-3 series) which is a fatty acid constituting seed oil such as perilla, egoma and flax. Hereinafter referred to as α-LNA), γ-linolenic acid (C18: 3 n-6 system, hereinafter referred to as γ-LNA) which is a constituent fatty acid of fats and oils of breast milk and evening primrose, arachidonic acid which constitutes fats and oils of animal organ tissues ( C20: 4 n-6 system, hereinafter referred to as AA), eicosapentaenoic acid (C20: 5 n-3 system, hereinafter referred to as EPA), docosahexaenoic acid (C22), which is a constituent fatty acid of fish oil such as bonito and tuna, and egg yolk oil. : 6 n-3 system, hereinafter referred to as DHA). These are mainly constituents of lipids contained in food raw materials, but also exist as constituent fatty acids in oils in microalgae and microbial tissues.
[0004]
In addition, as physiological functions of these polyunsaturated fatty acids, α-LNA improves memory learning ability (JP-A-1-153629), antiallergic action, serum lipid improving action, etc., AA platelet aggregation action, etc. Decreased blood lipids of DHA, including cholesterol-reducing effects of EPA, decreased platelet adhesion and increased red blood cell deformability (Yasu Tamura et al., “Food Science”, 161, 33-39, 1991) Katsuaki Imaizumi, “Clinical Nutrition”, Volume 83, No. 4, 440, 1993, etc.), platelet aggregation inhibition (Yuzo Hikawa et al., “Blood and Vascular”, Volume 15, No. 2, No. 138-141 (1984), improvement of memory and learning ability (A. hucas et al., The Lancet, 339 , 261, 1992, etc.), anti-dementia (M. Soderberg et al., Lipids, 26 (6), 421, 1991), antitumor (Tomio Narusawa et al., “Ayumi of Medicine”, 145, 911, 1988), antiallergy (M. Shikano et al., J. Immunology, 150 , 3525, 1993) and the like, and application to various uses including the use in therapeutic drugs and foods has been proposed.
[0005]
By the way, there is conjugated polyunsaturated fatty acid as a kind of polyunsaturated fatty acid. In general, conjugated unsaturated fatty acids are more chemically reactive than their corresponding non-conjugated unsaturated fatty acids, are easily oxidized, and form a quick-drying film. Application of eleostearic acid (cis-9, trans-11, trans-13-octadecatrienoic acid), which is a decomposed fatty acid, to the paint and paint fields has been conventionally performed. In addition, dairy products such as butter and cheese, and beef are conjugated linoleic acid, which is a conjugated isomer of linoleic acid (positional isomers and geometries of double bonds mainly composed of cis-9, trans-11-octadecadienoic acid). (A mixture of isomers) is known to exist in a small amount. Recently, conjugated linoleic acid obtained by conjugating linoleic acid has been put on the market as a food material to reduce body fat (Lipids, 31 , 853 (1997)), blood cholesterol reducing action, antitumor activity (MA Belury, Nutr. Res., 53, 83 (1995)) and the like have also been found.
[0006]
[Problems to be solved by the invention]
As described above, elucidation of the physiological actions of polyunsaturated fatty acids that constitute lipids and attempts to apply them to the treatment and prevention of various diseases have been made. Research on physiological functions of conjugated unsaturated fatty acids Is a stage that has just started to be examined in detail recently, and there is hardly any knowledge about conjugated polyunsaturated fatty acids having 3 or more double bonds. In view of the current situation, the present invention elucidates the physiological function of the conjugated polyunsaturated fatty acids, particularly the influence on cancer cells and normal cells, and specifically and effectively inhibits the growth or kills cells. Therefore, it is an object to be solved to provide a material that can be applied to the prevention or treatment of cancer diseases, and a pharmaceutical composition or an edible composition comprising the same.
[0007]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors have intensively studied various types of conjugated polyunsaturated fatty acids, and as a result, conjugated polyunsaturated fatty acids having 3 to 6 double bonds are remarkable. It has been found that it has a cancer-killing cell effect and does not adversely affect normal cells, and the present invention has been completed. In the following, the conjugated polyunsaturated fatty acids are used in the meaning including a single or mixed composition of conjugated polyunsaturated fatty acids and various derivatives having conjugated polyunsaturated fatty acids as constituent fatty acids.
[0008]
That is, the specific cancer cell killing agent which is a problem to be solved in the present invention is achieved by a cancer cell killing agent comprising conjugated polyunsaturated fatty acids having 3 to 6 double bonds as an active ingredient. Here, the constituent fatty acids of the conjugated polyunsaturated fatty acids are more preferably those rich in conjugated triene fatty acids, and further α-LNA, EPA, docosapentaenoic acid (C22: 5 n-3 series, hereinafter referred to as DPA). And those obtained by conjugating one or more non-conjugated polyunsaturated fatty acids selected from the group consisting of DHA. In addition, a more desirable embodiment of the conjugated polyunsaturated fatty acids is one selected from the group consisting of carboxylic acids, esters and salts having conjugated polyunsaturated fatty acids having 3 to 6 double bonds as constituent fatty acids. Or two or more types. Here, the ester is preferably an ester of a lower monohydric alcohol, and more preferably an acylglyceride and / or glycerophospholipid.
[0009]
The conjugated polyunsaturated fatty acids used in the present invention can be prepared by conjugating a corresponding non-conjugated polyunsaturated fatty acid, but the conjugation treatment involves a double bond. Add 50 to 100 parts by weight of a diol solution containing 10 to 30% by weight of an alkali metal hydroxide with respect to 1 part by weight of 3 to 6 non-conjugated polyunsaturated fatty acids and add 5 to 100 at 170 to 190 ° C. It is desirable to use a method for producing conjugated polyunsaturated fatty acids rich in conjugated triene fatty acids, which is characterized in that a conjugation reaction is performed for 30 minutes. The cancer cells targeted by the specific cancer-killing cell agent of the present invention are human-derived and are colon cancer cells, liver cancer cells, lung cancer cells, breast cancer cells or gastric cancer cells, and normal. More preferably, the cells are human-derived lung fibroblasts, skin fibroblasts or embryonic fibroblasts.
[0010]
In addition, the pharmaceutical composition and edible composition of the present invention can be achieved by preparing a composition containing the specific cancer cell killing agent.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The specific cancer-killing cell agent of the present invention and the pharmaceutical composition or edible composition comprising the same are described in detail below. According to the present invention, first, a specific cancer cell killing agent comprising conjugated polyunsaturated fatty acids having 3 to 6 double bonds as an active ingredient is provided. The conjugated polyunsaturated fatty acid in the conjugated polyunsaturated fatty acid referred to in the present invention is a polyunsaturated fatty acid having 3 to 6 double bonds in one molecule of the fatty acid, It includes isomers that are partially or entirely conjugated. In addition, the geometric isomer of each double bond may be either cis type or trans type.
[0012]
Constituent fatty acids of the conjugated polyunsaturated fatty acids of the present invention are conjugated diene fatty acids (hereinafter sometimes referred to as conjugated diene isomers), conjugated triene fatty acids (hereinafter sometimes referred to as conjugated triene isomers), conjugated tetraene fatty acids (hereinafter also referred to as conjugated triene fatty acids). Hereinafter, it may be classified into a conjugated tetraene body), a conjugated pentaene fatty acid (hereinafter sometimes referred to as a conjugated pentaene body), and a conjugated hexaene fatty acid (hereinafter sometimes referred to as a conjugated hexaene body). Examples of conjugated diene fatty acids are cis-9, trans-11, cis-15-octadecatrienoic acid, cis-5, trans-7, cis-11, cis-14-eicosatetraenoic acid, cis-5, trans -7, cis-11, cis-14, cis-17-eicosapentaenoic acid, cis-4, trans-6, cis-10, cis-13, cis-16, cis-19-docosahexaenoic acid, etc. it can. Further, as conjugated triene fatty acids, cis-9, trans-11, trans-13-octadecatrienoic acid (eleostearic acid), cis-6, trans-8, trans-10-octadecatrienoic acid, cis-8, Trans-10, cis-12-eicosatrienoic acid, cis-5, trans-7, trans-9, cis-14, cis-17-eicosapentaenoic acid, cis-4, cis-6, trans-8, cis -13, cis-16, cis-19-docosahexaenoic acid and the like. Cis-9, trans-11, trans-13, trans-15-octadecatetraenoic acid (parinaric acid), cis-5, trans-7, cis-9, cis-11-eicosatetraene as conjugated tetraene fatty acids Acid, cis-5, trans-7, trans-9, cis-11, cis-17-eicosapentaenoic acid, cis-4, cis-6, trans-8, trans-10, cis-16, cis-19- Examples thereof include docosahexaenoic acid. Similarly, cis-5, trans-7, trans-9, trans-11, trans-13-eicosapentaenoic acid, cis-4, trans-6, cis-8, trans-10, trans-12, cis-19 -Conjugated tetraenoic acid such as docosahexaenoic acid, conjugated hexaenoic acid such as cis-4, trans-6, trans-8, trans-10, trans-12, trans-14-docosahexaenoic acid are also conjugated polyunsaturated fatty acids of the present invention. It can be included in a category.
[0013]
The conjugated polyunsaturated fatty acids according to the present invention are a mixture of conjugated isomers as described above, but it is important that the number of double bonds is 3 to 6. In the case of conjugated linoleic acid having two double bonds, the action of cancer-killing cells, which is the desired effect of the present invention, is small, while seven or more conjugated polyunsaturated fatty acids themselves and their raw materials are available. Difficult and impractical. In addition, the constituent fatty acids of the conjugated polyunsaturated fatty acids are preferably rich in conjugated triene fatty acids, and the desired effect is more remarkably exhibited in the conjugated polyunsaturated fatty acids having such a composition. become. What is rich in conjugated triene fatty acids contains at least 5% of conjugated triene fatty acids (based on weight, hereinafter the same unless otherwise specified) with respect to the total weight of fatty acids constituting the conjugated polyunsaturated fatty acids, More desirably, it contains 10% or more. Such conjugated polyunsaturated fatty acids can be used regardless of the number of carbon atoms of the conjugated polyunsaturated fatty acids constituting them, and the position isomers and geometric isomers of the double bond may be of any composition. It doesn't matter what it is.
[0014]
In the present invention, as an aspect of the conjugated polyunsaturated fatty acids, the conjugated polyunsaturated fatty acid is composed of a carboxylic acid, an ester, and a salt, each of which includes a single or mixture of conjugated polyunsaturated fatty acids having 3 to 6 double bonds. The 1 type (s) or 2 or more types selected from a group are employable. Here, the salt is preferably an alkali metal salt or alkaline earth metal salt such as potassium, sodium, calcium or magnesium. Esters may be arbitrary, but esters having about 1 to 5 carbon atoms such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, etc. are desirable, and acyls such as monoglycerides, diglycerides, triglycerides, etc. Acyl glycerophospholipids such as glycerides, monoacyl or diacyl phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines are more desirable. Such acylglycerides and acylglycerophospholipids can be used alone or as a mixture of two or more thereof, and the above carboxylic acids, salts and lower monohydric alcohol esters may be used in combination as appropriate.
[0015]
Various methods can be employed to obtain the conjugated polyunsaturated fatty acids of the present invention, for example, a method of conjugating a non-conjugated polyunsaturated fatty acid as a raw material, tissues such as animals, plants, microorganisms, and algae. Alternatively, there is a method of collecting the product by separating and purifying it from the product.
[0016]
The former method comprises non-conjugated polyunsaturated fatty acids having 3 to 6 double bonds, more preferably α-LNA, EPA, DPA and DHA which are desirable n-3 fatty acids from the viewpoint of physiological function. One or two or more selected from the group or the above-mentioned esters or salts containing these as constituent fatty acids, or n-6 fatty acids γ-LNA, dihomo-γ-linolenic acid (C20: 3) and A single or mixed fatty acid selected from the group consisting of AA or the like, or an ester or salt thereof, or a mixture of the n-3 fatty acid and n-6 fatty acid or an ester or salt containing these as a constituent fatty acid. And
[0017]
Then, using this as a catalyst, alkali metal hydroxides such as potassium hydroxide and sodium hydroxide, iodine, potassium iodide, sulfur compounds such as sulfur dioxide and thiols, etc., while irradiating ultraviolet rays without catalyst, The conjugation reaction is carried out in an inert gas atmosphere such as nitrogen gas at 150 to 250 ° C. for 5 minutes to 1 hour, and if necessary, purification treatment such as catalyst removal is performed to achieve the desired conjugated polyvalent impurities. Saturated fatty acids can be produced.
[0018]
In the conjugation treatment, a diol containing 10 to 30% by weight, more preferably 15 to 25% by weight of an alkali metal hydroxide (as described above), more preferably water-soluble, relative to 1 part by weight of the raw material. 50 to 100 parts by weight of a solution of ethylene glycol, propylene glycol or the like is added, and a conjugation reaction is performed in an inert gas atmosphere similar to the above for 5 to 30 minutes, more preferably 5 to 15 minutes at 170 to 190 ° C. Thus, it is possible to produce conjugated polyunsaturated fatty acids rich in conjugated triene fatty acids with specific high cancer-killing cell activity.
[0019]
In the latter method, fats and oils that use conjugated polyunsaturated fatty acids as constituent fatty acids, such as ground oil, cherries and spinach seed oils, are squeezed in a conventional manner. The target conjugated polyunsaturated fatty acids can be collected by performing various treatments such as transesterification, hydrogenation, saponification decomposition, and purification as they are or as appropriate. The constituent fatty acid of the drill oil is eleostearic acid, and parinaric acid can be obtained from the seed oils of cherries and spinach. These conjugated polyunsaturated fatty acids can be used alone or in combination as an active ingredient of the specific cancer cell killing agent of the present invention.
[0020]
The specific cancer-killing cell agent of the present invention can be prepared by using the conjugated polyunsaturated fatty acids themselves as a single component or as a mixed component by appropriately combining them. Further, when the conjugated polyunsaturated fatty acid is used as an active ingredient and other ingredients, that is, a food and / or a pharmaceutical product are produced, it is acceptable and does not inhibit the expression of the desired effect of the present invention. It is also possible to prepare the specific cancer-killing cell agent of the present invention by appropriately using known additives, stabilizers, activators and the like. Examples of this combination component or raw material include amino acids, peptides, proteins, various sugars, starches and their degradation products, fats and oils, ascorbic acid, vitamin E, tocopherol, phytosterols, minerals, polyphenols such as catechins, and their derivatives. However, the present invention is not limited to these. The amount of the active ingredient contained in the specific cancer cell killing agent of the present invention is difficult to define uniformly depending on conditions such as the form of the cancer cell killing agent, purpose of use, application, operation and workability, but is generally 20 wt. % Or more, more preferably 30% by weight or more. If it is less than 20% by weight, there may be a lack of convenience in use, for example, a large amount of a cancer-killing cell agent must be added when preparing a use composition described later.
[0021]
The form of the specific cancer-killing cell agent of the present invention is usually an oily liquid when only the conjugated polyunsaturated fatty acids according to the present invention are used, but is adapted to the physical properties when using the above concomitant substances. Various forms can be adopted. That is, when the concomitant substance is oil-soluble, such as ascorbyl palmitate, phytosterol, vitamin E, etc., it is mixed with the conjugated polyunsaturated fatty acid according to the present invention to form a uniform state, and ascorbic acid, amino acid, In the case of water solubility or water dispersibility such as minerals and proteins, for example, the dry powder is kneaded with the conjugated polyunsaturated fatty acids according to the present invention to form a dispersed state, or water and optionally a surfactant. Can also be made into an emulsified state.
[0022]
The specific cancer-killing cell agent of the present invention exerts a remarkable killing effect on human tissue-derived cancer cells, particularly colon cancer cells, liver cancer cells, lung cancer cells, breast cancer cells or gastric cancer cells. This effect is remarkably greater than that of conjugated linoleic acid and its derivatives composed only of conjugated diene fatty acids when having two double bonds.
[0023]
The specific cancer-killing cell agent of the present invention inhibits or kills normal cells derived from human tissues, particularly lung fibroblasts, skin fibroblasts, and embryonic fibroblasts. Is not observed and does not adversely affect normal cells.
[0024]
In the present invention, as described above, a specific cancer cell killing agent comprising a specific conjugated polyunsaturated fatty acid as an active ingredient is provided, and a composition obtained by further blending it is also provided. As an embodiment of this composition, a pharmaceutical composition and an edible composition are suitable.
[0025]
The pharmaceutical composition of the present invention comprises the above-mentioned specific carcinostatic agent, that is, a conjugated polyunsaturated fatty acid having 3 to 6 double bonds as an active ingredient, and this is a known one that does not contradict the gist of the present invention. Additives and additives are added as necessary and processed into conventional preparations such as tablets, capsules, granules, powders, and injections. It is applied for the prevention or treatment of cancer diseases by oral intake, tube administration or injection administration. It is desirable to use the specific carcinostatic cell agent of the present invention to be blended in the preparation, which has been subjected to fractional purification treatment such as solvent fractionation and adsorbent treatment as appropriate to increase the content of conjugated triene fatty acid as much as possible. . The blending amount is generally 0.01 to 50% by weight, although it is difficult to uniformly define the formulation depending on the type, form, usage, dosage and the like of the formulation. The amount of ingestion when taken orally is not particularly limited, but as the conjugated polyunsaturated fatty acid which is an active ingredient, it is 0.01 to 10 g, more preferably 0.1 to 5 g per day for an adult (body weight 50 kg). . If it is less than this range, it will be difficult to achieve the desired effect of the present invention.
[0026]
Another preferred embodiment of the composition of the present invention is an edible composition. That is, the specific carcinostatic cell agent containing conjugated polyunsaturated fatty acids having 3 to 6 double bonds obtained as described above as an active ingredient is used as it is in a liquid, gel or solid food. For example, juice, soft drink, tea, soup, dressing, jelly, yogurt, pudding, sprinkle, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc. or dextrin as needed It can be processed into pellets, tablets, granules, etc. together with excipients such as lactose and starch, fragrances, pigments, etc., or coated with gelatin and formed into capsules for use as health foods, nutritional supplements and the like. The compounding amount of the cancer cell killing agent of the present invention in these foods or edible compositions is difficult to define uniformly depending on the type or state of the food or composition, but is about 0.01 to 50% by weight, more Preferably it is 0.1 to 30 weight%. If the blending amount is less than 0.01% by weight, the desired effect by oral intake is small, and if it exceeds 50% by weight, the flavor may be impaired or the food may not be prepared depending on the type of food. In addition, the specific cancer-killing cell agent of the present invention can be used for food as it is.
[0027]
【Example】
Example 1
Purified linseed oil (reagent manufactured by Crude Sigma, Inc., according to fatty acid composition analysis by GLC, 1 g of α-LNA: 67%) and 100 ml of ethylene glycol solution containing 21 wt% potassium hydroxide were added to the reaction vessel, and the air in the vessel was After substituting with nitrogen gas, it was sealed and subjected to a conjugation reaction at 180 ° C. for 10 minutes. Subsequently, the reaction product was repeatedly washed with ethanol to prepare a triglyceride (sample 1) having conjugated α-LNA as a constituent fatty acid. The content of the conjugated polyunsaturated fatty acid was measured by measuring an absorption spectrum at 220 to 360 nm using a spectrophotometer (manufactured by Shimadzu Corporation, VUV2400PC), and measured by AOAC Official Methods of Analysis (1990, 957.13). It was determined according to the described method (hereinafter the same unless otherwise specified). The conjugated diene body of the conjugated polyunsaturated fatty acid contained in Sample 1 was 19.5%, and the conjugated triene body was 63.0%. Sample 1 was directly used as the specific cancer-killing cell agent of the present invention.
[0028]
Example 2
1 g of Sample 1 separately prepared by the method of Example 1 was subjected to a hydrochloric acid hydrolysis reaction by a conventional method and purified to prepare a mixed fatty acid containing conjugated α-LNA (Sample 2). Conjugated diene product: 18.0%, conjugated triene product: 62.0%. Sample 2 was used as the specific cancer-killing cell agent of the present invention.
[0029]
Example 3
To the reaction vessel was added 1 g of fish oil-derived EPA (manufactured by Bizen Kasei Co., Ltd., GCL purity: 98%) and 100 ml of a propylene glycol solution containing 15 wt% sodium hydroxide, and the same procedure as in Example 1 was carried out at 170 ° C. for 20 minutes. A conjugation reaction was performed and purified to prepare conjugated EPA (Sample 3). Conjugated polyunsaturated fatty acid contained therein was conjugated diene body: 58.0%, conjugated triene body: 23.7%, conjugated tetraene body: 15.0%, and conjugated pentaene body: 2.5%. Sample 3 was used as the specific cancer-killing cell agent of the present invention.
[0030]
Example 4
To the reaction vessel was added 1 g of fish oil-derived DHA (manufactured by Bizen Kasei Co., Ltd., GLC purity: 99%) and 100 ml of an ethylene glycol solution containing 25 wt% potassium hydroxide, and the same procedure as in Example 1 was carried out at 180 ° C. for 15 minutes. A conjugation reaction was performed and purified to prepare conjugated DHA (Sample 4). Conjugated polyunsaturated fatty acid contained in this product was conjugated diene: 54.8%, conjugated triene: 22.0%, conjugated tetraene: 14.7%, and conjugated pentaene: 5.2%. Sample 4 was used as the specific cancer-killing cell agent of the present invention.
[0031]
Example 5
1 g of each of the sample 3 and sample 4 separately prepared by the methods of Examples 3 and 4 was placed in an esterification flask equipped with a condenser, and 60 ml of a sulfuric acid-ethanol solution was added, boiled for 1 hour, and cooled to room temperature. Thereafter, extraction was performed using 100 ml of water and 50 ml of petroleum ether, the extract was washed with water, and washing was repeated until the washing water did not show acidity with a methyl orange indicator. The petroleum ether solution was dehydrated with anhydrous sodium sulfate, and then the solvent was removed under reduced pressure to prepare ethyl ester (sample 5) containing conjugated EPA and conjugated DHA as constituent fatty acids. Conjugated polyunsaturated fatty acid contained in this product was conjugated diene: 56.0%, conjugated triene: 15.0%, conjugated tetraene: 18.0%, and conjugated pentaene: 3.5%. Sample 5 was used as the specific cancer-killing cell agent of the present invention.
[0032]
Example 6
1 g of each of the sample 3 and sample 4 prepared separately by the methods of Examples 3 and 4 was placed in a saponification flask equipped with a condenser, and 50 ml of 1 mol / liter potassium hydroxide-ethanol solution was added and heated to reflux for 1 hour. And cooled to room temperature. Thereafter, extraction and purification were performed using 50 ml of water and 100 ml of petroleum ether to prepare a potassium salt containing conjugated EPA and conjugated DHA as constituent fatty acids. Conjugated polyunsaturated fatty acid contained therein was conjugated diene body: 59.5%, conjugated triene body: 20.7%, conjugated tetraene body: 12.0%, and conjugated pentaene body: 1.5%.
[0033]
Example 7
According to the method described in Example 1, using 10 g of purified fractionated fish oil containing 27.5 wt% EPA and 18.3 wt% DHA and 400 ml of 15 wt% potassium hydroxide-containing ethylene glycol solution. The resulting product was subjected to a conjugation reaction at 180 ° C. for 25 minutes and purified to prepare triglyceride (sample 7) containing conjugated EPA and conjugated DHA as constituent fatty acids. The conjugated diene body of the conjugated polyunsaturated fatty acid contained therein was 47.0%, the conjugated triene body: 33.0%, the conjugated tetraene body: 10.5%, and the conjugated pentaenoic acid: 3.5%. Sample 7 was mixed with vitamin E and dextrin to obtain the specific carcinostatic cell agent of the present invention.
[0034]
Comparative Example 1
To the reaction vessel was added 1 g of linoleic acid (reagent manufactured by Sigma, USA, GLC purity: 99%) and 100 ml of an ethylene glycol solution containing 6 wt% potassium hydroxide, and the conjugation reaction was carried out at 180 ° C. for 25 minutes in the same manner as in Example 1. And purified to prepare conjugated linoleic acid. This thing was conjugated diene body: 90.6%. Subsequently, this conjugated linoleic acid was esterified according to the method described in Example 5 and purified to prepare an ethyl ester (comparative sample 1) having conjugated linoleic acid as a constituent fatty acid.
[0035]
Test example 1
The cancer-killing cell action of the various conjugated polyunsaturated fatty acids described above was evaluated by the method described below. That is, colon cancer cells (cell numbers (hereinafter the same): TKG0379, hereinafter referred to as DLD-1), which are five types of human-derived cancer cells distributed and obtained from the Cancer Cell Storage Facility attached to Tohoku University Institute of Aging Medicine. Liver cancer cells (TKG0205, hereinafter referred to as HepG2), lung cancer cells (TKG0184, hereinafter referred to as A549), breast cancer cells (TKG0479, hereinafter referred to as MCF7) and stomach cancer cells (TKG0228, hereinafter referred to as MKKN-7) were used. Each cell was seeded in a 96-well microplate, and when 80% confluent (cell fullness) was reached, 0.5% bovine serum albumin containing 15 μM conjugated polyunsaturated fatty acids was added, and 5% carbon dioxide. After 24 hours of incubation at 37 ° C. in an atmosphere, the number of viable cells was determined according to MTT (3- (4,5-dimethylazol-2-yl ) Was examined by 2,5-diphenyl -2H- tetrazolium bromide) method. The MTT method can be applied to most cells because it uses the enzyme activity in living cells as an index, and since this result is also relatively stable, it was adopted as a method for evaluating cell killing action. The results are shown in Table 1. Moreover, the result tested similarly about the fish oil (comparative sample 2) which uses nonconjugated EPA and nonconjugated DHA as a constituent fatty acid was written together.
[0036]
[Table 1]

[0037]
In Table 1, the numerical values represent the MTT activity (cell viability,%) when no test sample is added as 100, and the relative value at the test sample addition concentration (15 μM) is expressed as an average value ± standard deviation (n = 6). (The same applies hereinafter). The numerical value of each test sample was significantly different from the values of Comparative Samples 1 and 2 (P <0.05). From this data, the conjugated polyunsaturated fatty acids according to the present invention were found to have a cytocidal effect on colon cancer cells as compared to fish oil. Similar cell-killing effects were observed for liver cancer cells, lung cancer cells and gastric cancer cells.
[0038]
Test example 2
The effect of the various conjugated polyunsaturated fatty acids described above on normal cells was evaluated in the same manner as described in Test Example 1. Human-derived normal cells include lung fibroblasts (hereinafter referred to as MRC-5), skin fibroblasts (hereinafter referred to as TIG-103), embryonic fibroblasts (hereinafter referred to as KMS-) obtained from Human Science Research Resource Bank (Osaka). 6). The results are shown in Table 2 together with the results for conjugated linoleic acid ethyl ester and fish oil (unmodified). The meanings of numerical values and symbols in the table are the same as those in Table 1. From these data, conjugated polyunsaturated fatty acids such as α-LNA, EPA and DHA, which are one of n-3 fatty acids, have almost no effect on the growth (division, proliferation, etc.) of normal human cells. In particular, it has been clarified that the compound containing a large amount of conjugated triene has a strong effect.
[0039]
[Table 2]

[0040]
Example 8
Sample 4 or 7 conjugated polyunsaturated fatty acids 250 mg, refined soybean oil 30 mg, beeswax 10 mg and vitamin E 10 mg were heated to about 40 ° C. in a nitrogen gas atmosphere and mixed thoroughly to obtain a homogeneous liquid product. . This was supplied to a capsule filling machine, and a gelatin-coated capsule preparation having an inner volume of 300 mg was made as a prototype. These preparations can be used as pharmaceutical compositions or edible compositions.
[0041]
Example 9
To 1 liter of commercially available milk, 5 g of the conjugated polyunsaturated fatty acids of Sample 7 and 0.2 g of vitamin E were added and mixed well to produce an equivalent beverage. This product is not inferior in food suitability such as flavor, color and texture as compared with ordinary milk, and can be suitably used as a food for cancer prevention or the disease.
[0042]
【The invention's effect】
According to the present invention, a specific cancer cell killing agent comprising conjugated polyunsaturated fatty acids having 3 to 6 double bonds as an active ingredient is provided. This cancer cell killing agent has an effect of killing cancer cells such as human-derived colorectal cancer, liver cancer, lung cancer, breast cancer or stomach cancer, and does not adversely affect the growth of normal cells. Such an effect becomes more remarkable when the fatty acid constituting the conjugated polyunsaturated fatty acid contains a large amount of conjugated triene fatty acid. Incidentally, conjugated diene fatty acids such as conjugated linoleic acid having only two double bonds do not exhibit the above-mentioned cancer-killing cell action.
In addition, according to the present invention, a raw material is an n-3 non-conjugated polyunsaturated fatty acid such as α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc., which is rich in conjugated triene fatty acid. Provided is a conjugation method capable of efficiently conjugate isomerization to polyunsaturated fatty acids.
Furthermore, according to this invention, the pharmaceutical composition or edible composition formed by mix | blending the said specific cancer killing cell agent is provided. The composition can be used for prevention or treatment of cancer diseases.

Claims

Carbon - made as a conjugated multivalent active ingredient unsaturated fatty acids having 3 to 6 carbon-carbon double bonds, a specific and cancer cell agents which do not adversely affect the normal cells, wherein the conjugated multivalent The constituent fatty acid of the unsaturated fatty acid is obtained by conjugating one or more kinds of non-conjugated polyunsaturated fatty acids selected from the group consisting of eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, Here, the specific cancer-killing cell agent whose cancer cell is a colon cancer cell .

Conjugation process, a carbon - diol solution 50-100 wt containing 10-30 wt% alkali metal hydroxide relative to non-conjugated polyvalent 1 parts by weight of the unsaturated fatty acids having 3 to 6 carbon-carbon double bond parts was added, for 5 to 30 minutes at 170 to 190 ° C., specifically of claim 1 is due to the production method of the conjugated polyunsaturated fatty acid-rich conjugated triene fatty acids, characterized in that conjugating reaction Cancer cell killing agent.

Aspects of conjugated polyunsaturated fatty acids, carbon - a carboxylic acid to the conjugated polyunsaturated fatty acid configuration fatty acids having 3 to 6 carbon-carbon double bonds, one selected from the group consisting of esters and salts or der more thing is, wherein the ester is an ester of a lower monohydric alcohol, specific and cancer cell agent according to claim 1 or 2.

Lung fibroblasts of normal cells derived from human, specific and cancer cell agent according to any one of claims 1 to 3 which is a dermal fibroblasts or embryonic fibroblasts.

Pharmaceutical composition obtained by blending a specific and cancer cell agent according to any one of claims 1-4.