JP3714426B2 - Cancer metastasis inhibitor containing fucoidan oligosaccharide composition - Google Patents
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Description
【０００１】
【産業上の利用分野】
本発明はフコイダンを酸性条件下で加水分解することにより得られるフコイダンオリゴ
糖組成物を含有する癌転移抑制剤に関する。
【０００２】
【従来の技術】
フコイダンは褐藻類に含まれている硫酸化多糖の総称であり、硫酸化フコースを構成糖として含む物である。フコイダンは様々な褐藻類から分離され、癌増殖抑制活性、癌転移抑制活性、抗凝血活性、抗ウイルス感染活性等の生物活性が確認されている。これらの生物活性の検討に用いられた天然物由来のフコイダンは分子量分布が広く、かつ分子量は非常に大きな物である。
フコイダンの生物活性を検討する際、分子量分布が広いと、再現性に問題を生じ、また該巨大分子であるフコイダンは強い抗凝血活性を持つ。また巨大分子のフコイダンから夾雑するタンパク質分子を除去するのは極めて困難である。
フコイダンを低分子化し更に分子量分画により分子を比較的均一にする方法については、硫酸、酵素、ガンマ線、超音波等によりフコイダンを分解後、分子量分画する方法（特表平４−５０６０８９号）や、塩酸で加水分解後分子量分画する方法〔ジャーナル オブ アンドロロジー（Journal of Andrology) 、第１３巻、第５１９〜５２５頁（１９９２）〕などがある。しかしながら、前者は抗凝血活性の強いフコイダンオリゴ糖を得る方法であり、得られるオリゴ糖の分子量も５０００より大きい。また後者は、反応生成物として得られる微量のオリゴ糖を利用する方法に過ぎず、極めて収率が悪い。
【０００３】
【発明が解決しようとする課題】
フコイダンは種々の生理活性を有し、医薬としての開発が期待されている。しかしながら抗凝血活性が高いこと、分子量分布が広く、均一な製品の調製が困難なこと、巨大分子であり、夾雑するタンパク質の除去が困難であること等の問題点を有している。
本発明の目的は、フコイダンの生物活性を保持しながら、抗凝血活性が除去され、分子量分布的にも均一で、生物活性の再現性も良く、安全性の高い低分子化フコイダンを含有する癌転移抑制剤を提供することにある。
【０００４】
【課題を解決するための手段】
本発明を概説すれば、本発明は、下記の理化学的性質を有するフコイダンオリゴ糖組成物を含有することを特徴とする癌転移抑制剤に関する。
（１）分子量分布：５×１０3 以下〔セルロファインＧＣＬ−２５（生化学工業株式会社製）を用いたゲルろ過法による〕
（２) タンパク含量：検出されない
（３）抗凝血活性：５０００μｇ／ｍｌで抗凝血活性を示さない
【０００５】
本発明者らは、褐藻類由来のフコイダンが有機酸の存在下、加水分解処理により、極めて収率よく低分子化し、分子量分画により調製した本発明のフコイダンオリゴ糖組成物は医薬品として用いる際に問題となる抗凝血活性が実質的に除去され、かつ癌転移抑制活性は天然由来フコイダンと同等であり、更に、該組成物は抗真菌剤としても有用であるという新用途も見出し、本発明を完成した。
【０００６】
以下本発明に関して詳細に説明する。
本発明に使用されるフコイダンの種類は特に限定される物ではなく、例えばヒバマタ由来（シグマ社製）の物、マコンブ由来の物、その他すべての褐藻類由来の物を使用することができる。
【０００７】
フコイダンの有機酸による加水分解には、酢酸、ギ酸、プロピオン酸等の揮発性の有機酸、シュウ酸、クエン酸、コハク酸等の固体の有機酸等を使用することができる。
フコイダンの加水分解は、原料のフコイダン溶解液をこれらの有機酸でｐＨ調整し行う。フコイダンの溶解は通常の方法で行えば良く、溶解液中のフコイダン濃度はその最高溶解濃度でも良いが、通常はその粘性を考慮して決定すれば良い。フコイダン溶解液としては水、緩衝液等より目的に応じ選択すれば良い。溶解液のｐＨは通常ｐＨ５以下、好ましくはｐＨ１〜４の範囲に選定し、前記の有機酸により調整する。加水分解は該ｐＨ調整液を６０℃〜１２０℃、通常は１００℃で加温し、分子量５×１０3 以下の目的のフコイダンオリゴ糖組成物が調製できるまで行えば良い。また、加水分解して得られるフコイダンオリゴ糖の分子量は処理液のｐＨに依存し、例えば同じ酸を使用する場合ｐＨを３．５にするより２．５にした方が分子量の小さなフコイダンオリゴ糖が得られる。同じｐＨで処理する場合、酸の種類により低分子化の程度に差が生じる場合もあるが、生成物の分子量の制御は加水分解時間の調整により容易である。
【０００８】
次に低分子化フコイダンは分子量分画することにより、更に均一な分子量分布のフコイダンオリゴ糖組成物を調製することができる。分子量分画には通常よく使用されている方法を適用することができ、例えばゲルろ過法や分子量分画膜を使用すれば良い。得られた組成物は、必要に応じて脱塩処理、無菌処理を行い、凍結乾燥をすることにより、本発明のフコイダンオリゴ糖組成物の乾燥品を得ることもできる。
【０００９】
（Ａ）本発明のフコイダンオリゴ糖組成物の分子量の測定方法は次のとおりである。
セルロファインＧＣＬ−２５（生化学工業社製）のカラム（４．１×９７ｃｍ）に分子量標準物質として重合度２〜７までのマルトース及びプルラン（分子量約１，０００〜１０，０００のもの）を流し分子量と溶出ボリュウムの関係をグラフに表し、フコイダンオリゴ糖組成物の溶出ボリュウムからその分子量を求める。
本発明のフコイダンオリゴ糖組成物は分子量５×１０3 以下である。
【００１０】
（Ｂ）本発明のフコイダンオリゴ糖組成物のタンパク含量の測定方法は次のとおりである。
フコイダンオリゴ糖組成物を含有する試料溶液０．１ｍｌにプロテインアッセイキットＩ（バイオラッド社製）の染色液を５倍希釈した溶液５．０ｍｌを加え、かくはん後５分以上室温で放置した後、５９５ｎｍの吸光度を測定する。別に、牛血清アルブミン水溶液を用いて上記と同じ操作を行い、タンパク質の標準曲線を得、得られた標準曲線から試料溶液のタンパク含量を求める。
本発明のフコイダンオリゴ糖組成物はタンパクを含有しない。
【００１１】
（Ｃ）本発明のフコイダンオリゴ糖組成物の抗凝血活性の測定方法は次のとおりである。
抗凝血活性の測定方法（活性化部分トロンボプラスチン時間）
試薬は活性トロンボファックス、コントロール血漿としては、オーソ凝固コントロールＩ（共にオーソ・ダイアグノスティック・システムズ社製）を用い、方法はアグリカルチュラル アンド バイオロジカル ケミストリー（Agricultural and Biological Chemistry)、第５５巻、第７９１〜７９６頁（１９９１）記載の方法を参考に行う。
すなわち、１２×７５ｍｍの試験管に１００μｌのコントロール血漿、１０μｌのフコイダンオリゴ糖組成物の溶液（０．９％食塩水中）、及び１００μｌの活性トロンボファックスを加え混合し、３７℃の恒温槽中で３分間保持し、あらかじめ３７℃に保温しておいた２０ｍＭの塩化カルシウムを１００μｌを加えると同時にストップウォッチを始動させ激しく混和し３７℃で２０秒間保温する。次に試験管を恒温槽から取り出し、ゆっくりと前後に振りゲル塊の出現と同時にストップウォッチを止める。この時に要した時間を活性化部分トロンボプラスチン時間とする。
また結果の判定は、フコイダンオリゴ糖組成物を加えなかったときの活性化部分トロンボプラスチン時間と比較して有意に延長を認めないときに抗凝血活性がないと判定する。
【００１２】
フコイダン（シグマ社製）は５０μｇ／ｍｌで抗凝血活性を有するが、本発明のフコイダンオリゴ糖組成物は５０００μｇ／ｍｌで抗凝血活性を示さず、本発明のフコイダンオリゴ糖組成物は実質的に抗凝血活性を保有しない。
【００１３】
本発明によるフコイダンオリゴ糖組成物は、癌転移抑制剤、抗真菌剤として使用することができる。
従来、真菌感染症の治療剤としては、アンホテリシンＢ、フルシトシン、ミコナゾール、フルコナゾールのほか多数あるが、効力及び毒性若しくは耐性菌の点で問題がある。特に、近年増加傾向にある全身感染に有効な低毒性の薬剤は少ない。本発明のフコイダンオリゴ糖組成物は天然物由来で安全性も高く、新しいタイプの抗真菌剤である。
また親物質のフコイダンやヘパリンにも抗真菌作用が見出され、硫酸化多糖の新規用途が開発された。
【００１４】
本発明のフコイダンオリゴ糖組成物を有効成分とする癌転移抑制剤、抗真菌剤においては、該有効成分の含有率は対応する製剤の種類により異なるが、一般に有効成分として、０．１〜１００％含有することが好ましい。該有効成分を用いての癌転移抑制剤、抗真菌剤は通常の製剤、例えば錠剤、カプセル剤、丸剤、注射剤、シロップ剤等に製剤化して経口及び非経口的に投与することができる。
このための手段として、医薬品を製造するために用いる慣用の賦形剤及び添加剤を用いることができる。慣用の賦形剤としては、例えば水、生理食塩水、アルコール、ポリエチレングリコール、グリセロールエステル、ゼラチン、炭水化物、ステアリン酸マグネシウム、タルク等が挙げられる。また、慣用の添加剤として、防腐剤、滅菌剤、潤滑剤、コーティング剤、湿潤剤、乳化剤、着色剤、マスキングフレーバー、及び芳香剤等が挙げられる。
【００１５】
本発明による癌転移抑制剤、抗真菌剤はそれぞれ、その薬剤の投与形態、投与回数、患者の状態、患者の体重、病気の軽重により変化するが、投与量は体重１ｋｇ当り０．１ｍｇから１５０ｍｇ、好ましくは０．５ｍｇから１００ｍｇを、１日に数回に分けて投与することが好ましい。
投与回数も薬剤の形態、投与回数、患者の状態、体重、病気の軽重により定められるが、１日当り１〜３回の投与が好ましく、場合においては、連続静脈内滴注により投与することもできる。
【００１６】
本発明による癌転移抑制剤は癌転移の治療、予防に有効であり、抗真菌剤は真菌症の治療、予防に有効である。また本発明のフコイダンオリゴ糖組成物は、種々の食品にそれらの特性を変化させること無くして添加することもできる。この場合、例えば冷菓、パン、ゼリー等の固形状の食品ばかりでなく牛乳、果汁等の液状食品にも安定的に添加することが可能である。
【００１７】
また、本発明のフコイダンオリゴ糖組成物の１群６匹のマウス（Ｃ５７ＢＬ／６）における静脈内投与による急性毒性試験の結果では、後述する実施例記載の組成物は、いずれも５００ｍｇ／ｋｇの投与で全例共、全く毒性症状を示さなかった。したがって、本発明のフコイダンオリゴ糖組成物の静脈内投与によるＬＤ50（５０％致死量）は、いずれも５００ｍｇ／ｋｇ以上である。
【００１８】
【実施例】
以下に本発明を実施例をもって示すが、本発明が以下の実施例の範囲のみに限定されるものではない。
なお、下記実施例１〜３は、本発明で使用するフコイダンオリゴ糖組成物の例についての製造例と理化学的性質を示す参考例である。
【００１９】
実施例１
フコイダン（シグマ社製）２ｇを１００ｍｌの水に溶解しそのｐＨを酢酸にてｐＨ３．０に調整後１００℃で３時間処理した。この加水分解物をセルロファインＧＣＬ−２５（生化学工業社製）によるゲルろ過で分子量分画し分子量５，０００以下の画分をマイクロアシライザーＧ３（旭化成社製）により脱塩後凍結乾燥を行い１．９８ｇの生成物を得た。
本物質の抗凝血活性を測定したところ１０ｍｇ／ｍｌ以下では抗凝血活性を持たなかった。
【００２０】
実施例２
フコイダン（シグマ社製）２ｇを１００ｍｌの水に溶解し、そのｐＨをクエン酸にてｐＨ３．０に調整後１００℃で３時間処理した。この加水分解物をセルロファインＧＣＬ−２５によるゲルろ過で分子量分画し、分子量５，０００以下の画分を５分画（分子量５，０００〜３，０００超、３，０００〜２，０００超、２，０００〜１，０００超、１，０００〜５００超、５００以下）した。更にこれらの画分をマイクロアシライザーＧ３（旭化成社製）により脱塩後凍結乾燥を行い各画分それぞれ２９０、２４３、５１１、３２９，４３１ｍｇ得た。
これらの物質の抗凝血活性を測定したところ、分子量５，０００〜３，０００超のもの、及び３，０００〜２，０００超のものは５ｍｇ／ｍｌまで、その他の画分は１０ｍｇ／ｍｌ以下では抗凝血活性を持たなかった。
【００２１】
実施例３
フコイダン（シグマ社製）２ｇを１００ｍｌの水に溶解し、そのｐＨをギ酸にてｐＨ３．６に調整後１００℃で３時間処理した。この加水分解物をセルロファインＧＣＬ−２５によるゲルろ過で分子量分画し、分子量５，０００以下の画分を４分画（分子量５，０００〜３，０００超、３，０００〜２，０００超、２，０００〜１，０００超、１，０００以下）した。更にこれらの画分をマイクロアシライザーＧ３（旭化成社製）により脱塩後凍結乾燥を行い各画分それぞれ４６６、１６２、５００、２４４ｍｇを得た。
これらの物質の抗凝血活性を測定したところ、分子量５，０００〜３，０００超のもの、及び３，０００〜２，０００超のものは５ｍｇ／ｍｌまで、その他の画分は１０ｍｇ／ｍｌ以下では抗凝血活性を持たなかった。
【００２２】
実施例４
カンジダ アルビカンス（Candida albicans) ＴＩＭＭ １７６８株を３７℃、１夜培養後、集菌、生理食塩水に２×１０7 個／ｍｌとなるように懸濁した。フコイダン（シグマ社製）、実施例２で調製した分子量５，０００〜３０００超のフコイダンオリゴ糖組成物、ヘパリン（和光純薬社製）をそれぞれ生理食塩水に、８ｍｇ／ｍｌの濃度に溶解した。この各種溶液と上記菌懸濁液を１：１の割合で混ぜ、その０．２ｍｌ（１×１０6 個）をＩＣＲ系マウス（５週令、雌）の尾静脈より接種した。なお対照として、生理食塩水を用いた。
１４日間、マウスの生死を観察し、各サンプルの抗カンジダ活性を測定した。延命率は下記式（数１）により算出した。結果を表１に示す。
【００２３】
【表１】
【数１】
表１に示すようにカンジダ アルビカンスに対してはヘパリン、フコイダンとも明らかな延命効果がみられた。フコイダンの抗カンジダ作用はフコイダンオリゴ糖組成物においても保持され、フコイダンオリゴ糖組成物はフコイダンと同等の活性を示した。
【００２６】
実施例５
Ｂ１６メラノーマ細胞をＦＣＳ無添加のＲＰＭＩ−１６４０培地で十分に洗浄した後５×１０5 個／ｍｌとなるように、同培地に懸濁した。
フコイダン（シグマ社製）、実施例３記載の分子量５，０００〜３０００超のフコイダンオリゴ糖組成物、分子量２，０００〜１，０００超のフコイダンオリゴ糖組成物を、それぞれ生理食塩水に８ｍｇ／ｍｌ、０．８ｍｇ／ｍｌ、０．０８ｍｇ／ｍｌとなるように溶解、希釈した。この各種フコイダン溶液と上記細胞懸濁液を１：１の割合で混ぜ、その０．２ｍｌ（５×１０4 個）をＣ５７ＢＬ／６マウス（７週令、雌）の尾静脈より接種した。なお対照として生理食塩水を用いた。判定は細胞を接種してから１５日目に肺臓を摘出し、その表面に確認できるメラノーマの転移結節数をカウントし、下記式（数２）により癌転移抑制率を算出した。結果を表２に示す。
【００２７】
【数２】
【表２】
表２から明らかなように、本発明のフコイダンオリゴ糖組成物は明らかな癌転移抑制活性を示した。
【００３０】
実施例６
実施例１に記載の分子量５０００以下のフコイダンオリゴ糖組成物の凍結乾燥品２５ｍｇに、乳糖、でんぷん、ステアリン酸マグネシウムを加え、充分混合した後、カプセルに充てんし、カプセル剤を製造した（表３）。
【００３１】
【表３】
実施例２、３にそれぞれ記載されている各フコイダンオリゴ糖組成物についても同様にカプセル剤を製造した。
【００３３】
実施例７
実施例２に記載の分子量５０００〜３０００超のフコイダンオリゴ糖組成物の凍結乾燥品５００ｍｇにオートクレーブにより殺菌した生理食塩水を加えて溶解し、全量を１０ｍｌに調製した後、乾熱滅菌したアンプル瓶に封入し、１０ｍｌの液剤を製造した。
実施例２に記載の他のフコイダンオリゴ糖組成物、実施例１、３にそれぞれ記載の各フコイダンオリゴ糖組成物についても同様に液剤を製造した。
【００３４】
実施例８
実施例３に記載の分子量５０００〜３０００超のフコイダンオリゴ糖組成物の凍結乾燥品に、乳糖及びでんぷんを加えて混合した。１０％のでんぷんのりを混合物に加え、かくはんして顆粒を製造した。乾燥後、整粒し、これにタルク及びステアリン酸マグネシウムを混合し、常法により打錠して２００ｍｇ錠剤を製造した（表４）。
【００３５】
【表４】
実施例３に記載の他のフコイダンオリゴ糖組成物、実施例１、２にそれぞれ記載の各フコイダンオリゴ糖組成物についても同様に錠剤を製造した。
【００３７】
【発明の効果】
本発明により、抗凝血活性、また夾雑タンパクによる抗原性の除去されたフコイダンオリゴ糖組成物、及びその効率的な製造方法が提供される。該フコイダンオリゴ糖組成物は溶解性、生体への吸収性共に優れ、かつ、天然物由来で安全性も高く、癌転移抑制剤、抗真菌剤として有用であり、また食品への適用も可能な新規組成物である。[0001]
[Industrial application fields]
The present invention relates to a cancer metastasis inhibitor containing a fucoidan oligosaccharide composition obtained by hydrolyzing fucoidan under acidic conditions.
[0002]
[Prior art]
Fucoidan is a general term for sulfated polysaccharides contained in brown algae, and includes sulfated fucose as a constituent sugar. Fucoidan has been isolated from various brown algae and has been confirmed to have biological activities such as cancer growth inhibitory activity, cancer metastasis inhibitory activity, anticoagulant activity, and antiviral infection activity. Fucoidan derived from natural products used for studying these biological activities has a wide molecular weight distribution and a very large molecular weight.
When examining the biological activity of fucoidan, if the molecular weight distribution is wide, a problem occurs in reproducibility, and fucoidan, which is a macromolecule, has strong anticoagulant activity. It is also very difficult to remove contaminating protein molecules from the macromolecular fucoidan.
As for the method of reducing the molecular weight of fucoidan and making the molecule relatively uniform by molecular weight fractionation, the method of molecular weight fractionation after decomposing fucoidan with sulfuric acid, enzyme, gamma rays, ultrasonic waves, etc. (Japanese Patent Publication No. 4-506089) And a method of molecular weight fractionation after hydrolysis with hydrochloric acid [Journal of Andrology, Vol. 13, pp. 519-525 (1992)]. However, the former is a method for obtaining fucoidan oligosaccharide having strong anticoagulant activity, and the molecular weight of the resulting oligosaccharide is more than 5,000. The latter is only a method using a very small amount of oligosaccharide obtained as a reaction product, and the yield is extremely poor.
[0003]
[Problems to be solved by the invention]
Fucoidan has various physiological activities and is expected to be developed as a medicine. However, it has problems such as high anticoagulant activity, wide molecular weight distribution, difficulty in preparing a uniform product, and removal of contaminating proteins that are macromolecules.
It is an object of the present invention to contain a low-molecular-weight fucoidan that retains the biological activity of fucoidan, has anticoagulant activity removed, is uniform in molecular weight distribution, has good reproducibility of biological activity, and is highly safe. It is in providing a cancer metastasis inhibitor .
[0004]
[Means for Solving the Problems]
If it outlined present invention, the onset Ming, for cancer metastasis inhibitor which is characterized by containing a fucoidan oligosaccharide composition having the following physicochemical properties.
(1) Molecular weight distribution: 5 × 10 3 or less [by gel filtration using Cellulofine GCL-2 5 ( manufactured by Seikagaku Corporation)]
(2) protein content: not detected (3) anticoagulant activity: not such shown anticoagulant activity in 5000 [mu] g / ml [0005]
The present inventors reduced the molecular weight of fucoidan derived from brown algae by a hydrolysis treatment in the presence of an organic acid with extremely high yield, and the fucoidan oligosaccharide composition of the present invention prepared by molecular weight fractionation is used as a pharmaceutical product. The anticoagulant activity, which is a problem with the present invention, is substantially eliminated, and the activity of inhibiting cancer metastasis is equivalent to that of naturally-occurring fucoidan, and the composition is also useful as an antifungal agent. Completed the invention.
[0006]
The present invention will be described in detail below.
The kind of fucoidan used for this invention is not specifically limited, For example, the thing derived from Hibamata (made by Sigma), the thing derived from Macombu, and the thing derived from all other brown algae can be used.
[0007]
For the hydrolysis of fucoidan with an organic acid, a volatile organic acid such as acetic acid, formic acid, or propionic acid, or a solid organic acid such as oxalic acid, citric acid, or succinic acid can be used.
Hydrolysis of fucoidan is performed by pH adjustment fucoidan solution of raw materials in these organic acids. The fucoidan may be dissolved by an ordinary method, and the concentration of fucoidan in the solution may be the maximum dissolution concentration, but it is usually determined in consideration of its viscosity. The fucoidan solution may be selected from water, buffer solution, etc. according to the purpose. PH of solution is usually pH5 or less, preferably selected in the range of PH1～4, more adjusted to the organic acid of the. Hydrolysis 60 ° C. to 120 ° C. The pH adjustment solution, typically heated at 100 ° C., fucoidan oligosaccharide composition having a molecular weight of 5 × 10 3 or less of interest may be performed to be prepared. In addition, the molecular weight of fucoidan oligosaccharide obtained by hydrolysis depends on the pH of the treatment solution. For example, when the same acid is used, fucoidan oligosaccharide having a smaller molecular weight is obtained by setting the pH to 2.5 than to 3.5. Is obtained. When processing at the same pH, there is a case where the type of acid difference in the degree of low molecular weight is produced, the control of the molecular weight of the product is facilitated by adjustment of the hydrolysis time.
[0008]
Next, the low molecular weight fucoidan is subjected to molecular weight fractionation, whereby a fucoidan oligosaccharide composition having a more uniform molecular weight distribution can be prepared. For molecular weight fractionation, commonly used methods can be applied. For example, a gel filtration method or a molecular weight fractionation membrane may be used. The obtained composition can be desalted and aseptically treated as necessary, and lyophilized to obtain a dried product of the fucoidan oligosaccharide composition of the present invention.
[0009]
(A) The measuring method of the molecular weight of the fucoidan oligosaccharide composition of the present invention is as follows.
A column (4.1 × 97 cm) of Cellulofine GCL-25 (manufactured by Seikagaku Corporation) with maltose and pullulan (with a molecular weight of about 1,000 to 10,000) having a degree of polymerization of 2 to 7 as molecular weight standard substances. The relationship between the flowing molecular weight and the eluted volume is shown in a graph, and the molecular weight is obtained from the eluted volume of the fucoidan oligosaccharide composition.
The fucoidan oligosaccharide composition of the present invention has a molecular weight of 5 × 10 3 or less.
[0010]
(B) The method for measuring the protein content of the fucoidan oligosaccharide composition of the present invention is as follows.
After adding 5.0 ml of a 5-fold diluted solution of protein assay kit I (BioRad) to 0.1 ml of the sample solution containing the fucoidan oligosaccharide composition, the mixture was allowed to stand at room temperature for 5 minutes or more after stirring. The absorbance at 595 nm is measured. Separately, the same operation as described above is performed using an aqueous bovine serum albumin solution to obtain a protein standard curve, and the protein content of the sample solution is determined from the obtained standard curve.
The fucoidan oligosaccharide composition of the present invention does not contain protein.
[0011]
(C) The method of measuring the anticoagulant activity of the fucoidan oligosaccharide composition of the present invention is as follows.
Method for measuring anticoagulant activity (activated partial thromboplastin time)
The reagent is active thrombofax, and the control plasma is orthocoagulation control I (both manufactured by Orthodiagnostic Systems). The method is Agricultural and Biological Chemistry, Vol. 55, Vol. Reference is made to the method described on pages 791 to 796 (1991).
That is, 100 μl of control plasma, 10 μl of fucoidan oligosaccharide composition solution (0.9% in saline), and 100 μl of active thrombofax were added to a 12 × 75 mm test tube and mixed, and the mixture was placed in a 37 ° C. constant temperature bath. Hold for 3 minutes, add 100 μl of 20 mM calcium chloride that has been kept warm at 37 ° C., start the stopwatch and mix vigorously and keep at 37 ° C. for 20 seconds. Next, the test tube is taken out of the thermostatic bath, and is slowly shaken back and forth to stop the stopwatch simultaneously with the appearance of the gel lump. The time required at this time is defined as the activated partial thromboplastin time.
In addition, the determination of the result is that there is no anticoagulant activity when there is no significant extension compared to the activated partial thromboplastin time when no fucoidan oligosaccharide composition is added.
[0012]
Fucoidan (manufactured by Sigma) has anticoagulant activity at 50 μg / ml, but the fucoidan oligosaccharide composition of the present invention does not exhibit anticoagulant activity at 5000 μg / ml, and the fucoidan oligosaccharide composition of the present invention is substantially Does not possess anticoagulant activity.
[0013]
The fucoidan oligosaccharide composition according to the present invention can be used as a cancer metastasis inhibitor and an antifungal agent.
Conventionally, there are many other therapeutic agents for fungal infections besides amphotericin B, flucytosine, miconazole, fluconazole, but there are problems in terms of efficacy and toxicity or resistant bacteria. In particular, there are few low-toxic drugs effective for systemic infection that have been increasing in recent years. The fucoidan oligosaccharide composition of the present invention is a new type of antifungal agent derived from natural products and having high safety.
The parent substances fucoidan and heparin were also found to have antifungal activity, and new uses for sulfated polysaccharides were developed.
[0014]
In the cancer metastasis inhibitor and antifungal agent comprising the fucoidan oligosaccharide composition of the present invention as an active ingredient, the content of the active ingredient varies depending on the type of the corresponding preparation, but generally 0.1-100 as an active ingredient. % Content is preferable. The cancer metastasis inhibitor and antifungal agent using the active ingredient can be formulated into conventional preparations such as tablets, capsules, pills, injections, syrups and administered orally and parenterally. .
For this purpose, conventional excipients and additives used for producing pharmaceuticals can be used. Examples of conventional excipients include water, physiological saline, alcohol, polyethylene glycol, glycerol ester, gelatin, carbohydrate, magnesium stearate, talc and the like. Examples of conventional additives include preservatives, sterilants, lubricants, coating agents, wetting agents, emulsifiers, colorants, masking flavors, and fragrances.
[0015]
Each of the cancer metastasis inhibitor and antifungal agent according to the present invention varies depending on the administration form of the drug, the number of administrations, the condition of the patient, the weight of the patient, the severity of the disease, and the dosage is 0.1 mg to 150 mg per kg body weight. Preferably, 0.5 mg to 100 mg is administered in several divided doses per day.
The number of administrations is determined by the form of the drug, the number of administrations, the patient's condition, the body weight, and the severity of the disease, but preferably 1 to 3 times per day, and in some cases, it can be administered by continuous intravenous infusion. .
[0016]
The cancer metastasis inhibitor according to the present invention is effective for the treatment and prevention of cancer metastasis, and the antifungal agent is effective for the treatment and prevention of mycosis. The fucoidan oligosaccharide composition of the present invention can also be added to various foods without changing their properties. In this case, for example, it can be stably added not only to solid foods such as frozen desserts, bread and jelly, but also to liquid foods such as milk and fruit juice.
[0017]
In addition, as a result of acute toxicity test by intravenous administration of 6 mice (C57BL / 6) per group of the fucoidan oligosaccharide composition of the present invention, all the compositions described in Examples described later were 500 mg / kg. All patients showed no toxic symptoms after administration. Therefore, the LD 50 (50% lethal dose) by intravenous administration of the fucoidan oligosaccharide composition of the present invention is 500 mg / kg or more.
[0018]
【Example】
The present invention will be described below with reference to examples, but the present invention is not limited to the scope of the following examples.
In addition, the following Examples 1-3 are the reference examples which show the manufacture example about the example of the fucoidan oligosaccharide composition used by this invention, and a physicochemical property.
[0019]
Example 1
2 g of fucoidan (manufactured by Sigma) was dissolved in 100 ml of water, and the pH was adjusted to 3.0 with acetic acid, followed by treatment at 100 ° C. for 3 hours. This hydrolyzate is subjected to gel filtration using Cellulofine GCL-25 (Seikagaku Corporation), molecular weight fractionation is performed, and fractions having a molecular weight of 5,000 or less are desalted with a microacylizer G3 (Asahi Kasei Co., Ltd.) and freeze-dried 1.98 g of product was obtained.
When the anticoagulant activity of this substance was measured, it had no anticoagulant activity at 10 mg / ml or less.
[0020]
Example 2
2 g of fucoidan (manufactured by Sigma) was dissolved in 100 ml of water, adjusted to pH 3.0 with citric acid and then treated at 100 ° C. for 3 hours. This hydrolyzate is fractionated by gel filtration with Cellulofine GCL-25, and a fraction having a molecular weight of 5,000 or less is divided into 5 fractions (molecular weights of 5,000 to 3,000 or more, 3,000 to 2,000 or more). 2,000-1,000, 1,000-500, 500 or less). Furthermore, these fractions were desalted with a microacylizer G3 (manufactured by Asahi Kasei Co., Ltd.) and lyophilized to obtain 290, 243, 511, 329, 431 mg of the respective fractions.
When the anticoagulant activity of these substances was measured, those having a molecular weight of more than 5,000 to 3,000 and those having a molecular weight of more than 3,000 to 2,000 were up to 5 mg / ml, and other fractions were 10 mg / ml. The following did not have anticoagulant activity.
[0021]
Example 3
2 g of fucoidan (manufactured by Sigma) was dissolved in 100 ml of water, its pH was adjusted to pH 3.6 with formic acid and treated at 100 ° C. for 3 hours. This hydrolyzate is molecularly fractionated by gel filtration with Cellulofine GCL-25, and a fraction having a molecular weight of 5,000 or less is divided into four fractions (molecular weights of 5,000 to 3,000, 3,000 to 2,000 and more). 2,000 to over 1,000, 1,000 or less). Furthermore, these fractions were desalted with a microacylizer G3 (manufactured by Asahi Kasei Co., Ltd.) and lyophilized to obtain 466, 162, 500, and 244 mg, respectively.
When the anticoagulant activity of these substances was measured, those having a molecular weight of more than 5,000 to 3,000 and those having a molecular weight of more than 3,000 to 2,000 were up to 5 mg / ml, and other fractions were 10 mg / ml. The following did not have anticoagulant activity.
[0022]
Example 4
Candida albicans TIMM 1768 strain was cultured at 37 ° C. overnight, then collected and suspended in physiological saline at 2 × 10 7 cells / ml. Fucoidan (manufactured by Sigma), fucoidan oligosaccharide composition having a molecular weight of more than 5,000 to 3000 and heparin (manufactured by Wako Pure Chemical Industries, Ltd.) prepared in Example 2 were each dissolved in physiological saline at a concentration of 8 mg / ml. . These various solutions and the above-mentioned bacterial suspension were mixed at a ratio of 1: 1, and 0.2 ml (1 × 10 6 ) thereof was inoculated from the tail vein of an ICR mouse (5 weeks old, female). As a control, physiological saline was used.
The mice were observed for 14 days for life and death, and the anti-Candida activity of each sample was measured. The life extension rate was calculated by the following formula (Equation 1). The results are shown in Table 1.
[0023]
[Table 1]
[Expression 1]
As shown in Table 1, a clear life-prolonging effect was observed against Candida albicans for both heparin and fucoidan. The anti-candida action of fucoidan was retained in the fucoidan oligosaccharide composition, and the fucoidan oligosaccharide composition showed the same activity as fucoidan.
[0026]
Example 5
B16 melanoma cells were thoroughly washed with RPMI-1640 medium without addition of FCS and then suspended in the same medium so that the concentration was 5 × 10 5 cells / ml.
Fucoidan (manufactured by Sigma), fucoidan oligosaccharide composition having a molecular weight of 5,000-3,000 and fucoidan oligosaccharide composition having a molecular weight of 2,000-1,000 are described in Example 3 in physiological saline. It was dissolved and diluted so as to be ml, 0.8 mg / ml and 0.08 mg / ml. These various fucoidan solutions and the above cell suspension were mixed at a ratio of 1: 1, and 0.2 ml (5 × 10 4 cells) was inoculated from the tail vein of C57BL / 6 mice (7 weeks old, female). Saline was used as a control. Determination was made on the 15th day after cell inoculation, the lungs were excised, the number of metastatic nodules of melanoma that could be confirmed on the surface was counted, and the cancer metastasis inhibition rate was calculated by the following formula (Equation 2). The results are shown in Table 2.
[0027]
[Expression 2]
[Table 2]
As is clear from Table 2, the fucoidan oligosaccharide composition of the present invention showed clear cancer metastasis inhibitory activity.
[0030]
Example 6
Lactose, starch, and magnesium stearate were added to 25 mg of a freeze-dried product of fucoidan oligosaccharide composition having a molecular weight of 5000 or less described in Example 1, and mixed well, and then filled into capsules to produce capsules (Table 3). ).
[0031]
[Table 3]
Capsules were similarly prepared for the fucoidan oligosaccharide compositions described in Examples 2 and 3, respectively.
[0033]
Example 7
Ampoule bottles sterilized by dry heat after the lyophilized product of the fucoidan oligosaccharide composition having a molecular weight of 5000 to 3000 described in Example 2 was dissolved by adding physiological saline sterilized by autoclaving to a total volume of 10 ml. 10 ml of a liquid preparation was produced.
The liquid preparation was similarly manufactured about the other fucoidan oligosaccharide composition as described in Example 2, and each fucoidan oligosaccharide composition as described in Examples 1 and 3, respectively.
[0034]
Example 8
Lactose and starch were added to and mixed with the lyophilized product of the fucoidan oligosaccharide composition having a molecular weight of more than 5000 to 3000 described in Example 3. 10% starch paste was added to the mixture and stirred to produce granules. After drying, the particles were sized, talc and magnesium stearate were mixed therein, and tableted by a conventional method to produce 200 mg tablets (Table 4).
[0035]
[Table 4]
Tablets were similarly produced for the other fucoidan oligosaccharide compositions described in Example 3 and the respective fucoidan oligosaccharide compositions described in Examples 1 and 2.
[0037]
【The invention's effect】
INDUSTRIAL APPLICABILITY According to the present invention, a fucoidan oligosaccharide composition from which anticoagulant activity and antigenicity from contaminating proteins have been removed, and an efficient production method thereof are provided. The fucoidan oligosaccharide composition is excellent in solubility and absorbability to the living body, is naturally derived and highly safe, is useful as a cancer metastasis inhibitor and antifungal agent, and can be applied to foods. It is a new composition.
Claims (1)
（１）分子量分布：５×１０3 以下〔セルロファインＧＣＬ−２５（生化学工業株式会社製）を用いたゲルろ過法による〕
（２) タンパク含量：検出されない
（３）抗凝血活性：５０００μｇ／ｍｌで抗凝血活性を示さないA cancer metastasis inhibitor comprising a fucoidan oligosaccharide composition having the following physicochemical properties:
(1) Molecular weight distribution: 5 × 10 3 or less [by gel filtration using Cellulofine GCL-25 (manufactured by Seikagaku Corporation)]
(2) Protein content: not detected (3) Anticoagulant activity: No anticoagulant activity at 5000 μg / ml
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| JP02758994A JP3714426B2 (en) | 1994-02-01 | 1994-02-01 | Cancer metastasis inhibitor containing fucoidan oligosaccharide composition |
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| JP02758994A JP3714426B2 (en) | 1994-02-01 | 1994-02-01 | Cancer metastasis inhibitor containing fucoidan oligosaccharide composition |
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| JP2005172193A Division JP4341970B2 (en) | 2005-06-13 | 2005-06-13 | Antifungal agent containing fucoidan oligosaccharide composition |
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| EP2489375A1 (en) | 2011-02-16 | 2012-08-22 | Japan Institute of Leather Research | Type I-type IV collagen hybrid gel |
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| JP3605414B2 (en) * | 1995-04-28 | 2004-12-22 | タカラバイオ株式会社 | Sugar compounds |
| AU720004B2 (en) * | 1996-01-26 | 2000-05-18 | Research Institute For Glycotechnology | Apoptosis inducers |
| CA2248648A1 (en) | 1996-03-15 | 1997-09-18 | Takara Shuzo Co., Ltd. | A product of heat treatment of uronic acid, food, drink or drug including the product |
| EP0916269B1 (en) * | 1996-06-12 | 2006-06-14 | Takara Bio Inc. | Use of fucoidan |
| US6573250B2 (en) | 1996-06-12 | 2003-06-03 | Takara Shuzo Co., Ltd. | Food or beverage additive containing fucoidan and food and beverage containing fucoidan |
| FR2783523B1 (en) * | 1998-09-21 | 2006-01-20 | Goemar Lab Sa | FUCO-OLIGOSACCHARIDES, ENZYME FOR THEIR PREPARATION FROM FUCANES, ENZYME-PRODUCING BACTERIA AND APPLICATIONS OF FUCO-OLIGOSACCHARIDES FOR PLANT PROTECTION |
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| JP2009057283A (en) * | 2005-12-21 | 2009-03-19 | Konan Kako Kk | Angiopathy ameliorating agent comprising sulfated polysaccharide derived from green alga, and method for producing the same |
| US20080089941A1 (en) * | 2006-06-01 | 2008-04-17 | Mower Thomas E | Fucoidan compositions and methods |
| KR101486931B1 (en) | 2007-02-23 | 2015-01-27 | 박스터 인터내쇼날 인코포레이티드 | Process methods for fucoidan purification from seaweed extracts |
| JP5311327B2 (en) * | 2007-03-28 | 2013-10-09 | 国立大学法人鳥取大学 | Low molecular weight product of sulfated polysaccharide with suppressed elimination of sulfate group and method for producing the same |
| EP2326334A2 (en) | 2008-08-22 | 2011-06-01 | Baxter Healthcare SA | Methods for treating bleeding disorders |
| US8632991B2 (en) | 2010-01-14 | 2014-01-21 | Baxter International Inc. | Methods and compositions for treating bleeding disorders |
| WO2013012954A2 (en) | 2011-07-19 | 2013-01-24 | Baxter International Inc. | Resorption enhancers as additives to improve the oral formulation of non-anticoagulant sulfated polysaccharides |
| AU2013302871B2 (en) | 2012-08-14 | 2019-05-16 | Baxalta GmbH | Methods and systems for screening compositions comprising non-anticoagulant sulfated polysaccharides |
| US9265791B2 (en) | 2013-03-12 | 2016-02-23 | Baxalta GmbH | Resorption enhancers as additives to improve the oral formulation of low molecular weight heparins |
| JP6721947B2 (en) * | 2015-06-15 | 2020-07-15 | 理研ビタミン株式会社 | Biofilm formation inhibitor |
| CN109464295B (en) * | 2018-10-29 | 2021-12-28 | 名臣健康用品股份有限公司 | Skin care composition containing low molecular weight fucooligosaccharide |
| CN112618481B (en) * | 2021-02-22 | 2021-11-09 | 山东第一医科大学附属省立医院(山东省立医院) | Modified hydrogel, modified hydrogel microspheres and preparation method and application thereof |
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1994
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2489375A1 (en) | 2011-02-16 | 2012-08-22 | Japan Institute of Leather Research | Type I-type IV collagen hybrid gel |
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| JPH07215990A (en) | 1995-08-15 |
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