JP2004215506A - Sea weed extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composition containing a sea weed extract having an immunopotentiating action and a tumor-inhibiting effect. <P>SOLUTION: An anti-tumor agent contains the aqueous medium extract of Costaria costata (C. Agardh) Saunders as an active ingredient. An immunopotentiating agent contains the extract of Costaria costata (C. Agardh) Saunders as an active ingredient. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００３０】
インビトロでの免疫賦活作用（リンパ球混合培養による増殖反応、ｍｉｘｅｄ ｌｙｍｐｈｏｃｙｔｅ ｒｅａｃｔｉｏｎ、ＭＬＲ［藤原大美ら、免疫研究法ハンドブック、１１３−１１７（１９９６）］）
１）ヒトＭＬＲ
実験に用いたヒト末梢血リンパ球（ＰＢＬ）は、健常人ボランティア末梢血より分離した。一人のドナーのＰＢＬは、マイトマイシンＣを２０分間処理した後、１０^６細胞／ｍｌに調整し、刺激細胞として用いた。
もう一人のドナーから得たＰＢＬはナイロンウールカラムにアプライし、３７℃で１時間インキュベーションした。ナイロンウールに吸着しないＴ細胞を回収し、１０画^６細胞／ｍｌに調整した（以下、レスポンダーＴ細胞（ｒｅｓｐｏｎｄｅｒ Ｔ ｃｅｌｌｓという）。上記のＡＰＣとレスポンダーＴ細胞を１：１の比率で混合し、９６ウェル丸底プレートに分注した。その後各スルフォリピッドを添加した。
３−４日間培養後、^３Ｈ−チミジンを各ウェルに添加し、さらに２４時間培養した。ＳＫＡＴＲＯＮ ｈａｒｖｅｓｔｅｒで細胞を採取し、シンチレーションカウンターでレスポンダーＴ細胞の放射線活性を測定した。
【００３１】
２）抽出物の調製
１０ｍｇ／ｍｌの濃度になるように超純水で溶解後、５分間の煮沸で滅菌した。ＭＬＲにおける抽出物の最終濃度は１０〜５００μｇ／ｍｌとした。
【００３２】
活性成分である高分子多溶体の物理化学的性状の解明
インビトロでのスクリーニング（ＷＳＴ−１アッセイ）で効果の高かったスジメアルカリ抽出物を中心とし、スジメ水抽出物、アイヌワカメ水抽出物、参考として食用のナガコンブ水抽出物を供試試料として用いた。
【００３３】
各エキスの酸川水分解による構成糖の分析
エキス１ｍｇを水４ｍｌに溶解して、そのうち０．８ｍｌをアンプル管に移し、エバポレーターで乾固させた。２．５Ｍトリフルオロ酢酸０．８ｍｌを加え、ドライアイス−エタノール冷媒で凍結させた。アンプル管を真空下で冷媒から出し、内容物がゆっくり融けるのを待ち、液中の気泡を除いた。封管後、アルミブロックヒーターで１００℃、２４時間加熱した。アンプル管を開封後、内容物をエッペンドルフチューブに移し、エバポレーターで乾固させ、２０μｌの水で溶解した。これを１５００ｒｐｍ，３分間遠心分離して、上清を薄層クロマトグラフィー（ＴＬＣ）分析に供した。ＴＬＣにはシリカゲルを用いて、展開溶媒にはｎ−ブタノール：酢酸：水＝２：１：１（ｖ／ｖ／ｖ）、呈色試薬にはオルシノール硫酸を用いた。ＴＬＣ標品として、Ｄ−グルコース（和光純薬工業）および同条件で加水分解した試薬のアルギン酸（和光純薬工業）加水分解物を用いた。
【００３４】
各エキスのウロン酸含量の定量
各エキスは褐藻抽出物であるので、アルギン酸に特徴的なＤ−マンニュロン酸やＬ−グルロン酸などのウロン酸を大量に含むことが予想される。よって、各エキスのウロン酸含量を測定した。あらかじめ試薬Ａ（ホウ酸ナトリウム０．９５ｇを濃硫酸１００ｍｌに溶解した）および試薬Ｂ（カルバゾール０．１２５ｇを無水メタノール１００ｍｌに溶解した）を調製しておいた。１０μｇ／ｍｌの各エキス溶液１ｍｌを氷冷しながら、氷冷した試薬Ａ５ｍｌを加え、１００℃、１０分間加熱したあと氷冷した。試薬Ｂ０．２ｍｌを加え、１００℃、１５分間加熱したあと室温まで冷却し、５３０ｎｍで吸光値を測定した。検量線はＤ−グルクロン酸を用いて求めた。ウロン酸量は、Ｄ−グルクロン酸当量として求めた。
【００３５】
各エキスの多糖類の分別
１）アルギン酸
アルギン酸はナトリウム塩として得られるアルカリ抽出法を用いた。各エキスを適当量秤取した。０．２Ｎ硫酸５ｍｌを加え、室温で１夜振盪した。ろ紙（東洋濾紙Ｎｏ．１）を用いてろ過し、ろ液を除いた。残渣を水１ｍｌで洗浄後に、１％炭酸ナトリウム水溶液を５ｍｌ加えて、１夜振盪した。水２０ｍｌを加えたあとにろ過した。ろ液に２倍容のエタノールを加えてアルギン酸を析出させた。ガラス棒で粗アルギン酸ナトリウムを集めた。得られた粗アルギン酸ナトリウムを水に溶解後、３倍容のエタノールを加えてアルギン酸ナトリウムを析出させた。さらに２回同じ操作を繰り返した。得られたアルギン酸ナトリウムを乾燥して重量を測定した。
【００３６】
２）不溶性ラミナラン
不溶性ラミナランは塩酸抽出法を用いた。各エキスを適当量秤敢した。０．１２５Ｎ塩酸２．５ｍｌと３７％ホルマリン１μｌを加え、７０℃で攪拌した。ろ紙（東洋濾紙 Ｎｏ．１）を用いでろ過した。残渣をさらに７０℃の水０．２ｍｌで２回洗浄した。ろ液と洗浄液をあわせた。室温で３時間撹枠後に２目闇静置した。沈殿した粗不溶性ラミナランを遠心分離（３０００ｒｐｍｍ、５分間）で集めた。乾燥後にエタノールおよびジエチルエーテルそれぞれ０．２ｍｌで洗浄後、減圧下で乾燥させて重量を測定した。
【００３７】
３）可溶性ラミナラン
粗可溶性ラミナランは塩酸糊出法を用いた。各エキスを適当量秤取した。０．０９Ｎ 塩酸２．５ｍｌを加え、室温で２時間撹拝した。遠心分離（３０００ｒｐｍ，５分間）して上清を別の容器に移した包。沈殿をさらに０．０５Ｎ塩酸０．５ｍｌで２回洗浄した。上清液と洗浄液をあわせた。エタノールを８５％になるまで加えて沈殿を生成させた。沈殿を遠心分離（３０００ｒｐｍ，５分間）で集め、エタノールおよびジエチルエーテルそれぞれ０．５ｍｌで洗浄後、減圧下で乾燥させて重量を測定した。
【００３８】
４）フコイダン
フコイダンは塩酸抽出法を用いた。各エキスを適当量秤取した。０．１７Ｎ塩酸１．３ｍｌを加え、６５〜７０℃で１時間抽出した。抽出液を水酸化ナトリウムで中和した後、減圧濃縮した。残渣を水０．５ｍｌに溶解した。エタノールを１ｍｌ加えて、遠心分離で粗フコイダンを沈殿させた。乾燥後に水２．５ｍｌに溶解後に、さらに４Ｍ塩化カルシウム１ｍｌを加えて、アルギン酸カルシウムの沈殿を遠心分離で除いた。この上清に５％セチルピリジニウムクロリド（ＣＰＣ）水溶液３ｍｌを加えた。続いて塩化カルシウムの終濃度が０．５Ｍになるように水を加えて、一晩静置した。遠心分離（３０００ｒｐｍ、５分間）によってＣＰＣ複合体を沈殿させ、３Ｍ塩化カルシウム１．５ｍｌに溶解して、エタノール７ｍｌを加えた。生成した沈殿を遠心分離（３０００ｒｐｍ、５分間）で集め、減圧下で乾燥させて重量を測定した。
【００３９】
実験結果
海藻由来高分子多糖体の抽出と分画
乾燥原料１００ｇより実験室レベルで得られた高分子多糖体の粉末は１〜８ｇであった。結果を図２に示す。アルカリ抽出では水抽出よりもエキス抽出量はあまり多くはないが、これはろ過によるロスが増えているためで、実際のアルカリエキス抽出量はさらに多いと思われる。アルカリ抽出では粘性の高い溶液となる。酸抽出ではアルカリ抽出や水抽出よりもエキス抽出量は増えているが、高分子多糖体粉末としての収量は少なかった。この理由は加水分解により、低分子へと変化するものが多いと考えられる。
【００４０】
免疫賦活作用、抗腫瘍作用等に係る科学的評価
インビトロでのスクリーニング（ＷＳＴ−１アッセイ）
１）Ａ５４９の増殖に対するスジメ、アイヌワカメの抽出物の影響
いずれの試料も濃度が高くなると細胞の生存率は減少した。対照のフコイダン、クレスチンは、増殖を強く抑制した。フコイダンは、０．５ｍｇ／ｍｌで生存率３０％であり、クレスチンは１ｍｇ／ｍｌで１０％以下であった。また、褐藻類に含まれるアルギン酸は５ｍｇ／ｍｌで５０％程度であったが、海藻抽出物ではスジメは酸、アルカリ抽出物ともに５ｍｇ／ｍｌで５０％以下を示した。（図３）
【００４１】
ＭＫＮ２８の増殖に対するスジメ、アイヌワカメの抽出物の影響
フコイダン、クレスチンは１ｍｇ／ｍｌで４０％の生存率であったが、アルギン酸は濃度を高くしてもあまり効果がみられなかった。スジメ抽出物はいずれも濃度が高くなると生存率は減少し、５ｍｇ／ｍｌで５０％以下であった。アイヌワカメは酸、アルカリ抽出物５ｍｇ／ｍｌで５０％以下の生存率であった。（図４）
【００４２】
Ａ−４３１の増殖に対するスジメ、アイヌワカメの抽出物の影響
クレスチシは１ｍｇ／ｍｌで５０％の生存率であったが、スジメのアルカリ抽出物では０．５ｍｇ／ｍｌで５０％以下の生存率を示し、海藻抽出物の中では最も高い増殖抑制効果を示した。（図５）
【００４３】
インビボでの抗腫瘍作用および免疫賦活作用（マウス経口投与）
各被験物質投与終了目の翌日に、全ての動物から腫瘍を摘出し湿重量を測定した。その結果を図６−１に示す。対照群の摘出腫瘍重量は３２２４ｍｇであった。クレスチン１００及び３００ｍｇ／ｋｇ／ｄａｙ投与群の腫瘍重量は、それぞれ２４７９ｍｇ及び１７１７ｍｇを示し、対照群と比較していずれも有意な増殖抑制が認められた。腫瘍増殖抑制率は、それぞれ２３．１及び４６．７％と算出された。
スジメアルカリ抽出物３０，１００及び３００ｍｇ／ｋｇ／ｄａｙ、２１日間投与群の腫瘍重量は、それぞれ３０４３、２６８４及び２１７８ｍｇを示し、対照群と比較して１００ｍｇ／ｋｇ／ｄａｙ以上の投与で有意な増殖抑制が認められた。腫瘍増殖抑制率は、それぞれ５．６、１６．８及び３２．５％と算出された。
スジメアルカリ抽出物３００ｍｇ／ｋｇ／ｄａｙ、１４日間投与群の腫瘍重量は２８４６ｍｇを示し、対照群と比較して有意な抑制が認められたものの、腫瘍増殖抑制率は１１．８％と低かった。スジメ水抽出物１００及び３００ｍｇ／ｋｇ／ｄａｙ投与群の腫瘍重量は、それぞれ２７２９及び２２９８ｍｇを示し、対照群と比較していずれも有意な抑制が認められた。腫瘍抑制率は、それぞれ１５．４及び２８．７％と算出された。
【００４４】
インビトロでの免疫賦活作用（ＭＬＲ）
スジメ水抽出物は低濃度で増殖促進傾向がみられたが、高濃度（＞２００μｇ／ｍｌ）での培養は不可能であった。スジメアルカリ抽出物では、効果がみとめられなかった。スジメ酸抽出物は濃度依存的に増殖促進傾向（１０〜５００μｇ／ｍｌ）を示した。アイヌワカメ水抽出物では、増殖促進傾向は認められたが、バラツキが大きかった。アイヌワカメアルカリ抽出物では、効果が認められなかった。アイヌワカメ酸抽出物は低濃度（１０〜１００μｇ／ｍｌ）で増殖促進傾向を示した（図７）。
【００４５】
活性成分である高分子多糖体の物理化学的性状の解明
各エキスの酸加水分解による構成糖の分析
それぞれのエキス酸加水分解のＴＬＣ分析（図８）を行った。スジメアルカリ抽出物はアルギン酸加水分解物と一致したので、アルギン酸が主成分と決定できた。スジメ水抽出物は１個のスポットのみが検出されたが、同定には至らなかった。アイヌワカメ水抽出物はアルギン酸加水分解物に対応するスポットのほか、未同定のスポットが検出された。ナガコンブ水抽出物はアルギン酸加水分解物の他にＤ−グルコースに対応するスポットが検出されることから、アルギン酸の他に、ラミナランなどのグルカンが含まれていると予想される。
【００４６】
各エキスのウロン酸含量の定量
ウロン酸量はＤ−グルクロン酸量で求め、エキス中の重量％で示した（表２）。スジメアルカリ抽出物およびアイヌワカメ水抽出物でウロン酸の割合が高かった。これらのエキスはアルギン酸が多いと予想される。
【００４７】
表２ 各エキス中のウロン酸の含有率
【００４８】
【表２】

【００４９】
各エキスの多糖類の分別
スジメアルカリ抽出物は各多糖の含有率の合計は７３％程度であるが、その他のエキスは各多糖の含有率の合計が１００％を大きく超えた（表７）。これらの原因として、アルギン酸はＮａ塩で、可溶性ラミナランは粗収量で求めたこと、また本分画で得られたものをすべて当該多糖とみなしているため等の理由が考えられるが、おおまかな傾向は読み取れる。
スジメアルカリ抽出物はアルギン酸が主成分であった。スジメ水抽出物ではラミナランやフコイダンが比較的多かった。アイヌワカメ水抽出物ではアルギン酸が主成分であった。ナガコンブ水抽出物ではラミナランが多かった。
【００５０】
表３ 各エキス中の多糖の含有率（％）
【００５１】
【表３】

【００５２】
考察
海藻由来高分子多糖体の抽出と分画において、未利用資源であるスジメ、アイヌワカメを原料とし、乾燥物１００ｇより実験室レベルで得られた高分子多糖体の粉末は１〜８ｇであった。
【００５３】
アルカリ抽出では水抽出よりもエキス抽出量は多いと思われるが、粘性の高い溶液となる。
【００５４】
酸抽出ではアルカリ抽出や水抽出よりもエキス抽出量は増えているが、高分子多糖体粉末としての収量は少なくなっている。この理由は加水分解により、高分子多糖体が低分子へと変化するものが多いと考えられる。
【００５５】
今回、海藻抽出物（高分子多糖体粉末）各濃度につき、ＷＳＴ−１アッセイを用い、ヒト由来ガン細胞に対する生存率を求めた。対照としてクレスチン、アルギン酸ナトリウム、フコイダンを用い、比較検討を行った。
【００５６】
低濃度で効果が見られたのは対照試料のフコイダンやクレスチンである。海藻抽出物試料でも濃度依存的に生存率が低く（細胞増殖抑制が高い）なったが、低濃度で効果が見られた海藻抽出物試料はスジメのアルカリ抽出物のＡ−４３１（類表皮ガン細胞株）に対する効果であった。
【００５７】
試験結果
試験結果を図６−２に示した。スジメアルカリ抽出物では、投与量３０ｍｇ／ｋｇでは効果は認められなかったものの、投与量１００ｍｇ／ｋｇで１７％、投与量３００ｍｇ／ｋｇで３２％の有意な腫瘍増殖抑制効果が得られた（ｐ＜０．０１）。スジメ水抽出物でもスジメアルカリ抽出物と同様の効果が得られた。
【００５８】
移植後に投与した群（合計１４日間投与）は、移植７日前より投与した群（合計２１日間投与）よりも効果は低かった。これはスジメアルカリ抽出物を経口摂取することによって非特異的な免疫賦活活性が高められるものの、その効果が発揮されるには数日を要するものと考えられる。
【００５９】
なおクレスチンでは投与量３００ｍｇ／ｋｇで４７％の腫瘍増殖抑制効果が得られている。抗悪性腫瘍剤クレスチンの用法用量は３ｇ／日のため、６０ｋｇのヒトが１日３ｇ摂取すると、マウス投与量換算で約５０ｍｇ／ｋｇ／日となる。これは今回試験した腫瘍細胞Ａｄｅｎｏｃａｒｃｉｎｏｍａ７５５の腫瘍増殖抑制率から限って類推すると、スジメアルカリ抽出物投与量１００ｍｇ／ｋｇにほぼ相当するものと予測される。換言すればスジメアルカリ抽出物を１日６ｇ摂取すると、抗悪性腫瘍剤クレスチンを１日３ｇ服用したことと同等の効果に匹敵する。
【００６０】
インビトロによる免疫賦活作用では、レスポンダーＴ細胞がスジメ酸抽出物により濃度依存的に明らかな増殖促進傾向（１０〜５００μｇ／ｍｌ）を示した。アイヌワカメ酸抽出物も低濃度（１０〜１００μｇ／ｍｌ）で増殖促進傾向を示した。
【００６１】
特にスジメ酸抽出物には樹状細胞等の抗原提示細胞を刺激し、不細胞増殖を促進させる物質が含まれていることが示唆される。３２％の有意な腫瘍増殖抑制効果が得られたスジメアルカリ抽出物以上に、スジメ酸抽出物が抗腫瘍効果有することが期待できる。
【００６２】
スジメの抽出物（酸、アルカリ、水抽出物）についてインビボでの抗腫瘍作用および免疫賦活作用（マウス経口投与）
【００６３】
使用動物
５週齢のＳｌｃ：ＢＤＦ_１雄性マウスを日本エスエルシー株式会社より購入し、１週間の検疫期間を含む予備飼育の後、一般状態に異常が認められなかった動物を選択し試験に使用した。実験開始時の体重は、２５．０〜２９．８ｇであった。動物は、耳パンチ及び尾へのフェルトペンによる動物番号の記載により個体を識別した。また、収容したゲージには、実験中、処理群及び動物番号を記入したカードを付した。
【００６４】
飼育条件
予備飼育及び実験期間を通じ、温度２２±３℃、湿度５０±２０％、照明１２時間（８：００〜２０：００）、換気回数１３〜１７回／時間の環境下で、ステンレス製可動ラック（１７９０Ｗ×４７０Ｄ×１６５０Ｈｍｍ）に装着したポリサルフォン製ケージ（２６５Ｗ×４２７Ｄ×２０４Ｈｍｍ）に５〜６匹の集団で収容した。飼料は、ステンレス製固形飼料給餌器により固形飼料、ラボＭＲストック（日本農産工業株式会社）を、水は、ポリサルフォン製給水器（先管ステンレス製）により水道水を、各々自由に与えた。
【００６５】
試験方法
６週齢のＢＤＦ_１マウスを、表４の群構成に基づき使用した。投与７日目に、Ｃ５７ＢＬ／６マウスで継代維持した１０〜１４日目の腫瘍細胞を試験用マウスの腹側部皮下に１×１０^５細胞／０．０５ｍｌ／マウスの量で移植した。被験物質を１日１回経口投与した。被験物質投与最終日の翌日に、エーテル麻酔下にマウスの腹部大動脈より採血し、その後腫瘍を摘出しその重量を測定した。体重測定は、２回／週の割合で行った。得られた血液は、血漿を分画し、−８０℃で保存した。
【００６６】
【表４】

【００６７】
統計処理
各群で得られた数値の平均値及び標準誤差を算出した。各群間の統計分析は、Ｂａｒｔｌｅｔｔ法（有意水準５％）による等分散性の検定、等分散の場合は、さらに一元配置分散分析を行い、有意な場合には、Ｔｕｒｋｅｙ法により平均値の比較を行った。不等分散の場合は、Ｋｒｕｓｋａｌ−ＷａｌｌｉｓのＨ検定を行い、有意な場合はＴｕｒｋｅｙ法により平均順位の比較を行った。有意水準は、危険率５％及び１％とした。
【００６８】
また、平均摘出腫瘍重量について、対照群に対する被験物質投与群の割合（Ｔ／Ｃ）から、米国国立癌研究所（ＮＣＩ）の評価基準（Ｇｃｒａｎ， Ｒ．Ｉ． ｅｔ ａｌ． Ｃａｎｃｅｒ Ｃｈｅｍ． Ｒｅｐ． Ｐａｒｔ３， ｖｏｌ３ （１９７２））に従い、以下のように腫瘍抑制率（％）を算出し、抗腫瘍効果の判定を行った。
Ｔ／Ｃ（％）＝（被験物質投与群の平均摘出腫瘍重量（ｍｇ）／対照群の平均摘出
腫瘍重量（ｍｇ））×１００
腫瘍抑制率（％）＝１００−Ｔ／Ｃ≧３０％を抗腫瘍効果ありとした。
【００６９】
試験結果
試験結果を図６−２に示した。
１．スジメ酸抽出物
対照群の摘出腫瘍重量は、２３９０ｍｇであった。
スジメ酸抽出物３０、１００及び３００ｍｇ／ｋｇ／ｄａｙの２１日間投与群では、腫瘍重量は、それぞれ６０８、４９８及び３９２ｍｇを示し、１００ｍｇ／ｋｇ／ｄａｙ以上の投与で対照群と比較して有意な抑制が認められた。３００ｍｇ／ｋｇ／ｄａｙの１４日間投与群では、腫瘍重量は、８３４ｍｇ／ｋｇ／ｄａｙを示し、対照群と比較して抑制傾向が認められた。腫瘍抑制率は、３０、１００及び３００ｍｇ／ｋｇ／ｄａｙの２１日投与群では、それぞれ７４．６、７９．２及び８３．６％、３００ｍｇ／ｋｇ／ｄａｙの１４日間投与群では、６５．１％と算出され、いずれも抗腫瘍効果ありと判定された。
【００７０】
２．スジメアルカリ抽出物
スジメアルカリ抽出物３００ｍｇ／ｋｇ／ｄａｙの２１日間投与群では、腫瘍重量は、５６９ｍｇを示し、対照群と比較して有意な抑制が認められた。腫瘍抑制率は、７６．２％と算出され、抗腫瘍効果ありと判定された。
【００７１】
３．スジメ水抽出物
スジメ水抽出物３００ｍｇ／ｋｇ／ｄａｙの２１日間投与群では、腫瘍重量は、４１８ｍｇを示し、対照群と比較して有意な抑制が認められた。腫瘍抑制率は、８２．５％と算出され、抗腫瘍効果ありと判定された。
【００７２】
４．スジメ乾燥粉末
スジメ乾燥粉末３００ｍｇ／ｋｇ／ｄａｙの２１日間投与群では、腫瘍重量は、６８５ｍｇを示し、対照群と比較して有意な抑制が認められた。腫瘍抑制率は、７１．４％と算出され、抗腫瘍効果ありと判定された。
【００７３】
高分子多糖体の構造の解析
褐藻類にはアルギン酸、フコイダン、フコイダンなどの高分子多糖体が含まれる。一般にアルギン酸はうすいアルカリで、フコイダンはうすい塩酸で抽出されるといわれる。よってＴＬＣ分析、ウロン酸含量、多糖類の分別により高分子多糖体の構造の解析を行った。
【００７４】
スジメアルカリ抽出物はアルギン酸が主成分であり、スジメ水抽出物ではラミナランやフコイダンが比較的多かった。アイヌワカメ水抽出物ではアルギン酸が主成分であり、アイヌワカメアルカリ抽出物ではアルギン酸が主成分と考えられる。
【００７５】
インビトロによる免疫賦活作用で、レスポンダーＴ細胞が濃度依存的に明らかな増殖促進傾向を示した試料は、スジメアルカリ抽出物でなくスジメ酸抽出物であった。よって樹状細胞等の抗原提示細胞を刺激し、Ｔ細胞増殖を促進させる物質はラミナランもしくはフコイダンに関係するものと推測される。
【００７６】
本抽出物は、安全かつ廉価であり、その免疫賦活性及び腫瘍抑制効果を利用した機能性食品の原料として利用することができる。
【図面の簡単な説明】
【図１】海藻由来高分子多糖体製造フローである。
【図２】海藻由来高分子多糖体の抽出・分析結果である。
【図３】各種抽出物添加時のＡ５４９の生存率である。
【図４】各種抽出物添加時のＭＫＮ２８の生存率である。
【図５】各種抽出物添加時のＡ４３１の生存率である。
【図６】インビボでの抗腫瘍作用である。
【図７】インビボでの抗腫瘍作用である。
【図８】レスポンダーＴ細胞の増殖である。
【図９】海藻エキスの酸加水分解物の分析ＴＬＣである。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to an extract from brown algae, linen, which has an immunostimulating effect and / or an antitumor cell effect.
[0002]
[Prior art]
In eastern Hokkaido, seaweeds such as Ainu-Wakame, Sujime, Kalexa, and Hibamata grow naturally and can be collected in large quantities, but are not used as resources because they are not suitable for food.
[0003]
In addition, from the viewpoint of protecting useful resources such as kelp, the Habomai Fisheries Cooperative removes 600 tons of unused seaweeds such as Azalea and Ainu-Wakame, which grow near the sea near Nemuro. Disposal has become a serious problem.
[0004]
On the other hand, in recent years, research on physiologically active substances contained in seaweeds has been advanced, and it has been found that seaweeds contain high molecular polysaccharides such as alginic acid, fucoidan, and carrageenan. It is considered that this high molecular polysaccharide enhances the attack on cancer cells by activating immune cells such as macrophages and natural killer cells in a living body, and exhibits an antitumor effect (Patent Documents 1 to 3). In addition to seaweeds, high-molecular-weight polysaccharide components extracted from basidiomycetes, lentin of shiitake mushroom, krestin of kawatake mushroom, and schizophyllan sugar of shirohirotake mushroom have been used for cancer therapy for more than 20 years. (Patent Documents 4 and 5).
[0005]
However, studies on physiologically active substances derived from seaweed components, such as antitumor substances and immunostimulatory substances, are still few, and the necessity for future research is high.
[0006]
Thus, the present inventors have conducted intensive studies and, as a result, have found that an extract of linen has antitumor activity and immunostimulatory activity, and have completed the present invention.
[0007]
[Patent Document 1]
JP-A-10-158156
[Patent Document 2]
JP-A-5-139988
[Patent Document 3]
JP-A-6-256208
[Patent Document 4]
JP-A-7-215990
[Patent Document 5]
JP-A-10-195106
[0008]
[Problems to be solved by the invention]
The present invention provides a food or beverage comprising an extract of linen.
[0009]
[Means for Solving the Problems]
Examples of the seaweed used in the present invention include stripes and Ainu-Wakame, and stripes are particularly preferable. Although these seaweeds can be used as they are, they are preferably used as those that have been washed with water to remove dirt, dried, cut into slices, or crushed dry powders.
[0010]
An aqueous solvent is added to the washed dried seaweed to perform an extraction operation. Water is preferred as the aqueous solvent, but an acid such as hydrochloric acid or an alkaline aqueous solution such as sodium hydroxide can also be used for the water.
[0011]
The extraction temperature is from room temperature to the boiling point, preferably from 50 to 90 ° C, more preferably from 80 to 95 ° C. The extraction time is 5 minutes to 72 hours, preferably 30 minutes to 20 hours, more preferably 1 to 3 hours. In addition, the extraction operation may be performed once, but may be performed twice or more.
[0012]
The solid content is removed from the obtained extract by filtration, centrifugation, decantation or the like.
[0013]
The obtained extract can be used as it is or, if necessary, after neutralization, desalting and concentration, but it is usually preferable to carry out a more general purification treatment. As the purification treatment, precipitation using an organic solvent, salting out using ammonium sulfate, sodium chloride, potassium chloride, etc., dialysis, ultrafiltration, reverse osmosis treatment, gel filtration using a column filled with dextran, polyacrylamide gel, or the like should be used. Can be. Further, two or more of these processes can be performed in combination.
[0014]
The extract containing the active ingredient obtained by the above operation can be used as it is as an immunostimulant or an antitumor agent, but is usually further dried by spray drying, freeze drying, vacuum drying, hot air drying, or the like. be able to.
[0015]
Since the extract of the present invention has an advantage that it does not adversely affect the living body even when inoculated in large amounts, the extract itself or various nutrients are added thereto, or the immunostimulatory action or It can be provided as a functional food or health food having an antitumor effect. Specifically, by adding nutrients such as various vitamins and minerals, for example, nutritional drinks, soy milk, liquid foods such as soups and solid foods of various shapes, and further, as it is as a powder, or various It can also be used by adding to food. The content of the active ingredient in the extract of the present invention as such a functional food or health food is 10 to 1000 mg, preferably 30 to 360 mg per 1 kg of adult daily weight, and is divided into single doses or two to three times. Can be taken.
[0016]
【Example】
Experimental method Extraction and fractionation of seaweed-derived polysaccharide
FIG. 1 shows a flow of the operation. 100 g of the dried stripes were cut into slices, placed in 2000 g of water, and extracted at 80 to 95 ° C. for 2 hours. Suction filtration was performed using filter paper to obtain 1,265 g of a filtrate. This filtrate corresponded to 29.2 g as an extract extraction amount. 600 g of the above filtrate was dialyzed with an ultrafiltration device (fraction molecular weight 10,000) of Pellicon XL module Biomax-10 manufactured by Millipore. When the concentration of the concentrated solution reached 150 ml, a third volume of water was added to make the concentration 600 ml, and the second dialysis was performed. This operation was repeated to purify the high-molecular-weight polysaccharide by a total of four dialysis treatments.
[0017]
The fourth concentrated solution (150 ml) was concentrated under reduced pressure to about 50 ml, and a precipitate was precipitated with anhydrous ethanol.
A precipitate was precipitated in the same manner from the remaining 665 g of the filtrate, and the precipitate was combined with the previous precipitate, filtered, and dried under reduced pressure to finally obtain 2.3 g of an aqueous extract of polysaccharide polysaccharide. The recovery rate of the high molecular weight polysaccharide from the dried line was 2.3%.
[0018]
In addition, extract extraction was performed using a solvent of 0.1 M NaOH and 0.1 M HCl, and by the same fractionation treatment, an alkaline extract of polysaccharide polysaccharide and an acid extract of polysaccharide polysaccharide were prepared. Further, from the dried Ainu-Wakame, an Ainu-Wakame high-molecular-weight polysaccharide aqueous extract, an Ainu-Wakame high-molecular-weight polysaccharide alkaline extract, and an Ainu-Wakame high-molecular-weight polysaccharide acid extract were prepared. It is generally said that alginic acid is extracted with a thin alkaline solution and fucoidan is extracted with a thin hydrochloric acid solution (Nobuo Yamada, Science of Utilization of Seaweed (2000)).
[0019]
Scientific evaluation of immunostimulatory effects, antitumor effects, etc.
In vitro screening "WST-1 assay [Yoshi Shinmoto et al., Japan Society for Food Science and Technology, 64-68, 43 (1996)]"
1) Cells
The WST-1 assay is a cell proliferation assay using a microculture plate. By measuring the water-soluble formazan produced by living cells with a plate reader, cell proliferation can be rapidly measured.
This time, three human-derived adhesive cell lines (lung cancer cell line A549, stomach cancer cell line MKN28, epidermoid cancer A-431) obtained from the Human Science Research Resource Bank were used. The cells were cultured in a medium in which 10% fetal bovine serum was added to RPMI1640 (Asahi Techno Glass), EMEM (Giboo), DMEM (Giboo), respectively. Cells were harvested by trypsinization and subcultured every 2-4 days at a dilution of 1:10 to 1: 5. The cultivation was performed at 37 ° C., 5% carbon dioxide gas 95% air, and humidity 90% or more.
[0020]
2) Seaweed extract and control
The prepared seaweed-derived high-molecular-weight polysaccharides consisted of six types: a linen water extract, a linen alkali extract, a linen acid extract, an Ainu-Wakame water extract, an Ainu-Wakame alkali extract, and an Ainu-Wakame acid extract. As controls, antineoplastic agent krestin (PSK, Sankyo), sodium alginate (Wako Pure Chemical Industries), and fucoidan (Sigma) were used.
[0021]
3) Culture of cultured cells in the presence of seaweed extract
1 to 2.5 × 10 ⁴ / Ml of the cell suspension at a density of / ml was dispensed into a microculture plate, cultured at 37 ° C for 24 hours, the medium was removed, the seaweed extract dissolved in the medium was sterilized by filtration, and then 100 µl was added. Later, cell proliferation was measured in the WST-1 assay. As a control, a cell-free group and an extract-free group (medium only) were also measured.
[0022]
4) WST-1 assay
To a microculture plate in which cells have been cultured, 10 μ ユ of a WST-1 solution prepared with a cell counting kit (Dojin) is added, and the mixture is incubated at 37 ° C. for 2 hours. Then, the main wavelength is 450 nm and the reference wavelength is 450 nm using a microplate reader (Bio-Rad 550). The absorbance was measured at 630 nm, the absorbance of the cell-free group was subtracted, and the viability was determined with the absorbance of the extract-free group (medium only) as 100%.
[0023]
In vivo anti-tumor and immunostimulatory effects (oral administration to mice)
Screening for antineoplastic substances began in 1948 with the discovery of mouse lymphocytic leukemia L-1210. In the US National Cancer Institute (NCI) screening organization, in addition to L-1210, Sarcoma 180 and Ca755 were added. Surgical procedures using various types of tumor strains have been adopted since about 1959. Since then, many tumor lines have been used, but the most important in screening systems is to use systems that can predict the effects of clinically useful drugs (RI Geran). et al., Cancer Chem. Rep. Part 2, 3 (1972), Shigeru Tsukagoshi, Development and Evaluation of Cancer Drug Therapy, Realize, Tokyo, 21-27, 1985).
[0024]
Since almost all anticancer drugs currently used in clinical practice have been confirmed to be effective against Ca755, it is considered that studying the antitumor effect using Ca755 is useful (Shigeru Tsukakoshi (1985): Cancer Pharmaceutical Therapy Development and Effect Judgment, Realize, Tokyo, 21-27).
[0025]
1) Test substance
The analysis was carried out by selecting a lineme water extract and krestin as a subject, centering on a lineme alkali extract that was highly effective in in vitro screening (WST-1 assay).
[0026]
2) Animals used
Slc at 5-6 weeks of age: BDF ₁ Male mice were purchased from Nippon Kogyo SLC Co., Ltd., and after preliminary breeding including a quarantine period of one week, animals in which no abnormality was observed in the general condition were selected and used for the test. The body weight at the start of the experiment was 20.1-26.1 g. The animals were identified by ear punches and the animal numbers described with a felt pen on the tail. In addition, the housed cage was provided with a card in which the group and animal number were written during the experiment.
[0027]
3) Rearing conditions
Through the preliminary breeding and the experimental period, the stainless steel movable rack under the environment of temperature 22 ± 3 ° C, humidity 50 ± 20%, lighting 12 hours (8:00 to 20:00), ventilation rate 13 to 17 times / hour (1790 W x 470 D x 1650 H (mm)) and a group of 5 to 6 animals were housed in a cage made of polysalphon (outside diameter: 265 W x 427 D x 204 H (mm)). As a feed, a solid feed and a lab clothing stock (Nippon Agricultural Industry Co., Ltd.) were freely given by a stainless solid feed feeder, and tap water was freely given by a polysalphon waterer (a stainless steel tube).
[0028]
4) Test method
6-7 week old BDF ₁ The mice were grouped by stratified continuous randomization using body weight as an index and used based on the group composition in Table 1. In all groups except group G, mice were orally administered the test substance once a day for 7 days. On the 7th day after the administration, a 14th tumor cell adenocarcinoma 755 (hereinafter, abbreviated as Ca755) subcultured and maintained in C57BL / 6 mice was subcutaneously injected into the abdominal part of a test mouse at 1 × 10 5. ⁵ Cells were implanted at a rate of cells / 0.05 ml / mouse. After the tumor transplantation, the test substance was similarly orally administered for a total of 21 days for 14 consecutive days. Group G was orally administered once daily for 14 consecutive days from the day after tumor implantation. The day after the end of the administration of the test substance, blood was collected from the posterior vena cava using ether heparin as an anticoagulant under ether anesthesia, and the plasma was fractionated. After the blood collection, the tumor was excised and the wet weight was measured. IL-2, IFN-γ, IL-4 and IL-10 concentrations were quantified from the plasma of Groups A, C, F, I and K by ELISA (ENDOGEN, Inc.).
[0029]
[Table 1]

[0030]
In vitro immunostimulatory action (proliferation reaction by mixed lymphocyte culture, mixed lymphocyte reaction, MLR [Daimi Fujiwara et al., Handbook of Immunological Research, 113-117 (1996)])
1) Human MLR
Human peripheral blood lymphocytes (PBL) used in the experiment were separated from peripheral blood of healthy volunteers. One donor's PBL was treated with mitomycin C for 20 minutes, followed by 10 minutes. ⁶ Adjusted to cells / ml and used as stimulator cells.
PBL from another donor was applied to a nylon wool column and incubated at 37 ° C. for 1 hour. Collect T cells that do not adsorb to nylon wool and ⁶ (Responder T cells) The APC and the responder T cells were mixed at a ratio of 1: 1 and dispensed into a 96-well round bottom plate. The pid was added.
After culturing for 3-4 days, ³ H-thymidine was added to each well, and the cells were further cultured for 24 hours. The cells were collected with a SKATRON harvester, and the radioactivity of responder T cells was measured with a scintillation counter.
[0031]
2) Preparation of extract
After dissolving in ultrapure water to a concentration of 10 mg / ml, the mixture was sterilized by boiling for 5 minutes. The final concentration of the extract in the MLR was 10-500 μg / ml.
[0032]
Elucidation of physicochemical properties of polymer polysolution as active ingredient
As a test sample, a water lineage extract, a water extract of Ainu-Wakame seaweed, and an edible water extract of Nagakombu as a reference were used as test samples centering on a lineage alkali extract which was highly effective in in vitro screening (WST-1 assay).
[0033]
Analysis of constituent sugars by acid river water decomposition of each extract
1 mg of the extract was dissolved in 4 ml of water, of which 0.8 ml was transferred to an ampoule tube and dried by an evaporator. 0.8 ml of 2.5 M trifluoroacetic acid was added and frozen with a dry ice-ethanol refrigerant. The ampoule was removed from the refrigerant under vacuum and the contents were allowed to slowly melt, removing bubbles in the liquid. After sealing, the tube was heated at 100 ° C. for 24 hours using an aluminum block heater. After opening the ampoule, the contents were transferred to an Eppendorf tube, dried to dryness by an evaporator, and dissolved in 20 μl of water. This was centrifuged at 1500 rpm for 3 minutes, and the supernatant was subjected to thin layer chromatography (TLC) analysis. Silica gel was used for TLC, n-butanol: acetic acid: water = 2: 1: 1 (v / v / v) as a developing solvent, and orcinol sulfuric acid was used as a color reagent. D-glucose (Wako Pure Chemical Industries) and a hydrolyzate of alginic acid (Wako Pure Chemical Industries), a reagent hydrolyzed under the same conditions, were used as TLC samples.
[0034]
Determination of uronic acid content of each extract
Since each extract is a brown algae extract, it is expected that it contains a large amount of uronic acids such as D-mannuronic acid and L-guluronic acid, which are characteristic of alginic acid. Therefore, the uronic acid content of each extract was measured. Reagent A (0.95 g of sodium borate dissolved in 100 ml of concentrated sulfuric acid) and reagent B (0.125 g of carbazole dissolved in 100 ml of anhydrous methanol) were prepared in advance. While ice-cooling 1 ml of each 10 μg / ml extract solution, 5 ml of ice-cooled reagent A was added, heated at 100 ° C. for 10 minutes, and then ice-cooled. 0.2 ml of Reagent B was added, heated at 100 ° C. for 15 minutes, cooled to room temperature, and the absorbance was measured at 530 nm. The calibration curve was determined using D-glucuronic acid. The uronic acid amount was determined as D-glucuronic acid equivalent.
[0035]
Separation of polysaccharide of each extract
1) Alginic acid
Alginic acid used the alkali extraction method obtained as a sodium salt. An appropriate amount of each extract was weighed. 5 ml of 0.2N sulfuric acid was added, and the mixture was shaken at room temperature overnight. Filtration was performed using filter paper (Toyo Filter Paper No. 1), and the filtrate was removed. After the residue was washed with 1 ml of water, 5 ml of a 1% aqueous sodium carbonate solution was added, and the mixture was shaken overnight. After adding 20 ml of water, the mixture was filtered. Alginic acid was precipitated by adding twice the volume of ethanol to the filtrate. The crude sodium alginate was collected with a glass rod. After dissolving the obtained crude sodium alginate in water, 3 volumes of ethanol was added to precipitate sodium alginate. The same operation was repeated twice more. The obtained sodium alginate was dried and weighed.
[0036]
2) Insoluble laminaran
For the insoluble laminaran, a hydrochloric acid extraction method was used. An appropriate amount of each extract was weighed. 2.5 ml of 0.125N hydrochloric acid and 1 μl of 37% formalin were added and stirred at 70 ° C. It filtered using filter paper (Toyo filter paper No. 1). The residue was further washed twice with 0.2 ml of water at 70 ° C. The filtrate and the washing solution were combined. After stirring at room temperature for 3 hours, the mixture was allowed to stand still for the second time. The precipitated crude insoluble laminaran was collected by centrifugation (3000 rpm, 5 minutes). After drying, each was washed with 0.2 ml of ethanol and diethyl ether, dried under reduced pressure and weighed.
[0037]
3) Soluble laminaran
Crude soluble laminaran was used by hydrochloric acid sizing method. An appropriate amount of each extract was weighed. 2.5 ml of 0.09N hydrochloric acid was added, and the mixture was stirred at room temperature for 2 hours. A packet in which the supernatant was transferred to another container after centrifugation (3000 rpm, 5 minutes). The precipitate was further washed twice with 0.5 ml of 0.05N hydrochloric acid. The supernatant and the washing solution were combined. Ethanol was added to 85% to form a precipitate. The precipitate was collected by centrifugation (3000 rpm, 5 minutes), washed with 0.5 ml each of ethanol and diethyl ether, dried under reduced pressure and weighed.
[0038]
4) Fucoidan
Fucoidan used a hydrochloric acid extraction method. An appropriate amount of each extract was weighed. 1.3 ml of 0.17N hydrochloric acid was added, and the mixture was extracted at 65 to 70 ° C. for 1 hour. The extract was neutralized with sodium hydroxide and concentrated under reduced pressure. The residue was dissolved in 0.5 ml of water. 1 ml of ethanol was added, and crude fucoidan was precipitated by centrifugation. After drying and dissolving in 2.5 ml of water, 1 ml of 4M calcium chloride was further added, and the precipitate of calcium alginate was removed by centrifugation. To this supernatant, 3 ml of a 5% cetylpyridinium chloride (CPC) aqueous solution was added. Subsequently, water was added so that the final concentration of calcium chloride became 0.5 M, and the mixture was allowed to stand overnight. The CPC complex was precipitated by centrifugation (3000 rpm, 5 minutes), dissolved in 1.5 ml of 3M calcium chloride, and 7 ml of ethanol was added. The resulting precipitate was collected by centrifugation (3000 rpm, 5 minutes), dried under reduced pressure, and weighed.
[0039]
Experimental result
Extraction and fractionation of polysaccharides derived from seaweed
The amount of the powder of the high molecular weight polysaccharide obtained at the laboratory level from 100 g of the dry raw material was 1 to 8 g. FIG. 2 shows the results. The amount of extract extracted by alkali extraction is not much larger than that of water extraction, but this is because the loss due to filtration is increased, and it is considered that the actual amount of extracted alkali extract is even larger. Alkali extraction results in a viscous solution. In the acid extraction, the amount of extract extraction was larger than that of alkali extraction or water extraction, but the yield as a high molecular weight polysaccharide powder was small. It is considered that the reason for this is that many molecules are converted into low molecules by hydrolysis.
[0040]
Scientific evaluation of immunostimulatory effects, antitumor effects, etc.
In vitro screening (WST-1 assay)
1) Influence of extracts of linen and Ainu-Wakame on the growth of A549
Cell viability decreased at higher concentrations in all samples. The control fucoidan, krestin, strongly inhibited growth. Fucoidan had a 30% viability at 0.5 mg / ml and krestin had less than 10% at 1 mg / ml. Also, alginic acid contained in brown algae was about 50% at 5 mg / ml, but in seaweed extract, linen showed 50% or less at 5 mg / ml for both acid and alkali extracts. (Fig. 3)
[0041]
Influence of the extract of Sujime and Ainu-Wakame on the growth of MKN28
Fucoidan and krestin had a survival rate of 40% at 1 mg / ml, but alginic acid had little effect even at higher concentrations. The viability of all the linen extracts decreased with increasing concentration, and was less than 50% at 5 mg / ml. Ainu-Wakame had a survival rate of 50% or less at an acid / alkali extract of 5 mg / ml. (FIG. 4)
[0042]
Influence of the extract of the linen, Ainu-Wakame on the growth of A-431
Krestishi had a survival rate of 50% at 1 mg / ml, but an alkali extract of linen showed a survival rate of 50% or less at 0.5 mg / ml, and showed the highest growth inhibitory effect among seaweed extracts. Was. (FIG. 5)
[0043]
In vivo anti-tumor and immunostimulatory effects (oral administration to mice)
On the day after the end of each test substance administration, tumors were excised from all animals and the wet weight was measured. The results are shown in FIG. The weight of the removed tumor in the control group was 3,224 mg. The tumor weights of the krestin 100 and 300 mg / kg / day administration groups were 2479 mg and 1717 mg, respectively, and significant growth inhibition was observed in all cases as compared with the control group. The tumor growth inhibition rates were calculated to be 23.1 and 46.7%, respectively.
Tumor weights of the group treated with the alkaline extract 30, 100 and 300 mg / kg / day for 21 days were 3043, 2684 and 2178 mg, respectively, and were significantly increased by administration of 100 mg / kg / day or more compared to the control group. Suppression was observed. The tumor growth inhibition rates were calculated to be 5.6, 16.8 and 32.5%, respectively.
The tumor weight of the group treated with 300 mg / kg / day of the bamboo shoot alkali extract for 14 days was 2846 mg, and although a significant suppression was observed as compared with the control group, the tumor growth suppression rate was as low as 11.8%. The weight of the tumors in the administration groups of the linen water extract 100 and 300 mg / kg / day were 2729 and 2298 mg, respectively, and significant suppression was observed in both cases as compared with the control group. Tumor suppression rates were calculated to be 15.4 and 28.7%, respectively.
[0044]
In vitro immunostimulatory action (MLR)
The linen water extract tended to promote growth at low concentrations, but culture at high concentrations (> 200 μg / ml) was not possible. No effect was observed with the bark alkali extract. The slimeic acid extract showed a growth-promoting tendency (10-500 μg / ml) in a concentration-dependent manner. The Ainu-Wakame water extract showed a tendency to promote growth, but showed large variations. No effect was observed in the Ainu-Wakame alkaline extract. The Ainu-wakame acid extract showed a growth promoting tendency at a low concentration (10 to 100 μg / ml) (FIG. 7).
[0045]
Elucidation of physicochemical properties of high molecular weight polysaccharide as active ingredient
Analysis of constituent sugars by acid hydrolysis of each extract
TLC analysis (FIG. 8) of each extract acid hydrolysis was performed. Algae acid was determined to be the main component, since the slime alkali extract was consistent with the alginic acid hydrolyzate. Only one spot was detected in the line extract, but identification was not completed. In the Ainu-Wakame water extract, unidentified spots were detected in addition to spots corresponding to the alginic acid hydrolyzate. Since a spot corresponding to D-glucose is detected in the Nagakumbu water extract in addition to the alginic acid hydrolyzate, it is expected that glucans such as laminaran are contained in addition to alginic acid.
[0046]
Determination of uronic acid content of each extract
The amount of uronic acid was determined by the amount of D-glucuronic acid, and was shown as a percentage by weight in the extract (Table 2). The ratio of uronic acid was higher in the striped alkaline extract and the Ainu-wakame water extract. These extracts are expected to be high in alginic acid.
[0047]
Table 2 Content of uronic acid in each extract
[0048]
[Table 2]

[0049]
Separation of polysaccharide of each extract
The sum of the contents of polysaccharides of the slime alkali extract was about 73%, whereas the sum of the contents of polysaccharides of other extracts greatly exceeded 100% (Table 7). The possible causes are as follows: alginic acid is a Na salt, soluble laminaran was determined in a crude yield, and all of the fractions obtained in this fraction are regarded as the polysaccharide. Can be read.
Algaeic acid was the main component of the slime alkali extract. Laminaran and fucoidan were relatively abundant in the linen water extract. Alginic acid was the main component in the Ainu-Wakame water extract. The extract of Nagakombu was rich in laminaran.
[0050]
Table 3 Polysaccharide content in each extract (%)
[0051]
[Table 3]

[0052]
Consideration
In the extraction and fractionation of the seaweed-derived high molecular polysaccharide, the powder of the high molecular polysaccharide obtained at the laboratory level from 100 g of dried material was used as a raw material, which was used as an unused resource, such as line fish and Ainu-Wakame. .
[0053]
Although the amount of extract extraction seems to be larger in the alkaline extraction than in the water extraction, the solution becomes a viscous solution.
[0054]
Although the amount of extract extracted by acid extraction is larger than that of alkali extraction or water extraction, the yield as a high molecular weight polysaccharide powder is reduced. It is considered that the reason for this is that the high molecular weight polysaccharide often changes to a low molecular weight by hydrolysis.
[0055]
This time, the survival rate against human-derived cancer cells was determined using the WST-1 assay for each concentration of the seaweed extract (polymer polysaccharide powder). Krestin, sodium alginate and fucoidan were used as controls for comparison.
[0056]
The lower concentrations were effective for the control samples fucoidan and krestin. Although the survival rate of the seaweed extract sample was low (high cell growth inhibition) in a concentration-dependent manner, the seaweed extract sample which showed an effect at a low concentration was A-431 (an epidermoid carcinoma) of an alkali extract of linen. Cell line).
[0057]
Test results
The test results are shown in FIG. 6-2. Although no effect was observed at a dose of 30 mg / kg, a significant amount of tumor growth inhibitory effect of 17% at a dose of 100 mg / kg and 32% at a dose of 300 mg / kg was obtained with the alkaline extract of pomacedium (p. <0.01). The same effect as that of the alkaline extract of stripes was obtained with the aqueous extract of stripes.
[0058]
The group administered after transplantation (administration for a total of 14 days) was less effective than the group administered 7 days before transplantation (administration for a total of 21 days). This is thought to be due to the fact that the oral administration of the striped alkaline extract enhances the nonspecific immunostimulatory activity, but it takes several days for the effect to be exerted.
[0059]
In the case of krestin, a tumor growth inhibitory effect of 47% was obtained at a dose of 300 mg / kg. Since the dosage of the antineoplastic agent krestin is 3 g / day, if a 60 kg human takes 3 g / day, the dose will be about 50 mg / kg / day in terms of mouse dose. By analogy only with the tumor growth inhibitory rate of the tumor cell Adenocarcinoma 755 tested this time, it is expected that this will be substantially equivalent to the administration amount of 100 mg / kg of the slime alkali extract. In other words, taking 6 g of the alkaline slime extract daily is comparable to taking 3 g of the antineoplastic agent krestin daily.
[0060]
In the immunostimulatory effect in vitro, responder T cells showed a clear growth-promoting tendency (10 to 500 μg / ml) in a concentration-dependent manner by the stream acid extract. The Ainu-wakame acid extract also showed a tendency to promote growth at a low concentration (10 to 100 μg / ml).
[0061]
In particular, it is suggested that the smime acid extract contains a substance that stimulates antigen-presenting cells such as dendritic cells and promotes acellular proliferation. It can be expected that the slime acid extract has an antitumor effect more than the slime alkali extract from which a significant tumor growth inhibitory effect of 32% was obtained.
[0062]
In vivo anti-tumor and immunostimulatory activities of extracts (acid, alkali, water extracts) of linen (oral administration to mice)
[0063]
Animal used
5-week-old Slc: BDF ₁ Male mice were purchased from Japan SLC Co., Ltd., and after preliminary breeding including a quarantine period of one week, animals in which no abnormality was observed in the general condition were selected and used for the test. The body weight at the start of the experiment was 25.0-29.8 g. The animals were identified by ear punch and the animal number on the tail with a felt tip pen. The housed gauge was provided with a card indicating the treatment group and animal number during the experiment.
[0064]
Rearing conditions
Throughout the pre-breeding and experimental period, the stainless steel movable rack (22 ± 3 ° C., humidity 50 ± 20%, lighting 12 hours (8:00 to 20:00), ventilation frequency 13 to 17 times / hour, Groups of 5 to 6 were housed in polysulfone cages (265 W x 427 D x 204 Hmm) mounted on 1790 W x 470 D x 1650 Hmm). As a feed, a solid feed and a laboratory MR stock (Nippon Nosan Kogyo Co., Ltd.) were freely given by a stainless steel solid feed feeder, and tap water was freely given by a polysulfone waterer (a stainless steel pipe).
[0065]
Test method
6 week old BDF ₁ Mice were used based on the group composition in Table 4. On day 7 of administration, tumor cells from day 10 to 14 maintained in passage in C57BL / 6 mice were subcutaneously injected into the test mice at 1 × 10 5 subcutaneously on the ventral side. ⁵ Cells were transplanted in an amount of 0.05 ml / mouse. The test substance was orally administered once a day. On the day after the last day of administration of the test substance, blood was collected from the abdominal aorta of the mouse under ether anesthesia, and then the tumor was excised and weighed. Body weight measurements were taken twice a week. The obtained blood was fractionated from plasma and stored at -80 ° C.
[0066]
[Table 4]

[0067]
Statistical processing
The average value and standard error of the numerical values obtained in each group were calculated. Statistical analysis between each group was performed by testing for equal variance by the Bartlett method (significance level 5%). In the case of equal variance, one-way analysis of variance was performed, and when significant, comparison of mean values by the Turkey method was performed. Was done. In the case of unequal variance, a Kruskal-Wallis H test was performed, and in the case of significant variance, the average rank was compared by the Turkey method. The significance levels were 5% and 1%.
[0068]
The average excised tumor weight was evaluated based on the ratio of the test substance administration group to the control group (T / C) by the US National Cancer Institute (NCI) evaluation criteria (Gtran, RI et al. Cancer Chem. Rep. According to Part 3, vol3 (1972)), the tumor suppression rate (%) was calculated as follows, and the antitumor effect was determined.
T / C (%) = (average excised tumor weight (mg) of test substance administration group / average exclusion of control group)
Tumor weight (mg) x 100
Tumor suppression rate (%) = 100-T / C ≧ 30% was regarded as having an antitumor effect.
[0069]
Test results
The test results are shown in FIG. 6-2.
1. Slime acid extract
The weight of the removed tumor in the control group was 2390 mg.
In the 21-day administration group of 30, 100, and 300 mg / kg / day of the dimeic acid extract, the tumor weights were 608, 498, and 392 mg, respectively, and were significantly higher than the control group at the administration of 100 mg / kg / day or more. Suppression was observed. In the 14-day administration group of 300 mg / kg / day, the tumor weight was 834 mg / kg / day, indicating a suppression tendency as compared with the control group. The tumor suppression rates were 74.6, 79.2 and 83.6% in the 21-day administration group at 30, 100 and 300 mg / kg / day, and 65.1 in the 14-day administration group at 300 mg / kg / day, respectively. %, And all were determined to have an antitumor effect.
[0070]
2. Reed alkaline extract
In the 21-day administration group of 300 mg / kg / day of the bamboo alkali extract, the tumor weight was 569 mg, which was significantly suppressed as compared with the control group. The tumor suppression rate was calculated to be 76.2%, and it was determined that there was an antitumor effect.
[0071]
3. Striped water extract
In the group administered with 300 mg / kg / day of the water line extract for 21 days, the tumor weight was 418 mg, which was significantly suppressed as compared with the control group. The tumor suppression rate was calculated to be 82.5%, and it was determined that there was an antitumor effect.
[0072]
4. Line dry powder
In the 21-day administration group of 300 mg / kg / day of the dry powder of the stripe, the tumor weight was 685 mg, which was significantly suppressed as compared with the control group. The tumor suppression rate was calculated to be 71.4%, and it was determined that there was an antitumor effect.
[0073]
Analysis of structure of high molecular polysaccharide
Brown algae include high molecular polysaccharides such as alginic acid, fucoidan and fucoidan. It is generally said that alginic acid is extracted with a thin alkali and fucoidan is extracted with a thin hydrochloric acid. Therefore, the structure of the high molecular polysaccharide was analyzed by TLC analysis, uronic acid content, and fractionation of the polysaccharide.
[0074]
Alganic acid was the main component in the slimefish alkaline extract, and laminaran and fucoidan were relatively high in the slimewater extract. Alginic acid is considered to be the main component in the Ainu-Wakame water extract, and alginic acid is considered to be the main component in the Ainu-Wakame alkaline extract.
[0075]
The sample in which responder T cells showed a clear tendency to promote proliferation in a concentration-dependent manner due to in vitro immunostimulatory action was not a slime alkaline extract but a slime acid extract. Therefore, it is assumed that substances that stimulate antigen-presenting cells such as dendritic cells and promote T cell proliferation are related to laminaran or fucoidan.
[0076]
This extract is safe and inexpensive, and can be used as a raw material for functional foods utilizing its immunostimulatory and tumor-suppressing effects.
[Brief description of the drawings]
FIG. 1 is a flow chart for producing a seaweed-derived polymer polysaccharide.
FIG. 2 shows the results of extraction and analysis of a high molecular weight polysaccharide derived from seaweed.
FIG. 3 shows the survival rate of A549 when various extracts were added.
FIG. 4 shows the survival rate of MKN28 when various extracts were added.
FIG. 5 shows the survival rate of A431 when various extracts were added.
FIG. 6 shows the antitumor effect in vivo.
FIG. 7 shows in vivo antitumor effects.
FIG. 8 shows expansion of responder T cells.
FIG. 9 is an analytical TLC of an acid hydrolyzate of seaweed extract.

Claims (4)

A food or beverage comprising an extract of linen. The food or beverage according to claim 1, wherein the extract of linen is an acid extract. The food or beverage according to claim 1, wherein the extract of linen is a water extract. 2. The food or beverage according to claim 1, wherein the extract of linen is an alkaline extract.

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