Biological Activities and Structural Analysis of Brown Coloring Matterin Foods

ヤーコンの塊根部は流通や貯蔵の過程で褐変化することによって商品価値が低下することがしばしば問題となる。これはヤーコンに含まれているポリフェノール化合物が酵素的反応によって重合体となり,褐変現象を引き起こすためである。しかしながら,この重合体はVesiculogenと呼ばれ,食品の色,香りと強く関係するのみならず,免疫賦活効果と関連することが指摘されるが,生理活性に関する研究報告は少なく,その生成機序などにも不明な点が多い。また,Vesiculogenはその生成過程で様々な要因(食品中の成分,温度,反応時間など)の影響を受けるため,食品中で既に生成したVesiculogenの構造解析や機能性の直接的な検討は困難であるため,本研究ではヤーコンに含有されるものと同等の市販のポリフェノール標準試薬を用いてVesiculogenの合成を試みるとともに,マウスリンパ細胞を用いたマイトジェン活性試験によりVesiculogenの生理活性を検討した結果,以下のとおりであった。1)ヤーコンに含まれる各種成分の分析ヤーコンを塩酸濃度が2Nになるように加えて調製された50%メタノールで抽出し,抽出物をさらに酢酸エチルで分配抽出した。抽出物はHPLC(ODS-C18カラム10.Omm×250mm,溶離液0.05%TFA/メタノールを用いた)で分離し,それぞれの分離ピークをFT-IR,LC-MS,NMR等の分析機器を用いて構造解析を行った。その結果,ヤーコン抽出液中から,フマル酸,クロロゲン酸及び3種類のジエン系化合物が検出された。フマル酸の定量値は可食部100gあたり537.2mgであった。また,ヤーコンにフマル酸が含有されることは未報告であり,ヤーコンには新たに疲労回復の効果が期待されることがわかった。ジエン系化合物については,いずれも文献等に未記載であり,新規化合物の可能性が強く示唆された。2)褐色色素群の合成と生理活性ヤーコンに含有される主なポリフェノールはクロロゲン酸のみであったことから,市販のクロロゲン酸標準品を用い,同じく西洋ワサビ由来の市販の酵素(HRP)並びに少量のH_2O_2を添加して室温で反応させ,Vesiculogenを合成した結果,クロロゲン酸50mg,HRP20mgおよびH_2O_2(市販品100倍希釈液)5mlを添加した場合に最も褐変濃度の高いVesiculogenを得た。次いで,生理活性の一つして免疫賦活効果を検討するために,マウスリンパ細胞を用いたマイトジェン活性試験を行い,リンパ球幼若化能を検討した。その結果,分子量が50kDa以上の高分子なVesiculogen画分にのみ顕著な活性が認められた。以上より,酵素的褐変色素はヤーコンをはじめとするポリフェノール含有食品において容易に生成されることが推測され,免疫賦活効果に深く関与することが示唆された。The presence of the poly-phenol compounds and some enzymes are concerned in the cause of brown coloring resulted from the polymerization in foods. These polymers of characteristic brown color in foods are designated as Vesiculogen, and are expected to have a important role of increasing immunological activities for our health. However, very little is known about the productive mechanisms and the biological activities of Vesiculogen. It is often caused some troubles that the value of articles was spoiled by enzymes on a process of circulation and storage in commodities of Yacon. Therefore, the studies on Vesiculogen were performed on following under the procedures using Yacon as the materials for these experiments. 1) Yacon was extracted with 50% methanol including HCl adjusted to 2N of a concentration. Small quantities of components in extract were separated by HPLC using ODS-C18 column (φ1.0cm×25.0cm) and methanol (prepared on acidity with addition of TFA) as a eluting solvent. Several peaks of compounds were detected and collected respectively. Structural analysis of each compound was performed using some instruments such as FT-IR, LC-MS and NMR. 1) Two peaks (A and C) of compounds were identified as Fumaric acid (using NMR) and Chlorogenic acid (using LC/MS), and other three were found to be the diene compounds using NMR. Still the chemical structure of the diene compounds could not be completely identified in these experiments, but it was suggested that the diene compounds had the possibility of new chemicals. The content of Fumaric acid was 537.2mg per 100g of Yacon from the result of a quantitative analysis. Because it is well-known that some organic acids such as Fumaric acid show the effect of a fatigue recovery, it is equally expected that the effect of a fatigue recovery was occurred with the ingestion of Yacon. On the other side, the presence of Chlorogenic acid as a kind of polyphenols in Yacon was reconfirmed certainly from these analysis. 2) The composition of Vesiculogen using the chlorogenic acid of a commercialized reagent was performed on following the procedure. When the enzymes (HRP) of several quantities were added to the mix of Chlorogenic acid prepared in several quantities and hydrogen peroxidase, the several Vesiculogen colored brown were constituted. In such a case that 50mg of chlorogenic acid and 5m1 of hydrogen peroxidase were mixed with 20mg of HRP, the brown coloring of Vesiculogen turned to be deepest. The Vesiculogen was dialyzed with distilled water using spectro pour tube (pore size of MW. 50, 000) and was separated two fractions of above 50kDa (V-N fraction) and less than 50KDa (V-D fraction) of molecular weight. The mitogenic assay for each fraction was examined using spleen cells of male mice of C3H/HeN. The mitogenic activity was found remarkably in a V-N fraction, but not in a V-D fraction. It was suggested that the active substance of Vesiculogen was dependent on the fraction of high molecular above 50kDa. However it is a big problem to decrease in those article value by brown coloring occurred among the processes of circulation and storage, the elucidation of general mechanisms for production of Vesiculogen is expected in future by our studies connected with the relationship between the activities.P(論文)特集application/pdfFEATURE ARTICLESdepartmental bulletin pape