In this study, the identification and purification of antioxidant compound from cherry leaf was proposed by a novel strategy of high-speed countercurrent chromatographic (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in cherry leaf extract are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of one targeted peak would be investigated for the two-phase solvent system (tert.-butyl methyl ether/acetonitrile/0.1% TFA in water, 2/2/3, V/V/V), and purified by HSCCC. The purification of this antioxidant (3.0mg) form cherry leaf extract (30mg) was performed by HSCCC with optimal two-phase solvent system. Using mass spectrometric and nuclear magnetic resonance analysis, this antioxidant was identified to 3-O-caffeoylquinic acid. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.

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Glutathione (reduced glutathione: GSH) is thiol antioxidant which is synthesized in animal cells. GSH plays an important role in the antioxidant mechanisms. In this study, we examined acute effects of intake of sucrose or honey on glutathione status in several organs with a mouse model. ICR female mice were divided into three groups. Control group took only tap water ad libitim for 16 hours. Sucrose group took 12% sucrose solution and honey group took 12% honey solution. In honey group, amount of GSH in liver and kidney increased as compared with control group. Sucrose group tended to increase with liver and kidney GSH. In addition, GSH/GSSG ratio in liver and kidney were high levels in these groups. In sucrose group and honey group, plasma GSH level and GSH/GSSG ratio increased. On the other hand, in erythrocyte, amount of GSH was reduced and GSSG was increased by intake of sucrose or honey. Therefore, intakes of sugars may be effective as temporary defense against oxidative stress because intakes of sugars contributed to increase GSH level and/or GSH/GSSG ratio in a liver playing a key role of the GSH synthesis.

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Ascophyllan and fucoidan, fucose-containing sulfated polysaccharides isolated from brown seaweed Ascophyllum nodosum, have various biological activities in vitro. Our previous studies have demonstrated that these polysaccharides induce tumor necrosis factor-α (TNF-α) and granulocyte-colony stimulating factor (G-CSF) secretion from murine macrophage cell line, RAW264.7 cells in a concentration-dependent manner. In this study, we investigated in vivo effects of ascophyllan and fucoidan in mouse model in addition to in vitro studies. In Sarcoma180 ascites tumor-bearing mouse model, the increases in the survival rate of tumor-bearing mouse treated with intraperitoneal (i.p.) administration of ascophyllan and fucoidan were observed as compared to the control mouse injected with saline alone. Since ascophyllan and fucoidan showed no significant cytotoxicity on Sarcoma 180 cells in vitro, these polysaccharides may suppress tumor growth through the activation of host immune system rather than a direct cytotoxic effect on tumor cells. In fact, i.p. injection of ascophyllan and fucoidan into mouse resulted in significant increase in the serum levels of TNF-α and G-CSF.

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A simultaneous method by GC-MS was developed for determination of pesticide residues in livestock and seafood products. A large-volume injection technique that employs a spiral inlet liner inside of a programmed temperature vaporizer was used. This technique could extremely reduce the consumption of organic solvent and time for sample preparation. Pesticides in samples were extracted with ethyl acetate, and extracted solutions were defatted by liquid-liquid separation using hexane and acetonitrile with saturated hexane, followed cleanup by PSA and Silica cartridge for the samples except for chicken liver samples. A cleanup with PSA, Silica and SAX cartridges was required for chicken liver samples. An addition of polyethylene glycol into sample solutions and standard solutions could reduce the effect of matrices from the samples on the GC-MS response of studied almost pesticides, and improve the accuracy and precision of the analysis. The quantitation limits of pesticides were 0.005μg/g, and were satisfied for the uniform residue limit (0.01ppm) in Japan. The results of validation study showed that one hundred pesticides among the examined 110 pesticides could be applied on the routine monitoring tests.

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The comprehensive IgE-binding capacities of proteins (allergenome) in buckwheat seeds were examined using immunoproteomic techniques. Salt-soluble proteins were extracted from buckwheat seeds, separated using one- and two-dimensional electrophoresis, and analyzed using western blotting with buckwheat-allergic patients' sera. A one-dimensional immunoblot analysis with the buckwheat-allergic patients' sera revealed some IgE-binding proteins, and multiple spots containing known and novel IgE-binding proteins were detected using two-dinensional immunoblotting. Some spots were newly identified as 13S globulin protein subunits or isoforms. Some spots that were homologous to vicilin-like proteins indicated the presence of newly identified vicilin-like proteins in buckwheat. These results obtained from an immunoproteomic analysis may contribute not only to the construction of a comprehensive IgE-binding protein map of buckwheat, but also the detection of isoforms of IgE-binding proteins in buckwheat variants.

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黄変米であるシクロクロロチン(CYC)の定量分析を行うため、精製用固相抽出(SPE)カートリッジおよびLC-MS/MSを用いた簡便かつ高感度な分析法を開発した。CYCをメタノール:水(20:80)の混合液で米サンプルから抽出した後、Oasis HLBカートリッジに負荷し、メタノールで溶出した。LC-MS/MS分析は、ESIポジティブモードの多重反応モニタリング(MRM)を用いて行った。本分析法は、米中のCYCを高感度で測定するのに有用であり、その定量限界は1ng/gであった。本分析法により、日本国内で購入した米20サンプルを分析した。結果、いずれのサンプルからもCYCは検出されなかった。

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Aspergillus sp.(FARM AP-21280),A.oryzae(NBRS4214),A.awamori(NBRS4122)またEurotium sp.(FARM AP-21291)で発酵したCamellia sinensis Lの茶葉には、市販の茶製品(緑茶、ウーロン茶、紅茶およびプーアール茶)においてほとんど検出されないフェノール成分、teadenol Aおよびteadenol Bが高含量で含まれていた。特に、Aspergillusで発酵した茶葉ではより高含量(teadenol A:1.01-1.79%;teadenol B:0.16-0.37%、乾燥重量あたり)で認められた。HPLC-TOFMS分析は、さまざまな茶加工製品の二次代謝成分の同定に非常に有用であった。

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