Small molecule antioxidants in marine organisms from the Great Barrier Reef, Japan and USA

Specimens of the zoanthid, Palythoa tuberculosa and the ascidian, Lissoclinum patella were collected using SCUBA from 2-4 m depth at Davies Reef in June 1990. Specimens were stored at -20°C until extracted. Coral trout (Plectropomus leopardus) were caught by handline at the same reef and the ocular lenses excised and frozen. Freeze dried red alga samples were sourced from 2 locations: Porphyra tenera was a gift from the Yamamoto Nori Research Institute, Ota-ku, Tokyo, and Mastocarpus stellatus, collected from Schoodic Point, Maine, USA, was provided by Professor JM Shick.Methanolic aqueous extracts containing mycosporine-like amino acids (MAAs) were prepared from tissue samples from each species and the MAAs were separated and quantified by reverse-phase, isocratic high-performance liquid chromotography (HPLC).Phosphatidylcholine peroxidation inhibition assay (PC-assays) were conducted using 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and soybean phosphatidylcholine (PC) as radical inititor and lipid substrate, respectively.AAPH-initiated oxidation reactions of MAAs in sample tissue extracts were conducted without phosphatidylcholine as a competing substrate.Mycosporine-Gly was fractionated from the Palythoa extract and after processing was quantified by photometric analysis using published molar absorptivity data. The antioxidant activity of mycosporine-Gly was determined with the addition of appropriate quantities of sample to give 15 and 30 µM concentrations of mycosporine-Gly in the PC-assay. Ascorbic and uric acids were used for comparison of PC peroxidation inhibition rates.In these sample extracts, mycosporine-glycine was reactive to peroxyl radicals whereas iminomycosporine-like amino acids (shinorine, porphyra-334, palythine, asterina-330 and palythinol) were oxidatively robust. Purified mycosporine-glycine inhibited peroxyl radical-initiated autoxidation of phosphaditylcholine in a concentration-dependent manner. These results suggest that mycosporine-glycine may function as a biological antioxidant in marine organisms. This research was undertaken to examine the antioxidant activities of extracts from tissues of different marine species by their peroxyl radical-trapping ability using the phosphatidylcholine peroxidation inhibition assay (PC-Assay).

1990年6月，研究人员借助水肺潜水（SCUBA）于戴维斯礁（Davies Reef）2~4米水深处采集了群海葵（zoanthid）*Palythoa tuberculosa* 与被囊动物（ascidian）*Lissoclinum patella* 的样本，所有样本均保存于-20℃环境直至提取。研究人员于同一礁区采用手钓方式捕获了珊瑚鳟（*Plectropomus leopardus*），摘取其眼球晶状体并冷冻保存。冻干红藻样本来源于两处：坛紫菜（*Porphyra tenera*）为山本紫菜研究所（Yamamoto Nori Research Institute, Ota-ku, Tokyo）所赠；而从美国缅因州斯库迪克角（Schoodic Point, Maine, USA）采集的星状掌状藻（*Mastocarpus stellatus*）则由JM Shick教授提供。研究人员从各物种的组织样本中制备了含有类菌孢素氨基酸（mycosporine-like amino acids, MAAs）的甲醇水提取物，并通过反相等度高效液相色谱（HPLC）对MAAs进行分离与定量。以2,2'-偶氮二(2-脒基丙烷)二盐酸盐（AAPH）作为自由基引发剂、大豆磷脂酰胆碱（PC）作为脂质底物，开展了磷脂酰胆碱过氧化抑制实验（PC-assays）。在未添加磷脂酰胆碱作为竞争底物的条件下，开展了样本组织提取物中MAAs经AAPH引发的氧化反应。研究人员从瘤形棒海葵提取物中分离得到菌孢素甘氨酸（mycosporine-Gly），经处理后采用已发表的摩尔消光系数数据通过光度分析法完成定量。在PC实验体系中分别添加适量的菌孢素甘氨酸标准品，使其终浓度分别为15 μM与30 μM，以此测定其抗氧化活性；同时以抗坏血酸与尿酸作为对照，比较其磷脂酰胆碱过氧化抑制速率。在上述样本提取物中，菌孢素甘氨酸可与过氧自由基发生反应，而亚胺类类菌孢素氨基酸（iminomycosporine-like amino acids, shinorine、porphyra-334、palythine、asterina-330及palythinol）则具有较强的氧化稳定性。纯化后的菌孢素甘氨酸可呈浓度依赖性地抑制过氧自由基引发的磷脂酰胆碱自氧化反应。上述结果表明，菌孢素甘氨酸可能作为生物抗氧化剂在海洋生物中发挥生理功能。本研究旨在通过磷脂酰胆碱过氧化抑制实验（PC-Assay），基于过氧自由基捕获能力，探究不同海洋物种组织提取物的抗氧化活性。