Studies on paralytic toxins in a ribbon worm adherent to cultured oysters in Hiroshima Bay, Hiroshima Prefecture, Japan

1998年，在日本广岛县广岛湾，人们发现附着在养殖牡蛎壳上的带状蠕虫“himomushi”样本含有毒素，对小鼠有很强的麻痹作用。这些标本被鉴定为原头刺属（原头刺属）的一个未描述种。全身最大毒性（如河豚毒素TTX）为14,734 MU/g。采用80%甲醇醋酸酸化，二氯甲烷脱脂的方法提取姬虫毒素（HMT）。采用活性炭对水层进行色谱分析，采用高效液相色谱和气相色谱-质谱联用技术对未结合和结合的毒性组分进行分析。出乎意料的是，这些结果揭示了HMT由TTX、4-epiTTX、无水TTX和未知毒素组成。通过活化活性炭处理，Bio-Gel P-2和Bio-Rex 70 （H^+形式）层析，酸化甲醇溶液结晶，从混合样品中分离出HMT的主要毒性成分。再结晶毒素的特异毒性为3520 MU/mg。电喷雾电离质谱（ESI-MS）分析表明，该毒素在M /z 320和302处分别出现（M+H）^+和（M+H- h_2o）^+离子峰。由此确定的TTX的分子量为319，与报道的分子量一致。该毒素的红外光谱在3353、3235、1666、1612和1076cm^<-1>处存在吸收带。该光谱与TTX无法区分。重结晶毒素的^1H-NMR谱与TTX的相同。以2.33 （J=10.0 Hz）和5.48ppm（J=10.0 Hz）为中心的双辐射偶联是河豚毒素的特征。通过气相色谱-质谱分析，该毒素的碱水解产物与TTX一样，显示出喹唑啉骨架（c_9碱）。这些发现是第一个直接证明TTX存在的证据，在广岛县广岛湾漂浮的牡蛎培养筏上的牡蛎壳表面发现了带状蠕虫。少

In 1998, in Hiroshima Bay, Hiroshima Prefecture, Japan, specimens of the ribbon worm, "himomushi" adherent to the shells of cultured oysters were found to contain toxins which showed strong paralytic action in mice. The specimens were identified as an undescribed species of the genus Procephalothrix (formerly Cephalothrix). The maximum toxicity (as tetrodotoxin, TTX) was 14,734 MU/g whole body. The "himomushi" toxin (HMT) was extracted from the worm with 80% methanol acidified with acetic acid and the extract defatted with dichloromethane. The aqueous layer was chromatographed on activated charcoal and the unbound and bound toxic fractions were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. It was rather unexpectedly revealed from these results that HMT was comprised of TTX, 4-epiTTX, anhydro TTX and unidentified toxins. The main toxic component of HMT was isolated from a pooled specimen by a method which consisted of treatment with activat … More ed charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H^+ form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3,520 MU/mg. This toxin showed (M+H)^+ and (M+H-H_2O)^+ ion peaks at m/z 320 and 302, respectively by electrospray ionization-mass spectrometry(ESI-MS). The molecular weight thus determined was 319 for TTX in accordance with its reported molecular weight. The absorption band at 3353, 3235, 1666, 1612 and 1076cm^<-1> were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX.The ^1H-NMR spectrum for the recrystallized toxin was the same as that for TTX.The pair of doublets centered at 2.33 (J=10.0 Hz) and 5.48ppm(J=10.0 Hz), which are characteristic of tetrodotoxin, were shown to be coupled by double irradiation. By GC-MS analysis, the alkali-hydrolyzate of this toxin, like TTX, showed the quinazoline skeleton (C_9-base).These findings are the first direct evidence of the existence of TTX in the ribbon worms found on the surface of the shells of oysters hanging onto floating oyster culture rafts in Hiroshima Bay, Hiroshima Prefecture. Less