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“希姆什”含有对小鼠有强烈麻痹作用的毒素。标本被鉴定为原头藻属(原头藻)的一个未描述的种。最大毒性(河豚毒素，TTX)为14734MU/g全身。用80%甲醇、冰醋酸酸化、二氯甲烷脱脂的方法，从虫体中提取出一种毒素(HMT)。水层用活性炭层析，结合组分和游离组分用高效液相色谱和气相色谱-质谱仪进行分析。从这些结果中相当出人意料地揭示，HMT由TTX、4-表TTX、无水TTX和不明毒素组成。HMT的主要毒性成分是通过活性…处理的方法从混合样品中分离出来的更多的木炭，Bio-Gel P-2和Bio-Rex 70(H^+形式)的层析，最后从酸化的甲醇溶液中结晶。重结晶毒素的特异性毒性为3520MU/mg。经电喷雾电离-质谱仪(ESI-MS)测定，该毒素在m/z 320和302处分别出现(M+H)^+和(M+H-H2O)^+离子峰。由此测定的TTX的相对分子质量为319，与报道的TTX分子量一致。在红外光谱中观察到3353、3235、1666、1612和1076 cm~(-1)的吸收带。重结晶毒素的~1H-核磁共振谱与河豚毒素的相同。河豚毒素的特征是以2.33(J=10.0赫兹)和5.48ppm(J=10.0赫兹)为中心的一对双峰通过双重辐照耦合在一起。通过GC-MS分析，这种毒素的碱解产物与TTX一样，显示了喹唑啉骨架(C9碱基)。这一发现首次直接证明了广岛县广岛湾漂浮牡蛎养殖筏上牡蛎壳表面发现的带状蠕虫的存在。较少

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