FURTHER STUDIES ON THE STRUCTURE OF HOLOTHURIN *

Some species of sea cucumbers (class Holothuroidea) contain a poisonous principle. I t has been suggested that it protects these animals against predators. Sea cucumbers of various species are eaten in some parts of the world, but occasional poisoning by this food is presumably not related to the toxin under discussion, which is ineffective by oral ingestion. A peculiar gland found in these species and named after Georges L. C. F. D. Cuvier is particularly rich in poison, which is also found in the brownish slimy integument. We have studied the chemical composition of the toxic substance from Cuvier’s gland of Actinopyga agassizi, a holothuria found in the Bahama Islands. In two preliminary notes’ we have reported that the toxic material holothurin, obtained from the water extract of Cuvier’s gland, appears to consist of a few steroid aglycones, bound individually to four molecules of monosaccharides. Analysis of purified holothurin, designated holothurin A, and of its hydrolytic products, is in accord with this view? This would place holothurin A into a class of cardiac glycosides or steroid saponins such as have hitherto been found only in plants, especially monocotyledons. It shares with the plant drugs its saponinlike character and neurotoxic and hemolytic properties, which are described elsewhere3 and by Friess et al. in this monograph. In addition, holothurin contains one molecule of sulfuric acid, bound in ester linkage, which suggests a relationship with steroid alcohols, such as scymnol and ranol, in the bile of the most primitive vertebrates. The infrared spectrum shows bands a t 5.72 and 6.14 p, indicative of a fiveor six-membered ring lactone and one double bond. The absence of a positive Legal’s test in conjunction with the ultraviolet spectrum excludes the presence of an a $-unsaturated lactone. Hydrogenation of the double bond fails under a variety of conditions. Elementary analysis of holothurin A leads to the formula C50-5~H8~-8&sSNa. Methoxyl determination indicates the presence of one such group in holothurin A. Acid hydrolysis of the neutral nonreducing holothurin A yields water-insoluble aglycones, sulfuric acid, and watersoluble reducing sugars. At least four individual steroid aglycones are obtainable from the hydrolysis of holothurin A. The elementary analysis and molecular weight determination of these aglycones show between 26 and 28 * The work described in this paper was supported in part by Contracts NONR-2266 and NR 107-543 from the Office of Naval Research, Department of the Navy, and by Research Grant C-5097 from the National Science Foundation, Washington, D.C.