Toxic compounds in cycads.

Abstract

The present-day cycads comprise the diverse, modified, remnants of a much larger group of gymnosperms which flourished in the Mezozoic era. The systematic position that the cycads occupy in the botanical hierarchy is significant in that they represent relatively unchanged survivors from prehistory. The present global complement of 182 species occur in tropical and mild temperate regions in both hemispheres. Despite the density of proliferation of species, about one-half of the extant taxa are considered endangered, vulnerable or rare. Apart from characteristic features such as differences in growth forms, variation in reproductive structures and anatomical details, cycads are distinguished from all other plant groups by the unique phytotoxins, azoxyglycosides, which they possess. The toxicity of cycads is well-documented in cases which refer to both man and animals. Cycasin, which together with macrozamin represent the major azoxyglycosides occurring in cycads, has been reported to elicit responses similar to those that have been observed during carcinogenicity, mutagenicity and neurotoxicity assays. It has become apparent that the mechanism by which azoxyglycosides manifest their toxicity involves deglucosylation, by enzyme systems, which releases the aglycone, methylazoxymethanol (MAM), and sugar moieties. Metabolic activation of MAM succeeds deglucosylation to generate methylene carbene units (: CH2) which are capable of methylating macromolecules including DNA, RNA and protein. During this investigation, macrozamin was extracted from seed kernels of Encephalartos transvenosus and cycasin was tentatively identified in seed kernels of Cycas thouarsii . The hexa-acetate derivative of macrozamin was prepared whereas the tetra-acetate derivative of cycasin was not secured in a pure form. The spectroscopic techniques employed for identification include DV-absorption, infra-red and nuclear magnetic resonance spectroscopy, all of which are useful for detecting signals which arise as a result of the azoxy function. A kinetic study was carried out to determine the rate of hydrolysis of macrozamin with 4M sulphuric acid, and to allow calculation of the activation energy for the process. A comparison of the kinetic parameters determined for the above process with those derived for the hydrolysis of cycasin and methylazoxymethanol under similar conditions followed. The rates of hydrolysis increase in the order macrozamin, cycasin, methylazoxymethanol since the molecules contain two, one and zero glycosidic linkages respectively. Additional glycosidic bonds are observed to decrease the rate of reaction. Consequently, activation energies for hydrolysis of the above molecules decrease in the above-stated order. Macrozamin and cycasin were quantified in cycad material by high performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) and by the chromotropic acid assay. The results of the quantitative analysis has highlighted certain limitations of the methods of detection, and has been found to be applicable to a taxonomic evaluation and a proposal for the biosynthesis of the azoxyglycosides.