Abstract

Glucosinolates (GLs), and particularly two classes of their degradation products - isothiocyanates (ITC) and indoles - released upon myrosinase catalyzed hydrolysis, belong to the most promising chemopreventive dietary components. Despite their wide use in plant research, studies on chemopreventive mechanisms and industrial utilization in the production of e.g. dietary supplements, the methods of reliable characterization of myrosinase-GLs system have suffered form the lack of readily available standards, as well as also reliable routine methods. The biological potential ascribed to ITC and indoles, in the case of actual Brassica plant samples, is still assessed based on GL content, though it is well known that these two types of derivatives are not the only ones that are formed following myrosinase action. This means that the relationship between content and composition of bioactive Brassica ingredients and biological effect cannot be properly assessed or predicted. The aim of our research was to optimize known or to develop novel methods that will ensure precise description of GLs-myrosinase system in any source, natural or processed, and will enable to relate the rate of formation of ITC/indoles to biological effects observed. The methods optimized or newly established include: determinations of GLs by HPLC-ESI-MS with published list of ions for most frequent GLs, myrosinase activity by pH-static and spectrophotometric methods, total ITC determination improved by adding SPE enrichment, indoles by HPLC with spectrophotometric (abundant compounds) and fluorescent (low-content compounds) detection, as well as novel derivatization of ITC to dithiocarbamates enabling their identification and sensitive quantitation by HPLC-DAD-MS. Also the DAD-HPLC-MS method of determination of free and bound flavonoids for Brassica plants have been optimized. The developed methods were applied to characterize an array of Brassica samples, the examples of which will be given. GL conversion rate to ITC and indoles was found to differ significantly not only between Brassica species but also between individual plant parts and was independent of myrosinase activity. The yield of conversion of individual GLs to ITC was also not identical. The biological activity in tests used turned out to be mostly related to indoles, some of which were particularly toxic. The developed set of methods should ensure better and safer exploitation of chemopreventive potential of Brassica plants.

Authors (5)