[Separation and identifying features of the cardiac aglycones and glycosides of Nerium oleander L. flowers by thin-layer chromatography].

The present work was aimed at extracting some cardiotonic glycosides from Nerium oleander L. (N.o.L.) flowers, free from phytosterols and their esters and at resolving themselves by thick- and thin-layer chromatography. This work may contribute to the scientific recognition of N.o.L. drugs in the field of forensic medicine. MATERIALS AND METHODS. Apparatus. Kieselgel F254 thin-layer (250 mu) and Kieselgel thick-layer (2 mm) glass plates were used. MATERIALS. Chromatographic solvents were used; detectors were p-toluene sulphonic acid (PTSA) and antimony trichloride (SbCl3). STANDARDS. The following standards were used: gitoxigenin, oleandrin, digitoxin, and stigmasterol. Standard solutions were prepared at a M-3 concentration. The solvent system is described in Table I. EXTRACT. The extract was prepared using a mixture of solvents (hexane-diethylether-acetic acis, 50:50:1 v/v). With this mixture we noted that, in thin-layer chromatography, the used standards remained at the origin, while phytosterols and their esters migrated. White flowers of N.o.L. were air dried in the shade at room temperature, powered, and macerated in hexane-diethylether acetic acid for 24 hours. After removal of the phytosterols the remacerating powder was further percolated using a chloroform-methanol mixture (1:1.7 v/v) in order to extract the aglycone and the cardiac glycosides. As the medium's thin-layer chromatogram showed the absence of corresponding spots to test samples, the chloroform-methanol media were combined and evaporated under vacuum. The residue was redissolved in the chloroform-methanol mixture. The obtained extract concentration consisted of 5 grammes of dried flowers in 1 milliliter of solution, called extract E (Fig. 1). RESULTS. The results are reported in Figures 2 and 4 and in Table II (see text for explanation of symbols). Extract E (Fig. 2) and the F1, F2, and F3 concentrated fractions were examined by thin-layer chromatography (Fig. 1 and 3). Only the F3 fraction revealed compounds with the same chromatographic path and colours (detected by PTSA and SbCL3) as the reference standards gitoxigenin, oleandrin, and digitoxin. This finding was confirmed by using five different solvent-systems (Fig. 4). DISCUSSION AND CONCLUSION. The present study shows that the cardiac steroids and other constituents of Nerium oleander's flowers are separated in three fractions by thick-layer chromatography free from phytosterols and their esters. This result may have useful implications in the fields of analytical toxicology and forensic medicine and potential application in clinical practice given that cytotoxic and antileukemic activities of the extracts from plants containing cardiac glucosides have been reported.