Rotation Locular Countercurrent Chromatography and Quantitative ¹H-Nuclear Magnetic Resonance Spectroscopy of the Phorbol Ester Constituents of Croton Oil

Abstract

The toxic components of the seed oil of Croton tiglium L. (Euphorbiaceae), croton oil, are known to be cocarcinogenic phorbol-12, 13-diesters and cryptic cocarcinogenic phorbol-12, 13, 20-triesters. For a rapid isolation of mixtures of these phorbol esters from the oil rotation locular countercurrent chromatography (RLCC) was used. The eluted fractions were analysed by thin layer chromatography (TLC) and ¹H-nuclear magnetic resonance spectroscopy (¹H-NMR). The phorbol diesters were separated from the triesters although no baseline separation could be achieved, and the diesters as well as the triesters were completeley extracted from the oil. In a simple methanolic extraction procedure, on the contrary, the phorbol triesters were partially lost. A qualitative as well as a quantitative physico-chemical evaluation method was developed for the phytochemical screening of these cocarcinogenic and cryptic cocarcinogenic compounds in biological material using ¹H-NMR spectroscopy. It was possible to quantify the phorbol esters in mixtures, in semi-purified samples, or even in the complete oil, and to determine at the same time the proportion of diand triesters. Because of its speed and accuracy, this technique provides a valuable alternative for the less versatile biological methods, to screen for known classes of compounds (like e. g. phorbol esters).

1 L. A. Pieters is a research assistant of the Belgian National Foundation for Scientific Research (NFWO).