Histochemical and Immunocytochemical Localization of Tropane Alkaloids in Erythroxylum coca var. coca and E. novogranatense var. novogranatense

Abstract

The tropane alkaloids of Erythroxylum coca var. coca and Erythroxylum novogranatense var. novogranatense were localized using histochemical, cytochemical, and immunocytochemical probes. At the tissue level, these alkaloids were localized in leaves, stems, and fruits with Dragendorff's reagent. Alkaloids were found in the mesophyll, including palisade, spongy, and vascular parenchyma cell layers, and in some cells of the collenchyma. No alkaloids were detected in the epidermis of either Erythroxylum species. Alkaloids were also detected in the endosperm and embryos of orange and mature red fruits but not in tissues of immature green fruits. Quantitative HPLC reveals that embryos had 2.5-5 times more cocaine (w/w%) than endosperms, but 40 times less than leaves. In contrast to leaves, cocaine was a minor alkaloid present in fruits. Dragendorff's reagent was used to develop thin-layer chromatography plates and specifically stained the alkaloids extracted from leaves of both species. Complexing of alkaloids with tannins resulted in aggregates in the vacuole of E. coca leaf cells as visualized by transmission electron microscopy. Immunocytochemical localization, using anticocaine polyclonal antibodies on leaf tissues, proved that these compounds are localized in vacuoles of both photosynthetic and vascular parenchyma, as demonstrated by the use of Dragendorff's reagent. The alkaloids were associated with intravacuolar globules and appear to be aggregates with a core formed by phenolic compounds and a periphery enriched in alkaloids. The vacuolar localization of the cocaine alkaloids indicate that they may be complexed with phenols in vivo, thereby rendering them relatively immobile. The implications of the cellular and tissue localization of tropane alkaloids in Erythroxylum are discussed.