Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans

Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents - methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products.