In vitro anti-plasmodial activity of Dicoma anomala subsp. gerrardii (Asteraceae) : identification of its main active constituent, structure-activity relationship studies and gene expression profiling
Crampton, Bridget Genevieve; Maharaj, Vinesh J.; Mancama, Dalu; Becker, John V.W.; Van der Merwe, Marina M.; Van Brummelen, A.C. (Anna Catharina); Pillay, Pamisha; Mmutlane, Edwin M.; Parkinson, Chris; Van Heerden, Fanie R.; Crouch, Neil R.; Smith, Peter J.
BACKGROUND: Anti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The
resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative
to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for
investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile.
METHODS: Standard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column
chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The
crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and
X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite
lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional
groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary
features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications
in vitro using the pLDH assay. The effects of the pure compound on the P. falciparum transcriptome were
subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by
oligonucleotide microarray- and data analysis.
RESULTS: The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type
sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 1.865 μM against a chloroquine-sensitive
strain (D10) of P. falciparum. Synthetic analogues of the compound confirmed an absolute requirement that the amethylene
lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This
feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified
572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis
identified various biological processes and molecular functions that were significantly affected. Comparison of the
dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene
lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the
artemisinins have differentiated modes of action.
CONCLUSIONS: The novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an
ongoing role in advancing anti-plasmodial drug discovery efforts.