Phytomedicine 20 (2013) 270–274 Contents lists available at SciVerse ScienceDirect Phytomedicine jou rn al hom epage: www.elsevier.de/phymed Antitrypanosomal compounds from the essential oil and extracts of Keetia leucantha leaves with inhibitor activity on Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase J. Bero a,∗ , C. Beaufay a , V. Hannaert b , M.-F. Hérent a , P.A. Michels b , J. Quetin-Leclercq a a Université catholique de Louvain, Louvain Drug Research Institute, Pharmacognosy Research Group, Avenue E. Mounier, B1.72.03, B-1200 Brussels, Belgium b Université catholique de Louvain, de Duve Institute, Research Unit for Tropical Diseases, Avenue Hippocrate 74, B1.74.01, B-1200 Brussels, Belgium a r t i c l e i n f o Keywords: Keetia leucantha Trypanosoma brucei Glyceraldehyde-3-phosphate dehydrogenase Essential oil a b s t r a c t Keetia leucantha is a West African tree used in traditional medicine to treat several diseases among which parasitic infections. The dichloromethane extract of leaves was previously shown to possess growth- inhibitory activities on Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania mexicana mexicana with low or no cytotoxicity (>100 g/ml on human normal fibroblasts) (Bero et al. 2009, 2011). In continuation of our investigations on the antitrypanosomal compounds from this dichloromethane extract, we analyzed by GC-FID and GC–MS the essential oil of its leaves obtained by hydrodistillation and the major triterpenic acids in this extract by LC–MS. Twenty-seven compounds were identified in the oil whose percentages were calculated using the normalization method. The essential oil, seven of its constituents and the three triterpenic acids were evaluated for their antitrypanosomal activity on Try- panosoma brucei brucei bloodstream forms (Tbb BSF) and procyclic forms (Tbb PF) to identify an activity on the glycolytic process of trypanosomes. The oil showed an IC 50 of 20.9 g/ml on Tbb BSF and no activ- ity was observed on Tbb PF. The best antitrypanosomal activity was observed for ursolic acid with IC 50 of 2.5 and 6.5 g/ml respectively on Tbb BSF and Tbb PF. The inhibitory activity on a glycolytic enzyme of T. brucei, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also evaluated for betulinic acid, olenaolic acid, ursolic acid, phytol, -ionone and -ionone. The three triterpenic acids and -ionone showed inhibitory activities on GAPDH with oleanolic acid being the most active with an inhibition of 72.63% at 20 g/ml. This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of Keetia leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitrypanosomal activity on bloodstream forms of Tbb as compared to procyclic forms, namely geranyl acetone, phytol, -ionone, -ionone, ursolic acid, oleanolic acid and betulinic acid. The four last compounds were proven to be inhibitors of trypanosomal GAPDH, which may in part explain these antitrypanosomal activities. © 2012 Elsevier GmbH. All rights reserved. Introduction Human African trypanosomiasis or sleeping sickness is a vector- borne parasitic disease whose vector is Trypanosoma brucei, a protist parasite transmitted to humans by tsetse flies (Glossina genus). Sleeping sickness which may evolve into a neurological dis- ease and be lethal, threatens millions of people in 36 countries in sub-Saharan Africa (World Health Organisation (WHO) 2012). The number of actual cases is currently estimated at 30,000 (Drugs for Neglected Diseases initiative 2011). Resistance, toxicity and variable efficacy between strains or species of most of the drugs used as well as, for some of them, ∗ Corresponding author. Tel.: +32 2 764 7292; fax: +32 2 764 7293. E-mail address: joanne.bero@uclouvain.be (J. Bero). the need for a long course of parenteral administration show how the search for new antitrypanosomal compounds is needed, partic- ularly from plants used in traditional medicine, as a source of new leads with new mechanisms of action (Hoet et al. 2004b). In our search for more effective drugs against Trypanosoma bru- cei and as a continuation of our investigations on traditional plants used as antiparasitics in Benin (Bero et al. 2011), a special attention was devoted to Keetia leucantha which is a West African tree used in traditional medicine to treat parasitic diseases. The dichloromethane extract of leaves of K. leucantha was previously shown to have in vitro antitrypanosomal activity (IC 50 = 24.4 g/ml on Trypanosoma brucei brucei) with a good selec- tivity (SI = 2.7) (Bero et al. 2011). As triterpenic acids were identified in the twigs, we searched for this type of compounds in the leaves extract (Bero et al. 2011) but also analyzed its essential oil (obtained by hydrodistillation) as its components may also be found in 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.10.010