In vitro investigation of Brazilian Cerrado plant extract activity against Plasmodium falciparum, Trypanosoma cruzi and T. brucei gambiense

Abstract The threatened Brazilian Cerrado biome is an important biodiversity hotspot but still few explored that constitutes a potential reservoir of molecules to treat infectious diseases. We selected eight Cerrado plant species for screening against the erythrocytic stages of Plasmodium falciparum, human intracellular stages of Trypanosoma cruzi and bloodstream forms of T. brucei gambiense, and for their cytotoxicity upon the rat L6-myoblast cell line. Bioassays were performed with 37 hexane, ethyl acetate and ethanol extracts prepared from different plant organs. Activities against parasites were observed for 24 extracts: 9 with anti-P. falciparum, 4 with anti-T. cruzi and 11 with anti-T. brucei gambiense activities. High anti-protozoal activity (IC50 values < 10 μg/mL) without obvious cytotoxicity to L6 cells was observed for eight extracts from plants: Connarus suberosus, Blepharocalyx salicifolius, Psidium laruotteanum and Myrsine guianensis. Overall, studies of plant extracts will contribute to increase the biodiversity knowledge essential for Cerrado conservation and sustainable development. Graphical abstract


Introduction
The Cerrado is the second largest biome in Brazil with an estimated biodiversity of more than 13,000 plant species (Braga et al. 2008), and an endemism of 44% (Myers et al. 2000). This exceptionally rich biome is classified as a biodiversity hotspot (Myers et al. 2000) caused by the high impact of the human activity. Jones et al. (2008) linked such anthropogenic changes to the emerging or re-emerging of infectious diseases.
Although the treatments of malaria, Chagas disease and sleeping sickness are based on chemotherapies, for Chagas disease or sleeping sickness, there are few chemotherapeutics available with serious side effects and malaria parasite strains evolve drug resistance to the most effective drugs. The situation could be considered critical and urgently requires new effective drugs with new mechanisms of action. Natural products were and continue to be important sources of therapeutic drugs as exemplified for malaria (Cragg & Newman 2013;Pohlit et al. 2013).
In efforts to discover natural anti-protozoal products, here we report the antiparasitic extract activity of eight Cerrado plants on three tropical human protozoa: P. falciparum, the main responsible of the estimated 655,000 malaria deaths in 2010 (WHO 2011); Trypanosoma cruzi, the etiologic agent of Chagas disease with more than 10,000 deaths annually (WHO 2010); and T. brucei gambiense, responsible for a chronic form of sleeping sickness with 10,000 new cases reported in 2009 (WHO 2010).

Results and discussion
The eight plant species were selected according to ethno-medicinal information and their chemotaxonomic relationship to known active plants exhibiting biological properties, including anti-oxidant, antitumoural, anti-inflammatory, antispasmodic, anti-viral, fungicidal, antibacterial and anti-protozoal effects. Hexane, ethyl acetate and ethanol extractions of different plant organs (stem and root bark; wood, leaves and aerial parts) resulted in 37 crude extracts with yields ranging from 0.25 to 28.23% (w/w) ( Table S1).
As a preliminary screening, each extract was tested at fixed concentrations of 100, 10 and 1 μg/mL as described (Nguyen-Pouplin et al. 2007) against: (1) the erythrocytic stages of the chloroquine-resistant P. falciparum FcB1 strain; (2) the development of intracellular amastigote forms of the Tulahuen T. cruzi strain and (3) T. brucei gambiense bloodstream forms. Cytotoxic activity was subsequently investigated upon the rat L6-myoblast cell line. The IC 50 and IC 90 values were then determined for plant extracts demonstrating significant activity at 10 μg/mL (when percentages of parasite growth were <80% for P. falciparum and T. cruzi and <40% for T. brucei gambiense), together with cytotoxicity parameters (TC 50 and TC 90 ) ( Table 1).
Connarus suberosus (Connaraceae) is a typical species of the Brazilian Cerrado biome (Denardi et al. 2012). The ethyl acetate extract fractionation resulted in the isolation of a mixture of the alkyl benzoquinone rapanone and a previously unreported compound -suberonone active against Leishmania (L.) amazonensis promastigotes (da Costa et al. 2014). Here, the hexane extracts displayed high anti-P. falciparum and anti-T. brucei gambiense activities for the root wood (IC 50 values of 1.2 and 1.7 μg/mL, respectively) and the root bark (IC 50 values of 1.2 and 1.8 μg/mL, respectively) ( Table 1).
Amongst the Myrsinaceae family, the compound myrsinoic acid B from the Rapanea ferruginea ethanol stem bark extract presented activity against L. (Viannia) brasiliensis . Moreover, the isolated compound rapanone of Myrsine guianensis showed potential anti-oxidant and anti-inflammatory profiles (Ospina et al. 2001). From this species, we observed that only the hexane stem wood extract exhibited high anti-plasmodial activity (IC 50 = 5 μg/mL).
In relation to Psidium laruotteanum (Myrtaceae), the hexane and ethyl acetate leaf extracts revealed significant activity against T. brucei gambiense with IC 50 values of 3.9 and 6.8 μg/ mL, respectively (Table 1). These extracts were non-toxic to the L6 cells (TC 50 > 100 μg/ mL), resulting in a high selectivity index (>25.64 and >14.7, respectively) regarding this parasite, which is responsible for African sleeping sickness whereas, essential oil and polyherbal product from Psidium guajava L. leaves showed activity against Toxoplasma gondii (Lee et al. 2013) and rodent malaria species (Tarkang et al. 2014), respectively.
For Blepharocalyx salicifolius, also a member of the Myrtaceae family, the strongest activity against P. falciparum (IC 50 = 5.1 μg/mL) with the highest selectivity index (16.8) was observed for the ethyl acetate stem bark extract. It was also observed that the leaf extracts demonstrated high activities against P. falciparum and T. brucei gambiense -hexane extract: IC 50 = 8.1 and 3.5 μg/mL, respectively; ethanol extract: IC 50 = 9.3 and 7.2 μg/mL, respectively -without cytotoxicity upon L6 cells (Table 1). Futhermore, B. salicifolius displayed great bactericidal effects against Staphylococcus aureus and Escherichia coli species and fresh leaves had antispasmodic effects (Limberger et al. 2001). It was also suggested to have a great potential for isolation of compounds active against Leishmania with low toxicity (de Siqueira et al. 2010).
Results for Astronium fraxinifolium (Anacardiaceae) showed that the ethanol root wood extract presented a moderate activity against P. falciparum with IC 50 = 23.3 μg/mL, in addition to the hexane stem bark extract against T. brucei gambiense with an IC 50 value of 16.4 μg/mL, without cytotoxicity to L6 cells (Table 1). However, the leaf ethanolic extract was able to reduce L. (Viannia) braziliensis growth in vitro and in vivo (de Lima et al. 2014). We can notify that volatile oil of A. fraxinifolium leaves dominated by (E)-β-ocimene and α-terpinolene and high concentration of oxygenated compounds, demonstrated high antibacterial activity (Montanari et al. 2012).
Of the Fabaceae, Chamaecrista repens had a high anti-oxidant activity (David et al. 2007) and C. nictitans showed anti-viral activity against herpes simplex virus and non-cytotoxicity (Uribe et al. 2004). The ethanol aerial part extract of Chamaecrista desvauxii, selected in our study, did not exhibit antiparasitic activity (Table 1), while decoctions of its leaves and stem barks are used against malaria (Traore et al. 2013). Nonetheless, the hexane root wood extract of Vatairea macrocarpa showed activity against T. cruzi with IC 50 = 36.2 μg/mL, and no apparent cytotoxicity to L6 cells, with IC 50 > 100 μg/mL (Table 1).

Conclusion
From the 37 tested extracts, 9 extracts were active against P. falciparum; 4 against T. cruzi and 11 against T. brucei gambiense. It is interesting to highlight the activity of seven leaf extracts against T. brucei gambiense, an organ easily obtained from these species whose collection does not endanger the plant conservation. Most of these extracts were without any obvious cytotoxicity to rat myoblast-derived L6 cells. Eight extracts from C. suberosus, B. salicifolius, P. laruotteanum and M. guianensis proved to have high activity against at least P. falciparum or T. brucei gambiense (IC 50 < 10 μg/mL). We expect that the investigation of these four plants will contribute to Cerrado conservation, which is considered to be one of most threatened regions on Earth. The discovery of compounds to treat parasitic tropical diseases also warrants protection of the biome.