Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds

Abstract

Background: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum.

Methods: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926.

Results: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines.

Conclusions: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.

Fig. 1

Chemical structure of vernolide and vernodalol.

Fig. 2

Plasmodium berghei GFPcon oocysts on mosquito mid-gut, 10 days after infection.

Fig. 3

Exflagellation centres, vibrating accumulations of red blood cells attaching to microgametocytes with extruding motile, flagellate gametes. Photograph was taken after incubation of gametocytaemic blood in exflagellation medium for 20 min.

Fig. 4

Early sporogonic stages: spherical zygote (white arrow) and elongated ookinetes (red arrows).

Fig. 5

Proportion of mature ookinetes with respect to total early sporogonic stages. The test was conducted using ethanolic Vernonia amygdalina leaf extract (Ver-EtOH) at different concentration and solvent control (dimethyl sulfoxide). Each bar in the graph refers to readings from 3 and 6 microplate wells in experiment 1 and 2, respectively.

Fig. 6

Impact of organic solvent fractions from Vernonia amygdalina leaves (100 µg/mL) on sporogonic development. Mosquitoes were membrane fed on blood from three donors with Plasmodium falciparum gametocytaemia values of: a 160 gametocytes/μL, b 184 gametocytes/μL and c 80 gametocytes/μL. Each point represents the oocyst number from an individual mosquito. In a the data point of one midgut on which 94 oocysts were recorded, is not plotted in the graph (largely out of scale).