Solomon M Abay1,2, Leonardo Lucantoni3,4, Nisha Dahiya5, Geme Dori6, Edson G Dembo7, Fulvio Esposito8, Guilio Lupidi9, Sonny Ogboi10, Robert K Ouédraogo11,12, Annamaria Sinisi13, Orazio Taglialatela-Scafati14, R Serge Yerbanga15, Massimo Bramucci16, Luana Quassinti17, Jean Bosco Ouédraogo18, George Christophides19, Annette Habluetzel20. 1. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. solomonabay@gmail.com. 2. School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia. solomonabay@gmail.com. 3. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. l.lucantoni@griffith.edu.au. 4. Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD, 4111, Australia. l.lucantoni@griffith.edu.au. 5. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. nisha.dahiya@unicam.it. 6. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. gemeurge@yahoo.com. 7. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. edembo@gmail.com. 8. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. fulvio.esposito@unicam.it. 9. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. giulio.lupidi@unicam.it. 10. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. ogboijb@yahoo.com. 11. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. oued.kossivi.robert@gmail.com. 12. Institut de Recherche enSciences de la Santé, Direction Régionale de l'Ouest, Bobo-Dioulasso, Burkina Faso. oued.kossivi.robert@gmail.com. 13. Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131, Naples, Italy. annamaria.sinisi@unina.it. 14. Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131, Naples, Italy. scatagli@unina.it. 15. Institut de Recherche enSciences de la Santé, Direction Régionale de l'Ouest, Bobo-Dioulasso, Burkina Faso. yrserge@yahoo.fr. 16. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. massimo.bramucci@unicam.it. 17. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. luana.quassinti@unicam.it. 18. Institut de Recherche enSciences de la Santé, Direction Régionale de l'Ouest, Bobo-Dioulasso, Burkina Faso. jbouedraogo@gmail.com. 19. Department of Life Sciences, Imperial College London, London, UK. g.christophides@imperial.ac.uk. 20. School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032, Camerino, MC, Italy. annette.habluetzel@unicam.it.
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.
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.
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