Biological assays-in vitro and in vivo.
The IC50s of spilanthol and UDA were determined against the chloroquine-resistant K1 strain and the mildly chloroquine-resistant strain PFB of Plasmodium falciparum using a previously established method (Desjardins et al., 1979). The growth inhibition was assessed in triplicate by comparison of the radioactivity incorporated into the treated culture with that in control culture from the same plate. The in vivo experiments were carried out with 5 week old male and female Swiss mice weighing 23.2 g (±0.67) (Elevage Janvier, La Genest Saint Isle, France) were housed in standard environmental conditions (24±1.8 C, 55% humidity). The experiments were conducted in accordance with the EEC Directive of 1986 (86/609/EEC) on laboratory animals. Briefly, extracts of Spilanthes acmellla and spilanthol (25 mg/kg and 2.5 mg/kg twice daily, respectively) in a PBS methylcellulose solution (1%) were injected intraperitonealy into each animal 2 hours after inoculation with 107 Plasmodium yoelii yoelii parasites. Injections were continued twice a day for 4 days and experimental groups consisted of 5 mice (1 male and 4 females) per group. Parasitaemia was monitored daily by microscopic examination of Giemsa stained-thin blood smears.