Transmission blocking effects of Azadirachta indica limonoids on early sporogonic development of Plasmodium: activity and bioavailability of seed fractions and isolated compounds

Azadirachta indica (Meliaceae) possesses a wide spectrum of biological properties, conferred to the plant by secondary metabolites. A. indica seeds contain abundantly limonoid molecules such as azadirone, nimbin, salanin and azadirachtins (A to L), azadirachtin A (AzaA) being one of the most bio-active molecules. AzaA has been shown to inhibit Plasmodium berghei microgamete formation and an AzaA rich commercial kernel extract (NeemAzal®) was found to completely block the transmission of P. berghei to Anopheles stephensi females when administered to gametocytemic mice at an AzaA dose of 50 mg/kg before exposure to mosquitoes. The present study was aimed at i) elucidating early sporogonic stage specific effects of A. indica seed fractions and their main constituents; ii) assessing the bioavailability of a fraction rich in AzA and the isolated AzaA molecule through a biological response-based assay. Ex vivo and in vitro assays were performed with the murine malaria parasites P. berghei ANKA strain and P. berghei CTRPp.GFP. Fractions were obtained from A. indica seeds collected in Burkina Faso and from NeemAzal® (NA, provided by Trifolio-M GmbH, Lahnau, Germany) by column chromatography. Constituents were identified by NMR spectroscopy. NA, AzaA, nimbin and salannin rich fractions from unripe seeds tested at 50 µg/ml, revealed inhibitory activity on early sporogonic stages in vitro. Nimbin and salannin were found to interfere with ookinete maturation while NA and AzaA showed multiple effects on early sporogonic development. The IC50 value determined for NA was 6.8 µg/ml (CI95: 5.95- 7.86), about half of that of AzaA IC50 (12.4 µg/ml; CI95: 11.0- 14.04). The stronger activity of NA, when compared to AzaA, appeared not to be due to an additive or synergistic effect of other azadirachtins (B, D and I) present in NA, since the addition of these compounds at 50 µM to AzaA did not evidence any decrease of the IC50. Also, bioavailability of AzaA, administered as constituent of NA, compared to pure AzaA appeared to be increased. Ex vivo exflagellation tests using blood sampled from mice treated with NA at an AzaA dosage of 150 mg/kg, revealed a half life of NA anti-plasmodial compounds of up to 7 hours. Accumulated evidence on bioavailability and anti-plasmodial activity of limonoids against Plasmodium stages developing in the human and mosquito host, suggests A. indica as a valid resource for the design of limonoid dosed, transmission blocking phytomedicines.