title: In vitro and ex vivo activity of an Azadirachta indica A.Juss. seed kernel extract on early sporogonic development of Plasmodium in comparison with azadirachtin A, its most abundant constituent.
abstract: BACKGROUND
  NeemAzal®(NA) is a quantified extract from seed kernels of neem, Azadirachta indica A.Juss. (Meliaceae), with a wide spectrum of biological properties, classically ascribed to its limonoid content. NA contains several azadirachtins (A to L), azadirachtin A (AzaA) being its main constituent. AzaA has been shown to inhibit microgamete formation of the rodent malaria parasite Plasmodium berghei, and NA was found to completely inhibit the transmission of Plasmodium berghei to Anopheles stephensi mosquitoes when administered to gametocytemic mice at a corresponding AzaA dose of 50mg/kg before exposure to mosquitoes.
  
  
  OBJECTIVE
  The present study was aimed at i) assessing the pharmacodynamics and duration of action of NA and AzaA against P. berghei exflagellation in systemic circulation in mice and ii) elucidating the transmission blocking activity (TBA) of the main NA constituents.
  
  
  METHODS
  The NA and AzaA pharmacodynamics on exflagellation were assessed through ex vivo exflagellation assays, while TBA of NA constituents was evaluated through in vitro ookinete development assay.
  
  
  METHODS
  Pharmacodynamics experiments: Peripheral blood from P. berghei infected BALB/c mice with circulating mature gametocytes, were treated i.p. with 50mg/kg and 100mg/kg pure AzaA and with NeemAzal®(Trifolio-M GmbH) at the corresponding AzaA concentrations. The effect magnitude and duration of action of compounds was estimated by counting exflagellation centers, formed by microgametocytes in process of releasing flagellated gametes, at various time points after treatment in ex vivo exflagellation tests. Ookinete Development Assay: The direct effects of NeemAzal®and AzaA on ookinete development were measured by fluorescence microscopy after incubation of gametocytemic blood with various concentrations of test substances in microplates for 24h.
  
  
  RESULTS
  The exflagellation tests revealed an half-life of NA anti-plasmodial compounds of up to 7h at a NA dose corresponding to 100mg/kg equivalent dose of AzaA. The ookinete development assay showed an increased activity of NA against early sporogonic stages compared to that of AzaA. The IC50value determined for NA was 6.8µg/ml (CI95: 5.95-7.86), about half of the AzaA IC50(12.4µg/ml; CI95: 11.0-14.04).
  
  
  CONCLUSIONS
  The stronger activity of NA, when compared to AzaA, could not be explained by an additive or synergistic effect by other azadirachtins (B, D and I) present in NA. In fact, the addition of these compounds at 50µM concentration to AzaA did not evidence any decrease of the IC50against early sporogonic stages to that obtained with AzaA alone. It is likely that other non-limonoid compounds present in NA may contribute to AzaA activity and enhanced pharmacodynamics against exflagellation both in vitro and in vivo.
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: nisha.dahiya@unicam.it.
authors.forename: Nisha
authors.initials: N
authors.lastname: Dahiya
authors.affiliation: Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy. Electronic address: g.chianese@unina.it.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Chianese
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy; School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia. Electronic address: solomonabay@gmail.com.
authors.forename: Solomon Mequanente
authors.initials: SM
authors.lastname: Abay
authors.affiliation: Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy. Electronic address: scatagli@unina.it.
authors.forename: Orazio
authors.initials: O
authors.lastname: Taglialatela-Scafati
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: fulvio.esposito@unicam.it.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Esposito
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: giulio.lupidi@unicam.it.
authors.forename: Giulio
authors.initials: G
authors.lastname: Lupidi
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: massimo.bramucci@unicam.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bramucci
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: luana.quassinti@unicam.it.
authors.forename: Luana
authors.initials: L
authors.lastname: Quassinti
authors.affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom.. Electronic address: g.christophides@imperial.ac.uk.
authors.forename: George
authors.initials: G
authors.lastname: Christophides
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy. Electronic address: annette.habluetzel@unicam.it.
authors.forename: Annette
authors.initials: A
authors.lastname: Habluetzel
authors.affiliation: School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy; Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, 4111 Queensland, Australia. Electronic address: l.lucantoni@griffith.edu.au.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Lucantoni
mesh_terms: D000818:Animals; D000852:Anopheles; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D031601:Azadirachta / Q000737:chemistry*; D002460:Cell Line; D005260:Female; D006801:Humans; D036701:Limonins / Q000494:pharmacology*; D008288:Malaria / Q000469:parasitology* / Q000635:transmission; D008807:Mice, Inbred BALB C; D010936:Plant Extracts / Q000494:pharmacology*; D010962:Plasmodium berghei / Q000187:drug effects*; D012639:Seeds / Q000737:chemistry
chemical_list: D000981:Antiprotozoal Agents; D036701:Limonins; D010936:Plant Extracts; C010329:azadirachtin
keywords: Azadirachtin A; Exflagellation; NeemAzal(®); Pharmacodynamics; Plasmodium