Transmission blocking activity of a standardized neem (Azadirachta indica) seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi

Abstract

BackgroundThe wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal®, an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi.MethodsAnopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal® on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined.ResultsNeemAzal® completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule organizing centres and with the assembly of cytoskeletal microtubules, which are both fundamental processes in Plasmodium gametogenesis and ookinete formation.ConclusionsThis work demonstrated in vivo transmission blocking activity of an azadirachtin-enriched neem seed extract at an azadirachtin dose compatible with 'druggability' requisites. These results and evidence of anti-plasmodial activity of neem products accumulated over the last years encourage to convey neem compounds into the drug discovery & development pipeline and to evaluate their potential for the design of novel or improved transmission-blocking remedies.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species

“…that are structurally similar to those known to be gametocytocidal either in vitro, in vivo or ex vivo Adjalley et al, 2011;D'Alessandro et al, 2013;Duffy and Avery, 2013;Jones et al, 1994;Lucantoni et al, 2013Lucantoni et al, , 2010Saenz et al, 2013;Sun et al, 2013;Yerbanga et al, 2014). Using these two parallel selection strategies, the ethnobotanical set was narrowed down to a hundred plant species from which eight were collected and screened in the current study.…”

“…The three plant species investigated namely, L. leonurus, L. leonurus ex Hort and Olea europaea (Oleaceae) were selected based on previously reported activity against the intraerythrocytic asexual parasites of P. falciparum (Table 1) (Clarkson et al, 2004). Khaya anthotheca (Meliaceae) was selected as the Meliaceae family, has been shown to produce limonoids that are potent against gametocytes (Jones et al, 1994;Lucantoni et al, 2010;Yerbanga et al, 2014). The remainder of plant species was selected based on both activity and natural compound production (Table 1).…”

“…By contrast, less effort has been channeled towards examining extracts of plants for their activity against the sexual stages of the parasite. Currently, only three plant species have been directly screened for their malaria transmission blocking capacity: Azadirachta indica (Meliaceae) (Dhar et al, 1998;Jones et al, 1994;Lucantoni et al, 2010;Udeinya et al, 2006Udeinya et al, , 2008Yerbanga et al, 2014), Vernonia amygdalina (Asteraceae) and Guiera senegalensis (Combretaceae) (Yerbanga et al, 2014). A. indica has been comprehensively studied and…”

“…shown to have good gametocytocidal activity in vitro (Dhar et al, 1998;Jones et al, 1994;Udeinya et al, 2008Udeinya et al, , 2006, in vivo (Lucantoni et al, 2010) and ex vivo (Yerbanga et al, 2014). The active components are a diverse range of limonoid compounds (Jones et al, 1994;Lucantoni et al, 2010;Yerbanga et al, 2014).…”

“…The active components are a diverse range of limonoid compounds (Jones et al, 1994;Lucantoni et al, 2010;Yerbanga et al, 2014). V. amygdalina gametocytocidal activity was demonstrated in vivo with the active principles identified as the sesquiterpene lactones vernadalol and vernolide .…”