School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, (MC) Italy.
Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy.
Phytomedicine. 2016 Dec 15;23(14):1743-1752. doi: 10.1016/j.phymed.2016.10.019. Epub 2016 Oct 28.
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.
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.
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.
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.
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 IC value determined for NA was 6.8µg/ml (CI: 5.95-7.86), about half of the AzaA IC (12.4µg/ml; CI: 11.0-14.04).
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 IC against 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.
NeemAzal（NA）是一种从印楝种子仁中提取的定量提取物，印楝，Azadirachta indica A.Juss.（楝科），具有广泛的生物特性，经典上归因于其柠檬素含量。NA 含有几种印苦素（A 至 L），印苦素 A（AzaA）是其主要成分。已证明 AzaA 可抑制啮齿动物疟原虫 Plasmodium berghei 的小配子形成，并且当在暴露于蚊子之前以对应于 50mg/kg 的 AzaA 剂量向配子细胞血症小鼠给药时，NA 完全抑制 Plasmodium berghei 向按蚊的传播。
本研究旨在 i）评估 NA 和 AzaA 在系统性循环中对 P. berghei 出芽的药效学和作用持续时间，以及 ii）阐明 NA 主要成分的传播阻断活性（TBA）。
通过体外出芽试验评估 NA 和 AzaA 对出芽的药效学，通过体外卵囊发育试验评估 NA 成分的 TBA。
药效学实验：用循环成熟配子的 P. berghei 感染 BALB/c 小鼠的外周血，通过腹腔内注射 50mg/kg 和 100mg/kg 纯 AzaA 和 NeemAzal（Trifolio-M GmbH）以达到相应的 AzaA 浓度。通过在体外出芽试验中在治疗后不同时间点计数正在释放有鞭毛配子的小配子细胞中形成的出芽中心，来估计化合物的药效和作用持续时间。卵囊发育试验：通过荧光显微镜测量 NeemAzal 和 AzaA 对卵囊发育的直接影响，将含有各种浓度测试物质的配子细胞血症血液在微孔板中孵育 24 小时。
出芽试验显示，NA 抗疟化合物的半衰期长达 7 小时，剂量相当于 100mg/kg 的 AzaA 当量剂量。卵囊发育试验表明，NA 对早期孢子发生阶段的活性增加，与 AzaA 相比。确定的 NA 的 IC 值为 6.8µg/ml（CI：5.95-7.86），约为 AzaA 的 IC 值的一半（12.4µg/ml；CI：11.0-14.04）。
与 AzaA 相比，NA 更强的活性不能用 NA 中存在的其他印苦素（B、D 和 I）的相加或协同作用来解释。事实上，将这些化合物以 50µM 浓度添加到 AzaA 中并没有使对早期孢子发生阶段的 IC 值降低到仅用 AzaA 获得的值。可能是 NA 中存在的其他非柠檬素化合物有助于 AzaA 活性和增强对体外和体内出芽的药效学。
