In vitro and in vivo antimalarial activity of green synthesized silver nanoparticles using Sargassum tenerrimum - a marine seaweed.

Green nanotechnology has recently attracted a lot of attention as a potential technique for drug development. In the present study, silver nanoparticles were synthesised by using Sargassum tenerrimum, a marine seaweed crude extract (Ag-ST), and evaluated for antimalarial activity in both in vitro and in vivo models. The results showed that Ag-ST nanoparticles exhibited good antiplasmodial activity with IC50 values 7.71±0.39 µg/ml and 23.93±2.27 µg/ml against P. falciparum and P. berghei respectively. These nanoparticles also showed less haemolysis activity suggesting their possible use in therapeutics. Further, P. berghei infected C57BL/6 mice were used for the four-day suppressive, curative and prophylactic assays where it was noticed that the Ag-ST nanoparticles significantly reduced the parasitaemia and there were no toxic effects observed in the biochemical and haematological parameters. Further to understand its possible toxic effects, both in vitro and in vivo genotoxicological studies were performed which revealed that these nanoparticles are non-genotoxic in nature. The possible antimalarial activity of Ag-ST may be due to the presence of bioactive phytochemicals and silver ions. Moreover, the phytochemicals prevent the nonspecific release of ions responsible for low genotoxicity. Together, the bio-efficacy and toxicology outcomes demonstrated that the green synthesized silver nanoparticles (Ag-ST) could be a cutting-edge alternative for therapeutic applications.

Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

Standard Selection Treatments with Sulfadiazine Limit Plasmodium yoelii Host-to-Vector Transmission.

Some antimalarial drugs that have lost clinical usefulness have been repurposed for experimental applications. One example is sulfadiazine, an analog of p-aminobenzoic acid (pABA), which inhibits the parasite's folate synthesis pathway to block DNA synthesis. Sulfadiazine treatment of mice infected with Plasmodium yoelii and P. berghei is routinely used to enrich for gametocytes by killing asexual blood-stage parasites, but it is not well known if there are downstream effects on transmission. To determine if there was a significant effect of sulfadiazine exposure upon transmission, we transmitted Plasmodium yoelii (17XNL strain) parasites to Anopheles stephensi mosquitoes and evaluated the prevalence and intensity of infection under different sulfadiazine treatment conditions. We observed that there was a reduction in both the number of mosquitoes that became infected and in the intensity of infection if parasites were exposed to sulfadiazine in the mouse host or mosquito vector. Sulfadiazine treatment could be marginally overcome if mosquitoes were provided fresh pABA. In contrast, we determined that gametocytes exposed to sulfadiazine could develop into morphologically mature ookinetes in vitro, thus sulfadiazine exposure in the host may be reversible if the drug is washed out and the parasites are supplemented with pABA in the culture media. Overall, this indicates that sulfadiazine dampens host-to-vector transmission and that this inhibition can only be partially overcome by exposure to fresh pABA in vivo and in vitro. Because gametocytes are of great interest for developing transmission-blocking interventions, we recommend the use of less disruptive approaches for gametocyte enrichment. IMPORTANCE In this work, we have uncovered a substantial problem with how many studies of the sexual stages of rodent malaria parasites are conducted. Briefly, the isolation of sexual blood-stage Plasmodium parasites, or gametocytes, is essential to study pretransmission and transmission-stage biology of malaria. A routine method for the isolation of this specific stage in rodent-infectious malaria models is drug treatment with sulfadiazine, an antifolate that selectively kills actively replicating asexual blood-stage parasites but not gametocytes. Thus, researchers use this as a convenient way to produce highly enriched gametocyte samples. However, in this work, we describe how this standard drug selection with sulfadiazine not only kills asexual blood-stage parasites but also substantially impacts host-to-vector transmission.

Suppressive, curative, and prophylactic potentials of an antimalarial polyherbal mixture and its individual components in Plasmodium berghei-Infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria remains one of the most prevalent infectious diseases in tropical regions of the world, particularly in sub-Saharan Africa, where it remains epidemiologically holoendemic. The absence of effective vaccines and Plasmodium resistance to antimalarial drugs have been the major challenges to malaria control measures. An alternative strategy could be the application of validated and standardized herbal formulations. AIM OF THE STUDY: To evaluate the antimalarial activity of a polyherbal mixture (APM) and compare it to those of its individual constituent plants. METHODS: APM consisted of stem barks of Mangifera indica (MI), Azadirachta indica (AI), Nauclea latifolia (and roots, NL) and roots of Morinda lucida (ML). Dihydroartemisinin-piperaquine (DHP) and pyronaridine-artesunate (PA) served as positive controls. Antimalarial activity was evaluated using suppressive, curative and prophylactic assays in mice infected with Plasmodium berghei. RESULTS: All the herbal mixtures, individually and in combination, showed significant (p < 0.05) antiplasmodial activities in the various assays. They produced considerable parasite suppression (>50%), substantial clearance (>70%), and notable prophylaxis (>60%, except for NL: 35%). APM (95.4-98.7%) and AI (92%), respectively, elicited greater and comparable suppression relative to DHP (88%) and PA (87.3%). However, all the herbal decoctions, individually (72-93.6%) and in combination (82.5-91%), showed lower parasite clearance than DHP (100%) and PA (99.5%). Meanwhile, APM showed relatively greater suppression and prophylaxis than its constituent plants, suggesting that the combination produced synergistic or additive effects. CONCLUSION: These findings could substantiate the use of these plants, singly or in combination, as traditional remedies for malaria. Further studies are recommended to evaluate their clinical usefulness.

Neemazal ® as a possible alternative control tool for malaria and African trypanosomiasis?

BACKGROUND: Research efforts to identify possible alternative control tools for malaria and African trypanosomiasis are needed. One promising approach relies on the use of traditional plant remedies with insecticidal activities. METHODS: In this study, we assessed the effect of blood treated with different doses of NeemAzal ® (NA, neem seed extract) on mosquitoes (Anopheles coluzzii) and tsetse flies (Glossina palpalis gambiensis) (i) avidity to feed on the treated blood, (ii) longevity, and (iii) behavioural responses to human and calf odours in dual-choice tests. We also gauged NeemAzal ® toxicity in mice. RESULTS: In An. coluzzii, the ingestion of NA in bloodmeals offered by membrane feeding resulted in (i) primary antifeedancy; (ii) decreased longevity; and (iii) reduced response to host odours. In G. palpalis gambiensis, NA caused (i) a knock-down effect; (ii) decreased or increased longevity depending on the dose; and (iii) reduced response to host stimuli. In both cases, NA did not affect the anthropophilic rate of activated insects. Overall, the most significant effects were observed with NA treated bloodmeals at a dose of 2000 µg/ml for mosquitoes and 50 µg/ml for tsetse flies. Although no mortality in mice was observed after 14 days of follow-up at oral doses of 3.8, 5.6, 8.4 and 12.7 g/kg, behavioural alterations were noticed at doses above 8 g/kg. CONCLUSION: This study revealed promising activity of NA on A. coluzzii and G. palpalis gambiensis but additional research is needed to assess field efficacy of neem products to be possibly integrated in vector control programmes.

Evaluation of herbal antimalarial MAMA decoction-amodiaquine combination in murine malaria model.

CONTEXT: Co-administration of amodiaquine with MAMA decoction (MD), an herbal antimalarial drug comprising the leaves of Mangifera indica L. (Anacardiaceae), Alstonia boonei De Wild (Apocynaceae), Morinda lucida Benth (Rubiaceae) and Azadirachta indica A. Juss (Meliaceae) was investigated. The practice of concurrent administration of herbal medicines with orthodox drugs is currently on the increase globally. OBJECTIVE: The study was designed to investigate the possible enhancement of the antimalarial potency as well as possible herb-drug interaction resulting from concurrent administration of MAMA decoction with amodiaquine (AQ). MATERIALS AND METHODS: Combinations of MD with AQ were investigated in chloroquine (CQ)-sensitive Plasmodium berghei NK 65 in varying oral doses (mg/kg) at: sub-therapeutic [MD30 + AQ1.25], therapeutic [MD120 + AQ10] and median effective [MD40 + AQ3.8], using chemosuppressive and curative antimalarial test models. Secondly, P. berghei ANKA (CQ-resistant)-infected mice were orally treated with MD 120, 240, [MD120 + AQ10] and [MD240 + AQ10] mg/kg, using both models. The survival times of mice were monitored for 28 d. RESULTS: ED50 values of MD and AQ were 48.8 and 4.1 mg/kg, respectively. A total parasite clearance of CQ-sensitive P. berghei NK65 was obtained with the therapeutic combination dose in the curative test giving an enhanced survival time. In CQ-resistant P. berghei ANKA-infected mice, [MD120 + AQ10] and [MD240 + AQ10] mg/kg gave comparable activities with AQ (10 mg/kg) in both models. CONCLUSION: The therapeutic combination dose gave total parasite clearance of CQ-sensitive P. berghei NK65, whereas none of the doses tested showed notable activity against CQ-resistant P. berghei ANKA.

Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase.

Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples.