Evaluation of two sexual-stage antigens as bivalent transmission-blocking vaccines in rodent malaria.

BACKGROUND: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens used individually. METHODS: A chimeric protein composed of fragments of Pbg37 and PSOP25 was designed and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with individual recombinant proteins Pbg37 and PSOP25, mixed proteins (Pbg37+PSOP25), or the fusion protein (Pbg37-PSOP25), the antibody titers of individual sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins in the parasite. The transmission-blocking activity of the different immunization schemes was assessed using in vitro and in vivo assays. RESULTS: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote, and ookinete stages. The mixed proteins or the fusion protein induced antibodies with significantly stronger transmission-reducing activities in vitro and in vivo than individual antigens. CONCLUSIONS: There was no immunological interference between Pbg37 and PSOP25. The bivalent vaccines, which expand the portion of the sexual development during which the transmission-blocking antibodies act, produced significantly stronger transmission-reducing activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

Intervention of standardized ethanol leaf extract of Annickia polycarpa, (DC.) Setten and Maas ex I.M. Turner. (Annonaceae), in Plasmodium berghei infested mice produced anti-malaria action and normalized gross hematological indices.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a global public health burden due to large number of annual infections and casualties caused by its hematological complications. The bark of Annickia polycarpa is an effective anti-malaria agent in African traditional medicine. However, there is no standardization parameters for A. polycarpa. The anti-malaria properties of its leaf are also not known. AIM OF THE STUDY: To standardize the ethanol leaf extract of A. polycarpa (APLE) and investigate its anti-malaria properties and the effect of its treatment on hematological indices in Plasmodium berghei infected mice in the Rane's test. MATERIALS AND METHODS: Malaria was induced by inoculating female ICR mice with 1.0 × 107P. berghei-infected RBCs in 0.2 mL (i.p.) of blood. Treatment was commenced 3 days later with APLE 50, 200, 400 mg/kg p.o., Quinine 30 mg/kg i.m. (Standard drug) or sterile water (Negative control) once daily per group for 4 successive days. Anti-malarial activity and gross malaria indices such as hyperparasitemia, mean change in body weight and mean survival time (MST) were determined for each group. Changes in white blood cells (WBCs), red blood cells (RBCs), platelets (PLT) counts, hemoglobin (HGB) concentration, hematocrit (HCT) and mean corpuscular volume (MCV) were also measured in the healthy mice before infection as baseline and on day 3 and 8 after inoculation using complete blood count. Standardization was achieved by UHPLC-MS chemical fingerprint analysis and quantitative phytochemical tests. RESULTS: APLE, standardized to its total alkaloids, phenolics and saponin contents, produced significant (P < 0.05) dose-dependent clearance of mean hyperparasitemia of 22.78 ± 0.93% with the minimum parasitemia level of 2.01 ± 0.25% achieved at 400 mg/kg p.o. on day 8. Quinine 30 mg/kg i.m. achieved a minimum parasitemia level of 6.15 ± 0.92%. Moreover, APLE (50-400 mg/kg p.o.) evoked very significant anti-malaria activity of 89.22-95.50%. Anti-malaria activity of Quinine 30 mg/kg i.m. was 86.22%. APLE also inverse dose-dependently promotes weight gain with the effect being significant (P < 0.05) at 50 mg/kg p.o. Moreover, APLE dose-dependently increased the MST of malaria infested mice with 100% survival at 400 mg/kg p.o. Quinine 30 mg/kg i.m. also produce 100% survival rate but did not promote (P > 0.05) weight gain. Hematological studies revealed the development of leukocytopenia, erythrocytosis, microcytic anemia and thrombocytopenia in the malaria infected mice which were reverted with the treatment of APLE 50-400 mg/kg p.o. or Quinine 30 mg/kg i.m. but persisted in the negative control. The UHPLC-MS fingerprint analysis of APLE led to identification of one oxoaporphine and two aporphine alkaloids (1-3). Alkaloids 1 and 3 are being reported in this plant for the first time. CONCLUSION: These results indicate that APLE possessed significant anti-malaria, immunomodulatory, erythropoietic and hematinic actions against malaria infection. APLE also has the ability to revoke deleterious physiological alteration produced by malaria and hence, promote clinical cure. These properties of APLE are due to its constituents especially, aporphine and oxoaporphine alkaloids.

Potential antimalarial activity of Coccinia barteri leaf extract and solvent fractions against Plasmodium berghei infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Coccinia barteri (Hook. F.) is traditional used in Southeast of Nigeria in management of fever. This study aimed to evaluate the antimalarial activities of hydro-methanol crude extract and solvent fractions of Coccinia barteri leaf. MATERIALS AND METHODS: Two animal models employed for the study were, 4-day suppressive and curative assays against chloroquine sensitive Plasmodium berghei NK65. Level of parasitaemia, mean survival time (MST), anal temperature and weight loss were measured to assess antimalarial efficacy of the extract/fractions. Chloroquine (10 mg kg-1) was used as positive control. Chemo-profile of extract was evaluated using GC-MS, HPLC techniques and standard phytochemical analysis. Preliminary toxicity test was done using modified Lorke's method. RESULTS: The crude extract (100-400 mg kg-1) and solvent fractions (20-80 mg kg-1) demonstrated antimalarial activity in both models compared to controls. Semi purified fractions of the extract produced stronger percentage chemosuppression and inhibition of parasite. The % inhibition of the fractions, hexane, chloroform, ethyl acetate and aqueous at 80 mg kg-1 were 96.0 0, 95.29, 89.86 and 96.00% respectively on day 8 (D8). While on D14, 100% parasite clearance, indicating cure was obtained for hexane, chloroform and aqueous fraction treatment groups, no death occurred in these groups. Ethyl acetate fraction treated groups lived longer but were not fully protected. Some marker compounds were identified. CONCLUSIONS: These results support the use of C. barteri as malaria remedy and potential source of antimalarial templates. Long acting parasitaemia reduction effect indicates its possible combination potential in poly-herbal combination therapy.

Peyer's patch-immunomodulating glucans from sugar cane enhance protective immunity through stimulation of the hemopoietic system.

The aim of the present study was chemical clarification of in vitro Peyer's patch-immunomodulating polysaccharides in sugar cane molasses, and evaluation of in vivo modulating activity on immune function of T lymphocytes in Peyer's patches and on microenvironment of hemopoietic system. Five kinds of glucans, comprising of dextranase-sensitive and activity-related d-glucosyl moieties, were purified as in vitro Peyer's patch-immunomodulating polysaccharides from the molasses. Oral administration of a glucan-enriched subfraction induced IL-2 and GM-CSF-producing T lymphocytes in Peyer's patches, resulting in enhancement of IL-6 production in a hemopoietic microenvironment to boost neutrophil numbers in the peripheral blood stream. Oral administration of purified glucan or glucan-enrich sub-fraction of sugar cane reduced the number of Plasmodium berghei- or P. yoelii-infected erythrocytes in a murine infection model, using polysaccharide alone or via co-administration with the antimalarial drug, artesunate. These results suggested that Peyer's patch-immunomodulating glucans enhanced protective immunity through axis of Peyer's patches-hemopoietic system.

Development Insights of Surface Modified Lipid Nanoemulsions of Dihydroartemisinin for Malaria Chemotherapy: Characterization, and in vivo Antimalarial Evaluation.

BACKGROUND: The use of dihydroartemisinin (DHA) for effective malaria treatment is challenged by its poor aqueous solubility and inadequate bioavailability leading to treatment failures and emergence of resistant strains. A review of some novel drug delivery systems developed to address these challenges and their patents revealed that no study has reported the application of surface modified lipid nanoemulsions for improved antimalarial activity of DHA. OBJECTIVE: The main thrust of this study is to develop oral dihydroartemisinin formulations solubilized in surface modified lipid nanoemulsions, characterize, and evaluate their activity against murine malaria. METHOD: Lipid nanoemulsions containing dihydroartemisinin were formulated by high pressure homogenization using soybean oil, and polyethylene glycol 4000 was employed for surface modification. The formulations were characterized for droplet size, surface charge, pH, fouriertransform infrared spectroscopy, and surface morphology, viscosity and drug content efficiency. In vitro haemolytic study as a function of cytotoxicity using red blood cells as well as in vivo anti-malarial study using murine malaria model was also investigated. RESULTS: Nanoemulsions recorded droplet sizes ranging from 26 - 56 nm, and zeta potential in the range of -28 to -35 mV. The formulations were slightly acidic (pH 4.4 - 5.8) with the drug molecularly dispersed as seen using infrared spectroscopy. The formulations showed non- Newtonian flow with significant drug content efficiency in the range of 77-96%. The formulations did not induce haemolysis of cells and showed good clearance of parasitaemia. CONCLUSION: Surface-modified lipid nanoemulsion is a perfect carrier system for improving the anti-malarial activity of dihydroartemisinin.

Zingiber Officinale Roscoe and Echinops Kebericho Mesfin Showed Antiplasmodial Activities against Plasmodium Berghei in a Dose-dependent Manner in Ethiopia.

BACKGROUND: The emergence and spread of Plasmodium falciparum resistance to antimalarial drugs necessitated the search for new drugs from natural products. Zingiber officinal Roscoe and Echinops Kebericho Mesfin are traditional herbal medicines widely used for the treatment of malaria in Ethiopia. The aim of the study was to assess the toxicity profile and in vivo antiplasmodial activities of 70% methanol crude extracts of both plant materials against Plasmodium berghei. METHODS: Healthy male Swiss Albino mice of age 4-5 weeks and weight 25-36 g were infected by P. berghei. The extracts were administered orally at doses 5000, 2500 and 1250 mg/kg for acute toxicity of E. kebericho Mesfin. Graded doses at 1000, 500 and 250 mg/kg used for four days suppressive studies. Parasitemia, body weight, packed cell volume (PCV) and survival time were determined. SPSS Version 20 was used for the analysis of data of parasitemia, body weight, PCV, and survival times. Statistical significance was determined by one-way ANOVA. Independent t-test was used to compare results. Results were presented as a mean ± standard error of the mean (M ± SEM). All data were analyzed at a 95% confidence interval (α= 0.05). RESULTS: At the dose of 5000 mg/kg, E. kebericho Mesfin showed no toxic effects. The LD50 of extract could go beyond the dose used. In vivo antiplasmodial activity of extracts showed excellent chemo suppression at 500 and 1000 mg/kg in a dose dependent manner compared with the negative control. The chemo suppressions of the 1000 mg/kg of both plant extracts were 49.53 ± 1.90% and 32.83 ± 1.03%, respectively. The survival times of P. berghei infected mice were also a dose dependent manner while failed to prevent weight loss. CONCLUSION: The extracts of both medicinal plants showed antiplasmodial activities against P. berghei. It confirmed the literature findings and their traditional uses.

In vitro antiplasmodial activity and prophylactic potentials of extract and fractions of Trema orientalis (Linn.) stem bark.

BACKGROUND: Trema orientalis (T. orientalis Linn) has been used in the management of malaria in the western part of Nigeria and despite its application in ethnomedicine, there is dearth of scientific evidence to justify the acclaimed prophylactic antimalarial usage of the plant. The aim of this study is to assess the in vitro antiplasmodial cell-free assay and chemopreventive efficacy of the methanol extract of the stem bark of T. orientalis and its fractions as a prophylactic regimen for malaria prevention. Also, the antimicrobial activities of the extract and the fractions were investigated. METHOD: Vacuum liquid chromatography was used to obtain dichloromethane, ethylacetate and methanol fractions from the methanol extract of T. orientalis. The fractions were tested for their prophylactic and cell-free antimalarial activity using murine models and ß-hematin formation assay respectively. Disc diffusion method was used to determine the antibacterial activity of the extract and its fractions against both Gram-positive and Gram-negative bacteria. RESULTS: In the prophylactic experiment, dichloromethane (DCMF), methanol fraction (MF) and extract (ME) (in this order) showed significant chemopreventive effects against P. berghei invasion of the red blood cells when compared with both Sulfadoxine-Pyrimethamine (SP) and untreated controls. Results of the in vitro study showed that the DCMF had the highest effect in preventing the formation of ß-hematin when compared with other fractions. The DCMF also had the highest percentage inhibition of ß-hematin formation when compared with chloroquine. The extract and fractions showed a concentration dependent antibacterial activity. Methanol extract had a pronounced inhibitory effect on Enterobacter cloaca ATCC 13047 and Enterococcus faecalis ATCC 29212. Serratia mercescens ATCC 9986 and Pseudomonas aeruginosa ATCC 19582 were the most susceptible bacteria. CONCLUSION: The results obtained showed that both extract and fractions of T. orientalis possessed antiplasmodial and antimicrobial activity.

Antimalarial and antiplasmodial activity of husk extract and fractions of Zea mays.

CONTEXT: Zea mays L. (Poacae) husk decoctions are traditionally used in the treatment of malaria by various tribes in Nigeria. OBJECTIVE: To assess the antimalarial and antiplasmodial potentials of the husk extract and fractions on malaria parasites using in vivo and in vitro models. MATERIALS AND METHODS: The ethanol husk extract and fractions (187-748 mg/kg, p.o.) of Zea mays were investigated for antimalarial activity against Plasmodium berghei using rodent (mice) malaria models and in vitro activity against chloroquine sensitive (Pf 3D7) and resistant (Pf INDO) strains of Plasmodium falciparum using the SRBR green assay method. Median lethal dose and cytotoxic activities against HeLa and HEKS cells were also carried out. The GCMS analysis of the most active fraction was carried out. RESULTS: The husk extract (187-748 mg/kg, p.o.) with LD50 of 1874.83 mg/kg was found to exert significant (p < 0.05-0.001) antimalarial activity against P. berghei infection in suppressive, prophylactive and curative tests. The crude extract and fractions also exerted prominent activity against both chloroquine sensitive (Pf 3D7) and resistant (Pf INDO) strains of P. falciparum with the ethyl acetate fraction exerting the highest activity with IC50 values of 9.31 ± 0.46 µg/mL (Pf 3D7) and 3.69 ± 0.66 µg/mL (Pf INDO). The crude extract and fractions were not cytotoxic to the two cell lines tested with IC50 values of >100 µg/mL against both HeLa and HEKS cell lines. DISCUSSION AND CONCLUSION: These results suggest that the husk extract/fractions of Zea mays possesses antimalarial and antiplasmodial activities and these justify its use in ethnomedicine to treat malaria infections.

Antiplasmodial activity of Heinsia crinita (Rubiaceae) and identification of new iridoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Heinsia crinita is used in traditional medicine for the treatment of febrile illness and erectile dysfunction. Its stem bark powder is found in some peripheral markets in the Democratic Republic of the Congo (DRC) as a remedy against malaria. Investigations were conducted on crude extracts of leaves, fruits and stem barks in view to validate their use and to determine which plant part possesses the best antiplasmodial properties. MATERIALS AND METHODS: Different plant parts were extracted with methanol, ethanol and dichloromethane. Based on the preliminary assays, the dichloromethane extract of the stem bark was subjected to fractionation using preparative HPLC system and column chromatography. This step led to the isolation of two new iridoids which had their structures elucidated by NMR, UV, MS and FT-IR spectroscopic techniques. Extracts and pure compounds were tested in vitro against the 3D7 strain of Plasmodium falciparum. The inhibition of the parasite growth was evaluated in vitro by colorimetric method (p-LDH assay) and their cytotoxicity evaluated in vitro against the human non-cancer fibroblast cell line (WI38) through WST1 assay. The in vivo antiplasmodial activity was assessed by the inhibition of Plasmodium berghei growth in infected mice treated with the ethanol extract of H. crinita stem bark at the concentrations of 200 and 300mg/Kg/day per os, using a protocol based on the 4-d suppressive test of Peters and compared to a non-treated negative control group of mice (growth =100%). Finally the antioxidant activity of the same extract was evaluated using ABTS, DPPH and cell-based assays. RESULTS: A moderate in vitro antiplasmodial activity was observed for the dichloromethane extract of the stem bark of H. crinita (IC50 =29.2±1.39µg/mL) and for the two new iridoids, lamalbide 6, 7, 8- triacetate (IC50 =16.39±0.43µg/mL) as well as for its aglycone lamiridosin 6, 7, 8-triacetate (IC50 =0.44.56±1.12µg/mL). The ethanolic stem bark extract (200 and 300mg/kg/day, oral route) showed a moderate in vivo antimalarial activity in Plasmodium berghei-infected mice with 27.84±2.75% and 48.54±3.76% of inhibition of the parasite growth, respectively (p<0.01).). This extract displayed high cellular antioxidant activity using dichlorofluorescein-diacetate (DCFDA) on HL-60 monocytes. These crude extracts and pure compounds tested at the higher concentration of 100µg/mL did not show any cytotoxicity against WI38 cells. CONCLUSIONS: The results showed that H. crinita extracts possess antimalarial activity and contain some unusual iridoids with moderate antiplasmodial activity, therefore justifying to some extent its traditional use by the local population in DRC for this purpose. This is the first report of the isolation and antiplasmodial activity of these two new iridoids.

The Effects of Transdermally Delivered Oleanolic Acid on Malaria Parasites and Blood Glucose Homeostasis in P. berghei-Infected Male Sprague-Dawley Rats.

The present study investigated the effects of transdermally delivered oleanolic acid (OA) monotherapy and in combination with chloroquine (CHQ) on malaria parasites and glucose homeostasis of P. berghei-infected male Sprague-Dawley rats. Oral glucose test (OGT) responses to OA-pectin patch and CHQ-OA combination matrix patch were monitored in non-infected and infected rats. To evaluate the short-term effects of treatment, percentage parasitaemia, blood glucose, glycogen and plasma insulin were monitored in separate groups of animals treated with either OA-patch monotherapy or CHQ-OA combination pectin patch over a 21-days period. Animals treated with drug-free pectin and CHQ acted as untreated and treated positive controls, respectively. Infected control rats exhibited significantly increased parasitaemia which was accompanied by hypoglycaemia. Both OA monotherapy and CHQ-OA combination therapy reduced and cleared the malaria parasites within a period of 4 and 3 days, respectively. Compared to respective controls groups, OGT responses of animals treated with OA monotherapy or CHQ-OA combination therapy exhibited lower blood glucose levels at all time points. A once-off transdermal application of OA-patch or CHQ-OA combination patch significantly improved blood glucose concentrations inducing any changes in insulin concentration. Transdermal OA used as a monotherapy or in combination with CHQ is able to clear and reduce the malaria parasites within a shorter period of time without eliciting any adverse effects on glucose homeostasis of P. berghei-infected rats.

Natural products from Zanthoxylum heitzii with potent activity against the malaria parasite.

BACKGROUND: Zanthoxylum heitzii (Rutaceae) (olon) is used in traditional medicine in Central and West Africa to treat malaria. To identify novel compounds with anti-parasitic activity and validate medicinal usage, extracts and compounds isolated from this tree were tested against the erythrocytic stages of the human malaria parasite Plasmodium falciparum and for inhibition of transmission in rodent malaria parasite Plasmodium berghei. RESULTS: Hexane bark extract showed activity against P. falciparum (IC50 0.050 µg/ml), while leaf and seed extracts were inactive. Fractionation of the hexane bark extract led to the identification of three active constituents; dihydronitidine, pellitorine and heitziquinone. Dihydronitidine was the most active compound with an IC50 value of 0.0089 µg/ml (25 nM). This compound was slow acting, requiring 50 % longer exposure time than standard anti-malarials to reach full efficacy. Heitziquinone and pellitorine were less potent, with IC50 values of 3.55 µg/ml and 1.96 µg/ml, but were fast-acting. Plasmodium berghei ookinete conversion was also inhibited by the hexane extract (IC50 1.75 µg/ml), dihydronitidine (0.59 µg/ml) and heitziquinone (6.2 µg/ml). Water extracts of Z. heitzii bark contain only low levels of dihydronitidine and show modest anti-parasitic activity. CONCLUSIONS: Three compounds with anti-parasitic activity were identified in Z. heitzii bark extract. The alkaloid dihydronitidine is the most effective of these, accounting for the bulk of activity in both erythrocytic and transmission-blocking assays. These compounds may present good leads for development of novel anti-malarials and add to the understanding of the chemical basis of the anti-parasitic activity in these classes of natural product.

Antimalarial actions of Lawsonia inermis, Tithonia diversifolia and Chromolaena odorata in combination.

ETHNOPHARMACOLOGICAL RELEVANCE: Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis are medicinal plants used in treating malaria in traditional medicine system. Previous studies however showed that their dichloromethane, methanol (1:1) extracts were more active against Plasmodium parasite than the aqueous extracts. AIM OF THE STUDY: To determine the in vitro and in vivo antiplasmodial activity of dichloromethane, methanol (1:1) extracts of Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis in combination and evaluate their safety using acute limit toxicity test. MATERIALS AND METHODS: Dichloromethane, methanol (1:1) extracts of Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis leaves were combined at ratios 1:1, 1:3, 3:1, 1:5 and 5:1 using in vitro semi-automated microdilution technique against P. falciparum Chloroquine sensitive (D6) and Chloroquine resistant (W2) strains, with chloroquine and artemisinin as controls. The in vivo antiplasmodial activity of the crude extracts was carried out singly, and in combination at the different combination ratios on Plasmodium berghei Anka infected Swiss albino mice using Peters' 4-day suppressive test. Acute toxicity test was done in mice at 5000mg/kg. RESULTS: The in vitro combination of L. inermis and T. diversifolia (1:1) extracts against P. falciparum showed the highest synergy with IC50 of 0.43±0.02µg/mL and 2.55±0.19µg/mL against D6 and W2 respectively; while the combination of C. odorata with T. diversifolia and L. inermis were antagonistic. A synergy with chemosuppression of 83.6% against P. berghei infected mice was observed in L. inermis and T. diversifolia (1:1) treated animals. In contrast to the in vitro result, combination of C. odorata with T. diversifolia and L. inermis showed some degrees of synergy in vivo. Extracts were not toxic at the concentration tested. CONCLUSION: These findings rationalized the use of these plants in combination as antimalarials in traditional medicine. However, the combination of Chromolaena odorata with other medicinal plants should be used with caution because of its possible antagonistic effect.

Landolphia owariensis leaf extracts reduce parasitemia in Plasmodium berghei-infected mice.

Context Landolphia owariensis P. Beauv. (Apocyanaceae) leaf is used in southeast Nigeria to treat malaria. Objective This study evaluated the antiplasmodial activity of L. owariensis leaf extract and fractions, also the phytoconstituents were standardized and analyzed. Methods The effects of daily, oral administrations of 200, 400 and 800 mg/kg of L. owariensis leaf extract (LOE), its hexane (LOHF), ethyl acetate (LOEF) and methanol (LOMF) fractions on early, established and residual infections in Plasmodium berghei-infected albino mice were evaluated in vivo. The extract and fractions were subjected to phytochemical analysis and HPLC fingerprinting, and the acute toxicity of LOE was evaluated. Results The extract and fractions elicited 29-86, 18-95 and 75-96% significant (p < 0.001) suppression of parasitemia in early, established and residual infections, respectively. The ED50 values for suppressive activity of LOE, LOHF, LOEF and LOMF were 266.56, 514.93, 392.95 and 165.70 mg/kg, respectively. The post-day 30-survival index was 16.7-50, 16.7, 16.7-66.7 and 50-83.3% for LOE, LOHF, LOEF, and LOMF, respectively. Extract-treated mice significantly (p < 0.001) gained weight and had reduced mortality compared with negative control (untreated) mice. An oral LD50 value >5000 mg/kg in mice was established for LOE. The LOMF showed the greatest antiplasmodial activity in all the models, suggesting that the antimalarial activity of the plant may be attributed to alkaloids, flavonoids, saponins and tannins present in the fraction. Conclusion Results demonstrate the antiplasmodial activity of L. owariensis leaf, and provide a pharmacological rationale for its ethnomedicinal use as an antimalarial agent.

Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal rare microhomology-mediated end joining.

BACKGROUND: Genome editing of malaria parasites is key to the generation of live attenuated parasites used in experimental vaccination approaches. DNA repair in Plasmodium generally occurs only through homologous recombination. This has been used to generate transgenic parasites that lack one to three genes, leading to developmental arrest in the liver and allowing the host to launch a protective immune response. While effective in principle, this approach is not safe for use in humans as single surviving parasites can still cause disease. Here we use zinc-finger nucleases to generate attenuated parasite lines lacking an entire chromosome arm, by a timed induction of a double-strand break. Rare surviving parasites also allow the investigation of unconventional DNA repair mechanisms in a rodent malaria parasite. RESULTS: A single, zinc-finger nuclease-induced DNA double-strand break results in the generation of attenuated parasite lines that show varying degrees of developmental arrest, protection efficacy in an immunisation regime and safety, depending on the timing of zinc-finger nuclease expression within the life cycle. We also identify DNA repair by microhomology-mediated end joining with as little as four base pairs, resulting in surviving parasites and thus breakthrough infections. CONCLUSIONS: Malaria parasites can repair DNA double-strand breaks with surprisingly small mini-homology domains located across the break point. Timely expression of zinc-finger nucleases could be used to generate a new generation of attenuated parasite lines lacking hundreds of genes.

ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development.

The malaria parasite, Plasmodium, requires iron for growth, but how it imports iron remains unknown. We characterize here a protein that belongs to the ZIP (Zrt-, Irt-like Protein) family of metal ion transport proteins and have named ZIP domain-containing protein (ZIPCO). Inactivation of the ZIPCO-encoding gene in Plasmodium berghei, while not affecting the parasite's ability to multiply in mouse blood and to infect mosquitoes, greatly impairs its capacity to develop inside hepatocytes. Iron/zinc supplementation and depletion experiments suggest that ZIPCO is required for parasite utilization of iron and possibly zinc, consistent with its predicted function as a metal transporter. This is the first report of a ZIP protein having a crucial role in Plasmodium liver-stage development, as well as the first metal ion transporter identified in Plasmodium pre-erythrocytic stages. Because of the drastic dependence on iron of Plasmodium growth, ZIPCO and related proteins might constitute attractive drug targets to fight against malaria.

Azadirachta indica ethanolic extract protects neurons from apoptosis and mitigates brain swelling in experimental cerebral malaria.

BACKGROUND: Cerebral malaria is a rapidly developing encephalopathy caused by the apicomplexan parasite Plasmodium falciparum. Drugs currently in use are associated with poor outcome in an increasing number of cases and new drugs are urgently needed. The potential of the medicinal plant Azadirachta indica (Neem) for the treatment of experimental cerebral malaria was evaluated in mice. METHODS: Experimental cerebral malaria was induced in mice by infection with Plasmodium berghei ANKA. Infected mice were administered with Azadirachta indica ethanolic extract at doses of 300, 500, or 1000 mg/kg intraperitoneally (i.p.) in experimental groups, or with the anti-malarial drugs chloroquine (12 mg/kg, i.p.) or artemether (1.6 mg/kg, i.p.), in the positive control groups. Treatment was initiated at the onset of signs of brain involvement and pursued for five days on a daily basis. Mice brains were dissected out and processed for the study of the effects of the extract on pyramidal cells' fate and on markers of neuroinflammation and apoptosis, in the medial temporal lobe. RESULTS: Azadirachta indica ethanolic extract mitigated neuroinflammation, decreased the severity of brain oedema, and protected pyramidal neurons from apoptosis, particularly at the highest dose used, comparable to chloroquine and artemether. CONCLUSIONS: The present findings suggest that Azadirachta indica ethanolic extract has protective effects on neuronal populations in the inflamed central nervous system, and justify at least in part its use in African and Asian folk medicine and practices.

Brine shrimp toxicity and antimalarial activity of some plants traditionally used in treatment of malaria in Msambweni district of Kenya.

ETHNOPHARMACOLOGICAL RELEVANCE: In Kenya, most people especially in rural areas use traditional medicine and medicinal plants to treat many diseases including malaria. Malaria is of national concern in Kenya, in view of development of resistant strains of Plasmodium falciparum to drugs especially chloroquine, which had been effective and affordable. There is need for alternative and affordable therapy. Many antimalarial drugs have been derived from medicinal plants and this is evident from the reported antiplasmodial activity. AIM OF THE STUDY: The present study reports on the in vivo antimalarial activity and brine shrimp lethality of five medicinal plants traditionally used to treat malaria in Msambweni district, Kenya. MATERIALS AND METHODS: A total of five aqueous crude extracts from different plant parts used in traditional medicine for the treatment of malaria were evaluated for their in vivo antimalarial activity using Plasmodium berghei infected Swiss mice and for their acute toxicity using Brine shrimp lethality test. RESULTS: The screened crude plant extracts suppressed parasitaemia as follows: Azadirachta indica (L) Burm. (Meliaceae), 3.1%; Dichrostachys cinerea (L) Wight et Arn (Mimosaceae), 6.3%; Tamarindus indica L. (Caesalpiniaceae), 25.1%; Acacia seyal Del. (Mimosaceae) 27.8% and Grewia trichocarpa Hochst ex A.Rich (Tiliaceae) 35.8%. In terms of toxicity, A.indica root bark extract had an LC50 of 285.8 µg/ml and was considered moderately toxic. T.indica stem bark extract and G.trichocarpa root extract had an LC50 of 516.4 and 545.8 µg/ml respectively and were considered to be weakly toxic while A.seyal and D.cinerea root extracts had a LC50>1000 µg/ml and were therefore considered to be non toxic. CONCLUSIONS: The results indicate that the aqueous extracts of the tested plants when used alone as monotherapy had antimalarial activity which was significantly different from that of chloroquine (P≤0.05). The results also suggest that the anecdotal efficacy of the above plants reported by the study community is related to synergism of phytoconstituents since the assayed plant parts are used in combination with others to treat malaria. It is also evident that none of the screened plant extracts is toxic to the arthropod invertebrate, Artemia salina L. (Artemiidae) larvae, justifying the continued use of the plant parts to treat malaria. A.seyal, G.trichocarpa and T.indica have not been reported before for in vivo antimalarial activity and brine shrimp lethality.

In vivo antimalarial activity of Keetia leucantha twigs extracts and in vitro antiplasmodial effect of their constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: The West African tree Keetia leucantha (Rubiaceae) is used in traditional medicine in Benin to treat malaria. The twigs dichloromethane extract was previously shown to inhibit in vitro Plasmodium falciparum growth with no cytotoxicity (>100µg/ml on human normal fibroblasts). MATERIALS AND METHODS: The dichloromethane and aqueous extracts of twigs of K. leucantha were evaluated in vivo against Plasmodium berghei NK 173 by the 4-day suppressive test and in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity. Bioguided fractionations were realized and compounds were structurally elucidated using extensive spectroscopic analysis. RESULTS: The in vivo antimalarial activity of K. leucantha dichloromethane and aqueous twigs extracts were assessed in mice at the dose of 200mg/kg/day. Both extracts exhibited significant effect in inhibiting parasite growth by 56.8% and 53.0% (p<0.0001) on day 7-postinfection. An LC-MS analysis and bioguided fractionations on the twigs dichloromethane extract led to the isolation and structural determination of scopoletin (1), stigmasterol (2), three phenolic compounds: vanillin (3), hydroxybenzaldehyde (4) and ferulaldehyde (5), eight triterpenic esters (6-13), oleanolic acid and ursolic acid. The antiplasmodial activity of the mixture of the eight triterpenic esters showed an antiplasmodial activity of 1.66 ± 0.54 µg/ml on the 3D7 strain, and the same range of activity was observed for isolated isomers mixtures. CONCLUSIONS: This is the first report on the in vivo activity of K. leucantha extracts, the isolation of thirteen compounds and analysis of their antiplasmodial activity. The results obtained may partially justify the traditional use of K. leucantha to treat malaria in Benin.

Liver-stage malaria parasites vulnerable to diverse chemical scaffolds.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite's life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

Antimalarial potential of homeopathic medicines against schizont maturation of Plasmodium berghei in short-term in vitro culture

In vitro assessment of antimalarial drug susceptibility of Plasmodium has been a major research success, which has paved the way for the understanding of parasite and rapid screening of antimalarial drugs for their effectiveness. In the present study a preliminary screening to check the antiplasmodial activity of mother tincture (ϕ) and various potencies (6C, 30C, 200C) of homeopathic medicines Cinchona officinalis/china (Chin.), Chelidonium majus (Chel.) and Arsenicum album (Ars.) were done by assessing the in vitro schizont maturation inhibition assay. A significant reduction in the growth of intraerythrocytic stages of P. berghei was observed with decreasing dilution of ϕ and various potencies of Chin., Chel. and Ars. exhibiting a dose dependent effect. Maximum schizont maturation inhibition was observed by Chin. ϕ (1:1), Chin. 30 (1:1, 1:2) and Chel. 30 (1:1) i.e. 80%. The standard drug CQ at 10 µM concentration exhibited 95.4±1.6% inhibition of schizont maturation. Ars. 30 (1:1) also have been found to possess strong antiplasmodial efficacy with 75.5±2.6% schizont inhibition. The presence of free merozoites in Ars. 200 with weak schizonticidal inhibition activity (40-45%) also pointed towards the ability of parasite to survive in the given drug pressure.

O estudo in vitro da susceptibilidade de Plasmodium a drogras antimaláricas representa um grande avanço nas pesquisas, abrindo novas rotas para o entendimento do parasite e da efetividade de drogas antiomaláricas. Nesse trabalho, realizamos um estudo preliminar da atividade antiplasmódica da tintura mãe (ϕ) e várias potências (6 cH, 30 cH, 200 cH) dos medicamentos homeopáticos China officinalis (Chin), Chelidonium majus (Chel) e Arsenicum album (Ars), através do estudo in vitro da inibição da maturação de esquizontes. Observamos uma redução significativa do crescimento do estágio intra-eritrócito do P. berghei conforme a tintura mãe e demais potências de Chin, Chel e Ars foram diluídas, observando-se um efeito dependente da dose. O máximo de inibição na maturação dos esquizontes (80%) foi observado com Chin ϕ (1:1), Chin 30 cH (1:1, 1:2) and Chel 30 cH (1:1). A droga Cloroquina (CQ), usada como controle, em uma concentração de 10µM, exibiu (95.4 ± 1.6) % de inibição. Ars 30cH (1:1) também apresentou uma forte eficácia antiplasmódica com (75.5 ± 2.6) % de inibição de esquizontes. A presence de merozoites livres com Ars 200 cH e uma fraca atividade inibidora (40-45%) indicam a habilidade do parasita em sobreviver na presença dessa droga.