Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Bioactivity-directed evaluation of fruit of Kigelia africana (Lam.) benth. Used in treatment of malaria in Iwo, Nigeria.

ETHNOPHARMACOLOGICAL RELEVANCE: The ancient people of Iwo communities consisting of Ile-Ogbo, Olupona, Iwo and Ogbagba continue to engage in the traditional use of medicinal plants for the treatment and management of common diseases especially malaria. AIMS OF THIS STUDY: This study conducted an ethnomedicinal survey of plants used to treat malaria and feverish conditions by the people of Iwo, Nigeria. It also evaluated the antiplasmodial activity of the morphological parts of Kigelia africana (Lam.) Benth., and isolated, as well as characterised pure compounds from the semi-purified fractions of the fruit extract. MATERIALS AND METHODS: The ethnomedicinal survey was conducted using semi-structured questionnaires administered to only herb sellers in Iwo, Ile-Ogbo, Olupona, and Ogbagba areas of Osun State. Extracts of K. africana morphological parts; leaf, root, stem bark, and fruit were obtained by cold maceration in methanol, followed by assessment of acute toxicity (LD50) and antiplasmodial activity in Plasmodium berghei infected rats using the 4-day suppressive test model. The most active fruit extract was further subjected to activity-guided fractionation and purification using n-hexane, dichloromethane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and methanol (MeOH) in gradients to obtain the semi-purified fractions and two pure isolated compounds using various chromatographic and spectroscopic techniques. RESULTS AND DISCUSSION: From the survey, thirty-one plant species were identified for treating malaria in Iwo area. Azadirachta indica leaf was the most frequently used (78.3% of the respondents) while Manihot esculenta leaf (3.33%) was the least. The identified plants are distributed among 24 families, with Anacardiaceae and Asteraceae (11.67% each) been the most occurring families. Kigelia africana (Bignoniaceae) ranked the 6th position with 60% frequency of occurrence. The LD50 values obtained for the extracts were greater than 5000 mg/kg (p.o). The chemo-suppression activity of the extracts at 125 mg/kg was in the order of stem bark (26.59%), leaf (41.75%), root (43.95%), and fruit (54.54%). The semi-purified methanol fraction of the fruit showed the most antiplasmodial activity with a percent chemo-suppression of 69.94 and yielded 4-(2,3-dihydroxypropoxy)-3,5-dihydroxy-5-methylfuran-2-one and sucrose. CONCLUSION: The use of herbs and medicinal plants either singly or in combination for the treatment of malaria among the people of Iwo community in Nigeria is still well practised. Lack of formal education among most of the respondents and use of same local name for different plants species or plant parts; which often lead to wrong plant collection were among the constrains encountered. Kigelia africana has antiplasmodial activity in the order of fruit > root > leaf > stem bark.

Comparative antimalarial, toxicity and mito-protective effects of Diospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels on Plasmodium berghei infection in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Diospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels are traditionally used in Africa for the treatment of malaria. However, scientific evidence to substantiate this folkloric claim and their effects on liver mitochondria during malaria treatment have not been reported. AIM OF THE STUDY: This study investigated the efficacy of D. mespiliformis and M. whitei against chloroquine-sensitive and resistant strains of malarial parasites in mice. It also investigated the toxicity and protection against cellular organelles like mitochondria. MATERIALS AND METHODS: Male Swiss mice were infected with a chloroquine resistant (ANKA) strain of Plasmodium berghei and were treated via oral gavage with methanol extracts of D. mespiliformis and M. whitei reconstituted in diluted dimethylsulfoxide as vehicle (DMSO, 5% v/v) for five consecutive days. Percentage parasite load and clearance were assessed by microscopy. The infected control was treated with the vehicle. Hematological indices were assessed using standard procedures. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined using assay kits. Hepatic mitochondria were isolated via centrifugation, and their permeability transition (mPT), ATPase (mATPase) activity and lipid peroxidation (mLPO) were determined spectroscopically. Liver tissue histology was carried out by standard laboratory procedures. Phytochemical analysis of both extracts were performed using LC-MS to identify the most prominent compounds from each of the extracts. RESULTS: After treatment on day 5, D. mespiliformis and M. whitei at 400 mg/kg decreased mean values for: percentage parasitemia (5.0 ± 1.0, 2.0 ± 0.2), increased Packed Cell Volume (PCV) (36.0 ± 1.4, 36.0 ± 0.0%) and platelets (2.0 ± 1.4, 2.0 ± 2.8 × 105mm3) relative to the untreated control (20.0 ± 5.2; 30.0 ± 0.0%; 1.4 ± 1.4 × 105 mm3, respectively). At the same dose, D. mespiliformis and M. whitei decreased ALT (8.0 ± 3.8, 24.2 ± 4.0U/L), AST (6.2 ± 0.8, 8.0 ± 0.9U/L) and ALP (56.0 ± 0.7, 51.0 ± 1.0U/L) activities compared to the infected control (77.0 ± 10.9U/L, 14.0 ± 0.7U/L and 76.0 ± 6.0U/L, respectively). Both D. mespiliformis and M. whitei reversed mPT opening, decreased mATPase enhancement and mLPO, relative to the control. Histopathology of the liver showed extensive hemorrhagic lesions and severe disseminated congestion in the infected control while both D. mespiliformis and M. whitei were well tolerated at the highest dose. The LC-MS analysis of D. mespiliformis showed the presence of betulinic acid, tocopherol and kaempferol with antimalarial and antioxidant properties while the M. whitei sample contained coumarin and chlorogenic acid that have antimalarial and hepato-protective properties. CONCLUSIONS: D.mespiliformis and M. whitei show antimalarial effects against resistant Plasmodium berghei infection, enhanced cell viability, mito-protection and are not toxic in mice.

Regulated rutin co-administration reverses mitochondrial-mediated apoptosis in Plasmodium berghei-infected mice.

Malarial infection causes apoptosis in hepatocytes. However, it is not known if co-administration of antimalarial drug with rutin will reverse the apoptotic effects of malarial infection. Plasmodium berghei-infected mice were assigned into groups as follows: groups I to III were treated with the vehicle (Parasitised Untreated, PU), 10 mg/kg body weight of Artesunate-Mefloquine (AM) and Dihydroartemisinin-Piperaquine (DP) respectively. Groups IV to VII were treated with AM, DP but co-administered with 100, 200 mg rutin/kg body weight while groups VIII and IX received rutin (100 and 200 mg/kg body weight). Liver mitochondrial Permeability Transition (mPT) and ATPase (mATPase) were determined spectrophotometrically. Caspases 3 and 9 were assayed using ELISA while the levels of bax, cytochrome c release (CCR), p53 and bcl-2 expressions were assayed immunohistochemically. The mPT pore opening fold of 5 (PU), 16 (AM), 14 (AM + 100 mg rutin/kg body weight), 9 (AM + 200 mg rutin/kg body weight), 4(DP), were observed relative to calcium (24) while DP, rutin and their combinations did not open the pore. AM and DP significantly increased caspases 3 and 9 activities, enhanced mATPase activity but co-treatment with rutin (100 mg/kg) decreased these effects significantly. AM + rutin (100 mg/kg body weight) significantly decreased bax, p53, CCR and increased bcl-2 expression. The results showed that supplementing malarial treatment with rutin decreased apoptosis suggesting that rutin supplementation can minimise apoptosis in malarial infection.

Antimalarial Activity of Hydroalcoholic Root Extract of Acanthus polystachyus Delile (Acanthaceae) Against Plasmodium berghei-Infected Mice.

Failure of the efficacy of antimalarial drugs is recognized in different classes of medicines for treating malaria, which urges the need for new drugs. This study tried to check the in vivo antimalarial activity of the root extracts of Acanthus polystachyus Delile against Plasmodium berghei-infected mice. The study revealed that the methanolic crude extract of the root of Acanthus polystachyus Delile showed significant (P < .01) parasitemia suppressive activities in both models compared with the negative control. Parasitemia suppressive activities were 25.26%, 33.46%, and 51.48% in a 4-day suppressive test and 23.31%, 31.20%, and 43.54% in prophylaxis test at 100, 200, and 400 mg/kg of the extract, respectively, as compared to the negative control. Besides, the extract increases mean survival time significantly in all tested doses in a 4-day suppressive test, but in the prophylaxis model, only mice treated with 200 and 400 mg/kg significantly lived longer. Based on this finding, the root of Acanthus polystachyus Delile has strong antimalarial activity, which may be a good candidate for new antimalarial agents.

Evaluation of Antimalarial Activity of the 80% Methanolic Stem Bark Extract of Combretum molle Against Plasmodium berghei in Mice.

BACKGROUND: People are dependent on the traditional use of medicinal plants for the treatment of malaria without scientific validations. Therefore, this study aimed to evaluate the antimalarial activity of methanolic stem bark extract of Combretum molle in mice. METHODS: After being infected with Plasmodium berghei, the mice were randomly divided into 5 groups (n = 5). In all cases, group I mice were treated as negative control and received 3% Tween 80; group II mice were treated with 25 mg/kg chloroquine; and groups III, IV, and V mice were treated with 100 mg/kg, 200 mg/kg, and 400 mg/kg of crude extract, respectively. Data were analyzed using one-way analysis of variance followed by Tukey's post hoc analysis. RESULTS: In the chemosuppressive test, the parasitic suppression effect of the crude extract was found to be significant (P < 0.05) as compared with the negative control. In the curative experiment, the average parasitic level of those mice treated by the 3 doses of the crude extract was significantly suppressed at days 5, 6, and 7 of treatment (P < 0.001). Besides, the crude extract had been found to have a chemoprophylactic role as it inhibited the parasite level significantly relative to the negative control (P < 0.001). Moreover, the crude extract had preventive effects on packed cell volume reduction in the 3 tests (P < 0.001). CONCLUSIONS: The findings of the present study has supported the folklore use of the leaves of Combretum molle in the treatment of malaria. Therefore, further fractionation and characterization of the crude extract is necessary to identify the responsible lead compound(s) responsible for antiplasmodial activity.

In vitro and in vivo studies on anti-malarial activity of Commiphora africana and Dichrostachys cinerea used by the Maasai in Arusha region, Tanzania.

BACKGROUND: Traditional medicinal plants are one of the potential sources of anti-malarial drugs and there is an increasing interest in the use and development of traditional herbal remedies for the treatment of malaria and other ailments. This study was carried out with the aim to investigate the phytochemical screening, cytotoxic effect and antiplasmodial activities of Dichrostachys cinerea and Commiphora africana. Both plants are used by the Maasai in Tanzania in suspected malaria and other diseases. No previous work appears to have investigated the potential anti-malarial activity of the two plants. METHODS: This study aimed to investigate the in vitro anti-malarial activity of methanol and dichloromethane extracts of the two plants against chloroquine sensitive (D6) and chloroquine resistant (Dd2) strains of Plasmodium falciparum. The anti-malarial property was assessed by the lactate dehydrogenase method (pLDH). The in vivo anti-malarial study was carried out using the Peters' 4-day suppressive test in Plasmodium berghei in Balb/c mice. Cytotoxic tests were carried out using monkey kidney epithelial cell line in [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Qualitative phytochemical screening was carried out using standard methods of analysis. RESULTS: The phytochemical screening of plant extracts revealed the presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, glycosides and saponins. However, alkaloids were absent in most plant extracts. The dichloromethane extracts of C. africana (stem bark); D. cinerea (stem bark) and methanol extracts of D. cinerea (whole stem) all showed promising in vitro anti-malarial activities. All other extracts did not show any significant anti-malarial activity. The two most promising extracts based on in vitro studies, DCM extracts of C. africana (stem bark) and D. cinerea (stems bark), equally exhibited very significant anti-malarial activities in the mouse model. They exhibited parasite suppression rates of 64.24 and 53.12%, respectively, and considerable improvement in weight and survival rate. Most plant extracts were not cytotoxic except for DCM extract of D. cinerea (whole stem) CC50 (29.44 µg/mL). CONCLUSION: The findings of this study provide scientific evidence supporting the traditional use of the plants in the treatment of malaria by the Maasai in Arusha region, Tanzania. Consequently, further work including bioassay-guided fractionation and advanced toxicity testing may yield new anti-malarial drug candidates from the two plants.

Hypoglycemia induced by Plasmodium berghei infection is prevented by treatment with Tinospora crispa stem extract.

During Plasmodium malaria parasite infection in a human, the intraerythrocytic stages lead to the clinical manifestations of the disease, especially hypoglycemia. Hypoglycemia is a recognized feature of severe malaria and linked with a high risk of mortality for children. Hence, the present study aimed to investigate the protective effect of T. crispa stem extract on hypoglycemia induced by P. berghei infection tested with a mouse model. ICR mice were inoculated with 1 × 107 parasitized erythrocytes of P. berghei ANKA (PbANKA) by intraperitoneal injection and given 50, 100, and 200 mg/kg of ethanolic extract for 4-consecutive days. The results showed that T. crispa stem extract exerted a protective effect (100%) on hypoglycemia induced by PbANKA infection at doses of 100 and 200 mg/kg. A significantly (p < .05) prolonged mean survival time (28.0 ±â€¯1.9 days) of the extract treated mice was also observed. Additionally, no effect on blood glucose levels was seen in normal mice treated with all doses of extract. It can be concluded that T. crispa stem extract may have beneficial properties in protecting against hypoglycemia, and in increasing survival time during malaria infection.

Preparation and in vivo evaluation of anti-plasmodial properties of artemisinin-loaded PCL-PEG-PCL nanoparticles.

PURPOSE: Artemisinin (ART) has anti-inflammatory, antimicrobial, antioxidant, anti-amyloid, and anti-malarial effects, but its application is limited due to its low water solubility and poor oral bioavailability. In this study, the bioavailability, water solubility, and anti-plasmodial property of ART were improved by PCL-PEG-PCL tri-block copolymers. METHODS: The structure of the copolymers was characterized by 1H NMR, FT-IR, DSC, and GPC techniques. ART was encapsulated within micelles by a single-step nano-precipitation method, leading to the formation of ART-loaded PCL-PEG-PCL micelles. The obtained micelles were characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). The in vivo anti-plasmodial activity of ART-loaded micelles was measured against Plasmodium berghei infected Swiss albino mice. RESULTS: The results showed that the zeta potential of ART-loaded micelles was about -8.37 mV and the average size was 91.87 nm. ART was encapsulated into PCL-PEG-PCL micelles with a loading capacity of 19.33 ± 0.015% and encapsulation efficacy of 87.21 ± 3.32%. In vivo anti-plasmodial results against P. berghei showed that multiple injections of ART-loaded micelles could prolong the circulation time and increase the therapeutic efficacy of ART. CONCLUSION: These results suggested that PCL-PEG-PCL micelles would be a potential carrier for ART for the treatment of malaria.

The Antimalarial Effect of Curcumin Is Mediated by the Inhibition of Glycogen Synthase Kinase-3ß.

Curcumin, a bioactive compound in Curcuma longa, exhibits various pharmacological activities, including antimalarial effects. In silico docking simulation studies suggest that curcumin possesses glycogen synthase kinase-3ß (GSK3ß)-inhibitory properties. The involvement of GSK3 in the antimalarial effects in vivo is yet to be demonstrated. In this study, we aimed to evaluate whether the antimalarial effects of curcumin involve phosphorylation of host GSK3ß. Intraperitoneal administration of curcumin into Plasmodium berghei NK65-infected mice resulted in dose-dependent chemosuppression of parasitemia development. At the highest dose tested (30 mg/kg body weight), both therapeutic and prophylactic administrations of curcumin resulted in suppression exceeding 50% and improved median survival time of infected mice compared to control. Western analysis revealed a 5.5-fold (therapeutic group) and 1.8-fold (prophylactic group) increase in phosphorylation of Ser 9 GSK3ß and 1.6-fold (therapeutic group) and 1.7-fold (prophylactic group) increase in Ser 473 Akt in liver of curcumin-treated infected animals. Following P. berghei infection, levels of pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4 were elevated by 7.5-, 35.0-, 33.0-, and 2.2-fold, respectively. Curcumin treatment (therapeutic) caused a significant decrease (by 6.0- and 2.0-fold, respectively) in serum TNF-α and IFN-γ level, while IL-10 and IL-4 were elevated (by 1.4- and 1.8-fold). Findings from the present study demonstrate for the first time that the antimalarial action of curcumin involved inhibition of GSK3ß.

In vivo antimalarial activity of crude extracts and solvent fractions of leaves of Strychnos mitis in Plasmodium berghei infected mice.

BACKGROUND: Malaria is a major public health problem in the world which is responsible for death of millions particularly in sub-Saharan Africa. Today, the control of malaria has become gradually more complex due to the spread of drug-resistant parasites. Medicinal plants are the unquestionable source of effective antimalarials. The present study aimed to evaluate antiplasmodial activity and acute toxicity of the plant Strychnos mitis in Plasmodium berghei infected mice. METHODS: Standard procedures were employed to investigate acute toxicity and 4-day suppressive effect of crude aqueous and hydro-methanolic extracts of the leaves of Strychnos mitis against P. berghei in Swiss albino mice. Water, n-hexane and chloroform fractions, obtained from crude hydro-methanolic extract, were also tested for their suppressive effect against P. berghei. RESULTS: All crude extracts revealed no obvious acute toxicity in mice up to the highest dose administered (2000 mg/kg). All crude and solvent fractions of the leaves of Strychnos mitis inhibited parasitaemia significantly (p < 0.01). At the highest dose of 600 mg/kg, both aqueous and hydro-methanolic extracts demonstrated higher performance with 95.5 and 93.97% parasitaemia suppression, respectively. All doses of crude extracts and fractions of leaves of Strychnos mitis prolonged survival time of infected mice dose dependently. The highest two doses of the crude aqueous and hydro-methanolic extracts, and chloroform and aqueous fractions prevented weight loss in a dose dependent manner. Whereas, all doses of n-hexane fraction prevented loss of body weight but not in a dose dependent manner. The crude aqueous extract at the doses of 400 mg/kg and 600 mg/kg and hydro-methanolic extract at all dose levels significantly (p < 0.01) prevented packed cell volume reduction. Crude aqueous extract at a dose of 600 mg/kg and hydro-methanolic extract at all dose levels significantly prevented temperature reduction. Phytochemical screening of the crude aqueous and hydro-methanolic extracts revealed the presence of alkaloids, anthraquinones, glycosides, terpenoids, saponins, tannins and phenols. CONCLUSION: The results of this study provide support the traditional therapeutic use of Strychnos mitis for treatment of malaria. However, further in-depth study is needed to evaluate the potential of the plant towards the development of new antimalarial agent.

Antimalarial activity of Syzygium guineense during early and established Plasmodium infection in rodent models.

BACKGROUND: In Ethiopia, the leaves of Syzygium guineense have been found useful for the prevention and cure of malaria, and demonstrated antiplasmodial activity in vitro. Nevertheless, no scientific study has been conducted to confirm its antimalarial activity in vivo. Therefore, the objective of the study was to evaluate the antimalarial effect of Syzygium guineense leaf extract in mice. METHODS: Inoculation of the study mice was carried out by using the malaria parasite, Plasmodium berghei. The plant extract was prepared at 200, 400 and 600 mg/kg. Chloroquine and distilled water was administered to the positive and negative control groups respectively. Parameters like parasitaemia, survival time and body weight were determined following standard tests (4-day suppressive, Rane's and repository tests). RESULTS: Syzygium guineense crude leaf extract displayed considerable (p < 0.05) parasite suppression at doses of 600 and 400 mg/kg in a 4-day suppressive test with chemosuppressive value of 59.39 and 49.09% respectively. S. guineense crude leaf extract also showed dose-dependent schizontocidal activity in both the repository and curative tests. The extract also prevented body weight loss and prolonged survival date of mice significantly (P < 0.05) at the highest dose employed in the study. Qualitative chemical assay for S. guineense methanolic leaf extract revealed that the plant is endowed with different plant secondary metabolites exemplified by terpenoids, alkaloids, triterpenes, flavonoids, anthraquinones, tannins, glycosides, saponins and phenols. CONCLUSION: Syzygium guineense leaf extract possess antimalarial activity in mice. The test substance was found to be safe with no observable signs of toxicity in the study mice. The results of the present work confirmed the in vitro antiplasmodial finding and traditional claims in vivo in mice. Therefore, Syzygium guineense could be regarded as a potential source to develop safe, effective and affordable antimalarial agent.

Antiplasmodial activity of the ethanolic root bark extract of Icacina senegalensis in mice infected by Plasmodium berghei.

BACKGROUND: The root of Icacina senegalensis is used for the treatment of malaria and related conditions in southeastern Nigeria. METHODS: To establish its efficacy, the ethanolic root bark extract was investigated as antiplasmodial agent against Plasmodium berghei in mice. A 4-day suppressive test and the curative effect against established infection models of antiplasmodial studies were used. RESULTS: The root bark extract of I. senegalensis (50, 100, and 200 mg/kg) exhibited a significant (p<0.05) dose-dependent activity against the parasite based on suppressive and curative study. The antimalarial effect of I. senegalensis is compared with that of chloroquine (10 mg/kg), the standard drug. The ethanolic root bark extract also prolonged the survival time of infected mice. CONCLUSIONS: The results showed that the root bark extract possesses a potential antiplasmodial activity, which can be exploited for the possible development of new antimalarial agent.

Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes.

Plasmodium falciparum infections can cause severe malaria, but not every infected person develops life-threatening complications. In particular, carriers of the structural haemoglobinopathies S and C and infants are protected from severe disease. Protection is associated with impaired parasite-induced host actin reorganization, required for vesicular trafficking of parasite-encoded adhesins, and reduced cytoadherence of parasitized erythrocytes in the microvasculature. Here we show that aberrant host actin remodelling and the ensuing reduced cytoadherence result from a redox imbalance inherent to haemoglobinopathic and fetal erythrocytes. We further show that a transient oxidative insult to wild-type erythrocytes before infection with P. falciparum induces the phenotypic features associated with the protective trait of haemoglobinopathic and fetal erythrocytes. Moreover, pretreatment of mice with the pro-oxidative nutritional supplement menadione mitigate the development of experimental cerebral malaria. Our results identify redox imbalance as a causative principle of protection from severe malaria, which might inspire host-directed intervention strategies.

Tetrahydrobiopterin Supplementation Improves Phenylalanine Metabolism in a Murine Model of Severe Malaria.

Tetrahydrobiopterin (BH4) is an essential cofactor for both phenylalanine hydroxylase and nitric oxide synthase. Patients with severe malaria have low urinary BH4, elevated plasma phenylalanine, and impaired endothelium-dependent vasodilation, suggesting that BH4 depletion may limit phenylalanine metabolism and nitric oxide synthesis. We infected C57BL/6 mice with Plasmodium berghei ANKA to characterize BH4 availability and to investigate the effects of BH4 supplementation. P. berghei ANKA infection lowered BH4 in plasma, erythrocytes, and brain tissue but raised it in aorta and liver tissue. The ratio of BH4 to 7,8-BH2 (the major product of BH4 oxidation) was decreased in plasma, erythrocytes, and brain tissue, suggesting that oxidation contributes to BH4 depletion. The continuous infusion of sepiapterin (a BH4 precursor) and citrulline (an arginine precursor) raised the concentrations of BH4 and arginine in both blood and tissue compartments. The restoration of systemic BH4 and arginine availability in infected mice produced only a minor improvement in whole blood nitrite concentrations, a biomarker of NO synthesis, and failed to prevent the onset of severe disease symptoms. However, sepiapterin and citrulline infusion reduced the ratio of phenylalanine to tyrosine in plasma, aortic tissue, and brain tissue. In summary, BH4 depletion in P. berghei infection may compromise both nitric oxide synthesis and phenylalanine metabolism; however, these findings require further investigation in human patients with severe malaria.

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

Antiprotozoal screening of the Cuban native plant Scutellaria havanensis.

CONTEXT: Scutellaria havanensis Jacq. (Lamiaceae) is a native medicinal herb with a history of use in Cuba. OBJECTIVE: This study screens the antiprotozoal activity of S. havanensis. MATERIALS AND METHODS: Chloroform and methanol extracts from leaves and stems were evaluated in vitro at doses between 0.015 and 200 µg/mL against protozoan parasites: Plasmodium berghei, Trichomonas vaginalis and Leishmania amazonensis. Chloroform and methanol extracts were characterized by GC/MS. Cytotoxicity against mouse peritoneal macrophages was tested in parallel. RESULTS: Scutellaria havanensis extracts exhibited IC50 values between 7.7 and 32.2 µg/mL against trophozoites of P. berghei and T. vaginalis; while the extracts were inactive against L. amazonensis promastigotes. Trichomonicidal activity of methanol extract exhibited the best selectivity but chloroform extract showed the highest antiplasmodial, trichomonicidal and cytotoxic activity. The majority of compounds in the chloroform extract were hydroxy and/or methoxyflavones (77.96%), in particular, wogonin (48.27%). In methanol extract, wogonin (5.89%) was detected. Trichomonicidal effect of wogonin was moderate (IC50 = 56 µM) and unspecific with respect to macrophages (SI = 2). On the contrary, antiplasmodial activity of wogonin were particularly active (IC50 = 15 µM) demonstrating a higher selectivity index (SI = 7.4). CONCLUSIONS: Wogonin is an active principle compound of the chloroform extract of S. havanensis against P. berghei and T. vaginalis trophozoites, whereas the methanol extract of S. havanensis should be investigated more deeply as a trichomonicide. Our findings suggest that wogonin is potentially useful for the development of antimalarial alternative treatments.

Antipyretic and antimalarial activities of Solenostemon monostachyus.

CONTEXT: Solenostemon monostachyus P. Beauv (Lamiaceae) is an important herb used traditionally in the treatment of malaria, fever, and other diseases. OBJECTIVES: Antiplasmodial and antipyretic activities of S. monostachyus aerial extract were evaluated to ascertain the folkloric claim of its antimalarial and antipyretic activities. MATERIALS AND METHODS: The extract (75-225 mg/kg) and fractions (chloroform and aqueous; 150 mg/kg) of S. monostachyus were investigated for suppressive, prophylactic, and curative antiplasmodial activities against chloroquine-sensitive Plasmodium berghei infections in Swiss albino mice and for antipyretic activity against 2,4-dinitrophenol and yeast-induced pyrexia. Artesunate (5 mg/kg) and pyrimethamine (1.2 mg/kg) were used as positive controls for antiplasmodial models. Thin films made from tail blood of each mouse were used to assess the level of parasitaemia of the mice. RESULTS: The extract/fractions progressively reduced parasitaemia induced by chloroquine sensitive P. berghei infection in prophylactic (28.48-71.72%), suppressive (12.52-72.47%), and curative (22.4-82.34%) models in mice. These reductions were statistically significant (p < 0.01-0.001). They also improved significantly (p < 0.01-0.001) the mean survival time (MST) from 12.26 to 25.63 d relative to control (11.36 d). The activities of extract/fractions were incomparable with that of the standard drugs used (artesunate and pyrimethamine). The extract exerted prominent inhibition of pyrexia on dinitrophenol (87.33-90.11%, 5 h) and yeast (56.22-65.33, 5 h) induced pyrexia. Inhibition was significant (p < 0.05-0.001) from 3 to 5 h post-administration of extract and in a dose-dependent fashion. CONCLUSION: The plant may possess antiplasmodial and antipyretic effects which may in part be mediated through the chemical constituents of the plant.

N-acetyl cysteine and mushroom Agaricus sylvaticus supplementation decreased parasitaemia and pulmonary oxidative stress in a mice model of malaria.

BACKGROUND: Malaria infection can cause high oxidative stress, which could lead to the development of severe forms of malaria, such as pulmonary malaria. In recent years, the role of reactive oxygen species in the pathogenesis of the disease has been discussed, as well as the potential benefit of antioxidants supplementation. The aim of this study was to investigate the effects of N-acetyl cysteine (NAC) or mushroom Agaricus sylvaticus supplementation on the pulmonary oxidative changes in an experimental model of malaria caused by Plasmodium berghei strain ANKA. METHODS: Swiss male mice were infected with P. berghei and treated with NAC or AS. Samples of lung tissue and whole blood were collected after one, three, five, seven or ten days of infection for the assessment of thiobarbituric acid reactive substances (TBARS), trolox equivalent antioxidant capacity (TEAC), nitrites and nitrates (NN) and to assess the degree of parasitaemia. RESULTS: Although parasitaemia increased progressively with the evolution of the disease in all infected groups, there was a significant decrease from the seventh to the tenth day of infection in both antioxidant-supplemented groups. Results showed significant higher levels of TEAC in both supplemented groups, the highest occurring in the group supplemented with A. sylvaticus. In parallel, TBARS showed similar levels among all groups, while levels of NN were higher in animals supplemented with NAC in relation to the positive control groups and A. sylvaticus, whose levels were similar to the negative control group. CONCLUSION: Oxidative stress arising from plasmodial infection was attenuated by supplementation of both antioxidants, but A. sylvaticus proved to be more effective and has the potential to become an important tool in the adjuvant therapy of malaria.

Evaluation of antimalarial activity of leaves of Acokanthera schimperi and Croton macrostachyus against Plasmodium berghei in Swiss albino mice.

BACKGROUND: Malaria is one of the most important tropical diseases and the greatest cause of hospitalization and death. Recurring problems of drug resistance are reinforcing the need for finding new antimalarial drugs. In this respect, natural plant products are the main sources of biologically active compounds and have potential for the development of novel antimalarial drugs. A study was conducted to evaluate extracts of the leaves of Croton macrostachyus and Acokanthera schimperi for their in vivo antimalarial activity. METHODS: The plants were selected based on their ethnomedicinal information. Acute and sub- acute toxicity studies of the crude extracts were carried out in Swiss albino mice. To assess the effect of extracts of the plants on the parasite, a 4-day suppressive standard test was performed using Plasmodium berghei (ANKA strain). Data were analyzed using paired t-test and ANOVA. RESULTS: In acute toxicity study, the two plants extracts did not show any sign of toxicity up to 2000 mg/kg. In sub-acute toxicity study, both plants did not exhibit any hematological change and mortality throughout the observation period up to the highest dose of 1000 mg/kg given daily. Extracts of the leaves of both plants significantly (P < 0.05) suppressed parasitaemia in dose dependent manner at all dose levels. CONCLUSIONS: The findings may support the traditional use of the plants to treat malaria. Further pharmacological, toxicological and phytochemical studies are, however, required to evaluate the potential of the plants towards the development of new antimalarial agent.