Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Actividad anti-plasmodial de especies de Solanaceae presentes en Cuba / Antiplasmodial activity of Solanaceae species present in Cuba

Introducción: el control de la malaria depende en gran medida de una terapia efectiva. Muchos de los anti-maláricos actuales son de origen natural. Especies de la flora cubana contienen metabolitos anti-Plasmodium. En este estudio, se identifican extractos de Solanaceae con actividad antiplasmodial promisoria. Objetivo: evaluar la actividad esquizonticida frente a Plasmodium berghei de 31 extractos de 7 especies, correspondientes a 5 géneros de plantas de Solanaceae, colectadas en el occidente de nuestro país y sin antecedentes de un estudio similar. Métodos: se prepararon 31 extractos hidroalcohólicos (90 y 30 por ciento etanol) de diferentes órganos de: Brunfelsia undulata Sw., Datura stramonium L. var. tatula (L.) Torr., Physalis solanaceus (Schltdl.) Axelius, Solandra longiflora Tuss., Solanum myriacanthum Dunal, Solanum seaforthianum And. ySolanum umbellatum Mill.La actividad de los extractos se evaluó in vitro frente a P. berghei y se determinó su citotoxicidad frente a fibroblastos humanos MRC-5. Resultados: los extractos deB. undulata y S. umbellatumfueron inactivos.El extracto de tallos de S. seaforthianummostró la actividad antiplasmodial más potente (CI50 = 3,9µg/mL) con excelentes electividad (18,2). Conclusiones: se demostró la actividad anti-plasmodial in vitro de extractos de cinco especies de Solanaceae sin antecedentes de esta acción farmacológica. Se identificó un extracto con potente actividad esquizonticida frente a P. berghei y con excelente selectividad. Este resultado nos anima a continuar el estudio de la preparación vegetal de S. seaforthianum(AU)

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidospermaplants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorumextracts, the plant activity against Plasmodium falciparumwas evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium berghei strain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

Síntesis y evaluación preliminar de la posible actividad antimalárica y antineoplásica de derivados 7-cloroquinolina-4-sustituidos / Synthesis and preliminary evaluation of antimalarial and antineoplastic possible activity of 7-chloroquinoline-4-substituted derivatives

En el presente trabajo se describe la síntesis y la evaluación de la posible actividad Antimalárica y Antineoplásica de una serie de derivados 7-cloroquinolina-4-sustituidos. La estrategia empleada para la síntesis comienza con las obtención de los intermediarios clave 1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)etanona (2 y 3) mediante una sustitución nucleofílica aromática entre la 4,7-dicloroquinolina y la 3 y/o 4-amino acetofenona. Los derivados (E)-1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)-3-(fenilsustituido)prop-2-eno-1-ona (4 y 5), se sintetizaron a través de una condensación aldólica de Claisen-Schmidt entre los intermediarios clave y diferentes benzaldehídos sustituidos. Los derivados 7-cloro-N-(3 ó 4-(4,5-dihidro-5-(fenilsustituido)-1H-pirazol-3-il)fenil)quinolin-4-amina (6 y 7) y los 1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)-3-(fenilsustituido)propano-1-ona (8 y 9) se diseñaron por modificación molecular de la cetona a,b-insaturada de los compuestos finales 4 y 5, (metodología clásica de la Química Medicinal) para obtener dichos derivados rígidos 6 y 7, mediante la formación de un anillo D2-pirazolina y flexibles 8 y 9, a través de su reducción. La síntesis de los derivados 6 y 7 se realizó mediante una reacción de ciclo-condensación con hidrazina monohidratada y los derivados 8 y 9, se obtuvieron a través de una hidrogenación catalítica. En la evaluación de la actividad Antimalárica in vitro se evidenció que los derivados 4, 5, 6 y 7, mostraron actividades inhibitorias la formación de la b hematina importantes (superior al 70 %), siendo los más activos: 4l, 5g, 5c, 5g y 6e, 6f con valores comparable al de la CQ. En la evaluación Antimalárica in vivo se encontró que el derivado 4e fue el más activo con 26,4 días de sobrevivencia post-infección (230 % de incremento) y una parasitemia de 2,4 % (96 % de reducción). Con respecto a los resultados obtenidos en el efecto de estos derivados sobre la viabilidad y proliferación de las líneas celulares neoplásicas Jurkat E6.1, HL60, MCF-7 y A549, los compuestos 4a, 4g, 4l, 4m y 6e mostraron la mayor actividad inhibitoria del crecimiento de las células leucémicas HL60 después de 24h de tratamiento con valores de CI50 de 1,19 µM, 1,08 µM, 0,59 µM, 0,43 µM y 0,94 µM (hasta 3 y 100 veces más activos que la doxorubicina y que la CQ, respectivamente). En lo referente a la evaluación de la actividad proapoptótica en las líneas celulares neoplásicas Jurkat E6.1, HL60, MCF-7 y A549, se evidenció que los derivados 4, 5 y 6, al igual que los controles, generaron un aumento en el porcentaje de células positivas para la Anexina V/FITC dependiente de la dosis (apoptosis temprana y tardía). Ninguno de estos derivados indujo el proceso de necrosis en estas células.

The present investigation describes the synthesis and evaluation of the Antimalarial and Antineoplastic activity possible a series of derivatives of 7-substituted-4-chloro-quinoline. The strategy employed for the synthesis begins with preparation of the key intermediate 1-(3 or 4-(7-chloroquinolin-4-ylamino) phenyl)ethanone (2and 3) by a nucleophilic aromatic substitution between 4,7-dichloroquinoline and the 3 and/or4-amino acetophenone. The derivatives (E)-1-(3 or 4-(7-chloroquinolin-4-ylamino) phenyl)-3-(substitutedphenyl)prop-2-en-1-one (4and 5), were synthesized a through aldol condensation Claisen-Schmidt among different key intermediates and substituted benzaldehydes. The resulting 7-chloro-N-(3 or 4-(4,5-dihydro-5-(substitutedphenyl)-1H-pyrazol-3-yl)phenyl)quinolin-4-amine (6 and 7) and 7-chloro-4-[(3 or 4-(substituted phenyl)ethylcarbonyl)phenyl]aminoquinoline(8 and 9) were designed for the molecular modification , -unsaturated ketone of the final compounds 4and 5 (classic methodology Medicinal Chemistry) for said rigid derivatives 6and 7, through the formation of a 2-pyrazoline ring flexible and 8and 9, through its reduction. The synthesis of derivatives 6and 7were performed using a cycle-condensation reaction with hydrazine monohydrate and 8and 9derivatives were obtained via a catalytic hydrogenation. In the assessment of antimalarial activity in vitro was demonstrated that derivatives 4, 5, 6and 7showed inhibitory activities forming the major hematin (above 70%), being more active: 4l, 5g, 5c, 5g, 6eand 6f,with values comparable to that of CQ. In vivoantimalarial evaluation found that the derivative 4ewas most active with survival 26.4 dayspost-infection (230% increase) and a parasitemia of 2.4% (96% reduction). With regard to the results on the effect of these derivatives on the viability and proliferation of neoplastic cell lines Jurkat E6.1, HL60, MCF-7 and A549, compounds 4a, 4g, 4l,4mand 6eshow greater activity growth inhibitory HL60 leukemia cells after 24 h of treatment with IC50values of 1.19µM, 1.08µM, 0.59µM, 0.43µMand 0.94 M (to 3 and 100 times more active than doxorubicin and the CQ, respectively). Regarding the evaluation of pro-apoptotic activity on neoplastic cell lines Jurkat E6.1, HL60, MCF-7 and A549, was demonstrated that derivatives 4, 5and 6, like the controls, an increase in generated percentage of cells positive for Annexin V/FITC dose dependent (early andlate apoptosis). None of these derivatives induced necrosis process in these cells.

Evaluation of effectiveness of ethanolic extract of Artemisia aucheri, individually and in combination with chloroquine, on chloroquine - sensitive strain of Plasmodium berghei in sourian mice

Drug resistance in malaria parasites is extending in the world particularly in chemical synthesized drugs such as 4- aminoquinolines and aminoalcoholes. Employing herbal extracts is encouraged by WHO in the malarious areas. In this study, the effectiveness of ethanolic extract of Artemisia aucheri individually and in combination with chloroquine, has been considered against chloroquine - sensitive strain of Plasmodium berghei. At the first stage, ED50 of A. aucheri and chloroquine on P. berghei was calculated using in vivo test. Then based on the ED50s combination of A. aucheri and chloroquine with ratios of 0/100, 10/90, 20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, 90/10 and 100/0 were tested against the parasite. For evaluating the adverse effect of A. aucheri on the mice, for two weeks 1000mg/kg of the extract was daily employed and the mice were followed up for fifty days. ED50s for chloroquine and A. aucheri were 1.6mg/kg and 1000mg/kg respectively. The outcome of two drugs combination on the mice showed antagonistic effects on the chloroquine - sensitive strain of parasite. Two weeks daily administration of A. aucheri had no toxic effect on the mice. A. aucheri individually can be effective in reducing the parasite while in combination with chloroquine loses its property

In vivo antioxidant and biochemical evaluation of Sphenocentrum jollyanum leaf extract in P. berghei infected mice

Recent approach in treatment and drug development suggested that the control of oxidative stress in malarial infected patients may be an added advantage. In this study, effect of methanolic leaf extract of Sphenocentrum jollyanum pier [S. jollyanum] on liver damage, markers of oxidative stress and alteration in lipid profile in P. berghei infected mice was assessed. Oxidative stress was induced by intravenously inoculation of mice with 1 x 107 sporozoites P. berghei. Treatment of parasitized mice with leaf extract of S. jollyanum had a significant [p<0.05] reductions in elevated levels of total protein, globulin, AST, ALT, ALP, GGT and total bilirubin, serum, kidney and liver malondialdehyde [MDA] concentrations, but caused a significant [p<0.05] increased in the activities of serum and liver catalase [CAT], superoxide dismutase [SOD] and glutathione [GSH] level when compared with parasitized non-treated group [PNT]. The extract treated group also showed significant [p<0.05] improvement in the levels of HDLc, total cholesterol, LDL and reduction in triglyceride compared with parasitized non treated group. Our results revealed that the protective capacity and antioxidant activity of the extract is dose dependant. The findings suggest that antioxidant property of Sphenocentrum jollyanum leave extract might be an added advantage to it anti-malarial activity

The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.

In vitro antimalarial activity of extracts of some plants from a biological reserve in Costa Rica

Treatment with the usual antimalarial drugs, have induced parasite resistance, reinforcing the need to finding natural antimalarial components that would be found on plants from the forest. Therefore, we decided to look for these components in Costa Rican plants from a protected forest area. Fresh and dry extracts of roots, bark, leaves, flowers and fruits of 25 plants from a biological reserve in Costa Rica, Reserva Biológica Alberto Manuel Brenes (REBAMB), were studied in vitro for the presence of substances with antimalarial activity. By studying the inhibition of P. berghei schizogony, we assessed the antimalarial activity of several plant extracts: Aphelandra aurantiaca, A. tridentata (Acanthaceae); Xanthosoma undipes (Araceae); Iriartea deltoidea (Arecaceae); Neurolaena lobata (Asteraceae); Senna papillosa, Pterocarpus hayessi, Lonchocarpus pentaphyllus (Fabaceae); Nectandra membranacea, Persea povedae, Cinamomum chavarrianum (Lauraceae); Hampea appendiculata (Malvaceae); Ruagea glabra, Guarea glabra (Meliaceae); Psidium guajava (Myrtaceae); Bocconia frutescens (Papaveraceae); Piper friedrichsthalii (Piperaceae); Clematis dioica (Ranunculaceae); Prunus annularis (Rosaceae); Siparuna thecaphora (Siparunaceae); Solanum arboreum, Witheringia solanácea (Solanaceae); Ticodendrum incognitum (Ticodendraceae); Heliocarpus appendiculatus (Tiliaceae) and Myriocarpa longipes (Urticaceae). We used different parts of the plants as well as fresh and dried extracts for testing IC50. The solid content of the extracts ranged from 1-71.9μg/mL. The fresh extracts showed stronger activity than the dry ones. Since the plants showing the strongest antimalarial activity are very common in Central America, and some similar genera of these plants have shown positives results in South America, we considered important to present these findings for discussion. On the other hand, this is the first systematic study of this kind ever realized in a circumscribed and protected area of Costa Rica. Rev. Biol. Trop. 60 (2): 881-891. Epub 2012 June 01.

El tratamiento con las drogas antimaláricas de uso común han inducido resistencia por parte del parásito, lo que obliga a buscar en las plantas de los bosques, componentes naturales con actividad en contra de esta enfermedad. Por lo tanto, decidimos buscar dichos componentes en plantas de una Reserva Forestal de Costa Rica. Extractos tanto frescos como secos de raíz, corteza, hojas, flores y frutos, de 25 plantas de la Reserva Biológica Alberto Manuel Brenes (REBAMB), fueron estudiados in vitro en busca de sustancias con actividad antimalárica. Las plantas estudiadas fueron: Aphelandra aurantiaca, A. tridentata (Acanthaceae); Xanthosoma undipes (Araceae); Iriartea deltoidea (Arecaceae); Neurolaena lobata (Asteraceae); Senna papillosa, Pterocarpus hayessi, Lonchocarpus pentaphyllus (Fabaceae); Nectandra membranacea, Persea povedae, Cinamomum chavarrianum (Lauraceae); Hampea appendiculata (Malvaceae); Ruagea glabra, Guarea glabra (Meliaceae); Psidium guajava (Myrtaceae); Bocconia frutescens (Papaveraceae); Piper friedrichsthalii (Piperaceae); Clematis dioica (Ranunculaceae); Prunus annularis (Rosaceae); Siparuna thecaphora (Siparunaceae); Solanum arboreum, Witheringia solanacea (Solanaceae); Ticodendrum incognitum (Ticodendraceae); Heliocarpus appendiculatus (Tiliaceae) y Myriocarpa longipes (Urticaceae). Los extractos frescos y secos de las diferentes partes de las plantas fueron estudiadas y se determinó la IC50, el cual osciló entre 1-71.9mg/mL; los extractos frescos mostraron mayor actividad antimalárica. Las plantas que presentaron mayor actividad son muy comunes en Centroamérica y algunos géneros similares, aunque no las mismas especies, han sido encontrados positivos en América del Sur; por esta razón consideramos importante estos resultados como información y materia de discusión en este tema. Además este es el primer estudio sistemático de esta naturaleza realizado en un área boscosa circunscrita y protegida de Costa Rica.

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics / Alteración en el metabolismo de la hemoglobina y actividad antimalárica in vivo de azoles antimicóticos

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5 percent, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0 percent and 55.3 ± 3.6 percent, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia ( percentP) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0 percent compared to 26.6 ± 3.7 percent, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Los parásitos del género Plasmodium degradan la hemoglobina hospedera obteniendo aminoácidos libres para su síntesis proteica. Durante este evento, unidades de hemo libre tóxicas cristalizan espontáneamente formando un pigmento no tóxico denominado ß-hematina. En este trabajo, se investigó la capacidad de un grupo de azoles antimicóticos: clotrimazol (CTZ), ketoconazol (KTZ) y fluconazol (FCZ), en inhibir la síntesis de ß-hematina y la proteólisis de la globina. La síntesis de ß-hematina se registro por espectrofotometría a 405 nm y la proteólisis de la hemoglobina se determino por SDS-PAGE 15 por ciento seguido por análisis densitométrico de las bandas de hemoglobina intactas. Los compuestos fueron también ensayados in vivo en un modelo de malaria murina. CTZ y KTZ inhibieron la síntesis de ß-hematina con CI50 entre 10 y 15 µM y bloquearon la proteólisis de la hemoglobina (80.01 ± 2.04 por ciento y 55.33 ± 3.57 por ciento, respectivamente). En relación directa con los resultados encontrados in vitro, el CTZ redujo la parasitemia de ratones infectados en forma significativa, así como prolongó lo días de sobrevivencia post-infección en comparación con animales controles no tratados. Se sugiere así que la inhibición del metabolismo de la hemoglobina por los antimicóticos azólicos pudiera ser el mecanismo responsable de su actividad antimalárica.

Modification of oxidative status in Plasmodium berghei-infected erythrocytes by E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline compared to chloroquine

E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline (IQ) is a new quinoline derivative which has been reported as a haemoglobin degradation and ß-haematin formation inhibitor. The haemoglobin proteolysis induced by Plasmodium parasites represents a source of amino acids and haeme, leading to oxidative stress in infected cells. In this paper, we evaluated oxidative status in Plasmodium berghei-infected erythrocytes in the presence of IQ using chloroquine (CQ) as a control. After haemolysis, superoxide dismutase (SOD), catalase, glutathione cycle and NADPH + H+-dependent dehydrogenase enzyme activities were investigated. Lipid peroxidation was also assayed to evaluate lipid damage. The results showed that the overall activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were significantly diminished by IQ (by 53.5 percent and 100 percent, respectively). Glutathione peroxidase activity was also lowered (31 percent) in conjunction with a higher GSSG/GSH ratio. As a compensatory response, overall SOD activity increased and lipid peroxidation decreased, protecting the cells from the haemolysis caused by the infection. CQ shared most of the effects showed by IQ; however it was able to inhibit the activity of isocitrate dehydrogenase and glutathione-S-transferase. In conclusion, IQ could be a candidate for further studies in malaria research interfering with the oxidative status in Plasmodium berghei infection.

Variation of the chloroquine resistance transporter [crt] gene in chloroquine-resistant and chloroquine-sensitive plasmodium berghei

The emergence and spread of chloroquine resistant Plasmodium falciparum in the world stimulated some investigators to consider different aspects of chloroquine resistance in human and rodent Plasmodia. Using animal Plasmodia, particularly primate and rodent Plasmodia can be useful model for human Plasmodia studies. In this study we have tried to consider and compare the sequence of chloroquine resistance transporter [crt] gene among chloroquine-resistant and chloroquine-sensitive strains of Plasmodium berghei. This experimental study was performed at the Malaria Laboratory of School of public health. DNA was extracted from two strains of P. berghei which their resistance and sensitivity had been demonstrated in mice with treatment by chloroquine. By using specific primer for crt gene some parts of this gene were amplified by PCR, and obtained fragments were then sequenced and compared. There were considerable differences in crt gene between two strains. Sequenced 1212 bp of crt gene fragment in the two strains showed 43 differences at nucleotides level and 16 differences in presumed coding amino acids. crt can be addressed as a considerable gene which involves in induction of resistance to chloroquine in P. berghei, as P. falciparum. The results increased such a promise that considering crt gene in chloroquine-sensitive and chloroquine-resistant P. berghei can prepare suitable and helpful fields for more understanding the molecular aspects of chloroquine-resistance in Plasmodia and reversing the effectiveness of 4-aminoquinolines particularly chloroquine for treatment of drug resistant Plasmodia

Anti-malarial activity of Eclipta alba against Plasmodium berghei infection in mice.

The anti-malarial activity of Eclipta alba leaves extract was evaluated against Plasmodium'berghei ANKA strain in mice. A standard inoculum of 1 x 10(6) infected erythrocytes was used. The methanolic leaf extract (250-750 mg/kg) produced a dose-dependant chemosupression or schizontocidal effect during early and established infection and high mean survival time (m.s.t.) values particularly in the group administered 750 mg/kg/day of extract. The plant extract also exhibited repository activity. The results of the preliminary studies carried out with E. alba are encouraging, which can be exploited in malaria therapy.

Chloroquine efficacy in Plasmodium berghei NK65-infected ICR mice, with reference to the influence of initial parasite load and starting day of drug administration on the outcome of treatment.

We examined whether the initial number of parasites inoculated and the starting day of medication post-infection influenced the antimalarial efficacy of chloroquine (CQ) against Plasmodium berghei NK65 infection in ICR mice. Male ICR mice were inoculated intraperitoneally with 1 x 10(5), 1x10(6), 1 x 10(7), 1 x 10(8) P. berghei NK65-parasitized erythrocytes (pRBC). In the treated group, all mice received an oral dose of 20 mg/kg of CQ base for 4 days starting on day 0 after infection. From day 3, Giemsa-stained thin blood smears from tail vein blood were used to assess parasitemia. Mice in the untreated control in each group showed a progressive increase in parasitemia leading to death. Treatment of mice, inoculated with 1 x 10(5), 1 x 10(6) and 1 x 10(7) pRBC, with CQ showed a marked effect. All the mice survived during the experiment. During the observation period, malaria parasites could not be detected on microscopic examination. Conversely, mice inoculated with 1 x 10(8) pRBC showed little response to CQ treatment, and all mice showed a progressive increase in parasitemia and ultimately died. In another experiment, mice infected with 1 x 10(3) and 1x 10(5) pRBC were treated with an oral four-day dosage of 20 mg/kg of CQ base from days 2, 3 or 4 post-infection. Treatment of mice, inoculated with 1 x 10(3) pRBC, with CQ from days 2 and 3 showed a marked effect. All mice survived during the experiment. However, treatment from day 4 showed a limited derease in parasitemia and all the mice ultimately died. On the other hand, treatment from day 2 showed a marked effect against 1 x 10(5) P. berghei NK65-infected mice, but treatment from days 3 or 4 was only slightly effective and all the mice died with an increasing parasitemia. The present results indicate that in in vivo antimalarial drug-assay systems, several factors, sush as initial parasite load and starting time of treatment may influence the drug response in the host.

Evaluación química y biológica del efecto de extractos de plantas contra Plasmodium berghei / Biological and chemical evaluation of the effect of plant extracts on Plasmodium berghei

Extracts from thirteen species of plants were evaluated by(r)in vivo(r)antimalarial test against plasmodium berghei effects. Significant activities were observed in the ethyl acetate and aqueous extracts, elaborated of Cedrela tonduzii leaves, Trichilia havanensis and Trichilia americana barks, Neurolaena lobata and liricidia sepium leaves and Duranta repens fruits. Compounds identified include flavanoids, coumarins, mellilotic acid and iridoids which some kind of biodynamic activity has previously been reported. The flavone uercetin 1 purified from C. tonduzii gave strong antimalarial activity, however, its respective glucosides (quercetin 3-glucoside 2 y robinine 7) showed little significant activity

Evolucao da malaria por Plasmodium berghei em ratos jovens alimentados com racoes com diferentes teores de ferro / Evolution of the malaria by Plasmodium berghei in young rats alimented with rations with differents iron proportions

Evidencias clinicas e experimentais sugerem que hospedeiros com deficiencia de ferro sao menos susceptiveis a malaria grave e que a suplementacao de ferro poderia agravar a infeccao na tentativa de justificar estes fatos, dois experimentos foram realizados. Os resultados obtidos sugerem que os animais com deficiencia de ferro apresentam clareamento das parasitemias mais tardio e que o desenvolvimento de P. berghei em ratos nao e suprimido pela deficiencia de ferro. A suplementacao de ferro antes e durante a infeccao nao aumentou as parasitemias. Alem disso, a replecao de ferro durante a infeccao produziu incrementos em parametros hematologicos em animais com anemia ferropriva