Actividad antiplasmodial de plantas usadas en la medicina tradicional contra la malaria en Cuba / Antiplasmodial activity of plant species used in traditional medicine for the control of malaria in Cuba

Introducción: Las plantas utilizadas en la medicina tradicional se exploran internacionalmente como fuentes valiosas de nuevos agentes antipalúdicos. Objetivos: Evaluar la actividad inhibidora in vitro frente a Plasmodium berghei de extractos de 27 especies de plantas utilizadas en el siglo pasado contra la malaria en Cuba, valorando los precedentes de estudios científicos de estas especies. Métodos: Se prepararon extractos etanólicos de 27 especies de plantas que se evaluaron in vitro a través de la inhibición de la esquizogonia de P. berghei ANKA. Se realizó una revisión basada en la consulta de artículos científicos para la clasificación de actividad basada en rangos de CI50. Resultados: Dieciséis especies mostraron precedentes de actividad inhibidora in vitro o su utilización en la medicina tradicional de otros países; siete especies no mostraron actividad coincidente. Solamente los extractos hidroalcohólicos de Euphorbia tithymaloides L. (partes aéreas) y Swietenia mahagoni (L.) Jacq. (corteza) fueron activos con CI50 ≤ 5 µg/mL, mientras para Colubrina arborescens (Mill.) Sarg. (corteza), la CI50 fue 14,3 ± 1,9 µg/mL. Extractos de 11 especies se probaron por el interés de su utilización en Cuba, sin precedentes etnobotánicos y experimentales disponibles. Entre estos, las partes aéreas de Baccharis halimifolia L. var. angustior DC; y de Oxandra lanceolata (Sw.) Baill. constituyeron fuentes antiplasmodiales de valores moderados de CI50. El resto de los extractos no fueron activos. Conclusiones: Estos resultados apoyan el uso en medicina tradicional en Cuba contra malaria de E. tithymaloides, S. mahagoni, C. arborescens, B. halimifolia y O. lanceolata, lo cual estimula a ampliar su estudio.

Introduction: Plants used in traditional medicine are studied worldwide as valuable sources of new antiplasmodial agents. Objective: To evaluate the in vitro inhibitory activity against Plasmodium berghei of extracts from 27 plant species used in the last century against malaria in Cuba and assess the previous scientific studies on these species. Methods: Ethanolic extracts from 27 plant species were prepared and evaluated in vitro by inhibiting the schizogony of P. berghei ANKA. A review of scientific papers was conducted to classify the activity based on IC50. Results: Sixteen species showed precedents of inhibitory activity in vitro or of their use in traditional medicine in other countries; seven species did not show coincident activity. Only hydroalcoholic extracts from Euphorbia tithymaloides L. (aerial parts) and Swietenia mahagoni (L.) Jacq. (bark) displayed activity at IC50 ≤ 5 µg/mL, while for Colubrina arborescens (Mill.) Sarg. (bark) IC50 was 14.3 ± 1.9 µg/mL. Extracts from 11 species, with no ethnobotanical and experimental precedents available, were evaluated given the interest of their use in Cuba. Of these species, the aerial parts of Baccharis halimifolia L. var. angustior DC and Oxandra lanceolata (Sw.) Baill. constituted antiplasmodial sources of moderate IC50 values. The rest of the extracts were not active. Conclusions: These results support the use of E. tithymaloides, S. mahagoni, C. arborescens, B. halimifolia y O. lanceolata in traditional medicine against malaria in Cuba, which stimulate further studies.

Resistance of C57BL/6 Mice to Different Artemisinin-sensitive Strains of PbK173 / 中国实验方剂学杂志

ObjectiveThe tolerance of C57BL/6 mice to artemisinin-sensitive and -resistant strains of Plasmodium berghei （Pb） K173 and the differences in blood parameters， spleen coefficient and spleen structure during infection were compared to explore whether the artemisinin resistance of Pb would aggravate malaria infection. MethodPbK173 artemisinin-sensitive and -resistant strains were tested in parallel. C57BL/6 mice were randomly divided into 1 control group， 4 artemisinin-sensitive strain groups and 4 artemisinin-resistant strain groups by body weight. Each infection group was simultaneously inoculated （ip） with 1×107 infected red blood cells （iRBCs） of sensitive/resistant strain. For the mice in the survival test group， the body weight was recorded every day post infection， and the tail vein blood smear was collected to calculate the Pb infection rate. In the other infection groups， peripheral blood and spleen were collected on 2， 5 and 9 d after infection. Peripheral blood parameters， spleen coefficient， pathological section of spleen and spleen cells were detected in each group. ResultOn 1-3 d after infection， the infection rate of the resistant strain （0.4±0.0， 0.8±0.1， 1.9±0.4）% was always higher than that of the sensitive strain （0.2±0.1， 0.4±0.1， 1.1±0.3）% （P<0.01）. From the 4th d of infection， the infection rate of the two groups gradually approached. The survival period of the sensitive strain group （20.5±1.2） d was shorter than that of the resistant strain group （23.3±1.4） d （P<0.01）. On the 9th d， the white blood cell count of the sensitive strain group （16.2±1.1）×109 cells/L was higher than that of the resistant strain group （10.6±1.8）×109 cells/L （P<0.01）. Flow cytometry analysis of spleen cells showed that the sensitive strain group （3.6±0.4） demonstrated a higher CD4+/CD8+ value than the resistant strain group （2.3±0.2） on the 9th d （P<0.01）. The spleen of C57BL/6 infected mice was gradually enlarged during infection， and on the 9th d， the resistant strain group （3.1±0.1）% showed a higher spleen coefficient than the sensitive strain group （2.7±0.2）% （P<0.01）. In the early stage of C57BL/6 infected mice， the red pulp of spleen was hyperemic and swollen. On the 9th d， the marginal area of the spleen disappeared and the structure of the red and white pulp was destroyed. ConclusionWithout drug treatment， the protective immune responses of peripheral blood and spleen of C57BL/6 mice were more sensitive to PbK173 artemisinin-sensitive strain. The artemisinin-resistant strain of PbK173 bred with mouse-to-mouse blood transmission and increased artemisinin dose exhibited shortened growth period and reduced toxicity.

Effect of pregnancy malaria on T cells in immune organ of offspring mice / 中华地方病学杂志

Objective:To observe the influence of pregnant mice having malaria on T cell function of offspring mice, and to study the changes of cellular immune response in offspring mice exposed to malaria infection in uterus.Methods:Adult Kunming mice of clean grade were selected after mating, on the 14th day of pregnancy, pregnant mice were randomize assigned into experimental group ( n = 5) and control group ( n = 5) according to the method of random number table. Each mouse in the experimental group was intraperitoneally inoculated with 1 × 10 6 red blood cells infected with Plasmodium berghei ( P.b), and same volume of normal saline was given to control group. After birth, the changes of CD4/CD8 T cell subsets in their thymuses and spleens of the two group neonatal mice were analyzed by flow cytometry at day 0, 1, 3, 5 and 4-week-old. Then the 4-weeks-old neonatal mice were intraperitoneally inoculated with 1 × 10 6P.b. On the third day, the changes of CD4/CD8 T cells subsets in their thymuses and spleens were observed, respectively, and the immune response of spleen cells stimulated by P.b antigen or mitogen [concanavalin A (Con A)] was detected. Results:Compared with the control group, the proportions of CD3 +CD4 +CD8 - T cells in thymus and spleen of the offspring of the experimental group (0, 1, 3, 5 days) were higher ( P < 0.05), while the proportions of CD3 +CD4 -CD8 + T cells in thymus were lower ( P < 0.05). For 4-week-old offspring and after infection of P.b, the proportions of CD3 +CD4 +CD8 - T cells in thymus and spleen of the experimental group were both significantly higher than those of control group ( P < 0.05), in contrast, the proportions of CD3 +CD4 -CD8 + T cells in thymus and spleen were both significantly lower than those of control group ( P < 0.05). The spleen cells of 4-week-old mice were stimulated by P.b antigen or mitogen ConA in vitro, compared with the control group, there were no significant differences in the proportions of CD3 +CD4 +CD8 - T cells and CD3 +CD4 -CD8 + T cells in the experimental group ( P > 0.05). Conclusion:During pregnancy, the maternal infection of P.b could significantly affect the ratio of CD4/CD8 T cell subsets in thymus and spleen of offspring mice; and could change the cellular immune response of offspring to P.b infection.

Antimalarial activity of the aqueous extract of Euphorbia cordifolia Elliot in Plasmodium berghei-infected mice

Objective: To evaluate the antimalarial activity of the aqueous extract of Euphorbia (E.) cordifolia Elliot against Plasmodium (P.) berghei-infected mice. Methods: Thirty healthy Swiss mice were intraperitoneally inoculated with 200 μL of P. berghei parasitized-erythrocytes and divided into five groups, and then daily treated for 5 d with single dose of 10 mL/kg of distilled water for malaria control, 10 mg/kg of chloroquine for the chloroquine control and 100, 200 and 400 mg/kg of the aqueous extract of E. cordifolia for the three test groups. Parasitaemia was monitored by Giemsa-staining. At the end of the treatment, animals were sacrificed, and blood was collected for haematological and biochemical analyses. Organs were collected for biochemical and histopathological analyses. Statistical significance (P<0.05) was evaluated by analysis of variance followed by the Tukey post-test using Graphpad prism 7.0. Results: E. cordifolia extract decreased the parasite load to 2.46%, with an effective dose (ED

Antimalarial activity of the aqueous extract of Euphorbia cordifolia Elliot in Plasmodium berghei-infected mice

Objective: To evaluate the antimalarial activity of the aqueous extract of Euphorbia (E.) cordifolia Elliot against Plasmodium (P.) berghei-infected mice. Methods: Thirty healthy Swiss mice were intraperitoneally inoculated with 200 μL of P. berghei parasitized-erythrocytes and divided into five groups, and then daily treated for 5 d with single dose of 10 mL/kg of distilled water for malaria control, 10 mg/kg of chloroquine for the chloroquine control and 100, 200 and 400 mg/kg of the aqueous extract of E. cordifolia for the three test groups. Parasitaemia was monitored by Giemsa-staining. At the end of the treatment, animals were sacrificed, and blood was collected for haematological and biochemical analyses. Organs were collected for biochemical and histopathological analyses. Statistical significance (P<0.05) was evaluated by analysis of variance followed by the Tukey post-test using Graphpad prism 7.0. Results: E. cordifolia extract decreased the parasite load to 2.46%, with an effective dose (ED50) of 113.07 mg/kg compared to the malaria group where the parasite load increased to (46.46±10.28)%. E. cordifolia extract prevented hypoglycaemia, anaemia, leucocytosis and thrombocytopenia, attenuated the increase of transaminases activities, bilirubin and creatinine rate, and improved catalase and superoxide dismutase activities, while reducing malondialdehyde contents in the liver and kidney. E. cordifolia extract significantly prevented histological damages observed in the malaria control group. No acute toxicity sign was observed in mice with plant extract at the dose up to 5 000 mg/kg. Conclusions: E. cordifolia extract at 200 and 400 mg/kg showed significant antimalarial effects. This results support its traditional use in the treatment of malaria.

In vitro and in vivo antimalarial activity of the volatile oil of Cyperus articulatus (Cyperaceae)

Malaria is a disease of global tropical distribution, being endemic in more than 90 countries and responsible for about 212 million cases worldwide in 2016. To date, the strategies used to eradicate this disease have been ineffective, without specific preventive measures such as vaccines. Currently, the existing therapeutic arsenal is limited and has become ineffective against the expansion of artemisinin-resistant Plasmodium, demonstrating the need for studies that would allow the development of new compounds against this disease. In this context, we studied the volatile oil obtained from rhizomes of Cyperus articulatus (VOCA), a plant species commonly found in the Amazon region and popularly used as a therapeutic alternative for the treatment of malaria, in order to confirm its potential as an antimalarial agent by in vitro and in vivo assays. We cultured Plasmodium falciparum W2 (chloroquine-resistant) and 3D7 (chloroquine-sensitive) strains in erythrocytes and exposed them to VOCA at different concentrations in 96-well microplates. In vivo antimalarial activity was tested in BALB/c mice inoculated with approximately 106 erythrocytes infected with Plasmodium berghei. VOCA showed a high antimalarial potential against the two P. falciparum strains, with IC50 = 1.21 µg mL-1 for W2 and 2.30 µg mL-1 for 3D7. VOCA also significantly reduced the parasitemia and anemia induced by P. berghei in mice. Our results confirmed the antimalarial potential of the volatile oil of Cyperus articulatus. (AU)

Phytochemical Analysis and Antiplasmodial (curative) Activities of Methanolic Leaf Extract of Morinda lucida (Ewe Oruwo) in Male Swiss Mice Infected with Plasmodium berghei NK65

Aim:Medicinal plants have been used for the treatment of many infections and diseases including malaria. The study was conducted to determine the effect of in vivoanti-plasmodialand antioxidant properties of the methanolic leaf extract of Morinda lucidain male Swiss albino mice infected with Plasmodium Berghei NK65. Study Design and Methodology:Phytochemical, GC-MS and AAS analyses were determined in the plant. Swiss albino mice were inoculated intraperitoneally with Plasmodium bergheiNK65. Thirty-five (35) mice were grouped into seven groups, five per group. Group A were not infected with P.bergheiNK65.Group B, C and D served as the negative and positive control groups while Group E, F and G mice were treated with 400, 600 and 800 mg/kg body weight of methanolic leaf extract of M. lucida. Haematological parameters were determined in the whole blood using BC-3200 Auto Hematology Analyzer. TP, MDA, CAT, SOD % inhibition, SOD unit and vitamin A were all determined in the liver homogenateusing standard procedures.Results:The GC-MS result of the M. lucidashows the presence of five bioactive compounds. It was also observed that the plant contains the following minerals: iron, magnesium, potassium, phosphorus and copper. Acute toxicity shows that the LD50>000mg/Kg b.wt. The extract caused 30.96%, 32.93% and 67.23% reduction in parasitemia at 400, 600 and 800 mg/kg body weight respectively while chloroquine exerted 96.53% and artesunate exerted 92.03% reduction at 10 mg/kg body weight respectively. The Haematological parameters showed that the plant extractis nothaematotoxic since it significantly (P<0.05) reduced WBC count, and increase RBC, HGB, and HCT values in the treated mice compared to the infected untreated mice. This study shows that the mean lipid peroxidation (MDA) level was significantly decreased in the malaria treated mice (group C, D, E, F and G) compared to the untreated mice (group B). There was also a significant increase in the total protein, catalase, SOD % inhibition, SOD unit and Vitamin A levels in the liver homogenate of animals treated with chloroquine, artesunate and extract of M. lucidacompared to the untreated mice. Conclusions: The study shows that Morinda lucidapossess antiplasmodial activity in male Swiss mice infected with Plasmodium berghei NK 65.

Antimalarial activity of a novel series of artemisinin-derived 1, 2, 3-triazole dimers

Objective: To obtain suitable artimisinin-based drug candidates with high antimalarial activity. Methods: Three different reaction schemes were used to synthesize a total of 15 artemisinin-based compounds. The first synthetic scheme involved the synthesis of diazido aliphatic and aromatic compounds from commercially available dihalides and azido derivatives of artemisinin. The second scheme consisted of the reaction of dibromoaliphatic compounds with sodium azide in dimethylformamide which yielded the desired compounds. Artemisinin-based compounds on treatment with sodium azide and bromotrimethylsilane in dichloromethane produced the most potent compound GB-2. Another potent compound GB-1 was synthesized from artemisinin by treatment with alcohols in the presence of Aberlyst-15 in anhydrous dichloromethane. The third scheme involved the Huisgen 1,3-dipolar cycloaddition between the synthesized aliphatic and aromatic diazides and two alkyne derivatives of artemisinin to obtain the desired artemisinin dimers with average yields. Results: The best in vitro antiplasmodial activity was shown by the compound GB-2 registering IC

Antimalarial activity of a novel series of artemisinin-derived 1, 2, 3-triazole dimers

Objective: To obtain suitable artimisinin-based drug candidates with high antimalarial activity. Methods: Three different reaction schemes were used to synthesize a total of 15 artemisinin-based compounds. The first synthetic scheme involved the synthesis of diazido aliphatic and aromatic compounds from commercially available dihalides and azido derivatives of artemisinin. The second scheme consisted of the reaction of dibromoaliphatic compounds with sodium azide in dimethylformamide which yielded the desired compounds. Artemisinin-based compounds on treatment with sodium azide and bromotrimethylsilane in dichloromethane produced the most potent compound GB-2. Another potent compound GB-1 was synthesized from artemisinin by treatment with alcohols in the presence of Aberlyst-15 in anhydrous dichloromethane. The third scheme involved the Huisgen 1,3-dipolar cycloaddition between the synthesized aliphatic and aromatic diazides and two alkyne derivatives of artemisinin to obtain the desired artemisinin dimers with average yields. Results: The best in vitro antiplasmodial activity was shown by the compound GB-2 registering IC50 value 0.066 μg/mL against chloroquine-sensitive and 0.865 μg/mL against chloroquine-resistant strains of Plasmodium falciparum. It suppressed 59.0% parasitaemia in vivo of rodent malaria parasite Plasmodium berghei in Swiss albino model at 50 μg/kg body weight dosage. Molecular docking interactions of Plasmodium falciparum ATP6 (PfATP6) protein revealed strong bonding of GB-2 with Thr255 residue which is likely to be the reason for excellent antimalarial activity of this compound. Conclusion: Two compounds GB-1 and GB-2 exhibited excellent in vitro antiplasmodial activity and fair in vivo antimalarial activity. Of the two, GB-2 showed better activity which could be attributed to its strong bonding interactions with Thr255 as evidenced from the molecular docking study. Study helped in identifying artemisinin analogues possessing good antimalarial properties and further research in structural alterations of the selected molecules should be carried out which may result in obtaining potent drug candidates against the malarial parasite.

Previous Infection with Plasmodium berghei Confers Resistance to Toxoplasma gondii Infection in Mice

Both Plasmodium spp. and Toxoplasma gondii are important apicomplexan parasites, which infect humans worldwide. Genetic analyses have revealed that 33% of amino acid sequences of inner membrane complex from the malaria parasite Plasmodium berghei is similar to that of Toxoplasma gondii. Inner membrane complex is known to be involved in cell invasion and replication. In this study, we investigated the resistance against T. gondii (ME49) infection induced by previously infected P. berghei (ANKA) in mice. Levels of T. gondii-specific IgG, IgG1, IgG2a, and IgG2b antibody responses, CD4+ and CD8+ T cell populations were found higher in the mice infected with P. berghei (ANKA) and challenged with T. gondii (ME49) compared to that in control mice infected with T. gondii alone (ME49). P. berghei (ANKA) + T. gondii (ME49) group showed significantly reduced the number and size of T. gondii (ME49) cysts in the brains of mice, resulting in lower body weight loss compared to ME49 control group. These results indicate that previous exposure to P. berghei (ANKA) induce resistance to subsequent T. gondii (ME49) infection.

Characterization of Plasmodium berghei Homologues of T-cell Immunomodulatory Protein as a New Potential Candidate for Protecting against Experimental Cerebral Malaria

The pathogenesis of cerebral malaria is biologically complex and involves multi-factorial mechanisms such as microvascular congestion, immunopathology by the pro-inflammatory cytokine and endothelial dysfunction. Recent data have suggested that a pleiotropic T-cell immunomodulatory protein (TIP) could effectively mediate inflammatory cytokines of mammalian immune response against acute graft-versus-host disease in animal models. In this study, we identified a conserved homologue of TIP in Plasmodium berghei (PbTIP) as a membrane protein in Plasmodium asexual stage. Compared with PBS control group, the pathology of experimental cerebral malaria (ECM) in rPbTIP intravenous injection (i.v.) group was alleviated by the downregulation of pro-inflammatory responses, and rPbTIP i.v. group elicited an expansion of regulatory T-cell response. Therefore, rPbTIP i.v. group displayed less severe brain pathology and feverish mice in rPbTIP i.v. group died from ECM. This study suggested that PbTIP may be a novel promising target to alleviate the severity of ECM.

Induction of Angiogenesis by Malarial Infection through Hypoxia Dependent Manner

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×10⁶ parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.

In vivo antiplasmodial potential of three herbal methanolic extracts in mice infected with Plasmodium berghei NK65 / 中草药·英文版

Objective: The present study deals with the investigation of antiplasmodial potential of leaf methanolic extract of Aegle marmelos, Aristolochia indica and Cassia auriculata against Plasmodium berghei (NK65) infected mice. Methods: The chloroquine-sensitive parasites P. berghei (1 × 106) were inoculated into Swiss albino mice intraperitoneally. The methanol extracts of three herbal plants were orally administered in P. berghei infected mice which were further assessed using the four-day suppressive test at different doses of 150, 300 and 600 mg/kg per day. Chloroquine (CQ) was used as the standard drug with of 1.25, 2.5 and 5 mg/kg concentrations and was orally administered. Results: The leaves of A. marmelos, A. indica, and C. auriculata were found to suppress P. berghei parasitaemia in Swiss albino mice by (67.0 ± 4.02)%, (72.0 ± 8.44)% and (52.7 ± 2.06)% at 600 mg/kg/d with ED50 values of 284.73, 233.77 and 562.48 mg/kg, respectively. These herbal plants increased the mean survival time of infected mice and prevented body weight loss. GC-MS analysis revealed the presence of hentriacontan-16-one (C31H62O) in A. indica extract. The histopathology study showed non-toxic to kidney and liver at 600 mg/kg/body weight. Conclusions: Overall results revealed that herbal plants may be active in the development of novel and cheap antimalarial compounds.

Antimalarial Activity of Crude Extract and Fractions of Phyllanthus amarus in Plasmodium berghei-Infected Mice

Aim: This study evaluated the antimalarial activity of the crude extract and fractions of Phyllanthus amarus in Plasmodium berghei-infected mice. Place and Duration of Study: Department of Biochemistry, Federal University of Technology, Minna, Niger State, Nigeria, between February 2016 and August 2016. Methodology: Mice infected with Plasmodium berghei were administered orally with the crude extract of Phyllanthus amarus whole plant 72 hours post infection at doses ranging from 100-500 mg/kg/day, for five consecutive days. Chloroquine (5 mg/kg/day).and artesunate (50 mg/kg/day) were used as controls, while distilled water was administered to the negative control groups. N-hexane, chloroform, ethyl acetate and aqueous fractions, obtained from crude aqueous methanolic extract, were also evaluated for their inhibitory effect against P. berghei at doses ranging from 50-200 mg/kg/day. Level of parasitaemia, survival time, variations in the values of body weight and % PCV were monitored throughout the study period. Results: Crude extract of Phyllanthus amarus whole plant showed significant (P < 0.05) antiplasmodial activity in dose dependent pattern with 76.74% inhibition of parasite growth. Aqueous fraction at a dose of 200 mg/kg demonstrated significant antiplasmodial activity with %inhibition of parasite growth of 56.40. The variations in the values of weight and %PCV before and after treatment were not significant in both the crude and aqueous fraction. Significant inhibition of parasite growth by the crude extract and aqueous fraction resulted in longer mouse survival relative to the control, as confirmed in the mean survival time of the mice (27.67±1.45, 22.67±0.67, 29.33±0.67 and 6.67±0.88 days) for the crude extract (500mg/kg), aqueous fraction (200 mg/kg), chloroquine and negative control groups respectively. Phytochemical screening of the extracts revealed the presence of alkaloids, flavonoids, phenol, tannins, steroids, terpenoids and saponins. Conclusion: The results indicate that the whole plant extract and fractions of Phyllanthus amarus have antimalarial property which can serve as a novel source for the development of new and affordable antimalarial agent.

Antimalarial effect of two photo-excitable compounds in a murine model with Plasmodium berghei (Haemosporida: Plasmodiidae) / Efecto antimalárico de dos compuestos foto-excitables en un modelo murino con Plasmodium berghei (Haemosporida: Plasmodiidae)

Abstract Malaria represents a major health problem worldwide, affecting around 198 million people in 2016 according to WHO database. For decades, anti-malarial drug therapy has been used in the battle against this disease and its uncontrolled usage in endemic areas has developed the appearance of the drug resistance. Thus, it has emerged the necessity of finding new treatments that could be used as an alternative cure to malaria infection. The aim of this work was the evaluation of two photo-excitable compounds: Compound 1, which is (2E)-3-(4-dimethylamino-phenyl)-1-(4-imidazol-1-yl-phenyl)prop-2-en-1-one) and Compound 2, (1E,4E)1-[4-(dimethylamino)phenyl]-5-(4-methoxyphenyl)-1,4-pentadiene-3-one) as possible anti-malaria drugs with Plasmodium berghei ANKA strain in BALB/c mice as murine model. Cytotoxicity effect was evaluated by a cell proliferation by colorimetry assay (MTS); and the drug incorporation into the parasite was assessed in vitro with Indirect Immunofluorescence Assay (IFA) to determine the localization of the drugs into the parasitized red blood cells (RBCs). Finally, the curative effect of compounds no-radiation (fundamental state) and ration drugs were evaluated by oral drug administration of this drugs in BALB/c mice and chloroquine was used as positive control. This curative effect was determined daily by the parasitemia percentage. The results showed that both compounds were cytotoxic in fundamental state. Furthermore, cytotoxic effect was increased after radiation into the Solar Simulator, and compound 2 was more cytotoxic than compound 1. Curative assays showed that both compounds in fundamental state were non effective as anti-malarial drug. However, in the curative assays in the mice treated with compound 2, when this was ration showed a survival rate of 33 % and a parasitemia percentage decrease in compare to compound 1. Although the compounds did not show a similar or better anti-malarial effect than Chloroquine, Compound 2 presented certain anti-malarial effect after solar radiation. Rev. Biol. Trop. 66(2): 880-891. Epub 2018 June 01.

Resumen La malaria representa un importante problema de salud en todo el mundo, afectando a alrededor de 198 millones de personas en 2016 según la base de datos de la OMS. Durante décadas, se ha utilizado la terapia con fármacos anti-malpricos en la lucha contra esta enfermedad y su uso incontrolado en las zonas endémicas ha desarrollado la aparición de resistencia a los fármacos. Por lo tanto, se ha surgido la necesidad de encontrar nuevos tratamientos que podrían ser utilizados como una cura alternativa para la infección por el paludismo. El objetivo de este trabajo fue evaluar dos compuestos foto-excitables: El compuesto 1, que es (2E) -3- (4-dimetilamino-fenil) -1- (4-imidazol-1-ilfenil) prop-2 1-ona) y el Compuesto 2, (1E, 4E) -1- [4- (dimetilamino) fenil] -5- (4-metoxifenil) -1,4-pentadieno-3-ona) como posibles drogas antimaláricas con la cepa ANKA de Plasmodium berghei en ratones BALB / c como modelo murino. El efecto de la citotoxicidad se evaluó mediante una proliferación celular con el ensayo de colorimetría (MTS); y la incorporación del fármaco en el parásito se evaluó in vitro con Ensayo de Inmunofluorescencia Indirecta (IFA) para determinar la localización de los fármacos en los glóbulos rojos parasitados (RBCs). Finalmente, se evaluó el efecto curativo de los compuestos sin radiación (estado fundamental) y los fármacos irradiados mediante la administración oral de los fármacos en los ratones BALB / c, y se usó cloroquina como control positivo de cura. Este efecto curativo se determinó diariamente por el porcentaje de parasitemia. Los resultados mostraron que ambos compuestos eran citotóxicos en estado fundamental. Además, el efecto citotóxico se incrementó después de la radiación en el Simulador Solar, y el compuesto 2 fue más citotóxico que el compuesto 1. Los ensayos curativos mostraron que ambos compuestos en estado fundamental no eran eficaces como fármacos antimaláricos. Sin embargo, en los ensayos curativos en los ratones tratados con el compuesto 2, cuando fue irradiado, se observó una tasa de supervivencia del 33 % y una disminución del porcentaje de parasitemia en comparación con el compuesto 1. Aunque los compuestos no mostraron un efecto similar o mejor antimalárico que la cloroquina, el compuesto 2 presentó cierto efecto antimalárico después de la radiación solar.

Nano chloroquine delivery against Plasmodium berghei NK65 induced programmed cell death in spleen

Objective:To compare the protective effects of chitosan-trypolyphosphate (CS-TPP) nanoparticle conjugated chloroquine(CQ) with effect of CQ alone on the reversal of splenic damages and induction of apoptosis.Methods:Different researches have been carried out to explore the potential role of chitosan based drug delivery system against parasitic diseases. After successive Plasmodium berghei NK65 parasiste infection by intraperitoneal injection in Swiss mice and subsequent parasite development, the ROS generation, anti-apoptotic and pro apoptotic protein levels in spleen were measured. To analyze caspases, flow cytometry study was performed with annexin V -FITC and with PI staining.Results:The results revealed that ROS mediated caspase 3 and 9 activation and the induction of apoptosis occurred during the parasitic infection. However, CS-TPP conjugated CQ was relatively better in reversing the splenic damage compared with similar effects of CQ alone.Conclusions:This study indicates that Plasmodium berghei NK65 induces apoptosis in the spleen. The study further shows that CS-TPP nanoparticles conjugation with CQ have positive influence on the recovery of damaged host’s system towards maintenance of normal homeostasis, and this is shown to be selective to CS-TPP conjugated CQ treated animals only.

Nano chloroquine delivery against Plasmodium berghei NK65 induced programmed cell death in spleen

Objective: To compare the protective effects of chitosan-trypolyphosphate (CS-TPP) nanoparticle conjugated chloroquine(CQ) with effect of CQ alone on the reversal of splenic damages and induction of apoptosis. Methods: Different researches have been carried out to explore the potential role of chitosan based drug delivery system against parasitic diseases. After successive Plasmodium berghei NK65 parasiste infection by intraperitoneal injection in Swiss mice and subsequent parasite development, the ROS generation, anti-apoptotic and pro apoptotic protein levels in spleen were measured. To analyze caspases, flow cytometry study was performed with annexin V-FITC and with PI staining. Results: The results revealed that ROS mediated caspase 3 and 9 activation and the induction of apoptosis occurred during the parasitic infection. However, CS-TPP conjugated CQ was relatively better in reversing the splenic damage compared with similar effects of CQ alone. Conclusions: This study indicates that Plasmodium berghei NK65 induces apoptosis in the spleen. The study further shows that CS-TPP nanoparticles conjugation with CQ have positive influence on the recovery of damaged host's system towards maintenance of normal homeostasis, and this is shown to be selective to CS-TPP conjugated CQ treated animals only.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei

Objective: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. Methods: Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. Results: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P < 0.01), and IgG1 and IgG2a all increased (P < 0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P < 0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P < 0.05), and antiserum ookinete numbers (cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. Conclusions: GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.