Pharmacological potential of parasitic Angiosperms against microorganisms / Potencial farmacológico de Angiospermas parásitas contra microorganismos

Parasitic habit plants arouse great interest for their extraordinary growth adaptations and produce different secondary metabolites that allow them to survive the extreme conditions in which they develop. This can provide a range of compounds with multiple uses. However, those plants have been poorly explored and there is very little information about their natural compounds, some healing properties attributed to several communities have been scientifically supported. Articles were consulted in databases (Google Scholar, PubMed, Scielo, Science Direct, Scopus) considering the biological activity reported until June 2021. Parasitic plants present mainly antibacterial activity, some species antifungal moderate activity, and to a lesser extent were found reports of antiprotozoal activity. The composition of parasitic plants should continue to be investigated, which could be an alternative to combat various microorganisms that cause diseases, even resistant to traditional medicines.

Las plantas parásitas despiertan gran interés por sus adaptaciones extraordinarias de crecimiento, y producir diversos metabolitos secundarios que les permiten desarrollarse en condiciones extremas, lo que puede brindar un abanico de compuestos con múltiples usos. Sin embargo, estas plantas han sido pobremente exploradas y es muy poca la información que se tiene de sus compuestos naturales, se han soportado científicamente algunas propiedades curativas, atribuidas por diferentes comunidades. Se consultaron artículos en bases de datos (Google Scholar, PubMed, Scielo, Science Direct, Scopus), teniendo en cuenta la actividad biológica reportada hasta agosto de 2019. Las plantas parásitas presentan principalmente actividad antibacteriana, algunas especies con actividad antifúngica moderada, y en menor proporción reportes de actividad antiprotozoaria. Debe continuarse indagando la composición molecular de estas plantas, lo que podría ser una alternativa para combatir diversos microorganismos causantes de enfermedades, incluso aquellos resistentes a los medicamentos tradicionales.

Seasonal variance in the chemical composition of essential oils from Lantana camaraaccessions and their trypanocidal activity on Phytomonas serpens / Variación estacional en la composición química de los aceites esenciales de las accesiones de Lantana camaray su actividad tripanocida en Phytomonas serpens

The objective of this study was to investigate the seasonal variance of the content and chemical composition of the essential oil from Lantana camara accessions at two harvest times, and to analyze the trypanocidal activity on Phytomonas serpens. Essential oil content ranged from 0.13 to 0.29% in the rainy season and from 0.13 to 0.33% in the dry season. The compounds E-caryophyllene, α-humulene, α-curcumene and germacrene D defined the formation of four chemical clusters in the rainy and dry seasons, classified as: Cluster 1 (E-caryophyllene + germacrene D); Cluster 2 (germacrene D + E-caryophyllene); Cluster 3 (α-humulene + E-caryophyllene); and Cluster 4 (α-curcumene + E-caryophyllene). All L. camara essential oils, representing the four chemical clusters, inhibited P. serpenswith low concentrations, considering the following IC50 values: 18.34±6.60 µg/mL (LAC-018, Cluster 1); 9.14±3.87 µg/mL (LAC-027, Cluster 2); 14.56±3.40 µg/mL (LAC-037, Cluster 3); and 14.97±2.68 µg/mL (LAC-019, Cluster 4).

El objetivo de este estudio fue investigar la variación estacional del contenido y la composición química del aceite esencial de accesiones de Lantana camara en dos tiempos de cosecha y analizar la actividad tripanocida en Phytomonas serpens. El contenido de aceite esencial osciló entre 0,13% y 0,29% en la temporada de lluvias y entre 0,13% y 0,33% en la temporada seca. Los compuestos E-cariofileno, α-humuleno, α-curcumeno y germacreno D definieron la formación de cuatro grupos químicos en las estaciones lluviosa y seca, clasificados como: Grupo 1 (E-cariofileno + germacreno D); Grupo 2 (germacreno D + E-cariofileno); Grupo 3 (α-humuleno + E-cariofileno); y Grupo 4 (α-curcumeno + E-cariofileno). Todos los aceites esenciales de L. camara, que representan los cuatro grupos químicos, inhibieron P. serpens con bajas concentraciones, considerando los siguientes valores de CI50:18,34 ± 6,60 µg / mL (LAC-018, grupo 1); 9,14 ± 3,87 µg / ml (LAC-027, grupo 2); 14,56 ± 3,40 µg / ml (LAC-037, grupo 3); y 14,97 ± 2,68 µg / ml (LAC-019, grupo 4).

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

The heterologous expression of Escherichia coli MutT enzyme is involved in the protection against oxidative stress in Leishmania braziliensis

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Salvadora persica protects mouse intestine from eimeriosis / Salvadora persica protege o intestino do rato da eimeriose

Abstract Eimeriosis is a global poultry health problem. In the current study, we investigated the role of Salvadora persica leaf extracts (SE) against murine eimeriosis induced by Eimeria papillata. The infection induced an oocyst output of 6242 ± 731 oocysts/g feces. After treatment with 300 mg⁄kg SE, the oocysts expelled in feces decreased by approximately 3-fold. In addition, the total number of E. papillata in the parasitic stage decreased in the jejunum of mice after treatment with SE. In addition, SE significantly reduced the number of apoptotic cells by approximately 2-fold in the infected jejunum. SE ameliorated the changes in glutathione, malondialdehyde, and catalase due to E. papillata infection. Finally, SE regulated the cytokine genes, interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α, and the apoptotic genes, B-cell lymphoma-2, Bax, and Caspase-3. SE protects the jejunum from E. papillata induced injury and may have potential therapeutic value as a food additive during eimeriosis.

Resumo A eimeriose é um problema global de saúde avícola. No presente estudo, investigou-se o papel dos extratos de folhas de Salvadora persica (SE) contra a eimeriose murina induzida por Eimeria papillata. A infecção induziu uma produção de oocistos de 6242 ± 731 oocistos/g de fezes. Após o tratamento com 300 mg⁄kg SE, os oocistos eliminados nas fezes diminuíram em aproximadamente 3 vezes. Além disso, o número total de E. papillata no estágio parasitário diminuiu nos jejunos de camundongos após o tratamento com SE. Da mesma forma, o SE reduziu significativamente o número de células apoptóticas em aproximadamente 2 vezes no jejuno infectado. O estudo mostrou que o SE melhorou as alterações na glutationa, malonaldeído e catalase devido à infecção por E. papillata. Finalmente, o SE regulou os genes das citocinas, interleucina (IL) -1β, IL-6, interferon-γ e fator de necrose tumoral α, e os genes apoptóticos, linfoma-2, Bax e Caspase-3. Assim, o SE protegeu os jejunos das lesões induzidas por E. papillata e pode ter potencial valor terapêutico como aditivo alimentar durante a eimeriose.

Antiprotozoal action of synthetic cinnamic acid analogs

Abstract INTRODUCTION Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds. METHODS In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined. RESULTS Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed. CONCLUSIONS Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.

Antiplasmodial and antileishmanial activities of compounds from Piper tuberculatum Jacq fruits

Abstract INTRODUCTION This study assessed the activity of compounds from Piper tuberculatum against Plasmodium falciparum and Leishmania guyanensis. METHODS The effects of compounds from P. tuberculatum fruits on P. falciparum and L. guyanensis promastigote growth in vitro were determined. Hemolytic action and cytotoxicity in HepG2 and J774 cells were measured. RESULTS Three compounds showed strong antiplasmodial activity and one compound showed strong antileishmanial activity. Two compounds were non-toxic to HepG2 cells and all were toxic to J774 cells. The compounds showed no hemolytic activity. CONCLUSIONS The tested compounds from P. tuberculatum exhibited antiparasitic and cytotoxic effects.

Toxicidad, genotoxicidad y actividad anti-Leishmania de aceites esenciales obtenidos de cuatro (4) quimiotipos del género Lippia / Toxicity, genotoxicity and anti-Leishmania activity of essential oils obtained of four chemotypes of Lippia genus

Los aceites esenciales (AEs), pertenecientes al género Lippia, son candidatos interesantes de formulaciones tópicas en el tratamiento de la leishmaniasis cutánea (LC). El objetivo de este trabajo fue determinar el perfil toxicológico y la actividad anti-Leishmania de AEs obtenidos de plantas colombianas del género Lippia. Ratones BALB/c fueron tratados tópica u oralmente con AEs obtenidos de L. alba quimiotipo citral (AE1) y de L. origanoides quimiotipos timol (AE2), carvacrol (AE3) y felandreno (AE4). El efecto del tratamiento en la irritación de la piel, la toxicidad aguda oral, la genotoxicidad (prueba cometa y micronúcleos), los cambios en la función hepática y renal, la inducción de reacción de hipersensibilidad de contacto y en la actividad contra L. (V) panamensis y L. (V.) braziliensis fueron determinados. Todos los AEs presentaron un perfil toxicológico similar a los parámetros normales, exceptuando los aceites AE2 y AE3 los cuales fueron irritantes y presentaron algunos signos de toxicidad aguda oral al ser utilizados en altas concentraciones (concentraciones bajas no fueron tóxicas). El AE2 mostró actividad antiparasitaria en las formas parasitarias evaluadas. Concentraciones bajas de los AEs podrían utilizarse de forma segura como componentes de formulaciones farmacológicas en LC.

Essential oils (EOs) belonging to the genus Lippia are interesting candidates in pharmaceutical systems for cutaneous leishmaniasis (CL). The aim of this work was to determine both toxicological and antileishmanial activities of EOs obtained from different species of Lippia, a widely distributed Colombian plants. BALB/c mice were treated topically or orally with EOs obtained from L. alba citral chemotype (EO1) and L. origanoides thymol (EO2), carvacrol (EO3) and phellandrene (EO4) chemotypes. The skin irritation, oral acute toxicity, genotoxicity (comet assay and micronucleus test), liver and renal adverse effects, All the EOs showed a toxicological profile similar to the normal parameters, except for oils EO2 and EO3 which were irritant and showed some signs of acute oral toxicity at high concentrations (low concentration were safe). The EO2 showed antiparasitic activity. Low concentrations of the EO could be used safely as components of pharmacological formulations in CL.

Ascorbate peroxidase overexpression protects Leishmania braziliensis against trivalent antimony effects

Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.

In vitro and in vivo antileishmanial activity of Artemisia annua L. leaf powder and its potential usefulness in the treatment of uncomplicated cutaneous leishmaniasis in humans

ABSTRACT INTRODUCTION: Cutaneous leishmaniasis (CL) is a tropical disease that affects millions of individuals worldwide. The current drugs for CL may be effective but have serious side effects; hence, alternatives are urgently needed. Although plant-derived materials are used for the treatment of various diseases in 80% of the global population, the validation of these products is essential. Gelatin capsules containing dried Artemisia annua leaf powder were recently developed as a new herbal formulation (totum) for the oral treatment of malaria and other parasitic diseases. Here, we aimed to determine the usefulness of A. annua gel capsules in CL. METHODS: The antileishmanial activity and cytotoxicity of A. annua L. capsules was determined via in vitro and in vivo studies. Moreover, a preliminary evaluation of its therapeutic potential as antileishmanial treatment in humans was conducted in 2 patients with uncomplicated CL. RESULTS: Artemisia annua capsules showed moderate in vitro activity in amastigotes of Leishmania (Viannia) panamensis; no cytotoxicity in U-937 macrophages or genotoxicity in human lymphocytes was observed. Five of 6 (83.3%) hamsters treated with A. annua capsules (500mg/kg/day) for 30 days were cured, and the 2 examined patients were cured 45 days after initiation of treatment with 30g of A. annua capsules, without any adverse reactions. Both patients remained disease-free 26 and 24 months after treatment completion. CONCLUSION: Capsules of A. annua L. represent an effective treatment for uncomplicated CL, although further randomized controlled trials are needed to validate its efficacy and safety.

Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro

BACKGROUND Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells.

Screening of the in vitro antileishmanial activities of compounds and secondary metabolites isolated from Maytenus guianensis Klotzsch ex Reissek (Celastraceae) chichuá Amazon

Abstract INTRODUCTION Maytenus guianensis is a member of the Celastraceae family that is used in traditional medicine, particularly for its anti-parasitic and anti-cancer effects. To explore the ethnopharmacological potential of this plant, the present study was designed to screen the in vitro antileishmanial activities of extracts and compounds isolated from M. guianensis. METHODS Maytenus guianensis stems and leaves were extracted in acetone, followed by the preparation of eluates and isolation of secondary metabolites using chromatography on a glass column with silica gel as the fixed phase. The chemical components were identified using spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance of hydrogen-1 and carbon-13, mass spectroscopy, and infrared spectroscopy. The anti-Leishmania amazonensis activities of these eluates and compounds were evaluated by direct promastigote counting and viability assays. RESULTS It was found that the hexane bark eluate produced the strongest anti-L. amazonensis effect, with 90-100% inhibition of the promastigote form. The isolated metabolite that produced the best result was tingenone B, followed by a compound formed by the union of tingenone and tingenone B (80-90% inhibition). CONCLUSIONS Maytenus guianensis shows anti-parasite activity that warrants further investigation to determine the mechanisms underlying this antileishmanial effect and to evaluate the pharmacological potential of these eluates and isolated secondary metabolites, while minimizing any adverse effects.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract

Abstract INTRODUCTION: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. METHODS The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. RESULTS The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. CONCLUSIONS The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 µg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 µg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.