Cell death in amastigote forms of Leishmania amazonensis induced by parthenolide.

BACKGROUND: Leishmania amazonensis infection results in diverse clinical manifestations: cutaneous, mucocutaneous or visceral leishmaniasis. The arsenal of drugs available for treating Leishmania infections is limited. Therefore, new, effective, and less toxic leishmaniasis treatments are still needed. We verified cell death in amastigote forms of Leishmania amazonensis induced by the sesquiterpene lactone parthenolide. RESULTS: The tested compound was able to concentration-dependently affect axenic and intracellular amastigotes, with IC50 values of 1.3 µM and 2.9 µM, respectively after 72 h incubation. No genotoxic effects were observed in a micronucleus test in mice. Parthenolide induced morphological and ultrastructural changes in axenic amastigotes, including a loss of membrane integrity, swelling of the mitochondrion, cytoplasmic vacuoles, and intense exocytic activity in the region of the flagellar pocket. These results led us to investigate the occurrence of autophagic vacuoles with monodansylcadaverine and the integrity of the plasma membrane and mitochondrial membrane potential using flow cytometry. In all of the tests, parthenolide had positive results. CONCLUSIONS: Our results indicate that the antileishmanial action of parthenolide is associated with autophagic vacuole appearance, a reduction of fluidity, a loss of membrane integrity, and mitochondrial dysfunction. Considering the limited repertoire of existing antileishmanial compounds, the products derived from medicinal plants has been one the greatest advances to help develop new chemotherapeutic approaches.

Intramuscular and topical treatment of cutaneous leishmaniasis lesions in mice infected with Leishmania amazonensis using coumarin (-) mammea A/BB.

Treatment of cutaneous leishmaniasis remains limited to a few available options. Recent studies showed in vitro antileishmanial activity of (-) mammea A/BB, a coumarin isolated from leaves of Calophyllum brasiliense. Moreover, the dichloromethane crude extract and hexane fraction from this plant demonstrated in vivo activity in mice infected with Leishmania amazonensis. We evaluated the antileishmanial activity of (-) mammea A/BB in the L. amazonensis BALB/c mice model. The animals were given intramuscular and topical treatment with (-) mammea A/BB for 30 consecutive days. The results demonstrated that 18mg/kg/d intramuscularly or 0.2% topically of (-) mammea A/BB significantly reduced the size of skin lesions in footpads of mice compared with those in the control group (p<0.05). The activity of Glucantime(®) (corresponding to 27mg/kg/d of pentavalent antimony) administered intramuscularly was similar to that of (-) mammea A/BB (p<0.05) by both routes of administration. The histopathological evaluation showed no changes in the organs analyzed. These results indicate that the coumarin obtained from C. brasiliense is the antileishmanially active compound and can be used to control the development of cutaneous leishmaniasis lesions caused by L. amazonensis.

Triagem fitoquímica e avaliação das atividades antimicrobiana e citotóxica de plantas medicinais nativas da região oeste do estado do paraná / Phytochemical screening and evaluation of antimicrobial and cytotoxic activities of native medicinal plants from west region of paraná state

Muitas plantas não apresentam suas potencialidades terapêuticas comprovadas por meio de estudos científicos. Assim, o objetivo deste trabalho foi realizar um estudo fitoquímico, antimicrobiano e citotóxico das espécies vegetais Eupatorium serratum (Asteraceae), Myrcianthes pungens (Myrtaceae), Urera nitida (Urticaceae), Campomanesia xanthocarpa (Myrtaceae) e duas variedades de Psidium cattleyanum (Myrtaceae). Para tanto, as plantas foram secas e extraídas com diferentes solventes (hexano, metanol e acetato de etila) obtendo-se 18 extratos brutos. Métodos específicos foram usados para identificação de alcalóides, saponinas, flavonóides, taninos e esteróides. Para determinar as concentrações inibitória mínima (CIM) e bactericida mínima (CBM) utilizaram-se bactérias Gram positivas (Staphylococcus aureus e Micrococcus luteus) e Gram negativas (Escherichia coli e Salmonella typhi). O método de difusão em disco foi usado contra leveduras (Candida albicans and Saccharomyces cerevisiae). C. xanthocarpa, M. pungens (extratos acetato de etila e hexano) e uma variedade de P. cattleyanum (extrato acetato de etila), apresentaram CIM ? 62,5 µg/ml contra bactérias Gram-positivas, o que pode estar relacionado aos taninos encontrados nas folhas dessas plantas. O extrato hexânico de M. pungens mostrou a mais forte inibição, apresentando CBM de mesma concentração. Os extratos metanólicos de C. xanthocarpa e M. pungens mostraram boa atividade antifúngica. O ensaio citotóxico verificou o efeito hemolítico e o extrato hexânico de uma variedade de P. cattleyanum apresentou alta citotoxicidade nas concentrações testadas.

Many plants do not present its proven therapeutic potentialities through scientific studies. Thus, the aim of this work was to accomplish a phytochemical, antimicrobial and cytotoxic study of the vegetal species Eupatorium serratum (Asteraceae), Myrcianthes pungens (Myrtaceae), Urera nitida (Urticaceae), Campomanesia xanthocarpa (Myrtaceae) and two varieties of Psidium cattleyanum (Myrtaceae). The plants were dried and extracted with different solvents (hexane, methanol and ethyl acetate) resulting in 18 crude extracts. Specific methods had been used for identification of alkaloids, saponnins, flavonoids, tannins and steroids. To determine the minimum inhibitory (MIC) and bactericidal (MBC) concentrations were used Gram-positive (Staphylococcus aureus and Micrococcus luteus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria. The method of disc diffusion was used against yeasts (Candida albicans and Saccharomyces cerevisiae). C. xanthocarpa, M. pungens (ethyl acetate and hexane extracts) and a variety of P. cattleyanum (ethyl acetate extract), presented MIC ? 62.5 µg/ml against Gram-positive bacteria, which may be related to tannins in the leaves of these plants. The hexanic extract of M. pungens showed the strongest inhibition, presenting MBC of equal concentration. The methanolic extracts of C. xanthocarpa and M. pungens showed good antifungal activity. The cytotoxic assay verified the hemolytic effect and the hexanic extract of a variety of P. cattleyanum presented high citotoxicity at the concentrations tested.

Synergistic effects of parthenolide and benznidazole on Trypanosoma cruzi.

Parthenolide previously isolated from Tanacetum vulgare was tested for its in vitro combinatory effect with benznidazole against Trypanosoma cruzi. Parthenolide showed a strong synergistic activity against epimastigote forms, reducing 23-fold the concentration of benznidazole necessary to inhibit 50% of cell growth (IC(50) of 1.6 to 0.07 µg/ml) when in combination with parthenolide. In addition, an additive effect against trypomastigote forms (FIC 1.06), followed by an antagonistic effect on the cytotoxicity (FIC 2.36), was observed for the combination of both drugs. Parthenolide induced morphological alterations in the body shape of trypomastigote forms, causing rounding and shortening of the parasite and loss of integrity of the plasma membrane, as previously described by other workers.

Effects of medicinal plant extracts on growth of Leishmania (L) amazonensis and Trypanosoma cruzi

Este estudo descreve a triagem de extratos obtidos de 19 espécies de plantas usadas na medicina tradicional brasileira para o tratamento de várias doenças. Os extratos foram testados contra formas amastigota axênica e promastigota de Leishmania (L.) amazonensis, e formas epimastigota de Trypanosoma cruzi in vitro na concentração de 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, e Tanacetum vulgare apresentaram efeito significante contra um ou ambos parasitas, com a porcentagem de inibição de crescimento entre 49,5 e 99%. Os extratos não mostraram efeito citotóxico em hemácias de carneiro. Essas plantas medicinais podem ser fontes alternativas de novos compostos clinicamente ativos contra L. amazonensis e T. cruzi.

Antileishmanial activity of parthenolide, a sesquiterpene lactone isolated from Tanacetum parthenium.

The in vitro activity of parthenolide against Leishmania amazonensis was investigated. Parthenolide is a sesquiterpene lactone purified from the hydroalcoholic extract of aerial parts of Tanacetum parthenium. This isolated compound was identified through spectral analyses by UV, infrared, (1)H and (13)C nuclear magnetic resonance imaging, DEPT (distortionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multiple-quantum coherence), and electron spray ionization-mass spectrometry. Parthenolide showed significant activity against the promastigote form of L. amazonensis, with 50% inhibition of cell growth at a concentration of 0.37 microg/ml. For the intracellular amastigote form, parthenolide reduced by 50% the survival index of parasites in macrophages when it was used at 0.81 microg/ml. The purified compound showed no cytotoxic effects against J774G8 macrophages in culture and did not cause lysis in sheep blood when it was used at higher concentrations that inhibited promastigote forms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gelatin as the substrate showed that the enzymatic activity of the enzyme cysteine protease increased following treatment of the promastigotes with the isolated compound. This finding was correlated with marked morphological changes induced by parthenolide, such as the appearance of structures similar to large lysosomes and intense exocytic activity in the region of the flagellar pocket, as seen by electron microscopy. These results provide new perspectives on the development of novel drugs with leishmanicidal activities obtained from natural products.