These results indicate that parthenolide induced amastigote cell death by autophagy, but other mechanisms of cell death cannot be dismissed, such as apoptosis and necrosis. Considering the limited repertoire of existing antileishmanial compounds, continuously developing new leishmanicidal compounds is essential. In the ongoing search for the best antileishmanial compounds, products derived from plants are gaining ground. The isolation and purification of the active components of medicinal
plants has been one the greatest advances. Additionally, delineation of the biochemical mechanisms involved in mediating effect of these compounds would help develop new chemotherapeutic approaches. Methods #Adriamycin clinical trial randurls[1|1|,|CHEM1|]# Drugs find more Parthenolide (minimum 90%) was purchased from Sigma-Aldrich (Steinheim, Germany). Amphotericin B (Cristália, Produtos Químicos Farmacêuticos Ltda, Itapira, SP, Brazil) was used as a positive control. In all of the tests, 0.05% dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) was used to dissolve the highest dose of the compounds and had no effect on the parasites’ proliferation or morphology.
Axenic amastigotes Promastigotes of the Leishmania species differentiate to amastigotes with the combination of low pH and high temperature . The WHOM/BR/75/Josefa strain of Leishmania amazonensis, isolated by C.A. Cuba-Cuba (University of Brasília, Brasília, Distrito Federal, Brazil) from a human case of diffuse cutaneous leishmaniasis, was used in the present study. Axenic amastigote cultures were obtained by the in vitro differentiation of promastigotes from the stationary phase in 25 cm2 tissue culture flasks by progressive temperature increase and pH decrease . The cultures were maintained at 32°C in Schneider’s insect medium (Sigma, St. Louis, MO, USA), pH 4.6,
with 20% fetal bovine serum through weekly serial sub-culturing for further studies. Antiproliferative effect For the parasite growth inhibition assays, L. amazonensis axenic amastigotes were harvested during the exponential phase of growth, and 106 cells were added to each well of a 24-well plate and treated with different concentrations of parthenolide Tolmetin and amphotericin B. Medium alone and 0.05% DMSO were used as negative controls. For each treatment, the parasites were observed and counted daily using a Neubauer chamber with an optical microscope. Each experiment was performed in duplicate and twice on different occasions. The antiproliferative effect (percentage of growth inhibition) was evaluated with 5 day treatment, and the data are expressed as the mean ± standard error of the mean (Microsoft Excel). The corresponding 50% and 90% inhibitory concentrations (IC50 and IC90) were determined from the concentration-response curves (Excel software).