Soulamarin Isolated from Calophyllum brasiliense (Clusiaceae) Induces Plasma Membrane Permeabilization of Trypanosoma cruzi and Mytochondrial Dysfunction

Abstract

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi. It has high mortality as well as morbidity rates and usually affects the poorer sections of the population. The development of new, less harmful and more effective drugs is a promising research target, since current standard treatments are highly toxic and administered for long periods. Fractioning of methanol (MeOH) extract of the stem bark of Calophyllum brasiliense (Clusiaceae) resulted in the isolation of the coumarin soulamarin, which was characterized by one- and two-dimensional 1H- and 13C NMR spectroscopy as well as ESI mass spectrometry. All data obtained were consistent with a structure of 6-hydroxy-4-propyl-5-(3-hydroxy-2-methyl-1-oxobutyl)-6″,6″-dimethylpyrane-[2″,3″:8,7]-benzopyran-2-one for soulamarin. Colorimetric MTT assays showed that soulamarin induces trypanocidal effects, and is also active against trypomastigotes. Hemolytic activity tests showed that soulamarin is unable to induce any observable damage to erythrocytes (cmax. = 1,300 µM). The lethal action of soulamarin against T. cruzi was investigated by using amino(4-(6-(amino(iminio)methyl)-1H-indol-2-yl)phenyl)methaniminium chloride (SYTOX Green and 1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-chloride (MitoTracker Red) as fluorimetric probes. With the former, soulamarin showed dose-dependent permeability of the plasma membrane, relative to fully permeable Triton X-100-treated parasites. Spectrofluorimetric and fluorescence microscopy with the latter revealed that soulamarin also induced a strong depolarization (ca. 97%) of the mitochondrial membrane potential. These data demonstrate that the lethal action of soulamarin towards T. cruzi involves damages to the plasma membrane of the parasite and mitochondrial dysfunction without the additional generation of reactive oxygen species, which may have also contributed to the death of the parasites. Considering the unique mitochondrion of T. cruzi, secondary metabolites of plants affecting the bioenergetic system as soulamarin may contribute as scaffolds for the design of novel and selective drug candidates for neglected diseases, mainly Chagas disease.

Killing of Trypanozoon Parasites by the Equine Cathelicidin eCATH1

“…Trypanosomatids have a single mitochondrion exhibiting unique structural and functional characteristics that are remarkably distinct from those of host mammalian cells. These features make this organelle an important target for new pharmaceutical agents (32,39). In the present study, after incubation with eCATH1, the parasites showed a rapid dose-dependent decrease of rhodamine fluorescence, revealing mitochondrial membrane potential disruption.…”

“…Accordingly, the 50% inhibitory concentration (IC 50 ) of 9.5 M found for eCATH1 against T. b. brucei, T. evansi, and T. equiperdum allows us to consider eCATH1 as an effective AMP with a score of 2ϩ. However, comparisons to other IC 50 s of AMPs should be carefully analyzed, given the different protocols described in the literature for their evaluation (26,(31)(32)(33). The concentrations of eCATH1 used in this study that revealed trypanocidal activity in vitro were 2-fold to 15-fold higher than those sufficient to kill various Grampositive and Gram-negative bacteria (34).…”

Antiprotozoal activity of extracts and isolated triterpenoids of ‘carnauba’ (Copernicia prunifera) wax from Brazil

“…Amastigotes were added to macrophages at 10:1 ratio (amastigotes:macrophage) and incubated for 24 h. Non-internalized parasites were removed by washing twice with medium and the cells were then incubated with the test extracts or compounds 1-3 up to 150 lg/mL highest concentration for 120 h at 37 C under 5% CO 2 humidified atmosphere, using miltefosine as standard drug. At the end of the assay, the cells were fixed in MeOH, stained with Giemsa and observed under a light microscope to determine the number of infected macrophages out of 400 cells (Rea et al 2013). …”

“…Despite this knowledge, few studies have evaluated biological activity of 'carnauba' wax, including defensive proteins with antifungal activity (Cruz et al 2002). As part of our continuous project involved in the discovery of antiprotozoal compounds from Brazilian plant species (Grecco et al 2012;Rea et al 2013;Morais et al 2014;Costa-Silva et al 2015), the present work was aimed to identify antiprotozoal metabolites of C. prunifera wax (types 1 and 4) as well as the evaluation of their mammalian cytotoxicity. As a result, three bioactive dammarane-type triterpenoids: (24R Ã )-methyldammar-25-ene-3b,20-diol (carnaubadiol, 1), (24R Ã )-methyldammara-20,25-dien-3-one (2) and (24R Ã )-methyldammara-20,25-dien-3a-ol (3) were characterized.…”

Antiparasitic activity of natural and semi-synthetic tirucallane triterpenoids from Schinus terebinthifolius (Anacardiaceae) – Structure/activity relationships

“…Considering the limited and highly toxic therapeutic arsenal, the study of alternative therapies is essential. In continuation of the investigation of bioactive compounds from Brazilian flora [19][20][21][22], the present study was undertaken to determine the effectiveness and cytotoxicity of three main compounds isolated from leaves extract of S. terebinthifolius (E-and Z-masticadienoic acids and Z-schinol) against promastigotes and intracellular amastigotes of Leishmania (L.) infantum, as well as trypomastigote forms of Trypanosoma cruzi. Aiming to establish relationships between the chemical structures and the antiparasitic activity, nine semi-synthetic tirucallane derivatives were obtained and tested against parasites and mammalian cells.…”