Soulamarin isolated from Calophyllum brasiliense (Clusiaceae) induces plasma membrane permeabilization of Trypanosoma cruzi and mytochondrial dysfunction.

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 (1)H- and (13)C 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

Evaluation of the 50% Inhibitory Concentration of soulamarin against trypomastigotes and intracellular amastigotes.

<p>The viability of trypomastigotes was determined with MTT at 550 nm and the hemolytic activity was determined at 550 nm. The efficacy of soulamarin against intracellular amastigotes was determined using light microscopy counting.</p><p>95%CI – 95% confidence interval; IC₅₀ – 50% inhibitory concentration. IC₅₀ of benznidazole against trypomastigotes - 440.7 µM (95%CI 272.4–478.4 µM) and against intracellular amastigotes - 319.7 µM (95%CI 283.8–360.1 µM).</p

Mitochondrial membrane potential of <i>T. cruzi</i> trypomastigotes treated with soulamarin* (p<0.05).

<p>(<b>A</b>): Microplate spectrofluorimeter data showing soulamarin-treated <i>T. cruzi</i>, positive control (FCCP), and negative control (untreated cells). (<b>B–D</b>): Fluorescence microscope images, <b>B</b> - soulamarin-treated <i>T. cruzi</i>; <b>C</b> - untreated <i>T. cruzi</i> (negative control); <b>D</b> – FCCP (positive control). Panels <b>I</b> - images with blue fluorescence channel (DAPI); panels <b>II</b> - images with red fluorescence channel (MitoTracker Red); panels <b>III</b> - merged images (excitation and emission wavelengths of 540 and 595 nm; 1000×magnification).</p

Fluorescence measurements (SYTOX Green) of <i>T. cruzi</i> after incubation with soulamarin, reflecting the modified permeability of the plasma membrane.

<p>Parasites were treated with soulamarin (IC₁₀₀ = 386 µM, IC₅₀ = 219 µM and IC₂₅ = 103 µM) and compared to Triton X-100 (100% permeability = positive control) as well as an untreated negative control* (p<0.05).</p

Chemical structure of soulamarin.

<p>Chemical structure of soulamarin.</p