Hest activity were then selected and tested on L. (L.) major and L. (V.) braziliensis promastigote assays. As observed in table 2, the IC50 determined for these compounds were similar for the three species tested, except for TPM 6, which showed a lower activity against L. (V.) braziliensis than that observed for L. (L.) amazonensis.Intracellular amastigote assayThe compounds selected from the promastigote assays were subsequently tested on intracellular amastigote assays. Table 3 summarizes the results obtained. TPM 6 was the most effective compound against intracellular amastigotes of L. (L.) amazonensis, followed by GV, TPM 9, TPM 1 and TPM 2. Similar findings were observed for L. (V.) braziliensis, except for GV, which it was not tested against this species. The mean value of parasite growth inhibition observed with the control drug (0.2 mg/ml AmB) was 98 for L. (V.) braziliensis and 99.5 for L. (L.) amazonensis.CytotoxicityThe MTT assay was performed to Title Loaded From File determine the cytotoxicity of TPM 1, TPM 2, TPM 6, TPM 9 and GV. Table 3 summarizes the results of cytotoxicity assays against peritoneal macrophages fromTriphenylmethane Activity against LeishmaniasisTable 2. In vitro anti-leishmanial activity of TPM compounds expressed as IC50 (mM) on promastigotes assay.TPMIC50 (mM)L.(L.) amazonensisTPM 1 0.436 (0.375; 0.497) TPM 2 0.546 (0.462; 0.630) TPM 3 TPM 4 .1.0 .2.0 .1.0 0.031a (0.026; 0.036) TPM 7 TPM 9 .5.0 0.769 (0.614; 0.924) TPM 10 GV .4.0 0.025 (0.016; 0.034)L.(L.) major0.567 (0.521; 0.613) 0.764 (0.677; 0.851) n.d. n.d. n.d. 0.045a,b (0.041; 0.049) n.d. 0.734 (0.658; 0.810) n.d. 0.034 (0.029; 0.039)L.(V.) braziliensis0.492 (0.445; 0.539) 0.551 (0.476; 0.626) n.d. n.d. n.d. 0.063b (0.054; 0.072) n.d. 0.839 (0.745; 0.933) n.d. n.d.Figure 2. Dose-effect analysis of TPM 6 against L (L.) amazonensis. Promastigotes of L. (L.) amazonensis were plated in 24 well plates at a plating density of 16106 parasites/mL in Schneider’s medium supplemented with 20 FCS, pH 7.2. TPM 6 was diluted in the same medium and added to parasites suspension at 0.001; 0.005; 0.01 and 0.05 mM, in triplicate. After 48 h, the parasites were counted and compared to the controls containing parasites in absence of drugs. Three independent experiments were done and the results were analyzed with MiniTabH Program. Data are the mean 6 SD. doi:10.1371/journal.pone.0051864.gTPM 5 TPMBALB/c mice. The ratio of cytotoxicity to biological activity was used to determine the selectivity index (SI) of the compounds (Table 3). It is generally considered that biological efficacy is not due to in vitro cytotoxicity when this index is 10 [23]. The IC50 values observed in macrophage assays for TPM 1, TPM 2, and TPM 9 were higher than that observed for TPM 6 and GV, indicating that those compounds provide lower toxicity to macrophages. However, TPM 6 and GV presented higher selectivity indexes as compared to TPM 1, TPM 2 and TPM 9 for L. (L.) amazonensis infected macrophages. TPM 6 also presented the highest SI for L. (V.) braziliensis infected macrophages (Table 3).IC50 values correspond to mean and 95 CI of results Title Loaded From File obtained from triplicates; n.d., not determined; data obtained for linear regression on MiniTabH 15.1 software, a,b p,0,05 compared IC50 determined for L.(L.) amazonensis, L.(L.) major and L.(V.) braziliensis . doi:10.1371/journal.pone.0051864.tIn vivo assayThe quantification 12926553 of parasites within lesions was used to evaluate the efficacy of different treatments in BALB/.Hest activity were then selected and tested on L. (L.) major and L. (V.) braziliensis promastigote assays. As observed in table 2, the IC50 determined for these compounds were similar for the three species tested, except for TPM 6, which showed a lower activity against L. (V.) braziliensis than that observed for L. (L.) amazonensis.Intracellular amastigote assayThe compounds selected from the promastigote assays were subsequently tested on intracellular amastigote assays. Table 3 summarizes the results obtained. TPM 6 was the most effective compound against intracellular amastigotes of L. (L.) amazonensis, followed by GV, TPM 9, TPM 1 and TPM 2. Similar findings were observed for L. (V.) braziliensis, except for GV, which it was not tested against this species. The mean value of parasite growth inhibition observed with the control drug (0.2 mg/ml AmB) was 98 for L. (V.) braziliensis and 99.5 for L. (L.) amazonensis.CytotoxicityThe MTT assay was performed to determine the cytotoxicity of TPM 1, TPM 2, TPM 6, TPM 9 and GV. Table 3 summarizes the results of cytotoxicity assays against peritoneal macrophages fromTriphenylmethane Activity against LeishmaniasisTable 2. In vitro anti-leishmanial activity of TPM compounds expressed as IC50 (mM) on promastigotes assay.TPMIC50 (mM)L.(L.) amazonensisTPM 1 0.436 (0.375; 0.497) TPM 2 0.546 (0.462; 0.630) TPM 3 TPM 4 .1.0 .2.0 .1.0 0.031a (0.026; 0.036) TPM 7 TPM 9 .5.0 0.769 (0.614; 0.924) TPM 10 GV .4.0 0.025 (0.016; 0.034)L.(L.) major0.567 (0.521; 0.613) 0.764 (0.677; 0.851) n.d. n.d. n.d. 0.045a,b (0.041; 0.049) n.d. 0.734 (0.658; 0.810) n.d. 0.034 (0.029; 0.039)L.(V.) braziliensis0.492 (0.445; 0.539) 0.551 (0.476; 0.626) n.d. n.d. n.d. 0.063b (0.054; 0.072) n.d. 0.839 (0.745; 0.933) n.d. n.d.Figure 2. Dose-effect analysis of TPM 6 against L (L.) amazonensis. Promastigotes of L. (L.) amazonensis were plated in 24 well plates at a plating density of 16106 parasites/mL in Schneider’s medium supplemented with 20 FCS, pH 7.2. TPM 6 was diluted in the same medium and added to parasites suspension at 0.001; 0.005; 0.01 and 0.05 mM, in triplicate. After 48 h, the parasites were counted and compared to the controls containing parasites in absence of drugs. Three independent experiments were done and the results were analyzed with MiniTabH Program. Data are the mean 6 SD. doi:10.1371/journal.pone.0051864.gTPM 5 TPMBALB/c mice. The ratio of cytotoxicity to biological activity was used to determine the selectivity index (SI) of the compounds (Table 3). It is generally considered that biological efficacy is not due to in vitro cytotoxicity when this index is 10 [23]. The IC50 values observed in macrophage assays for TPM 1, TPM 2, and TPM 9 were higher than that observed for TPM 6 and GV, indicating that those compounds provide lower toxicity to macrophages. However, TPM 6 and GV presented higher selectivity indexes as compared to TPM 1, TPM 2 and TPM 9 for L. (L.) amazonensis infected macrophages. TPM 6 also presented the highest SI for L. (V.) braziliensis infected macrophages (Table 3).IC50 values correspond to mean and 95 CI of results obtained from triplicates; n.d., not determined; data obtained for linear regression on MiniTabH 15.1 software, a,b p,0,05 compared IC50 determined for L.(L.) amazonensis, L.(L.) major and L.(V.) braziliensis . doi:10.1371/journal.pone.0051864.tIn vivo assayThe quantification 12926553 of parasites within lesions was used to evaluate the efficacy of different treatments in BALB/.
