The trypanocidal effect of six lignan lactones, (-)-cubebin (1), (-)-O-methyl cubebin (2), (-)-O-benzyl cubebin (3), (-)-6,6'-dinitrohinokinin (4), (-)-hinokinin (5) and dimethoxymorelensin (6), previously synthesized by our research group, was evaluated in vitro and in vivo. The compounds with higher anti-epimastigote activity were screened against intracellular amastigote of Trypanosoma cruzi. Among these, compound 5 was selected to be assayed in vivo. It was observed that compounds 5, 6 and 2 showed higher trypanocidal activity against epimastigote forms of T. cruzi, displaying inhibitory concentration (IC(50)) values of 0.67, 3.89 and 31.35 muM, respectively. These compounds were also evaluated against intracellular amastigote forms of T. cruzi, with five displaying similar activity to benznidazole. In vivo assays showed significant reduction of parasitaemia after administration of five in mice infected.

We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P<0.01). The optimal final concentration of CPRG was 200 microM and the optimal incubation period was 6 h when parasites were incubated for 3 days. Once the assay was standardized, the trypanocidal activities of nifurtimox and benznidazole were determined both by CPRG assay and microscopic counting, demonstrating the methods utility for drug-screening. The efficacy obtained was comparable to that obtained with the manual method.

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm 2 pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract.Results The extract at concentrations of 250 and 125 mg/mL inhibited 80.39 and 54.27% of promastigote (LC 50 value ¼ 105.78 mg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC 50 value ¼ 110.69 mg/mL) and against epimastigote (LC 50 value ¼ 245.23 mg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 mg/mL with 95.41% (LC 50 value ¼ 141.01 mg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (5500 mg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities. ARTICLE HISTORY

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