In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

Abstract

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Fig. 1. : compounds isolated from Selaginella sellowii: amentoflavone (1) and robustaflavone (2).

,Fig. 2. : antileishmanial activity of the hydroethanolic extract from Selaginella sellowii (SSHE) on intracellular amastigotes. Peritoneal macrophages were infected with Leishmania amazonensis and treated with different concentrations of SSHE. Infection index was calculated 24, 48 and 72 h after treatment. Bars represent the mean ± standard deviation of quadruplicates. p < 0.01 (*) and p < 0.0001 (**) for the different concentrations compared to untreated cells (control) (Student’s t test).

Fig. 3. : chromatographic profile at 270 nm and ultraviolet absorption spectra (240-400 nm) of the hydroethanolic extract from Selaginella sellowii.

Fig. 4. : antileishmanial activity of amentoflavone (A) and robustaflavone (B) on intracellular amastigotes. Peritoneal macrophages were infected with Leishmania amazonensis and treated with different concentrations of the compounds. Infection index was calculated 72 h after treatment. Bars represent the mean ± standard deviation of six replicates. p < 0.01 (**) and p < 0.0001 (*) for the different concentrations compared to untreated cells (control) (Student’s t test).

Fig. 5. : nitric oxide release by L. amazonensis infected macrophages 72 h after treatment with hydroethanolic extract from Selaginella sellowii (A), amentoflavone (B) and robustaflavone (C). Bars represent the mean ± standard deviation of six replicates. p < 0.05 (*) for the different concentrations compared to untreated cells (Student’s t test).