Leishmanicidal Activity of (+)-Phyllanthidine and the Phytochemical Profile of Margaritaria nobilis (Phyllanthaceae)

Abstract

The effects of the Securinega alkaloid (+)-phyllanthidine on Leishmania (L.) amazonensis and the first chemical investigation of Margaritaria nobilis L.f. (Phyllanthaceae) are described. Treating the parasites with this alkaloid caused a dose-dependent reduction in promastigote growth of 67.68% (IC50 82.37 μg/mL or 353 µM) and in amastigote growth of 83.96% (IC50 49.11 μg/mL or 210 µM), together with ultrastructural alterations in the promastigotes. No cytotoxic effect was detected in mammalian cells (CC50 1727.48 µg/mL or CC50 5268 µM). Classical chromatographic techniques and spectral methods led to the isolation and identification of betulinic acid, kaempferol, corilagin, gallic acid and its methyl ester, besides (+)-phyllanthidine from M. nobilis leaves and stems. Margaritaria nobilis is another source of the small group of Securinega alkaloids, together with other Phyllanthaceae (Euphorbiaceae s.l.) species. The low toxicity to macrophages and the effects against promastigotes and amastigotes are suggestive that (+)-phyllanthidine could be a promising antileishmanial agent for treating cutaneous leishmaniasis.

Keywords: Leishmania (L.) amazonensis; alkaloid (+)-phyllanthidine; leishmanicidal activity.

Figure 1

Structures of some Securinega alkaloids.

Figure 2

Structures of compounds 1–6 isolated from Margaritaria nobilis.

Figure 3

Viability of macrophages treated with different concentrations of (+)-phyllanthidine, when measured by MTT reduction assay after 48 h of treatment. The viability of untreated cells was 100% and no differences were found in treated cells with different concentrations when compared with the control.

Figure 4

Growth curve of Leishmania (L.) amazonensis promastigotes that were treated with different concentrations of (+)-phyllanthidine and 0.5 µg/mL of Amphotericin B (AMP-B): (A) treated for 48 h and (B) treated for 96 h. *** p < 0.001 represents the difference between treated and untreated cells.

Figure 5

Effect of (+)-phyllanthidine on the amastigote survival for Leishmania (L.) amazonensis. Infected murine peritoneal macrophages were treated with 50 and 100 µg/mL of the drug for 48 h after infection. The results are shown in standard derivations of survival inhibition compared with the untreated control. The positive control is Amphotericin B (AMP-B). *** p < 0.001 represents the difference between treated and untreated cells.

Figure 6

Effect of (+)-phyllanthidine on the ultrastructure of Leishmania (L.) amazonensis promastigotes, which were observed by scanning (SEM) (A–C) and transmission (TEM) (D–F) electron microscopy (A) Control-promastigote form without treatment; (B) promastigote treated with 50 µg/mL, showing the septation of the cell body and the shortening of the flagellum (arrows); and (C) promastigotes treated with 100 µg/mL; observe the cellular debris and formation of rosettes; (D) Control without treatment, showing normal characteristics; (E) promastigote forms treated with 50 µg/mL of alkaloid. Note the swelling of the kinetoplast; and (F) treatment with 100 µg/mL. The M—Mitochondria (*) acidocalcisomes. Scale bars = 2 µm.