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.

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Context: New antileishmanias are needed because of toxicity, high cost and resistance problems associated with available drugs. Nectandra (Lauraceae) produces several classes of compounds but its essential oil has not previously been reported to have antileishmania activity.Objective: We evaluated the cytotoxicity and antileishmania activity of essential oils from Nectandra amazonum Nees, N. gardneri Meisn., N. hihua (Ruiz & Pav.) Rohwer and N. megapotamica (Spreng.) Mez.Materials and methods:Nectandra oils were extracted from stem bark/leaves by hydrodistillation and compounds were identified by GC-MS. Oils were tested against Leishmania infantum and L. amazonensis intracellular amastigotes and nitric oxide production was evaluated. Cytotoxicity was achieved on NIH/3T3 and J774.A1 cells for the selectivity index (SI).Results and discussion:Nectandra gardneri was active against L. infantum and L. amazonensis (IC50 =  2.7 ± 1.3/2.1 ± 1.06 μg/mL) and contained 85.4% sesquiterpenes, of which 58.2% was intermediol. Besides low cytotoxicity (SI >11.3), N. gardneri induced a significant increase in NO production by L. infantum-infected macrophages. Nectandra hihua had the best activity on L. infantum amastigotes (IC50 =  0.2 ± 1.1 μg/mL). This oil was 89.0% sesquiterpenes, with 28.1% bicyclogermacrene. The two specimens of N. megapotamica had different activities on amastigotes. The one richer in sesquiterpenes (49.9%) was active against both species (IC50 =  12.5 ± 1.4/21.3 ± 1.2) and had phenylpropanoid E-asarone as the main compound (42.4%). Nectandra amazonum showed moderate activity on both the species (IC50 =  31.9 ± 2.0/22.1 ± 1.3 μg/mL) and low selectivity (0.9 < SI >2.6), probably due to the major presence of β-caryophyllene (28.5%).Conclusions: Our data identify compounds that can now be isolated and used for the development of new antileishmanias.

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reported in vitro, the mechanisms involved in the parasite death, as well as its action in vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC50 2.3 ± 0.93 μM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro. The increase of ROS in vitro, associated with the reduction of NO and iNOS expression in vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.