Antileishmanial Activity, Toxicity and Mechanism of Action of Complexes of Sodium Usnate with Lanthanide Ions: Eu(III), Sm(III), Gd(III), Nd(III), La(III) and Tb(III).

Leishmaniases are neglected diseases with limited therapeutic options. Diffuse cutaneous leishmaniasis can occur in Brazil due to Leishmania amazonensis. This study details the antileishmanial activity and cytotoxicity of complexes of sodium usnate (SAU) with lanthanide ions ([LnL3 (H2O)x] (Ln = La(III), Nd(III), Gd(III), Tb(III), Eu(III) and Sm(III); L = SAU). All lanthanide complexes were highly active and more potent than SAU against L. amazonensis promastigotes and intracellular amastigotes (Pro: IC50 < 1.50 µM; Ama: IC50 < 7.52 µM). EuL3·3H2O and NdL3·3H2O were the most selective and effective on intracellular amastigotes, with a selectivity index of approximately 7.0. In silico predictions showed no evidence of mutagenicity, tumorigenicity or irritation for all complexes. Treatment with EuL3·3H2O triggered NO release even at the lowest concentration, indicating NO production as a mechanism of action against the parasite. Incubating promastigotes with the lanthanide complexes, particularly with SmL3·4H2O and GdL3·3H2O, led to a change in the mitochondrial membrane potential, indicating the ability of these complexes to target this essential organelle. The same complexes caused cell death through cell membrane disruption, but their relationship with early or late apoptotic processes remains unclear. Thus, the inclusion of lanthanide ions in SAU improves selectivity with a promising mechanism of action targeting the mitochondria.

In Vitro antileishmania activity of sesquiterpene-rich essential oils from Nectandra species.

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 ß-caryophyllene (28.5%). CONCLUSIONS: Our data identify compounds that can now be isolated and used for the development of new antileishmanias.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii.

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Hairless mice as an experimental model of infection with Leishmania (Leishmania) amazonensis.

HRS/J Hairless mice have been investigated as an experimental model in cutaneous leishmaniasis induced by Leishmania (Leishmania) amazonensis. The animals were inoculated with 10(6) promastigotes into the right hind footpad and the course of infection was followed up for 30, 60 and 90 days. BALB/c mice were infected and used as control. Hairless mice were susceptible to L. (L.) amazonensis infection and a progressive increase in number of parasites and footpad thickness was detected over time. Signals of dissemination and visceralization were confirmed by the presence of parasite in the draining lymph node of lesion and spleen, at different times post infection. IL-10 gene expression evaluated by RT-PCR was significantly higher in Hairless mice at 60 days post infection, corroborating the pattern of susceptibility. These results point this inbred strain as a promising susceptible model for the study of experimental infection induced by L. (L.) amazonensis. This model would allow the use of other infection sites that minimize secondary interference and best monitoring the skin lesion, as in the case of in vivo assays of potential drugs for LT.

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

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.

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

Amentoflavone as an Ally in the Treatment of Cutaneous Leishmaniasis: Analysis of Its Antioxidant/Prooxidant Mechanisms.

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.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

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

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.