Carla C. P. Arruda

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.

Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole Analogues of Neolignans Veraguensin, Grandisin and Machilin G

Sixteen 1,4-diaryl-1,2,3-triazole compounds derived from the natural products veraguensin, grandisin and machilin G were synthesized, with yields of 78-92%. Biological activity tests against Leishmania amazonensis promastigotes showed that three of these compounds were the most active, with maximum inhibitory concentration (IC50) values of 1.1, 3.71 and 7.23 µM. One compound was highly active against Leishmania infantum, with an IC50 value of 5.2 µM, and one derivative showed an IC50 value of 28.6 µM against Trypanosoma cruzi trypomastigotes. Regarding structure-activity relationship (SAR), hybrid 1,2,3-triazolic compounds containing a methylenedioxy group, showed the best antileishmanial and antitrypanosomal activities.

Antileishmanial Activity and Structure-Activity Relationship of Triazolic Compounds Derived from the Neolignans Grandisin, Veraguensin, and Machilin G

Sixteen 1,4-diaryl-1,2,3-triazole compounds 4–19 derived from the tetrahydrofuran neolignans veraguensin 1, grandisin 2, and machilin G 3 were tested against Leishmania (Leishmania) amazonensis intracellular amastigotes. Triazole compounds 4–19 were synthetized via Click Chemistry strategy by 1,3-dipolar cycloaddition between terminal acetylenes and aryl azides containing methoxy and methylenedioxy groups as substituents. Our results suggest that most derivatives were active against intracellular amastigotes, with IC50 values ranging from 4.4 to 32.7 µM. The index of molecular hydrophobicity (ClogP) ranged from 2.8 to 3.4, reflecting a lipophilicity/hydrosolubility rate suitable for transport across membranes, which may have resulted in the potent antileishmanial activity observed. Regarding structure-activity relationship (SAR), compounds 14 and 19, containing a trimethoxy group, were the most active (IC50 values of 5.6 and 4.4 µM, respectively), with low cytotoxicity on mammalian cells (SI = 14.1 and 10.6). These compounds induced nitric oxide production by the host macrophage cells, which could be suggested as the mechanism involved in the intracellular killing of parasites. These results would be useful for the planning of new derivatives with higher antileishmanial activities.

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.

Design, synthesis and anti-trypanosomatid activities of 3,5-diaryl-isoxazole analogues based on neolignans veraguensin, grandisin and machilin G

Using bioisosterism as a medicinal chemistry tool, 16 3,5‐diaryl‐isoxazole analogues of the tetrahydrofuran neolignans veraguensin, grandisin and machilin G were synthesized via 1,3‐dipolar cycloaddition reactions, with yields from 43% to 90%. Antitrypanosomatid activities were evaluated against Trypanosoma cruzi, Leishmania (L.) amazonensis and Leishmania (V.) braziliensis. All compounds were selective for the Leishmania genus and inactive against T. cruzi. Isoxazole analogues showed a standard activity on both promastigotes of L. amazonensis and L. braziliensis. The most active compounds were 15, 16 and 19 with IC50 values of 2.0, 3.3 and 9.5 μM against L. amazonensis and IC50 values of 1.2, 2.1 and 6.4 μM on L. braziliensis, respectively. All compounds were noncytotoxic, showing lower cytotoxicity (>250 μM) than pentamidine (78.9 μM). Regarding the structure–activity relationship (SAR), the methylenedioxy group was essential to antileishmanial activity against promastigotes. Replacement of the tetrahydrofuran nucleus by an isoxazole core improved the antileishmanial activity.

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

Abstract 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.

Characterization of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells

This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E‐NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+. A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E‐NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.