Estudo da atividade leishmanicida dos flavonóides glicosilados (tilirosídeo: canferol 3-O-β-D-(6’’-E-P-cumaroil) glicosídeo e di-raminosídeo : canferol 3,7-DI-O-α-L-raminopiranosídeo) isolados da Herissantia crispa L. (Brizicky)

Abstract

The therapeutic arsenal available for the treatment of Leishmaniasis is restricted to few drugs, which have severe side effects and limited clinical efficacy. In this sense, chemotherapy based on natural bioactive molecules has been a strategy adopted for the discovery of new leishmanicidal drugs. The objective of this work was to evaluate the leishmanicidal activity of two compounds isolated from Herissantia crispa L. (Brizicky) using experimental models in vitro. Bioactive compounds: Thiliroside and Dhiraminoside were solubilized in 1% DMSO. The activity of the glycosylated flavonoids on the promastigote forms of L. braziliensis cultured in vitro was evaluated by incubating the parasite in Schneider's medium containing concentrations of the Thiliroside or Dhiraminoside between 100 and 6.25 μg / mL. Dhiraminoside (IC 50 = 55.88 μg/mL) had a leishmanicidal effect on the promastigote forms of L. braziliensis when compared to the Thiliroside (IC 50 = 109.27 μg/mL). In the cytotoxicity assay using murine macrophages of the J774 A.1 lineage by the Alamar Blue (AB) bioassay, DIRA and TILI were considered non-cytotoxic (CC50 > 50 μg/mL). DIRA CC50 (168.94 μg/mL) or TILI (74.49 μg/mL) were compared to the IC 50 of the protozoa in order to establish the Selectivity Index (SI), demonstrating that the Dhiraminoside is 3,0 times more selective for the parasite when compared with Thiliroside SI 0,7. For in vitro evaluation of leishmanicidal activity against intracellular amastigote forms, J774 A.1 cells infected with L. braziliensis were used in a pre and post-treatment regimen with different concentrations of DIRA (100 to 6.25 μg/mL). Pretreatment with 100 μg / ml of dhiraminoside resulted in a 56.36% decrease in parasite load in infected macrophages, compared to the amphotericin B (77.78%) control group, considering treatment times it is observed that the parasite death was due to previous cellular stimulation mechanisms. In order to investigate the modulation capacity of DIRA to stimulate the production of NO via pre- and post-treatment, nitrite (NO2-) production was measured in the supernatants of the macrophages at 24, 48 and 72 hours, with and without LPS ( 50 μg/mL) and IFN-γ (1 μg/mL). Pre-treatment with DIRA (100 μg/mL) induced the production of NO2- (78 μg/mL), conferring leishmanicidal action, associated with NO production. In contrast, in the post-treatment regimen, DIRA did not present expressive levels of nitrite, evidencing possible inhibitory effects of nitric oxide in post-infection by L. braziliensis. In this context, the cellular immune response was evaluated by the production of Th1 and Th2 standard cytokines quantified in cell culture supernatant pre- and post-treated with 100 μg/mL DIRA and subsequently previously infected with L. braziliensis promastigotes. DIRA in the pretreatment showed an expressive increase of Th1, IL-12 and IFN- γ cytokines, outlining a mechanism of resistance to L. braziliensis infection. In the post-treatment, the hypothesis of inhibition of nitric oxide by the parasite this time is supported by the increase of IL-10 and consequent decrease in NO production. The results indicate that Dhiraminoside even having leishmanicidal and immunomodulatory action, requires in vivo studies to overcome the limitations of the in vitro assays employed.