Cestodes are etiological agents of neglected diseases such as echinococcosis and cysticercosis, which are major public health problems. Antiparasitic treatment relies on a small number of approved drugs, which are often only partially effective, poorly tolerated and require prolonged administration. In this study, the cestocidal activity of four South American Stevia species was analyzed using a motility assay on the laboratory cestode model, Mesocestoides vogae. The four Stevia extracts showed cestocidal activity, with S. alpina var. alpina as the most active. The results of this report show that Stevia species represent a source of new molecules with potential for the treatment of neglected tropical diseases caused by cestodes.
Methods
The cestocidal activity of Stevia species extracts and compounds was evaluated with a worm tracker device that quantifies M. vogae TTy (tetrathyridium larval stage) motility. Viability was evaluated with a worm tracker device (WMicrotracker MINI) with an algorithm adapted to measure the movement of M. vogae TTy. Briefly, the 24 h incubated TTy were distributed in U-shaped 96-well microplates (five TTy per well) with 150 μL of MvRPMI medium at 37 ◦C under 5% CO2 atmosphere and incubated for an additional 24 h. Incubations with extracts or compounds were performed the following day. Extracts were tested at concentrations of100, 500 and 1000 μg/mL and pure compounds at concentrations of 50, 100 and 500 μM. As positive controls, pre-treated parasites with ethanol 70% for 30 min and the antiparasitic drug praziquantel at 20 μM were used. All motility assays were performed using an equal amount of the drug vehicle (1% DMSO final concentration) and the corresponding negative control (1% DMSO). To determine the effects of the treatments, TTy were incubated for nine days without changing the medium in the same conditions as described above. Measurements of motility with the WMicrotracker and microscopical observations were performed before adding the compounds (day 0) and daily afterward. The data was collected from three independent biological replicates, each corresponding to TTy obtained from a different mouse, in quadruplicate for each tested condition.
Results
Stevia alpina var. alpina extract showed the highest cestocidal activity of the four species analyzed here. This extract showed an almost complete reduction of viability from day 1 at all concentrations tested (96–100%, p < 0.0001 days 1–9 for all concentrations) (Figure 3A). The other three extracts showed a dose-dependent response. At a high concentration (1000 μg/mL), they killed TTy from day 1 (96–100%, p < 0.001–p < 0.0001, days 1–9) (Figures 3A and 4A). S.multiaristata showed a high cestocidal activity also at 500 μg/mL (84–97%, p < 0.001–p < 0.0001, days 1–9) (Figure 3B).S. maimarensis and S. aristata, showed similar cestocidal activity. At 500 μg/mL, an initial reduction of viability was observed (S. maimarensis: 59%, p < 0.05, day 1; 78%, p < 0.01, day2; S. aristata: 78%, p < 0.0001, day 2) and a delayed high cestocidal action (days 3–9: S.maimarensis: 84–96%, p < 0.01–p < 0.001; S. aristata: 96–100%, p < 0.001–p < 0.0001). An unexpected stimulatory effect was displayed at 100 μg/mL (S. maimarensis: 26–37% of increase, p < 0.01–p < 0.001, days 2–9) (Figure 4A).
The cestocidal effect of the compounds was measured by quantifying worm motility using the worm microtracker device adapted to cestodes . This method allows the simultaneous evaluation of a high number of compounds, objectively and quantitatively. It also enables continuous, real-time and non-invasive measurements, facilitating the evaluation of parasitic viability on the same plate throughout the testing period. M. vogae is a cestode laboratory model that allows the implementation of systematic drug evaluation studies. The results obtained showed that all the tested extracts produced a high and significant reduction of M. vogae TTy viability at the highest concentration tested (1000 μg/mL). At lower concentrations, S. alpina var. alpina was the most potent, reducing parasite viability by more than 95% from the first day of incubation.
Molecules 2024, 29, 4430. https://doi.org/10.3390/molecules29184430
María del Pilar Cevasco Contreras, Jimena Borgo, Ana María Celentano, Orlando Germán Elso, Hernán Bach, Cesar Atilio Nazareno Catalán, Augusto Ernesto Bivona, Hugo Rolando Vaca, Mara Cecilia Rosenzvit and Valeria Patricia Sulsen.