Anti- Trypanosoma cruzi Properties of Sesquiterpene Lactones Isolated from Stevia spp.: In Vitro and In Silico Studies

Abstract

Stevia species (Asteraceae) have been a rich source of terpenoid compounds, mainly sesquiterpene lactones, several of which show antiprotozoal activity. In the search for new trypanocidal compounds, S. satureiifolia var. satureiifolia and S. alpina were studied. Two sesquiterpene lactones, santhemoidin C and 2-oxo-8-deoxyligustrin, respectively, were isolated. These compounds were assessed in vitro against Trypanosoma cruzi stages, showing IC₅₀ values of 11.80 and 4.98 on epimastigotes, 56.08 and 26.19 on trypomastigotes and 4.88 and 20.20 µM on amastigotes, respectively. Cytotoxicity was evaluated on Vero cells by the MTT assay. The effect of the compounds on trypanothyone reductase (TcTR), Trans-sialidase (TcTS) and the prolyl oligopeptidase of 80 kDa (Tc80) as potential molecular targets of T. cruzi was investigated. Santhemoidin C inhibited oligopeptidase activity when tested against recombinant Tc80 using a fluorometric assay, reaching an IC₅₀ of 34.9 µM. Molecular docking was performed to study the interaction between santhemoidin C and the Tc80 protein, reaching high docking energy levels. Plasma membrane shedding and cytoplasmic vacuoles, resembling autophagosomes, were detected by transmission microscopy in parasites treated with santhemoidin C. Based on these results, santhemoidin C represents a promising candidate for further studies in the search for new molecules for the development of trypanocidal drugs.

Keywords: 2-oxo-8-deoxyligustrin; Stevia alpina; Stevia satureiifolia var satureiifolia; molecular targets; prolyl oligopeptidase; santhemoidin C; sesquiterpene lactones; trypanocidal activity.

Figure 1

Two- and three-dimensional structures of isolated compounds: (A) santhemoidin C; (B) 2-oxo-8-deoxyligustrin.

Figure 2

Effect of isolated compounds on the three stages of T. cruzi. Both santhemoidin C and 2-oxo-8-deoxyligustrin were tested at 1–100 µg/mL concentrations in duplicates to evaluate: (A): Growth inhibition of T. cruzi epimastigotes. (B) Trypanocidal activity against bloodstream trypomastigotes; (C) inhibition of T. cruzi amastigotes. Results are expressed as the mean ± SEM.

Figure 3

Cytotoxicity evaluation of the STLs on mammalian cells. Vero cell viability was determined by the MTT method in the presence of either santhemoidin C or 2-oxo-8-deoxyligustrin. Cells were incubated for 48 h with increasing concentrations of the compounds. Results are expressed as a percentage of viability. Bars represent the means ± SEM.

Figure 4

Enzymatic activity of Tc80 was measured by the conversion of the fluorogenic substrate Z-Gly-Pro-7-AMC to AMC in the presence of santhemoidin C or 2-oxo-8-deoxyligustrin. (A) Initial reaction velocity (Vi) was determined as the slope (ΔRFU/Δtime) from the linear region in the RFU vs. time curve for tested concentrations of santhemoidin C (I) or 2-oxo-8-deoxyligustrin (II). (B) Dose-dependent inhibition of Tc80 (0.02 µg) by santhemoidin C. (C) Michaelis-Menten Plot was constructed by non-linear regression to estimate kinetic parameters (Km and V_max) in the presence or absence of 50 µM santhemoidin C. Results are expressed as the mean ± SEM. Asterisk indicates significant differences compared to vehicle (*** p < 0.001).

Figure 5

Homology modeling of Tc80. (A) Graphic representation of the 3D structure of the Tc80 protein of T. cruzi. The color pattern is in accordance with the quality of the estimated local model: in blue the similarity with the template is higher, and in red, the similarity is lower. (B) Ramachandran plot showing the ϕ and Ψ dihedral values for each amino acid of the Tc80 model.

Figure 6

(A) Best interaction pose for santhemoidin C (light gray and red spheres) with Tc80 (B) Best interaction pose for 2-oxo-8-deoxyligustrin (light grey and red spheres) with Tc80. The secondary structure of the protein is rendered in cartoon representation, as: α-helices (red), 𝛽-sheets (yellow) and loops and turns (green).

Figure 7

(A) Best interaction pose for santhemoidin C (in gray) with target Tc80. Five hydrogen bonds can be observed (yellow lines) with residues Trp 178, Trp 132, Lys 385, Leu 340 and Arg 384. (B) Best interaction pose for 2-oxo-8-deoxyligustrin (in grey) with target Tc80. One hydrogen bond formed can be observed with residue Trp178 (yellow lines).

Figure 8

(A,B) Untreated parasites displayed typical morphology. M, mitochondrion; K, kinetoplast; N, nucleus. (C) Epimastigotes treated for 48 h with 12 µM of santhemoidin C induced a plasma membrane shedding (black arrows). (D) Parasites treated with 18 µM for 48 h displayed the formation of cytoplasmic vacuoles, which resemble autophagosomes (asterisks) and (E) a detachment of plasma membrane (arrowhead).