S carvone

The following terpenes were obtained from Sigma-Aldrich: (−)-isopulegol (≥99%), γ-terpinene (97%), α-terpinene (≥95%), (−)-linalool (≥97%), (S)-(+)-carvone (≥96%), citral (≥95%) (mixture of isomers), (−)-α-phellandrene (≥90%) and TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine)). Acetic acid (ACS), FeCl₃ × 6H₂O, FeSO₄ × 7H₂O, phosphoric acid (ACS), hydrochloric acid (ACS), and metanol (analytic purity grade) were obtained from Polish Reagents (Gliwice, Poland). The standards trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, 97%), gallic acid (>98%), and the surfactant Tween 20 were purchased from Sigma-Aldrich.

The terpenes S (+) carvone, D (+) limonene, (−) caryophyllene oxide, and citral were purchased from Sigma-Aldrich (St. Louis, MO). The reference medication BNZ (Radanil®, Roche) was donated by Santander’s State Secretary of Health, and purified by Dr. Leonor Yamile Vargas, from the Environmental Chemistry Program at Universidad Santo Tomás de Aquino (Bucaramanga). Concentrations ranging from 1.85 to 50 μg/mL were used for epimastigote (Epi) and amastigote (Amas) assays and 0.39 to 3.12 μg/mL for trypomastigote (Tryp) assays. The oils (at a density of 0.9 g/mL) and terpene stock solutions were prepared in dimethyl-sulfoxide (DMSO, Sigma-Aldrich (St. Louis, MO)), to get a 10% (v/v) solution, without exceeding a DMSO final concentration of 0.1%, in any solution. Working solutions (3.7 to 300 μg/mL) were diluted immediately prior to use with Liver Infusion Tryptose (LIT, Becton Dickinson, FL, USA) media and Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technology, CA, USA) for T. cruzi cells and Vero lineage, respectively.

S-carvone and R-carvone were purchased from Sigma Aldrich (SIGMA-ALDRICH, St. Louis, MO, USA). Both substances were emulsified in 5% tween 80 (VETEC, Rio de Janeiro, Brazil), diluted in PBS and administered orally by single gavage at 10 mg/Kg. Briefly, each enantiomer emulsion was prepared by weighing 3 mg of the compound in test tubes. The substances were emulsified in with 0.15 mL of tween 80 and after homogenization, the volume was adjusted to 3 mL with PBS. Each animal received 0.1 mL of this emulsion per 10 g of body weight, corresponding to a dose of 10 mg/Kg. Equal volume of dexamethasone (1 mg/Kg) was administered orally by single gavage as a control drug. Ovalbumin (OVA, grade V) was purchased from SIGMA Chemical (St. Louis, MO, USA) and used in both sensitization and challenge protocols. Al(OH)₃ and Evans Blue were purchased from VETEC (Rio de Janeiro, RJ, USA). PBS containing 5% tween 80 (vehicle) was administered orally to the mice in both the control and OVA groups.