¹H and ¹³C NMR spectra were recorded at 300 and 75 MHz, respectively, on a Bruker Ultrashield 300 Advance III spectrometer (Bruker–Biospin, Germany). DMSO (Aldrich) was used as the solvent and as the internal standard. ESI–HRMS were obtained in a Bruker Daltonics microTOF-QII (ESI–QTOF positive mode). Silica gel (Merck, 230–400 mesh) and Sephadex LH-20 were used for the chromatographic column (CC) separation, while silica gel (SiO₂) 60 PF₂₅₄ (Merck) was used for analytical thin layer chromatography.

The leaves of C. sylvestris were collected from a single tree in the Atlantic Forest district of São Paulo City, SP, Brazil (coordinates 23 53′08.86″S, 46 40′10.45″O), in October 2012. Botanical identification was made by Dr. Roseli Buzanelli Torres, and the leaves were deposited into the IAC Herbarium with the voucher number IAC 55272. The research project was registered in the Sistema de Patrimônio Genético e Conhecimento Tradicional Associado (Sisgen) platform, with the registration number A90708B.

Dried and powdered leaves of C. sylvestris (290 g) were extracted using MeOH (5 × 1 L) at room temperature. After filtration and concentration under reduced pressure, the crude MeOH extract was resuspended in MeOH:H₂O (9:1, v/v) and partitioned with n-hexane, CH₂Cl₂, EtOAc, and n-BuOH. These extracts were stored under refrigeration in an inert atmosphere and absence of light until its fractionation. After that, the n-hexane phase was evaluated and exhibited anti–T. cruzi activity (100% of trypomastigotes death at 300 μg/ml), so it was selected for fractionation procedures.

The hexane phase (6.4 g) was submitted by CC using SiO₂ eluted with n-hexane with increased amounts of EtOAc to afford 10 fractions (I to X). Part of fraction V (1,076 mg) was subjected to CC over Sephadex LH-20 eluted with MeOH, to give four fractions (V-1 to V-4). Fraction V-2 (204.7 mg) was submitted to CC over SiO₂ eluted with increased amounts of acetone in CH₂Cl₂ to give pure compound 1 (35 mg).

T. cruzi trypomastigotes (Y strain) were cultivated in the Rhesus monkey kidney cells (LLC-MK2, ATCC CCL 7) and were maintained in the RPMI-1640 medium (Sigma-Aldrich) supplemented with 2% FBS at 37°C in a 5% CO₂ humidified incubator. Murine fibroblasts cells (NCTC clone L929, ATCC) and LLC-MK2 were maintained in the RPMI-1640 supplemented with 10% FBS at the same conditions described above. Macrophages were collected from the peritoneal cavity of BALB/c mice with RPMI-1640 supplemented with 10% FBS and were maintained in a 5% CO₂ humidified incubator at 37°C.

The solvent DMSO was used to dissolve the compounds. To avoid toxic effects in the parasites and mammalian cells, it was added at 0.5% (v/v) in the microplate wells and used as the internal control. The 50% inhibitory concentration (IC₅₀) was determined against T. cruzi trypomastigote (i) and amastigote (ii) forms, as follows:

i) Trypomastigotes: Trypomastigotes (1 × 10⁶/well) were seeded in 96-well plates and incubated with compound 1 up to 150 μM for 24 h at 37°C in a 5% CO₂ incubator. The parasite viability was determined using the resazurin (Alamar Blue^®) colorimetric method. Benznidazole was used as the standard drug, and the untreated cells were used as the negative control (Barbosa et al., 2020). The mortality of the parasites induced by fractions (bioguided fractionation) was performed using the incubation of trypomastigotes at a fixed concentration of 300 µg/ml for 24 h at 37°C in a 5% CO₂ incubator. The parasite viability was analyzed by light microscopy (× 400 magnification) by the motility and morphology of the parasites compared to the untreated group.

NCTC cells (6 × 10⁴ cells/well), murine fibroblasts (ATCC), were seeded in 96-well plates and incubated with serial dilutions (base 2-fold) of compound 1 (200–1.6 μM) for 48 h in a 5% CO₂ incubator at 37°C. The 50% cytotoxic concentration (CC₅₀) was determined by the MTT colorimetric method (Tada et al., 1986). The selectivity index (SI) value was calculated using the ratio: CC₅₀ against NCTC cells/IC₅₀ against T. cruzi amastigotes.

Trypomastigotes (2 × 10⁶ cells/well) were seeded in the 96-well black polystyrene microplates and incubated with 1 μM of SYTOX Green (Molecular Probes) in HANKS’ balanced salt solution (Sigma-Aldrich) supplemented with 10 mM d-Glucose (Sigma-Aldrich, HBSS + Glu) in the dark at 24°C. After 15 min, compound 1 was added at IC₅₀ concentration (18.2 μM) and the fluorescence was monitored for 4 h using a fluorimetric microplate reader (FilterMax F5-Molecular Devices) with excitation and emission wavelengths of 485 and 535 nm, respectively. Maximum permeabilization was obtained with 0.5% Triton X-100 (positive control), and untreated parasites were considered as the negative control (Mesquita et al., 2014).

Trypomastigotes (2 × 10⁶ cells/well) were treated with compound 1 (18.2 μM) in HBSS + Glu for 4 h at 37°C in a 5% CO₂ incubator. Subsequently, 0.2 µM of DiSBAC₂ 3) (Molecular Probes) were added and the samples were incubated at 37°C for 5 min. The fluorescence was measured using the Attune NxT flow cytometer (Thermo Fisher Scientific) with excitation and emission wavelengths of 488 and 574 nm, respectively. The parasites were treated with gramicidin D (0.5 µg/ml) (Sigma-Aldrich) (positive control) and the untreated parasites served as a negative control (Morais et al., 2019).

Trypomastigotes (2 × 10⁶ cells/well) were incubated with compound 1 (18.2 μM) in HBSS + Glu for 4 h at 37°C in a 5% CO₂ incubator. The parasites were stained with JC-1 dye (Thermo Fisher) at 10 μM in the dark for 20 min, and fluorescence levels were measured using the Attune NxT flow cytometer (Thermo Fisher Scientific) with an excitation filter wavelength of 488 nm, and emission wavelengths of 530 nm (BL-1) and 574 nm (BL-2). The ΔΨ_m was determined by the ratio BL-2/BL-1. CCCP (100 μM) (Sigma-Aldrich) treated parasites were used as a positive control and the untreated parasites as a negative control (Luque-Ortega and Rivas, 2010).

Trypomastigotes (2 × 10⁶ cells/well) were treated with compound 1 (18.2 μM) for 4 h in HBSS + Glu at 37°C in a 5% CO₂ incubator. H₂DCFDA (molecular probe) was added (5 μM), and the parasites were incubated for 15 min. Then, the fluorescence intensity was measured using a fluorimetric microplate reader (FilterMax F5 Multi-Mode, Molecular Devices) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Sodium azide (10 mM) was used as a positive control and the untreated parasites were used as a negative control (Keil et al., 2011).

Trypomastigotes (2 × 10⁶ cells/well) were pretreated with 5 μM of Fluo-4 AM (Molecular Probes) in PBS 1x, for 60 min at 37°C in the dark. Then, the parasites were washed and treated with compound 1 (18.2 μM). Fluorescence measurements were performed for 4 h using a fluorimetric microplate reader (FilterMax F5 Multi-Mode, Molecular Devices) with the excitation and emission wavelengths of 485 and 535 nm, respectively. Maximum calcium levels were obtained using 0.5% Triton X-100 and the untreated parasites were used as a negative control (Rea et al., 2013).

The 50% inhibitory concentrations and 50% cytotoxic concentration values were calculated using sigmoidal dose–response curves using GraphPad Prism 6.0 software. Unless stated, the reported data correspond to the mean ± standard deviation of at least two independent experiments performed with duplicate samples. The one-way ANOVA of variance with Tukey’s multiple comparison test was used for the significance test (p value).

Compound 1 was evaluated in vitro against T. cruzi. and displayed activity against amastigotes and trypomastigotes forms of parasite with IC₅₀ values of 15.8 and 18.2 µM, respectively (Table 1). Light microscopy analysis (×400 magnification) revealed that compound 1 eliminated the intracellular amastigotes with selectivity, killing >95% of the parasites at 30 µM (Figure 2), preserving the morphology of the host cells. At 15 μM, it was possible to observe 50% reduction of the amastigotes, when compared to the untreated macrophages (T.cruzi-infected) (Figure 2). The intracellular amastigotes are the clinically relevant forms of the parasite, and are mainly present in the chronic phase of the disease (Alonso-Padilla and Rodríguez, 2014); while the trypomastigotes is also of therapeutic importance, once this form is widely found at the acute phase of the human disease. If not completely eliminated during the drug treatment, trypomastigotes can lead to the reactivation of the parasitemia and disease transmission (Katsuno et al., 2015).

Regarding the mammalian cytotoxicity, no toxic profile of the cells was detected after treatment with compound 1 to the highest tested concentration. Using the ratio between the cytotoxicity and activity against amastigotes, the selectivity index (SI) of this sesquiterpene was >12. Benznidazole showed an IC₅₀ value against the intracellular amastigotes of 5.0 µM, and a mammalian cytotoxicity (CC₅₀) of 190.6 µM, resulting in a selective index of 38.1.

Essential oils containing the T-cadinol (1) from different plants such as Cymbopogon nardus (Norhayati et al., 2013), Kadsura longipedunculata (Mulyaningsih et al., 2010), and Humulus lupulus (Jeliazkova et al., 2018) have demonstrated the activity against the etiologic agent of the African trypanosomiasis (Trypanosoma brucei). Considering the promising activity of T-cadinol against both forms of T. cruzi and the lack of mammalian cytotoxicity, we suggest that this compound could be used as a hit candidate for future optimization studies on drug discovery against Chagas disease.

Considering the potent and selective activity of T-cadinol (1) against T. cruzi, we investigated the lethal action using the extracellular trypomastigotes. During drug discovery studies, the study of the target organelles of a hit candidate can provide significant information for the synthesis of new derivatives (Tiwari et al., 2018). The following assays were carried out in trypomastigotes at the 50% inhibitory concentration.

The plasma membrane is an essential regulator of ions and nutrient transport, as well as pH homeostasis, acting as a barrier between the extra and intracellular environments. In the present study, the plasma membrane permeability of T. cruzi parasites was analyzed spectrofluorimetrically, using the probe SYTOX Green. Our results showed that compound 1 induced no permeabilization of the membrane when compared to the untreated parasites (Figure 3), similar to the eudesmane sesquiterpene costic acid, isolated from Nectandra barbellata (Lauraceae) (Londero et al., 2020).

The membrane potential and the differences in ion concentrations between the intracellular and the extracellular millie determine the flow of ions in channels. Alterations in the potential can lead to an ionic imbalance and the formation of transmembrane pores, affecting the acquisition of crucial nutrients for proliferation and cellular activities (Morth et al., 2011). In the present work, using the fluorophore DiSBAC₂ in flow cytometry, it was possible to verify that the compound 1 caused no interference in the membrane when compared to untreated parasites (Figure 4). It suggests that T-cadinol enters the plasma membrane of the parasite without affecting the gradient of cations (Na⁺, Ca²⁺, and K⁺) and anions Cl⁻,that forces ions to passively move in one direction. Considering the lethal activity of T-cadinol (1) in the trypomastigotes, our initial approaches suggest other mechanisms of action of the compound than the parasite plasma membrane damage.

Protozoan parasites present membrane structures with considerable differences in lipids and proteins relative to the mammalian cells. These are essential for parasite specific processes, such as host cell invasion, nutrient acquisition or protection against the host immune system (Vial et al., 2003). Despite the attractive approach to target T. cruzi membrane with natural products, compound 1 showed no interference in this site and we proceeded to investigate other organelles.

From energy production to oxidative stress control, the mitochondria is responsible for innumerable metabolic processes. This organelle is involved in the growth and differentiation, being crucial to the cell survival. Additionally, unlike mammalian cells, trypanosomatids exhibit a unique mitochondria, which extends throughout the parasite length (Menna–Barreto and Medeiros, 2014). Due to these factors, the search for compounds that affect this essential organelle could be an interesting strategy for the search of new anti–T. cruzi therapies.

To analyze the mitochondrial membrane of T. cruzi trypomastigotes, the fluorescence of JC-1 dye was used in the flow cytometer. This fluorophore indicates changes in the mitochondrial membrane potential according to the formation of J-aggregates (BL-2 fluorescence) and monomers (BL-1 fluorescence). Our data showed an intense hyperpolarization of the mitochondrial membrane, leading to a significant enhance in the BL-2/BL-1 ratio when compared to the untreated parasites (Figure 5). Previous reports of two other natural sesquiterpenes, costic acid isolated from Nectandra barbellata (Londero et al., 2020) and deoxymikanolide isolated from the species of Mikania (Puente et al., 2019), demonstrated an imbalance of the T. cruzi mitochondria, but leading to a depolarization of this organelle. The mitochondria in trypanosomatid are unique and vital organelles, serving as target for several drugs and compounds. The current findings established the essential role of the mitochondria in protozoan parasites and their peculiarities as compared to the mammalian counterpart, which is being considered an attractive candidate for drug discovery (Sundar and Singh, 2018).

Mitochondria are the principal resource of reactive oxygen species such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Under normal conditions, the respiratory chain generates around 3–5% of ROS over the total oxygen consumed, and these species are used as signalization in the cell proliferation and growth. Therefore, derangements in the mitochondria can affect the production of these species, caused from interference in different biosynthetic pathways to lethal oxidative stress (Machado-Silva et al., 2016; Maldonado et al., 2020). Considering the effect of compound 1 in the mitochondria, the ROS levels in T. cruzi using the fluorescent probe H₂DCFDA was also investigated. The results demonstrated that compound 1 induced a slight decrease in the ROS levels, characterizing a reduced fluorescence when compared to the untreated parasites (Figure 6). This reduction can be attributed to the mitochondrial membrane hyperpolarization, since the breakdown of the respiratory chain can cause a disorder of the proton motive force and consequently, the alteration of the ROS production.

The function of the mitochondria is the production of metabolic energy in the form of ATP. Most of the oxygen consumed by the mitochondrial electron transport chain is reduced to water, and a small proportion is converted to ROS (1–2%) (Wiley et al., 2014). The reduction of ROS caused by the compound 1 suggests a damage to the bioenergetic activity of T. cruzi mitochondria, as corroborated by the studies of the ΔΨ _m.

Intracellular calcium is an important cell signaling component and plays a key role in trypanosomatids infectivity, mobility, and differentiation. These ions are concentrated mostly in the endoplasmic reticulum, mitochondria, and acidocalcisomes. Basically, failure in any Ca²⁺ regulation pathway or storage disarranges homeostasis and can be extremely harmful to the parasites. Additionally, variations in the mitochondrial calcium induce high conductance channel formation across the membrane, disturbing the electrical potential (Benaim et al., 2020a; Scarpelli et al., 2021).

In the present work, Ca²⁺ levels were spectrofluorimetrically evaluated employing the Fluo-4 AM dye. The obtained results showed that compound 1 caused no interference in the intracellular calcium levels of T. cruzi when compared to the untreated parasites (Figure 7). Based on these findings, it is possible to assume that the mitochondrial impairment caused by T-cadinol (1) has no direct relationship to a Ca²⁺ extrusion effect. Further studies should be performed to clarify if T-cadinol (1) has a direct effect in the mitochondria or can also affect other metabolic pathways at early time of incubation. Despite Ca²⁺ imbalance has been considered an attractive strategy against Trypanosoma cruzi parasites, both the single mitochondria and the acidocalcisomes have been considered important targets for drug discovery studies, as they play essential roles in the bioenergetics of these protozoan parasites (Benaim et al., 2020b).