BeWo cells were commercially obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), while HTR8/SVneo cells were a gift from Dr. Estela Bevilacqua (University of São Paulo, São Paulo, SP, Brazil). Both cells were cultured in RPMI 1640 medium (Cultilab, Campinas, SP, Brazil) supplemented with 100 U/mL penicillin (Sigma Chemical Co., St. Louis, MO, USA), 100 μg/mL streptomycin (Sigma) and 10% fetal bovine serum (FBS) (Cultilab) at 37°C and 5% CO₂.

Human third-trimester placentas (36 to 40 weeks of pregnancy), n = 4, were collected after elective cesarean deliveries at the Clinics Hospital of the Federal University of Uberlândia (HC-UFU), MG, Brazil. Placental tissues were collected based on exclusion criteria, as follows: pre-eclampsia, chronic hypertension, infectious diseases including toxoplasmosis, chorioamnionitis, chronic kidney disease, heart disease, connective tissue disease, pre-existing diabetes mellitus, gestational diabetes mellitus and other pathological manifestations. Briefly, placental tissues were washed in sterile PBS to remove excess blood, then aseptically dissected to remove endometrial tissue and fetal membranes up to 1 h after collection. Terminal chorionic villi containing five to seven free tips per explant were harvested and added to 96-well microplates (one villus per well) in 200 µL/well of fresh RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% FBS for 24 h at 37°C in a humidified atmosphere containing CO₂ (5%) (Silva et al., 2017).

Toxoplasma gondii tachyzoites (2F1 clone) constitutively expressing the β-galactosidase gene were maintained by serial passages in BeWo cells cultured in medium containing 2% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂. 2F1 clone is derived from the highly virulent strain RH and was a gift from Dr. Vern Carruthers, School of Medicine from the University of Michigan (USA).

The samples of oleoresin and leaf hydroalcoholic extract from C. multijuga were provided by Professor Carlos Henrique Gomes Martins from the Department of Microbiology (DEMIC), Institute of Biomedical Sciences (ICBIM), Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil. Authorization to carry out scientific studies with plant species from the Brazilian biodiversity was requested from the Council for Authorization and Information on Biodiversity (SIBIO/ICMBio/MMA/BRAZIL) and Genetic Heritage Management (CGEN/MMA/BRAZIL). Authorizations to carry out research activities with these plants were issued under numbers 35143-1 and 010225/2014-5, respectively.

Authentic oleoresin was collected in the North region, in the state of Amazonas, in the city of Manacapuru, by Jonas J. M. da Silva by drilling the tree trunks using a 2” drill bit. The exudates removed were stored in glass bottles. All the plant material collected was identified by Silvane Tavares Rodrigues, a voucher specimen (NID 03/2013 and 62/2013) was deposited in the Herbarium of the Brazilian Agricultural Research Corporation (Embrapa Eastern Amazon), by direct comparison with authentic herbarium vouchers, of which a taxonomic identity certificate is available upon request.

Air dried leaves (40 °C for 48 h) of C. multijuga (200 g) were grounded and then exhaustively extracted by maceration with 1.2 L of ethanol/H₂O (7:3) at room temperature for 48 h to afford 50 g of C. multijuga hydroalcoholic extract of leaves after lyophilization (Pimenta et al., 2019).

We verified the toxicity of the compounds selected in BeWo and HTR8/SVneo cells. For this purpose, we performed the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolin bromide)] colorimetric assay, following the protocol described by Mosmann (1983). BeWo (3×10⁴ cells/well/200 µL) and HTR8/SVneo (1.5×10⁴ cells/well/200 µL) cells were cultured in 96-well plates for 24 h in RPMI 1640 medium with 10% FBS at 37°C and 5% CO₂. Next, cells were treated in serial dilutions with increasing concentrations of oleoresin or hydroalcoholic extract from C. multijuga in a medium with 10% FBS for 24 h. In parallel, both cells were treated with DMSO (0.8%), the percentage used to obtain higher dilution for both oleoresin and hydroalcoholic extract. As control, cells were not treated and received only medium. Then, the supernatants were removed and the cells incubated with 10 µL MTT (5 mg/mL) plus 90 µL of RPMI 10% FBS medium for 4 h at 37°C and 5% CO₂. Formazan crystals resulting from cellular metabolism were solubilized by adding a solution containing 10% sodium dodecyl sulfate (SDS, Sigma) and 50% N,N-dimethyl formamide (Sigma) for 30 min (Mosmann, 1983). Optical densities were measured at 570 nm on a plate reader (Versa Max ELISA Microplate Reader, Molecular Devices, Sunnyvale, CA, USA). Data were expressed as the percentage of viable cells (cell viability %) in comparison to untreated cells (100% of cell viability). Three independent experiments were performed in eight replicates.

We selected different concentrations of oleoresin and hydroalcoholic extract from C. multijuga after MTT assay and tested their effect on T. gondii growth. BeWo (3×10⁴ cells/well/200 µL) and HTR8/SVneo (1.5×10⁴ cells/well/200 µL) cells were cultured in 96-well plates for 24 h in RPMI 1640 medium with 10% FBS at 37°C and 5% CO₂. Next, both cells were infected with T. gondii tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasites per cell). After 3 h, the medium was discarded, extracellular parasites were retired by washing with medium, and the cells were treated with non-toxic concentrations of oleoresins or hydroalcoholic extracts from C. multijuga for additional 24 h at 37°C and 5% CO₂ as follows: for BeWo cells, 4 to 32 µg/mL; for HTR8/SVneo, 4 to 16 µg/mL. Sulfadiazine and pyrimethamine (SDZ + PYR, 200 + 8 µg/mL for BeWo, and 100 + 4 µg/mL for HTR8/SVneo) was used as positive control (Silva et al., 2017). BeWo or HTR8/SVneo cells infected by T. gondii and non-treated were used as negative controls. After 24 h of treatment, the culture supernatants were collected and stored at -80°C for further measurement of cytokines. In parallel, T gondii intracellular proliferation was analyzed using a colorimetric β-galactosidase assay (Silva et al., 2017; Souza et al., 2021). We quantified T. gondii intracellular proliferation and calculated the number of tachyzoites in comparison with a standard curve containing free tachyzoites (1×10⁶ to 15,625×10³ parasites). Infected BeWo or HTR8/SVneo cells treated only with medium (negative control) represented uninhibited parasite growth. We calculated the means of untreated cells (medium) that corresponded to 100% of T. gondii proliferation. Then, all values about number of tachyzoites obtained from all treatments were compared to 100%, and data demonstrated in % of T. gondii proliferation. Three independent experiments were performed in eight replicates.

In order to evaluate the maintenance of the antiparasitic effects of oleoresins and hydroalcoholic extracts in T. gondii growth, we firstly performed the reversibility test (Teixeira et al., 2020). Briefly, BeWo (3×10⁴ cells/well/200 µL) or HTR8/SVneo (1.5×10⁴ cells/well/200 µL) cells were seeded in 96-well plates as above described. After 24 h in culture, cells were infected with T. gondii tachyzoites (3:1) for 3 h, washed to remove extracellular parasites and treated for two conditions as follows: (1) BeWo cells were treated with oleoresin or hydroalcoholic extract (both 16 or 32 µg/mL), HTR8/SVneo cells were treated with oleoresin or hydroalcoholic extract (both 8 or 16 µg/mL), or both cells received SDZ + PYR (positive control) or only medium (negative control), then parasite intracellular proliferation was performed after 24 h in culture conditions; (2) cells and parasites were cultured in the same conditions as previously described, but after 24 h of treatment, the cells were washed, the medium replaced by new RPMI 1640 medium without treatments, and the parasites were allowed to grow for additional 24 h. In both situations, we quantified the T. gondii proliferation using the β-galactosidase assay as mentioned above. Finally, we measured the percentage reversibility rate (treatment reversibility %) at 24 h after treatment removal compared to the untreated group (considered as 100% reversibility) and the corresponding treatment condition at 24 h of treatment (baseline for comparison). Three independent experiments were performed in eight replicates.

To corroborate the reversibility data, we investigated whether the treatment with oleoresin or hydroalcoholic extract in infected BeWo and HTR8/SVneo cells would interfere with the ability of these parasites to invade and replicate within new fresh cells. Briefly, BeWo and HTR8/SVneo cells were seeded at 1.0x10⁶ cells/well in 6-well plates. After 24 h, cells were infected with T. gondii tachyzoites (3:1) for 3 h at 37°C and 5% CO₂. Then, we treated the cells with oleoresin or hydroalcoholic extract at 16 or 32 µg/mL for BeWo; or 8 or 16 µg/mL for HTR8/SVneo. Either SDZ + PYR or only medium was added as controls. After 24 h, we collected the intracellular parasites from infected cells by multiple passages through a 21- and 26-gauge needle. Finally, T. gondii tachyzoites from each experimental condition were then allowed to reinfect previously seeded BeWo (3×10⁴ cells/well/200 µL) and HTR8/SVneo (1.5×10⁴ cells/well/200 µL) cells monolayers in 96-well plates. The ability to infect the cells was analyzed after 3 h, when the β-galactosidase assay was used to quantify the total number of tachyzoites (% of T. gondii invasion), as described above. Three independent experiments were performed in eight replicates.

We performed an adherence test (Oliveira et al., 2006; Borges et al., 2016; Teixeira et al., 2020). Cells were seeded at a density of 1.0 × 10⁵ (BeWo) and 6.0 × 10⁴ (HTR8/SVneo) in 24-well plates containing 13-mm coverslips in each well. Cells were fixed with paraformaldehyde (4%) for 30 min. They were then washed three times with 1x phosphate buffered saline (PBS). Subsequently, T. gondii tachyzoites were preincubated for 1 h with oleoresin, hydroalcoholic extract or SDZ + PYR. Pretreated tachyzoites with 16 or 32 µg/ml of oleoresin or hydroalcoholic extract were used to adhere BeWo cells; while preincubated parasites with 8 or 16 µg/ml of oleoresin or hydroalcoholic extract were used to adhere HTR8/SVneo cells. SDZ + PYR (200 + 8 μg/mL for later adhesion in BeWo, or 100 + 4 μg/mL for later adhesion in HTR8/SVneo), or only medium, were used as controls. Pretreated tachyzoites by 1 h, as described, were resuspended in medium and incubated with fixed cells for 3 h. Subsequently, the coverslips were incubated overnight with mouse anti-T. gondii monoclonal primary antibody [SAG1/p30] (Abcam TP3 #ab8313) (diluted 1:500 in PGN-0.01% saponin solution). Coverslips were then rinsed three times with 1x PBS and incubated with Alexa Fluor 488 conjugated anti-mouse IgG (Invitrogen, USA #A11001) (diluted 1:500 in PGN-0.01% saponin solution), phalloidin conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma, P1951) (diluted 1:50 in PGN+saponin) and TOPRO-3 Iodide (Life Techonologies) (diluted 1:500 in PGN+saponin) for 1 h in the dark at room temperature to label tachyzoites, F-actin and nuclei, respectively. We mounted coverslips on glass slides and samples were analyzed by confocal fluorescence microscopy (Zeiss, LSM 510 Meta, Germany) with an inverted microscope (Zeiss Axiovert 200 M). We analyzed the following parameters: the number of BeWo and HTR8/SVneo cells with adhered tachyzoites and the total number of adhered tachyzoites per cell in a total of 20 randomly chosen fields. Two independent experiments were performed in three replicates.

After checking the adhesion of pretreated parasites on BeWo and HTR8/SVneo cells, we also investigated the intracellular replication and invasion of pretreated parasites with oleoresins or hydroalcoholic extract (Castanheira et al., 2015; Teixeira et al., 2020). Then, T. gondii tachyzoites were preincubated for 1 h at 37°C and 5% CO₂ with oleoresin or ethanol extract (32 or 16 µg/mL for later infection in BeWo, or 16 or 8 µg/mL for later infection in HTR8/SVneo). In parallel, tachyzoites were also treated with SDZ + PYR (200 + 8 μg/mL for later infection in BeWo, or 100 + 4 μg/mL for later infection in HTR8/SVneo), or only medium. Subsequently, the pretreated parasites were added in previously adhered BeWo or HTR8/SVneo cells in 96-well plates. Finally, two different analyses were performed: (1) to verify the T. gondii invasion, both cells were maintained with pretreated tachyzoites for only 3 h; or (2) to verify parasite intracellular proliferation, the cell monolayers were washed after 3 h of infection, a fresh supplemented culture medium was added, and the culture maintained for another 24 h at 37°C and 5% CO₂. In both experiments, intracellular T. gondii was quantified using the β-galactosidase assay, as described above (Silva et al., 2017; Souza et al., 2021). Three independent experiments were performed in eight replicates.

Toxicity of oleoresin and hydroalcoholic extract in human villous explants was performed using viability assays with lactate dehydrogenase (LDH) and MTT, according to previous protocols (Teixeira et al., 2020). In both assays, explants were treated with oleoresin or hydroalcoholic extract from C. multijuga at 64, 128 or 256 µg/mL or SDZ + PYR (150 + 200 µg/mL) (Silva et al., 2017). As viability control, explants were treated with culture medium alone. After 24 h of incubation, the culture supernatants were collected and LDH concentration measured according to the manufacturer’s instructions (Castro-Filice et al., 2014), with minor modifications (LDH Liquiform, Labtest Diagnostica S.A., Lagoa Santa, MG, Brazil). This assay is based on the consumption and decreased absorption of NADH at 340 nm, measured by a microplate reader (Versa Max ELISA Microplate Reader, Molecular Devices, Sunnyvale, CA, USA). LDH released in the culture the medium was expressed in U/L of LDH enzymatic activity and was used as a marker of tissue integrity. In parallel, in the same experimental condition, as described above, the viability of the tissue was also evaluated by the MTT test (Teixeira et al., 2020). Tissue viability was expressed in percentages (% viability by MTT incorporation), and the absorbance of villi incubated with culture medium alone (untreated villous explants) was considered to be 100% viable. In addition, we performed a morphological analysis of the treated explants. Tissue sections were included in paraffin, stained with hematoxylin/eosin and examined under a light microscope (BX40 Olympus, Tokyo, Japan). Two independent experiments were performed in six replicates.

Also, we quantified the T. gondii intracellular proliferation in explants treated with oleoresin or hydroalcoholic extract from C. multijuga using the β-galactosidase colorimetric assay (Silva et al., 2017; Souza et al., 2021), with minor modifications. Explants were cultured in 96-well microplates (one villus per well/200 µl) in supplemented culture medium for 24 h at 37°C and 5% CO₂. Then, the villi were infected with T. gondii tachyzoites (1 × 10⁶ parasites per well/200 µL) and incubated for 24 h at 37°C and 5% CO₂. Subsequently, the villous explants were thoroughly rinsed with culture medium to remove non-adherent parasites. Following our tissue viability data (LDH and MTT assays), the villi were treated for additional 24 h with oleoresin or hydroalcoholic extract (256 and 128 µg/mL), or SDZ + PYR (150 + 200 µg/mL). Uninfected and untreated or infected and untreated explants were cultured with culture medium alone. Finally, the culture supernatants were collected and stored at -80°C for future cytokine measurement. In addition, the villous explants were collected and stored at −80°C for the following analyses: determination of protein content using Bradford reagent and T. gondii intracellular proliferation by β-galactosidase assay. Initially, frozen villous explants were homogenized by the addition of 150 µL of radioimmunoprecipitation assay buffer (RIPA) [50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% (w/v) sodium deoxycholate and 0.1% (w/v) sodium deoxycholate SDS, pH 7.5] containing protease inhibitor cocktail (Complete, Roche Diagnostic, Mannheim, Germany). Homogenate was centrifuged at 21,000 × g for 15 min at 4°C, and the supernatant was collected to measure the total amount of protein using the Bradford method (Bradford, 1976). T. gondii intracellular proliferation was carried out with 20 µL of supernatants from each sample (Teixeira et al., 2020), absorbance measured at 570 nm using a kinetic plate reader (Versa Max ELISA Microplate Reader, Molecular Devices, Sunnyvale, CA, USA), the number of tachyzoites was normalized according to total protein concentration (µg/mL) of each villus obtained by Bradford assay, and expressed by the number of parasites per µg of tissue. The data were presented as a percentage (% of T. gondii proliferation), as described above. Four independent experiments were performed in six replicates.

We measured cytokines in supernatants using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). ELISA for IL-6, IL-8, TNF, IL-10, and MIF were purchased from OpTEIA, BD Bioscience, San Diego, CA, USA; or Duoset R&D Systems, Minneapolis, MN, USA, and tests were performed according to the manufacturer’s instructions. The detection limits of each cytokine were 4.7 pg/mL for IL-6; 31.2 pg/mL for IL-8; 7.8 pg/mL for TNF, IL-10 and MIF.

For villous explants, the data were normalized according to the total protein of each villous. Then, the data about cytokines were obtained by the ratio between concentration of cytokines (pg/mL) and concentration of total protein from Bradford assay (µg/mL), resulting in pg/mg of tissue (Silva et al., 2017; Souza et al., 2021).

The influence of oleoresin and hydroalcoholic extract in ROS production was also verified in BeWo and HTR8/SVneo cells. The assay was based on the peroxide-dependent oxidation of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) to form the fluorescent compound 2’,7’-dichlorofluorescein (DCF), with some modifications. Briefly, BeWo (3×10⁴ cells/well/200 µL) or HTR8/SVneo (1.5×10⁴ cells/well/200 µL) cells were seeded in 96-well black clear-bottom plate. Then, cells were infected or not with T. gondii tachyzoites (3:1) for 3 h; they were then washed abundantly with culture medium and treated with oleoresin or hydroalcoholic extract (16 or 32 µg/mL) for BeWo and (8 or 16 µg/mL) for HTR8/SVneo cells, or both cells received SDZ + PYR cells (200 + 8 μg/mL for BeWo, and 100 + 4 μg/mL for HTR8/SVneo) or only medium, as controls. Hydrogen peroxide (H2O2) was used as a positive control for ROS production. After treatment for 24 h, cells were harvested, washed with 1×PBS and incubated with 150 μL of H2DCF-DA (10 μM; diluted in 1×PBS containing 10% FBS) for 45 min at 37°C and 5% CO2 in the dark. Finally, the DCF fluorescence intensity was detected using a microplate reader (GloMax^® Discover System, Promega Corporation) at excitation and emission wavelengths of 488 nm and 522 nm, respectively. The data are presented as median fluorescence intensity (MFI). Three independent experiments were performed in eight replicates.

The present research protocol using human tissue samples was approved by the Ethics Committee of the Federal University of Uberlandia, MG, Brazil, with approval number 3.679.426. A consent term was obtained from all subjects and/or their legal guardians.

All data were expressed as means ± standard deviations (SD) using GraphPad Prisma version 8.01. Significant differences were compared to controls by using One-way ANOVA, Bonferroni’s multiple comparisons post-test for the parametric data. Nonparametric data were analyzed by the Kruskal–Wallis test and Tukey’s multiple comparison post-test. Data were considered statistically significant when P < 0.05.

Firstly, we evaluated the cell viability in BeWo and HTR8/SVneo cells treated with hydroalcoholic extract or oleoresin in several concentrations.

Reduced cell viability was detected in BeWo cells exposed to hydroalcoholic extract or oleoresin from 64 to 512 µg/mL (^* P < 0.05, ^**** P < 0.00001) in comparison to untreated cells (medium) ( Figures 1A, B ). DMSO-treated cells did not show any change in cell viability in relation to untreated cells ( Figures 1A, B ). On the other hand, HTR8/SVneo diminished cell viability when treated with hydroalcoholic extract or oleoresin from 32 (^*** P < 0.0001, ^** P < 0.001) to 512 µg/mL (^**** P < 0.00001) when compared to untreated cells (medium) ( Figures 1C, D ). As observed for BeWo, DMSO also did not induce change in cell viability for HTR8/SVneo ( Figures 1C, D ).

In our cell viability assay, we established concentrations of hydroalcoholic extract and oleoresin that did not alter the viability of BeWo and HTR8/SVneo cells for the subsequent set up of experiments.

Both hydroalcoholic extract and oleoresin reduced significantly the parasite proliferation in BeWo ( Figure 2A ) and HTR8/SVneo ( Figure 2B ) cells in all concentrations tested in comparison to untreated cells (medium) (^*** P < 0.0001; ^***** P < 0.00001). In addition, sulphadiazine and pyrimethamine (SDZ + PYR) also diminished T. gondii replication in relation to untreated cells (^**** P < 0.00001). These results demonstrated that both the hydroalcoholic extract and oleoresin had an antiproliferative effect against T. gondii.

In order to determine whether the antiparasitic effects promoted by the hydroalcoholic extract and oleoresin from C. multijuga would be irreversible, we exposed infected BeWo and HTR8/SVneo cells to the hydroalcoholic extract or oleoresin for 24 h, then cell monolayers were rinsed and incubated with medium free of treatment for additional 24 h. At the same time, we quantified the parasite proliferation after 24 h of treatment as a baseline for comparison. For BeWo cells, we choose 16 and 32 µg/mL for both the hydroalcoholic extract and oleoresin; for HTR8/SVneo, 8 and 16 µg/mL were the choice.

As previously detected ( Figure 2 ), again both the hydroalcoholic extract and oleoresin reduced T. gondii intracellular proliferation after 24 h of treatment in BeWo ( Figure 3A ) and HTR8/SVneo cells ( Figure 3B ) when compared to untreated cells (medium) (^** P < 0.001, ^**** P < 0.00001). Also, SDZ + PYR decreased parasite replication in relation to untreated cells (^**** P < 0.00001) ( Figures 3A, B ). Interestingly, both the hydroalcoholic extract and oleoresin showed an irreversible effect on tachyzoites, since a reduced parasite growth remained active in BeWo and HTR8/SVneo cells in comparison to untreated cells (considered as 100% reversibility), even after removal of treatments for additional 24 h (^**** P < 0.00001) ( Figures 3A, B ). For both cells, SDZ + PYR also showed irreversible effect (^**** P < 0.00001) ( Figures 3A, B ). These results suggest that both the hydroalcoholic extract and oleoresin maintained their antiproliferative effect even after the elimination of the treatment.

In order to obtain more information about the effect triggered by both the hydroalcoholic extract and oleoresin on T. gondii tachyzoites, we evaluated whether intracellular parasites obtained directly from treated-BeWo and HTR8/SVneo cells could maintain their ability to infect new host cells. For this purpose, tachyzoites were collected from treated-BeWo and HTR8/SVneo cells and used to infect new monolayer cells. For BeWo cells, all concentrations of hydroalcoholic extract and oleoresin reduced the ability of tachyzoites to infect new BeWo cells when compared to untreated cells (medium) (^*** P < 0.0001; ^**** P < 0.00001) ( Figure 3C ). SDZ + PYR also reduced the ability of parasites to infect new BeWo cells in relation to medium (^**** P < 0.00001) ( Figure 3C ). Although the hydroalcoholic extract and oleoresin have downmodulated the ability of parasites to infect new BeWo cells, this effect was lower if compared to classical treatment (SDZ + PYR) (^& P < 0.05) ( Figure 3C ). Finally, for HTR8/SVneo cells, all treatments (hydroalcoholic extract, oleoresin and SDZ + PYR) dampened the capacity of T. gondii to infect new HTR8/SVneo cells in comparison to untreated cells (^* P < 0.05; ^** P < 0.001; ^**** P < 0.00001) ( Figure 3D ). Interestingly, parasites derived from hydroalcoholic extract-treated-cells at 16 µg/mL demonstrated lower ability to infect new cells when compared to SDZ + PYR (^& P < 0.05) ( Figure 3D ).

These findings indicate that the inhibitory effects promoted by the hydroalcoholic extract and oleoresin from C. multijuga dampen the ability of parasites to invade new host cells.

To assess whether hydroalcoholic extract and oleoresin have a direct action on tachyzoites, we performed a variety of assays. In the first set of experiments, we evaluated whether the hydroalcoholic extract and oleoresin treatments would affect parasite adhesion to host cells. For this purpose, T. gondii tachyzoites were pretreated for 1 h with oleoresin or hydroalcoholic extract and then incubated with previously fixed-BeWo or HTR8/SVneo cells.

As result for BeWo cells, only the hydroalcoholic extract or oleoresin at 32 µg/mL was able to reduce the number of cells with adhered parasites when compared to untreated parasites (medium) (^*** P < 0.0001, ^* P < 0.05) ( Figure 4A ). On the other hand, all concentrations of treatments, except for SDZ + PYR, reduced the total number of adhered parasites in relation to untreated parasites (^**** P < 0.00001, ^** P < 0.001) and SDZ + PYR-treated parasites (^& P < 0.05) ( Figure 4B ). Representative images showing the effect of the hydroalcoholic extract and oleoresin on BeWo cells incubated with pretreated-tachyzoites can be observed in Figures 4C-F .

Similarly, parasites pretreated with the hydroalcoholic extract or oleoresin diminished their ability to adhere in HTR8/SVneo cells, since the number of cells with adhered parasites as well as the total number of adhered parasites per field were significantly smaller when related to untreated parasites (medium) (^**** P < 0.00001) and SDZ + PYR-treated parasites (^& P < 0.05) ( Figure 5A, B ). Also, SDZ + PYR reduced the number of cells with adhered parasites (^*** P < 0.0001) and the total number of adhered parasites (^** P < 0.001) in relation to the medium ( Figure 5A, B ). Representative images showing the effect of the hydroalcoholic extract and oleoresin on HTR8/SVneo cells incubated with pretreated-tachyzoites can be observed in Figures 5C-F .

Considering that pretreated parasites had lower ability to adhere to host cells ( Figures 4 , 5 ), we now verified the capacity to invade and proliferate when also preincubated with the hydroalcoholic extract or oleoresin. Our data demonstrated that the pretreatment of T. gondii tachyzoites with all the hydroalcoholic extract or oleoresin concentrations triggered low rates of invasion and intracellular proliferation, regardless of the cell type (^*** P < 0.0001, ^**** P < 0.00001), in comparison to untreated parasites (medium) ( Figures 6A-D ). SDZ + PYR also reduced the invasion and replication of pretreated tachyzoites in BeWo and HTR8/SVneo cells in relation to the medium (^** P < 0.001, ^*** P < 0.0001, ^**** P < 0.00001) ( Figures 6A-D ).

These findings indicate that the hydroalcoholic extract and oleoresin from C. multijuga dampen the ability of parasites to adhere, invade and proliferate in host cells from maternal-fetal interface.

So far, our findings demonstrated that the hydroalcoholic extract and oleoresin from C. multijuga have a direct effect on the parasites, since treated parasites have reduced ability to adhere, invade and replicate into the cells. However, we could not exclude the possibility that these compounds may affect the host cell environment. Thus, we investigated the possible immunomodulatory effects of the hydroalcoholic extract and oleoresin by measuring cytokines in culture supernatant.

For BeWo cells, our results showed that in the absence of infection, concentrations of 32 µg/mL of the hydroalcoholic extract and oleoresin induced an increase in IL-6 levels in comparison to untreated cells (medium) (^* P < 0.05, ^** P < 0.001) and SDZ + PYR (^& P < 0.05) ( Figure 7A ). After T. gondii infection, levels of IL-6 were higher in untreated/infected cells (medium Tg) compared to untreated and uninfected cells (medium) (^# P < 0.05). However, SDZ + PYR reduced IL-6 in relation to untreated/infected cells (^** P < 0.001). In addition, all concentrations of the hydroalcoholic extract or oleoresin induced an upregulation of IL-6 in infected BeWo cells when compared to SDZ + PYR-treated cells (^& P < 0.05). Also, 32 µg/mL of oleoresin induced an increase of IL-6 in comparison to untreated/infected cells (^** P < 0.001) ( Figure 7B ).

Regarding IL-8, uninfected BeWo cells reduced or increased the level of this cytokine when treated with SDZ + PYR or 32 µg/mL, respectively (^*** P < 0.0001) in relation to untreated cells (medium) ( Figure 7C ). Interestingly, 32 µg/mL oleoresin triggered a higher release of IL-8 in comparison to SDZ + PYR-treated cells (^& P < 0.05) ( Figure 7C ). However, all concentrations of the hydroalcoholic extract and oleoresin downmodulated IL-8 in infected BeWo cells in comparison to untreated/infected cells (medium Tg) (^**** P < 0.00001) or SDZ + PYR-treated cells (^& P < 0.05) ( Figure 7D ).

For MIF production, uninfected BeWo cells augmented the release of this cytokine in all concentrations of the hydroalcoholic extract and oleoresin when compared to untreated cells (medium) (^* P < 0.01, ^** P < 0.001, ^**** P < 0.00001) or SDZ + PYR-treated cells (^& P < 0.05) ( Figure 7E ). However, in the presence of infection, untreated and 32 µg/mL oleoresin-treated cells upregulated or downmodulated MIF in comparison to medium (^# P < 0.05) or medium Tg (^*** P < 0.0001), respectively ( Figure 7F ).

Finally, we measured ROS in BeWo cells infected or not with T. gondii and treated or not with the hydroalcoholic extract or oleoresin. We just observed that uninfected cells treated with 32 µg/mL of the hydroalcoholic extract augmented the level of ROS in relation to untreated cells (medium) (^* P < 0.01) and SDZ + PYR-treated cells (^& P < 0.05) ( Figure 7G ). Additionally, infected BeWo cells did not change ROS release in presence of any treatment ( Figure 7H ).

TNF and IL-10 were not detected in BeWo cell supernatants under any experimental conditions (data not shown).

We also verified cytokine and ROS production in HTR8/SVneo cells infected or not with T. gondii and treated with the hydroalcoholic extract or oleoresin.

In the absence of infection, HTR8/SVneo cells increased IL-6 release when treated with the hydroalcoholic extract (8 µg/mL) or oleoresin (16 µg/mL) in comparison to untreated cells (medium) (^* P < 0.01, ^** P < 0.001) and SDZ + PYR-treated cells (^& P < 0.05) ( Figure 8A ). In the presence of infection, untreated HTR8/SVneo cells, as well as cells treated with the hydroalcoholic extract or oleoresin, increased the levels of IL-6 in relation to the medium or SDZ + PYR-treated cells, respectively (^# P < 0.05, ^& P < 0.05) ( Figure 8B ). Also, IL-6 was lower in SDZ + PYR-treated cells in comparison to untreated and infected cells (medium Tg) (^** P < 0.001) ( Figure 8B ).

Regarding IL-8 for uninfected cells, SDZ + PYR reduced IL-8 (^*** P < 0.0001) and all concentrations of the hydroalcoholic extract and oleoresin (^* P < 0.01, ^**** P < 0.00001) increased IL-8 when compared to the medium ( Figure 8C ). Also, the hydroalcoholic extract and oleoresin presented higher levels of IL-8 in comparison to SDZ + PYR (^& P < 0.05) ( Figure 8C ). When HTR8/SVneo cells were infected by T. gondii, IL-8 was augmented in relation to the medium (^# P < 0.05), SDZ + PYR decreased IL-8 in comparison to only infected cells (medium Tg) (^**** P < 0.00001), and all treatments with the hydroalcoholic extract or oleoresin upregulated IL-8 in relation to SDZ + PYR (^& P < 0.05) ( Figure 8D ).

The MIF production was not significantly changed in uninfected HTR8/SVneo cells, except for uninfected and SDZ + PYR-treated cells (^* P < 0.01) ( Figure 8E ). Although not statistically different, the hydroalcoholic extract and oleoresin showed an increase in MIF release in infected HTR8/SVneo cells, while only infected cells augmented MIF in comparison to untreated and uninfected cells (^# P < 0.05) ( Figure 8F ).

Finally, we measured ROS in HTR8/SVneo cells infected or not with T. gondii and treated or not with the hydroalcoholic extract or oleoresin. We observed that uninfected cells treated with the hydroalcoholic extract, oleoresin or SDZ + PYR presented low levels of ROS in relation to untreated cells (medium) (^* P < 0.01, ^** P < 0.001, ^*** P < 0.0001, ^**** P < 0.00001) ( Figure 8G ). Although not statistically different, the hydroalcoholic extract and oleoresin showed a reduction in ROS release in infected HTR8/SVneo cells, while only infected cells diminished ROS in comparison to untreated and uninfected cells (^# P < 0.05) ( Figure 8H ).

TNF and IL-10 were not detected in supernatants under any experimental conditions (data not shown).

First, to determine non-toxic concentration to use in the experiments, the villous explant viability after treatments with oleoresin or hydroalcoholic extract was performed by measuring LDH release and MTT assay. Our data showed that both oleoresin and hydroalcoholic extract did not alter tissue viability at any of the concentrations tested when compared to untreated explants (medium). Also, treatment with SDZ + PYR did not cause significant cytotoxicity in villous explants in relation to the untreated group ( Figures 9A, B ). In addition, treatments did not alter the tissue morphological structure, which was highlighted by the typical morphology of syncytiotrophoblast cells (black arrows) and mesenchyme (*) compared to untreated villous explants ( Figures 9C-F ).

We evaluated the effects of the hydroalcoholic extract and oleoresin (256 and 128 µg/mL) on the control of T. gondii intracellular proliferation in human chorionic villi using the β-galactosidase assay. We observed that treatments at both concentrations significantly reduced the percentage of intracellular parasites in comparison to untreated/infected explants (considered as 100% proliferation) (^**** P < 0.00001). As expected, treatment with SDZ + PYR also inhibited parasite growth (^**** P < 0.00001) when compared to the untreated group (medium) ( Figure 10 ).

In addition, we evaluated whether the hydroalcoholic extract and oleoresin would modulate the cytokine profile in chorionic villi. Our results showed that IL-6, IL-8, MIF and TNF showed no significant change in their production when uninfected explants were treated with both hydroalcoholic extract or oleoresin if compared to medium, except for TNF when 256 µg/mL hydroalcoholic extract was added in the samples (^* P < 0.05) ( Supplementary Figure 1 ). Finally, T. gondii infection reduced the levels of all cytokines in comparison to untreated explants (medium), regardless of hydroalcoholic extract and oleoresin treatments (^# P < 0.05) ( Supplementary Figure 1 ).