**3. Results**

### *3.1. Evaluation of Citotoxicity of V. macrophylla EO*

MTT assay was used to study cell viability and consequently the proliferative capacity of SKBR3 adenocarcinoma cells after treatment with *V. macrophylla* EO. This assay allows to evaluate the toxicity of a substance, through the comparison of cell viability indices obtained from treated cells compared to control. Figure 2 shows the results of the MTT assay obtained from treatments at 24, 48 and 72 h at different concentrations of *V. macrophylla* EO (1.25; 2.5; 5 and 10 μg/mL) on human breast adenocarcinoma cells SKBR3.

The results showed that *V. macrophylla* EO was able to reduce cell proliferation below 60%, even at the lowest concentrations (1.25 μg/mL) after 24 h of treatment. This trend was also observed at longer times (48 and 72 h) for all other concentrations (2.5; 5, and 10 μg/mL).

### *3.2. Immunoflorescence Microscopy Observations*

In the course of experimental analyses, we performed investigations by fluorescence microscopy that allowed us to evaluate the morphological alterations of cells after treatment with *V. macrophylla* EO at different times and concentrations.

After treatment with *V. macrophylla* EO, the SKBR3 cells were labeled both with Hoechst 33258, which is used to highlight morphological-ultrastructural alterations in the nucleus, and with FITC-phalloidin, to highlight changes in the cell cytoskeleton and actin filaments, induced by different treatments with EO (Figure 3). The micrographs obtained by fluorescence microscopy showed that at 1.25 and 2.5 μg/mL concentrations, the lowest concentrations of EO, after 24 h of treatment, the nuclei still possessed a morphology similar to those of the control cells, while the actin filaments of the cytoskeleton revealed a significant rearrangemen<sup>t</sup> of their ultrastructures. Moreover, in some cells, there were numerous vesicular structures, probably containing the same EO (Figure 4a,b, see arrowheads). Images of cells treated with the same concentration of EO for an incubation time of 48 h revealed a reduction of nuclei diameters of the cells. Moreover, the morphologies of the actin filament appeared altered, and numerous green fluorescence points were identified exclusively around the nucleus (Figure 4c,d). This result reveals that *V. macrophylla* EO is able to disorganize the actin filament network (see also Figure 3).

**Figure 2.** Evaluation of SKBR3 cell viability treated for 24, 48 and 72 h with different concentrations of EO obtained from *Vepris macrophylla* leaves. Cell viability was calculated considering the control value and standardized to 100%. Data represent mean ds (n = 3).

**Figure 3.** Images after double-cell staining with FITC-phalloidin (green) and Hoechst 33258 (blue) in SKBR3 control cells.

**Figure 4.** Immunofluorescence micrographs shown the morphological alterations on SKBR3 cells induced by *Vepris macrophylla* EO after treatment with 1.25 (**<sup>a</sup>**,**<sup>c</sup>**) and 2.5 μg/mL (**b**,**d**) concentrations at two incubation times 24 h (**<sup>a</sup>**,**b**) and 48 h (**<sup>c</sup>**,**d**). Arrowheads indicate probably vesicles filled with EO.

Immunofluorescence observations of SKBR3 cells treated with 5 and 10 μg/mL, at 48 h, were in agreemen<sup>t</sup> with results obtained by MTT assay (see Figure 2), showing both the same morphological alterations and numerous vesicles inside the cytoplasm of cancer cells (Figure 5a,b, arrowheads).

**Figure 5.** Morphological alterations of SKBR3 cells after treatment with 5 (**a**) and 10 (**b**) μg/mL of *Vepris macrophylla* EO at 48 h. Arrowheads indicate probably vesicles filled with EO.

## *3.3. SEM Analysis*

SEM observations of SKBR3 control cells at 24, 48 and 72 h showed numerous microvilli and random "ruffles" on the cell surface (Figure 6).

**Figure 6.** SEM images of SKBR3 control cells at 24, 48 and 72 h (**<sup>a</sup>**–**<sup>c</sup>** respectively).

After treatment with EO at different concentrations, cancer cells appeared very damaged. Figures 7–9 show important ultrastructural alterations of SKBR3 cells. The morphological alterations are clearly visible after only 24 h of treatment (Figure 7) with four different concentrations of EO (0.01; 0.1; 1.25 and 2.5 μg/mL, respectively). Moreover, at higher concentrations (1.25 and 2.5 μg/mL), SKBR3 cells appeared flat and adherent to the substrate, probably due the effect on cytoskeletal actin disorganization, as also shown by fluorescence microscopy analysis (Figure 4). When cells were treated with the lowest concentrations of EO (0.01 and 0.1 μg/mL), at 48 h, the plasma membranes were entirely destroyed (Figure 8a,b). Cells showed plasma membrane alterations like those observed 24 h after treatment with the highest concentration (2.5 μg/mL) of EO. Figure 8d shows cells treated with 2.5 μg/mL for 48 h, in which the plasma membranes are completely damaged. Finally, as a dose-dependent effect, after 72 h, these morphological alterations were clearly visible in all SKBR3 cells treated with *V. macrophylla* EO (Figure 9a–c).

**Figure 7.** SEM micrographs of SKBR3 cells after 24 h of V. macrophylla EO incubation (0.01 (**a**), 0.1 (**b**), 1.25 (**c**) and 2.5 (**d**) μg/mL, respectively).

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**Figure 8.** SEM micrographs of SKBR3 cells after 48 h of *V. macrophylla* EO incubation (0.01 (**a**), 0.1 (**b**), 1.25 (**c**) and 2.5 (**d**) μg/mL, respectively).

**Figure 9.** Micrographs of SKBR3 cells after 72 h of *V. macrophylla* EO incubation (0.01 (**a**), 0.1 (**b**), 1.25 (**c**) and 2.5 (**d**) μg/mL, respectively).

Figure 10 shows immunofluorescence and SEM images of the SKBR3 cells with some vesicles on and inside the cytoplasm (arrowheads). These structures, containing the stored EO of *V. macrophylla*, could slow the release of EOs, enhancing both the antiproliferative activity and alteration of cancer cell adhesion on the substrates by actin filaments.

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**Figure 10.** SKBR3 treated with *Vepris macrophylla* EO at 24 h with 1.25 μg/mL (**a**) fluorescence microscopy and (**b**) scanning electron microscopy micrographs. Arrowheads indicate probably vesicles filled with EO.
