*3.3. Light and Electron Microscopic Observation in HepG2 and Caco-2 Cells*

HepG2 and Caco-2 cells exposed to different concentrations of the extract experienced a significant impairment of cell growing which compromised the survival of the cells at high concentrations.

Under light microscope, unexposed HepG2 cells underwent normal mitotic processes (Figure 3A). However, when cells were treated with 15.6 and 31.2 μg/mL of the extract they revealed an intense lipid degeneration in the cytoplasm with vacuoles that tends to confluency (Figure 3B,C). Moreover, aberrant mitotic figures were also detected, which suggest that the extract was able to stop the cell growing at any step, including mitosis (Figure 3B,C).These morphological features were also observed under electron microscopy with cells showing big lipid drops (Figure 3D–F).

The damage observed in Caco-2 cells exposed to the extract was less profuse in comparison to HepG2 cells, although a marked vacuolization was shown as well as cell death. In fact, the presence of apoptotic nuclei was more frequent in the case of Caco-2 cells. Whereas cells underwent normal mitotic process in the control group (Figure 4A), aberrant mitosis can be observed from the lowest concentration assayed (13.9 μg/mL) (Figure 4B) showing the first stages in the process of apoptosis characterized by a continuous ring of condensed chromatin at the interior surface of the nuclear envelope (Figure 4C). Caco-2 cells were analyzed by transmission electron microscopy showing a nucleus with an irregular surface, decondensed chromatin, and very developed nucleoli with well-known fibrillar center. Autophagosomal vacuoles were observed in the cytoplasm (Figure 4D). Similar findings were observed in the ultrastructural study, with the lowest concentration showing lipid degeneration (Figure 4E,F). In addition, Caco-2 cells showed big citoplasmatic inclusions as a result of autophagic processes that lead to cell death (Figure 4E,F).

**Figure 3.** Morphology of HepG2 cells after 24 h of exposure to the extract observed by light microscopy (**A**–**C**, bars = 25 μm) and electron microscopy (**D**–**F**, bars = 2 μm). Unexposed control cultures (**A**) and HepG2 cells exposed to 7.79 μg/mL of the stilbene extract (45%) (**D**), 15.59 μg/mL of the extract (**B**,**E**) and 31.18 μg/mL of the stilbene extract (45%) (**C**,**F**). (**A**) Unexposed cells undergoing mitotic processes (arrow heads). (**B**) and (**C**) Lipid degeneration with confluent lipid drops (arrow) and aberrant mitotic figures (arrow head). (**D**) Cells showing nucleus (N) and nucleolus (n); mitochondria (m); lipid drops (L). (**E**) Big lipid drops (L); nucleus (N) and nucleolus (n) are also observed. (**F**) High amount of lipid drops that tends to confluency (L).

**Figure 4.** Morphology of Caco-2cells after 24 h of exposure to the stilbene extract (45%) observed by light microscopy (**A**–**C**, bars = 25 μm) and electron microscopy (**D**–**F**, bars = 2 μm). Unexposed control cultures (**A**,**D**), and Caco-2 cells exposed to 13.9 μg/mL of the extract (**B**,**E**) and 27.88 μg/mL of the extract (**C**,**F**). (**A**) Unexposed cells undergoing mitotic processes (black arrow). (**B**) Cells undergoing aberrant mitosis (black arrow). (**C**) Cells showing apoptotic nuclei condensation (arrow head). (**D**) Unexposed cells showing normal nucleus with an irregular surface (N), nucleolus (n) with large fibrillar center (white arrow), and autophagosomes (Ap) in the cytoplasm. (**E**) Damaged nucleus and mitochondria are observed as well as big lipid drops. Autophagosomes (Ap) evidenced degenerative process. (**F**) Lipid drops (L), autophagosomes (Ap), and autophagic vacuoles (AV) are shown.
