*3.7. Cell Cycle Studies*

The cell cycle tests by means of propidium iodide were performed to investigate the cell cycle corresponding to the Sub-G1 phase of the cells in contact with the interaction products prepared (PZQ–SEPet and PZQ–VHSet) and their raw materials (PZQ, SEP and VHS) (Figure 7). These studies allowed us to deduce if there is any alteration of any phase of the cell cycle despite the cell proliferation not being affected, after observing above that there is no significant loss in cell viability of the cells treated with the samples studied.

The control tests are shown in the upper part of the figure (Figure 7). Control tests demonstrated that, in comparison with cellular cycles of healthy, untreated cells ("not treated" cells in Figure 7), DMSO does not affect the cells, unlike etoposide (antineoplasic drug). As can be seen by the cell cycle analysis, the etoposide control group is severely affected and induces death to 64.3% of the cell population.

In Figure 7a, the results showed that the cell cycle was not affected when the cells were treated with PZQ, SEP and PZQ–SEPet, and only at high concentrations, the cell death was increased slightly, although not in a notable way. Furthermore, in none of the concentrations tested was it observed that the PZQ–SEPet interaction product produced a significantly higher percentage of cell death than that with the pure PZQ.

The results of PZQ, VHS and PZQ–VHSet samples are shown in Figure 7b. The PZQ–VHSet interaction product showed that the cellular cycle of the cells was not affected, and cell death was not observed at low sample concentration (0.8–4 μM). At high concentrations (20–100 μM), affectation of the cell cycle of the cells was observed as well as greater cell death. This damage in the cell cycle is very high at the 100 μM concentration. This is the same in pure VHS clay, therefore, this occurs in the interaction product due to the presence of the clay mineral.

In summary, the PZQ–SEPet interaction product showed that it does not affect, in any concentration, the cellular cycle of the cells, and its behavior is similar to the pure drug. However, the PZQ–VHSet interaction product showed that it affects, at high concentrations, the cellular cycle, so only the lower concentrations (<0.8–4 μM) do not affect the Sub-G1 phase of the cells (Figure 7).

**Figure 7.** Cell cycle of the HCT116 line treated with the PZQ, SEP and PZQ–SEPet (**a**), and with the PZQ, VHS and PZQ–VHSet (**b**). The percentages indicate the number of cells in Sub-G1 (apoptotic or necrotic). Negative controls (Not treated and DMSO) and positive control (Etoposide) are also included.
