## 3 Results and Discussion
### Evaluation of Cytotoxicity  
A dose and time-dependent effect of _S. aspera_ extract was observed for both cell lines. Interestingly, for TE671 cells, the cytotoxic effect was more prominent at 72 h of treatment, at the range of concentrations between 0.04-3.125 mg/mL. At 24 h, inhibition was observed at 0.78-3.12 mg/mL whereas after 48 h of treatment, the effect was observed at 0.39-3.12 mg/mL (Figure 1A). On the other hand, the range of the concentrations (0.39-3.12 mg/mL) that significantly inhibited the proliferation of A172 cells with respect to the control group after treatment with the extract of _S. aspera_, was the same for all time points. At the concentrations from 0.19 mg/mL to 0.02 mg/mL, no statistically significant effect was observed, while cells recovered at 0.04 mg/mL (Figure 1B). This effect was also evident through microscopical investigation. TE671 cells were confluent without any treatment (Figure 2A), while a similar behavior was observed for the 0.39 mg/mL concentration (Figure 2C). On the other hand, at 0.78 mg/mL cells were significantly reduced (Figure 2B). The same behavior was observed for the A172 cell line, where cells were confluent without any treatment (Figure 3A), while cells were also near-confluence at the 0.39 mg/mL concentration (Figure 3C). Finally, at 0.78 mg/mL cells were significantly reduced (Figure 3B), as in the case of TE671 cells. Figure 1: Dose dependent and time dependent effect after treatment with _S. aspera_ extract on TE671 (**A**) and A172 (**B**) cells. Data are presented as the mean \(\pm\) standard error of the mean (SEM) (with n = 8). The asterisk (*) indicates significant differences between untreated and treated cells. The grey color corresponds to 24 h of treatment, the pink to 48 h and the light blue to 72 h.  
Figure 2: Microscopy inspection of the TE671 rhabdomyosarcoma cells, grown for 72 h in DMEM with no other treatment (**A**), cells treated with 0.78 mg/mL of the extract (**B**) and cells treated with 0.39 mg/mL of the extract (**C**). Images were captured at \(\times\)200 magnification. In the case of TE671 cells, the lowest IC\({}_{50}\) was observed at the time point of 72 h and was calculated at 0.629 \(\pm\) 1.31 mg/mL. The IC\({}_{50}\) values for 24 and 48 h were calculated at 0.820 \(\pm\) 1.38 mg/mL and 1.044 \(\pm\) 1.22 mg/mL, respectively (Figure 4A). For the A172 cell line, the lowest IC\({}_{50}\) was calculated at 0.482 \(\pm\) 0.98 mg/mL 48 h after treatment. The IC\({}_{50}\) values for 24 and 72 h were calculated at 1.061 \(\pm\) 1.09 mg/mL and 0.749 \(\pm\) 1.18 mg/mL, respectively (Figure 4B). Various chemotherapeutic agents are available and are currently used to combat cancer [35; 36]. However, due to their high cost and the often-severe associated side effects their partial or even full replacement is a compelling research objective. Natural products  
Figure 4: IC\({}_{50}\) of _S. aspera_ extract on TE671 cells (**A**) and A172 cells at 24, 48 and 72 h (**B**). The lowest IC\({}_{50}\) value for TE671 cell line was 0.629 mg/mL, calculated at 72 h and 0.482 mg/mL for A172 cell line, calculated at 48 h. Cancer cell viability increases as concentration of the drug decreases. Figure 3: Microscopy inspection of the A172 glioblastoma cells, grown for 72 h in DMEM with no other treatment (**A**), cells treated with 0.78 mg/mL of the extract (**B**) and cells treated with 0.39 mg/mL of the extract (**C**). Images were captured at \(\times\)200 magnification. are promising substitutes and they are intensively studied for their anticancer activity. Cases of in vitro experiments in which cells were co-treated with a chemotherapeutic drug and a natural product have shown that natural products are able to decrease the dose of the first agent but also to counteract cell damage caused by chemotherapeutics due to increased reactive oxygen species (ROS) production [37].