*2.3. Cell Viability*

Macrophages play several roles in the biology of organisms, including development, homeostasis, and repair, as well as the immune response against pathogens [36]. The evaluation of the viability of RAW 264.7 murine macrophages has previously been used as an indicator of the activation of macrophages and, subsequently, for the characterization of the immunomodulatory potential of new compounds with biomedical applications [37]. Among these analyses, the capacity to reduce MTT to formazan crystals by mitochondrial dehydrogenases is an important measure for evaluating cell viability. Therefore, the effect of SP1 and SP2 on the ability of RAW 264.7 macrophages to reduce MTT after an exposure period of 24 h at concentrations ranging from 12.5 to 100 μg/mL was initially evaluated. As shown in Figure 4, a statistically significant (*p* < 0.05) increase in MTT reduction capacity was promoted by SP1 at concentrations of 12.5 and 25 μg/mL, whereas for the other concentrations, the values did not differ from that of the negative control. For SP2, the cell viability did not differ from that of the negative control, except at the highest concentration (100 μg/mL), which caused a statistically significant decrease (*p* < 0.05) in MTT reduction capacity of around 30%. In this case, the higher SP2 concentration may have interfered with mitochondrial metabolism and consequently led to a reduction of cell viability. These results sugges<sup>t</sup> that at concentrations less than 100 μg/mL, SP1 and SP2 do not cause considerable cytotoxic effects on RAW 264.7 macrophages. For the following analyses, the concentration of 100 μg/mL of SP2 was excluded because it promoted a reduction in cell viability and potentially had a cytotoxic effect.

**Figure 4.** Effects of SP1 and SP2 of *C. cupressoides* on the cell viability of RAW 264.7 macrophages. NC—negative control. The data are presented as the mean ± standard deviation (*n* = 3). Different letters represent statistically significant differences between the different SPs (*p* < 0.05). Different numbers represent statistically significant differences between the same concentrations of the two SPs (*p* < 0.05). \* represents samples that presented statistically significant differences in relation to the negative control (*p* < 0.05).
