*2.1. Cytotoxic Activity In Vitro*

#### 2.1.1. Cytotoxic Activity of Extracts and DOX

MAW and MAE showed moderate cytotoxic activities against A549 cells. After 72 h of treatment with extracts, MAW IC50 value was shown to be 56.36 ± 0.30 μg/mL, while MAE IC50 was 51.97 ± 3.27 μg/mL. The IC50 of DOX was 0.44 ± 0.02 μg/mL.

#### 2.1.2. Cytotoxic Activity of DOX in Combination with Extracts

The combined extracts and DOX e ffects were evaluated using an isobolographic analysis method. After incubating cells with subtoxic concentrations of extracts in combination with DOX, there was an increase in cytotoxicity compared to the controls (Table 1). The CI values ranged from 0.14 to 0.38 for DOX/MAW and from 0.12 to 0.4 for DOX/MAE treatment, suggesting strong synergism (Table 2).


**Table 1.** Concentrations of doxorubicin alone or in combination with extracts that induced 50% decrease in cell survival after 72 h of treatment.

IC50 values are presented as the mean ± standard deviation (SD) from three independent experiments; DOX:doxorubicin; MAW: water extract of *M. aquifolium*; MAE: ethanol extract of *M. aquifolium*.


**Table 2.** Isobolographic analysis.

CI: combination index.
