*4.2. Plant Material and Extraction*

*Ageratina havanensis* (Kunth) R. M. King & H. Robinson (Asteraceae) as authenticated by Prof. Iralys Ventosa, Instituto de Ecología y Sistemática, Havana, Cuba (voucher herbarium specimen number HAC-42498) was collected in Havana (East Havana, Alamar neighbourhood) during 2012 in flowering and vegetative stage. The secondary metabolites were extracted as previously reported [17,18].

#### *4.3. Cytotoxicity and P-glycoprotein Activity Assays*

The study was conducted using 4T1 cells (ATCC, Manasssas, VA, USA) cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and glutamine (2 mmol/L) at 37 ◦C in 5% CO2 humidified incubator.

Cytotoxicity was measured using the MTT reduction assay [49]. After extracts exposure, cells were washed and a 100 μL/well of MTT reagen<sup>t</sup> (5 mg/mL) was added. After a 4 h incubation period at 37 ◦C, supernatants were discarded. The dye was extracted with dimethyl sulfoxide and optical density was read at 540 nm. The inhibition of reduction of MTT was expressed as the percentage of viable cells referred to control cells, those incubated in presence of the vehicle only (100% viability value). IC50 values were calculated.

P-glycoprotein (P-gp) activity was evaluated by the Rhodamine-123 (Rho-123) accumulation assay [13]. Cells (4 × 10<sup>4</sup>/well) were treated for 24 h with sub-toxic concentrations of the extracts (10–200 μg/mL) or verapamil (20 μM), an inhibitor of P-gp activity (positive control). Cells were washed and incubated with 5 μg/mL Rho-123 for 2 h. Afterwards, the cells were lysed with 0.1% Triton X-100. In cell lysates fluorescence intensity was measured at the 485 nm excitation and 535 nm emission wavelengths. Data were expressed as percentage of fluorescence relative to control cells.
