*4.4. In Vitro Anti-Proliferative Assay*

Human cells were bought at ATCC (https://www.lgcstandards-atcc.org/). Human hepatocellular liver carcinoma cells (HepG2; ATCC®HB-8065™) were cultured in EMEM medium, human melanoma cells (A2058; ATCC®CRL-11147TM) were cultured in DMEM, adenocarcinomic human alveolar basal epithelial cells (A549; ATCC®CL-185TM) were cultured in F-12K medium. The media were supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin.

To evaluate the in vitro antiproliferative effects of the fractions, HepG2, A2058, and A549 cell lines were seeded in 96-well microtiter plates at a density of 1 <sup>×</sup> 104 cells/well and incubated at 37 ◦C to allow for cell adhesion in the plates. After 16 h, the medium was replaced with fresh medium containing increasing concentrations of the fractions (1, 10, and 100 μg/mL) for 72 h. Each concentration was tested at least in triplicate. After 72 h, cell viability was assessed using the MTT test (3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide; A2231,0001, AppliChem Panreac Tischkalender, Darmstadt, GmbH). Briefly, the medium was replaced with medium containing MTT at 0.5 mg/ml and the plates were incubated for 3 h at 37 ◦C. After incubation, cells were treated with isopropyl alcohol (used as MTT solvent) for 30 minutes at room temperature. Absorbance was measured at OD = 570 nm using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Fisher Scientific, Waltham, MA, USA). Cell survival was expressed as a percentage of viable cells in the presence of the tested samples, with respect to untreated control cultures.

## *4.5. Statistical Analysis*

Student's t-test was performed using GraphPad Prism statistic software, V4.00 (GraphPad Software, San Diego, CA, USA). Data were considered significant when p values were <0.05 (∗ for *p* < 0.05, ∗∗ for *p* < 0.01, and ∗∗∗ for *p* < 0.001).
