*4.3. Anti-Inflammatory Assay*

The anti-inflammatory assay was performed as in Lauritano et al. [12]. Briefly, <sup>∼</sup>106 human monocyte THP-1 cells/mL (ATCC®TIB-202TM) supplemented with 50 ng/mL phorbol 12- myristate 13-acetate (PMA, Sigma-Aldrich) were seeded in 96-well plates and incubated at 37 ◦C, 5% CO2 for 48 h in RPMI-1640 medium (Biochrom). After 72 h, 80 μL fresh RPMI medium and 10 μL/well of test sample were added. In particular, fractions A, B, C, D, and E were tested at 100 and 50 μg/mL, while 1-palmitoyl-sn-glycero-3-phosphocholine (Sigma L5254) was tested at 3.13, 6.25, 12.5, 25, and 50 μg/mL. The tests were performed at least in triplicate. After incubation for 1 h, all samples were incubated with 1 ng/mL lipopolysaccharide (LPS) for 6 h at 37 ◦C and plates were then frozen at −80 ◦C. Enzyme-linked immunosorbent assay (ELISA) was used to test TNF-α inhibition. ELISA was performed as in Lauritano et al. [12]. Results were read at 405 nm.
