*4.2. Extraction and Fractionation*

For extraction and fractionation, the protocol by Cutignano et al. [26] was used, but with some modifications. Briefly, a methanolic extract was firstly prepared by adding 5 mL methanol (MeOH) for each g of pellet (algae were cultured in three different occasions in triplicate batches), sonicating the samples for 30 s, centrifuging them at 4000 rpm for 5 min at room temperature and drying the supernatant with a rotovapor.

For the fractionation, columns were activated (column type: 6 mL/500 mg resin) with 6 mL methanol and 17 mL of distilled water. The resin used was a spherical poly(styrene-divinylbenzene) resin for SPE (Chromabond®HR-X, Düren, Germany). One mL of distilled water was added for each 20 mg of methanolic extract. Samples were sonicated to obtain a good suspension and added to the columns. The column was eluted as follows: (1) wash step with 2 mL of distilled water, throwing away 1.5 mL to eliminate salts, (2) addition of 18 mL of distilled water to obtain fraction A, (3) addition of 24 mL of methanol (CH3OH)/water (50:50) to obtain fraction B, (4) addition of 18 mL Acetonitrile

(CH3CN)/water (70:30) to obtain fraction C, (5) addition of 18 mL acetonitrile (100%) to obtain fraction D, (6) and finally addition of 18 mL of dichloromethane/methanol (CH2Cl2/CH3OH; 90:10) to obtain fraction E.
