*3.2. Seaweed Collection*

The green macroalgae *C. cupressoides* var. *flabellata* was collected in the city of Nísia Floresta, on the southern coast of the state of Rio Grande do Norte, Brazil. After the collection, the seaweed was transported to the Laboratório de Biotecnologia de Polímeros Naturais of the Biochemistry Department, Universidade Federal do Rio Grande do Norte, for the removal of epiphytic species, sediment, and encrusted organisms and subsequent extraction of their SPs. The material collection occurred under authorization of Brazilian National Management System Genetic Heritage and Associated Traditional Knowledge (loose translation) SisGen n◦ A0D4240.2.1.1.

#### *3.3. Extraction of SPs*

To obtain the SPs-rich extract, the seaweeds were dried, crushed, delipidated with ethanol, and submitted to proteolysis. The proteolytic digestion was conducted with 100 g of this powdered seaweed, 0.25 M NaCl (500 mL) pH = 8.0, and a mixture of alkaline proteases (Prolav 750, Prozyn Biosolutions, São Paulo, SP, Brazil) at 15 mg/g powdered seaweed; for 18 h at 60 ◦C. The SPs-rich extract was obtained after filtration and centrifugation (10,000× *g* for 20 min, 4 ◦C), and it was submitted to fractionation step as described by Costa et al. [23]. Briefly, 0.3 volumes of propanone (4 ◦C) was dropped to the SPs-rich extract under gentle agitation and maintained for 24 h. The material was centrifuged (10,000× *g* for 20 min, 4 ◦C), dried, and kept in the dark until further use. The fractionation was repeated by adding 0.5, 1.0, and 2.0 volumes of propanone to the supernatant. Based on the propanone volumes used, the fractions were named CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, respectively.

#### *3.4. Purification of SPs by Liquid Ion Exchange Chromatography*

The SPs of the CCB-F1.0 fraction were purified with a fast protein liquid chromatography (FPLC) system at ÄKTA-GE (Healthcare Life Sciences, Little Chalfont, Bucks, UK) using a 5 mL HiTrap DEAE FF column (GE Healthcare, Westborough, MA, USA). To prepare the sample, the CCB-F1.0 fraction was solubilized in 0.25 M NaCl at concentration of 5 mg/mL, filtered using 0.22 μm filters, and injected into the column (1 mL). The SPs bound to the column were eluted by applying a stepwise NaCl gradient (0.525, 0.675, and 0.9 M), based on a first elution with continuous gradient ranging from NaCl 0.25 to 3.0 M. All peaks were monitored by DMMB metachromasia and absorbance at 525 nm [60]. This procedure was repeated ten times. Then, the material (each peak) obtained was pooled and dialyzed (MW cut-off of 6 kDa) against distilled water and lyophilized.
