Algae were washed with distilled water, dried
in air-circulating ovens (Blue Mod1401440SC, USA)
at 45 °C for 5 h and crushed in industrial blender
(type TA-2 METVISA model, Brazil). The dried and
ground material from each collection and each alga was
mixed in equal amounts to dilute any possible seasonal
difference in chemical extracts to be obtained. To obtain
crude extracts, 500 g samples of dried seaweed were
suspended in 1000 mL dichloromethane, chloroform,
methanol, ethanol and water and macerated for 72 h with
three repetitions. Organic extracts were filtered and rotoevaporated
(Rotevaporator Buchii Heating Bath-B490,
Switzerland) at 25 and 40 °C, except for the aqueous
extract that was lyophilized (Edwards High Vacuum
Lyophilizer, ModE2MB, Brazil). The mass from these
extracts was measured and stored under refrigeration
for subsequent cytotoxicity assays. Dichloromethane
extracts from H. musciformis and P. gymnospora were
selected for fractionation by having higher yields:
11.86 g (2.58%) and 13.96 g (3.04%), respectively. For
fractionation by liquid-liquid partition, crude extracts
of H. musciformis (2.73 g) and P. gymnospora (1.90 g)
were suspended in methanol:water (3:1) and extracted
with hexane and chloroform respectively, resulting in
hexane and chloroform fractions to test for cytotoxic
activities. All solvents used were VETEC-Quimica
Fina (RJ-Brazil).