Extraction of Marine Invertebrate Metabolites

The extraction procedure was carried out by following the methods described by Shuchizadeh et al. with some modifications [26 (link)]. The gonad (5gm), and body wall (84.3gm) were submerged in reagent grade (99%) methanol, hexane, and ethyl acetate (PRA grade, ≤ 99.5%, Sigma-Aldrich) in 1:3 (w/v) ratio and constantly agitated on orbit shaker (Lab-line Orbit Shaker, Model 3520) for 96 hours at room temperature. The flasks were covered with aluminium foil to avoid photolysis and thermal degradation of secondary metabolites prior to extraction. The extract was then decanted and filtered with Whatman® Grade 3 Filter Paper (diameter 12.5cm). The resulting filtrate was concentrated using a rotary evaporator (BU-R134 Rotary Vap System, Switzerland) at reduced pressure and temperature (40–45°C). The concentrated crude residues were stored at 4°C for the subsequent investigations.