Isolation and Extraction of Bioactive Cyanobacterial Compounds

Initial screening assays led to the selection of two cyanobacterial strains, Cyanobium sp. LEGE 07175 and Nodosilinea sp. LEGE 06001 isolated from the Portuguese coast [11 (link)] and maintained in the LEGEcc in CIIMAR, Matosinhos, Portugal. Strains were cultured in Z8 medium supplemented with marine tropical salt (25 g/L), at 25 °C, with a photoperiod of 14 h/10 h light and dark, respectively, and at a light intensity of 10–30 μmol photons∙m⁻²∙s⁻¹. Cyanobium sp. LEGE 07175 cultures were grown in 20-L flasks with constant aeration and, at the exponential phase, cells were harvested through centrifugation, before being frozen and freeze-dried. For the Nodosilinea sp. LEGE 06001 strain, available freeze-dried biomass was used, which followed the same growth conditions. The biomass of LEGE 07175 (13 g) and LEGE 06001 (56.5 g) was extracted by repeated percolation with a warm mixture of CH₂Cl₂/MeOH (2:1, v/v), yielding a crude extract of 1.9 g and 8.74 g, respectively. Both crude extracts were fractionated by normal-phase (Si gel 60, 0.015–0.040 mm, Merck KGaA, Darmstadt, Germany) VLC (vacuum liquid chromatography) with an increasing polarity grade, from 90% n-hex to 100% EtOAc and 100% MeOH [12 (link)], giving a total of nine fractions each.