2.2 Isolation of compound
Approximately 500 g of the powdered cyanobacterial mass was defatted with hexane and Soxhlet-extracted at 65 ºC with chloroform for about 6 to 8 hrs. The chloroform extract was concentrated to 1/5th the volume using a rotary vacuum evaporator (IKA, Germany) at 150 rpm/40 ºC. Fatty acid synthase (FAS) inhibition assay gave positive result with the extract and it was marked as the property at every step involved in the purification of the compound/s. The concentrate was then added to excess of methanol in a separating funnel, stirred and kept overnight till a greenish precipitate was formed. The greenish precipitate was removed and re-dissolved in minimal amount of chloroform. This step was repeated until the precipitate turned pale white. The pale white waxy precipitate was further purified by 240 H silica gel column chromatography (5 x 30 cm column). The column was subjected to gradient elution by ethyl acetate to methanol and finally by dichloromethane. Further purification was carried out by vacuum liquid chromatography with TLC grade silica 60 H in 1.5 x 15 cm column. Elution was carried out from 100% dichloromethane to 100% methanol. All the chemicals used were from Merck, India. Final purification of the compound was done by reverse phase chromatography, using Agilent 1200 High Pressure Liquid Chromatographic (HPLC) system equipped with Extend-C18 column of 1.8 µm, 2.1 x 50 mm, Diode Array Detector in combination with Chem32, and Chemstation softwares. Gradient elution was performed with acetonitrile to water at a constant flow rate of 0.2 mL/min with a constant column temperature of 30 ºC.