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.