Then, the extract was divided into two 1.5 mL new tubes.
Next, 500 mL of chloroform:isopropanol (3:2) was added each tubes
and were centrifuged for 5 min at 12,000 rpm. The lower phasewas
transferred into 2 mL centrifuge tubes as the first extraction. The
remaining methanolic solutions were used for the second extraction
by adding 500 mL of chloroform. Then, the lower phases were
taken to the tubes. It was called as second extraction. Afterward,
Na2SO4 was added to the tubes and mixed by hand and centrifuged
at 12,000 rpm for 1 min in order to remove water. Extractions were
evaporated to dryness under gentle stream of nitrogen and finally
the volume was completed to 0.5 mL with HPLC grade methanol
and passed through a PTFE millipore membrane filter (0.22 mm).