The cartridges were washed with 1 mL of H2O and 1 mL of 3% MeOH sequentially and finally eluted with 2 mL of 25% MeOH.
The cartridges were washed with 2 mL of H2O and 1 mL of 10% MeOH sequentially and finally eluted with 2 mL of 50% MeOH.
A portion of the supernatant (4 mL) was transferred to a silica column (glass Pasteur pipet, 150 × 5 mm, filled to a height of 4 cm with silica gel 60), conditioned with MeOH/acetone (1 + 1, v/v) containing 1% formic acid (5 mL); the column was washed with the "conditioning" mixture (4 mL), then MeOH (2 mL), and finally MeOH containing 1% NH3 (8 mL).
Fraction 4 was further purified by silica gel column chromatography CHCl3-MeOH system, and finally yielding purified compound C (isorhamnetin), compound D (kaempferol), and compound E (quercetin).
The methanol extracts (320 g) were subjected to macroporous resin D101 column chromatography (CC) and eluted with the following gradient: MeOH/H2O (3:7, 6 4, 9 1) and finally MeOH, to give 4 fractions (Fr. 1 4).
They were then transferred to the column and eluted as above except the stepwise elution gradient was comprised of 500 ml of 0 1, 1 1 and 1 0 (v/v) CH2Cl2: hexane and finally MeOH, and 2.5 ml fractions were collected.
Finally, MeOH and MilliQ water were alternately used to wash 3 times.
Thereafter, the residue obtained was further fractionated with chloroform (CHCl3) (3 × 250 ml) (Merck, Darmstadt, Germany) and finally with methanol (MeOH) (3 × 250 ml) (Merck, Darmstadt, Germany).
Propolis was initially sequentially extracted with MeOH (high-polar solvent), then CH2Cl2 (medium-polar solvent) and finally hexane (non-polar solvent).
Alkaloids were eluted using n-hexane gradually enriched with ethylacetate (EtOAc), then gradually enriched with CHCl3 and finally with a mixture of EtOAc and CHCl3 gradually enriched with MeOH.
