2.8.1. Gradient Elution Column Chromatography

Selected extracts were combined and adsorbed on equal mass of silica (70 g of silica, 60–120 mesh) to make a loading sample. The sample was packed on a silica gel column, and the gradient elution method was used to run the column. The column was first eluted with n-hexane as the mobile phase, and the polarity of the mobile phase was increased by gradually adding 2.5% of ethyl acetate increments. After reaching 100% ethyl acetate, the polarity was further increased by gradually adding 2.5% methanol. Volumes of 250 mL fractions of the eluate were collected during the process. A total of 428 fractions were obtained from the column chromatography, and each fraction was concentrated using a rotary evaporator RII (BUCHI, Labortechnik AG, Switzerland). The fractions were dried to a constant mass under a fan in a fume hood cabinet. All fractions were stored in sterile screw top tubes at room temperature.