4.4.1. Silica Gel Column Chromatographic Separation

Forty-two grams of the silica gel 60 (Merck) was placed into a glass column with a silica height of 15 cm. The extract was eluted with 250 mL of the following organic solvents: hexane, dichloromethane, ethyl acetate, and methanol, in increasing orders of polarity. The separation occurred in a vacuum, with 0.5 g of the dry extract [82]. In the second step, the ethyl acetate fraction was eluted in a vacuum using, as a mobile phase, the gradients shown in Table 6. The ethyl acetate fraction was chosen based on the TLC and HPLC results.

**Table 6.** Gradient solvent system used in column chromatography.

