The BtD extract (5.5 g) was fractionated by successive open-column chromatography using silica gel as the stationary phase (60, F254, Merck KGaA, Darmstadt, Germany) and a gradient system n-hexane: ethyl acetate: methanol with ascending polarity. Fractions of 40 mL were collected and pooled into four subfractions (BtD1, BtD2, BtD3, and BtD4). BtD4 (116 mg) was acetylated and subfractioned into four subfractions (BtD4.1, BtD4.2, BtD4.3, and BtD4.4). Separation was monitored by TLC and HPLC. Aluminum plates coated with silica gel 60 F254 (normal phase, Merck) and silica gel 60 RP-18 F254S (reverse phase, Merck) were used. The plates were analyzed with an ultraviolet light lamp (UVGL-58, 254–365 nm UV) and specific developers. HPLC was performed with equipment consisting of a separation module chromatographic system (Waters 2695) and photodiode array detector (Waters 2996), as well as a 250 × 4 mm Licrosphere® 100 RP-18 Column (5 μm particle size). The mobile phase consisted of gradient water: acetonitrile. Samples were analyzed at 400 μg/mL, with a flow of 0.9 mL/min and a sample injection of 10 μL. The detection of compounds was carried out between 195–600 nm [12 (link)].
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