Column chromatography (CC) was carried out using silica gel 60 (63–200 μm, E. Merck, Sigma-Aldrich), while thin-layer chromatography (TLC) silica gel G F254 (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt) was employed for vacuum liquid chromatography (VLC). TLC was performed using pre-coated silica gel 60 GF254 plates (E. Merck, Darmstadt, Germany; 20 × 20 cm, 0.25 mm in thickness). Spots were visualized by heating at 110 °C, according to [43 ], after spraying with para-anisaldehyde (PAA) reagent (85:5:10:0.5 absolute EtOH: sulfuric acid: glacial acetic acid: para-anisaldehyde) [43 ].
In addition, all the kits used for the biological study were purchased by Biosystems SA Costa Brava 30, Barcelona (Spain) and DiaSys Diagnostic Systems GmbH, Holzheim, Germany.