Processing and ethanolic extraction of Propolis samples

Raw propolis was macroscopically screened to remove impurities (e.g., pollen, wood, and dead bees) prior to extraction. A mortar and pestle were used to fragment the samples of propolis. For extraction, 5 ml of ethanol was added to 50 mg of propolis sample and sonicated for 180 min, and reextracted thrice. A syringe filter (Acrodisc 0.45 μm) was used to filter the samples, and a nitrogen flow was used for drying the filtered solution. To obtain pure components, further fractionation, and purification of raw propolis was achieved by CC and SEC. An appropriate quantity of absolute ethanol (100 ml/g) was added to raw propolis and sonicated for an hour to obtain ethanol-based extract for fractionation. An appropriate quantity of ethanol was subsequently used to filter and re-extract two times, with subsequent filtering every time. After the extracts were merged, a rotary evaporator was used to evaporate and dry the solvent, followed by weighing. An extraction yield of 12.8869 g was obtained. A small quantity (1 ml) of ethyl acetate was used to re-dissolve the residue (2 mg) completely, followed by sonication to stimulate the dissolution of the residue. The extracted solution was poured into empty weighed vials and labeled. Crude propolis samples and purified fractions were analyzed by LC–MS, HPLC in association with a range of detectors, including ELSD, UV, and high resolution mass spectrometry, as well as NMR spectroscopy.