2.3 Saponification of the pumpkin seed oil
Forty-four batches (~40 g each) of PSO, 170 mL 10% potassium hydroxide in ethanol (w/v ) and 25 mL 1% pyrogallol in ethanol (w/v ) were placed in a three-necked flask equipped with a reflux condenser, a stopper and a glass gas inlet tube. The end of the tube dipped into the solution in order to purge the mixture with nitrogen to avoid oxidative tocochromanol degradation. The solution was heated and saponified for 4 h under reflux. After cooling the reaction flask on crushed ice, the complete solution was transferred into a separatory funnel and the flask was flushed twice with 10 mL water each. Then, 150 mL dest. water, 4 mL concentrated hydrochloric acid and 100 mLn ‑hexane were added to the separatory funnel and the solution was shaken vigorously for at least 1 min. The upper organic phase was separated and the lower polar phase was extracted three more times with another 100 mL n ‑hexane, respectively. The combinedn -hexane extracts were washed three times with 2% potassium hydroxide in water (w/v ), dried with sodium sulfate and filtrated through a folded filter. The solvent was removed by means of a rotary evaporator (40 °C, 200 mbar). The residue was transferred into a 10 mL brown glass vial by means of a Pasteur pipette. For this purpose, two fully drawn pipettes of n -hexane were added in the flask, the flask was closed with a stopper and vigorously shaken. After this, the entire solution was transferred with another Pasteur pipette into a 10 mL brown glass vial. This procedure was repeated five times and the residue was stored in the refrigerator at 4 °C until further use. Of the 44 repetitions, four batches (total initial amount ~160 g PSO), respectively, were combined to give between 0.8 and 1 g saponified PSO extract. These eleven samples of PSO extracts of 0.8-1.0 g sample weight were subsequently separated by CCC.