Two systems: ethanol extraction and aqueous extraction
were studied and compared. Frozen citrus peel powder (2 g)
was placed in a 50ml centrifuge tube, and 16 ml of solvent or
aqueous phase was added. The preparation was left to stand
at different temperature (various from 20 to 80 ◦C) for 3 h.
The mixtures were then centrifuged using a Mistral 1000
centrifuge (MSE Labsupply Pierce, Loughborough, Leicestershire,
UK)at 500×g for 10 min at room temperature. After centrifugation, the supernatants were filtered through Whatman
No. 42 filter paper (Whatman Inc., Clifton, NJ, USA).
Following filtration, a 10ml aliquot of the filtratewas concentrated
by evaporation of the solvent, using a rotary evaporator
(BU¨ CHI Rotavapor R-114, BU¨ CHI, Lausanne, Switzerland)
under partial vacuum at 40 ◦C until less than 1ml of filtrate
remained. The extractwas then re-dissolved in 10 ml of Milli-
Q water and stored at 4 ◦C prior to purification step. All the
extracts were prepared in triplicate.