Extraction was carried out as described by Shirahigue et al.
(2010) with a few modifications (schematic representation shown
in Fig. 1). Briefly, grinded grape pomace (20 g) was thawed and
placed on a glass flask where 80% (v/v) of ethanol/water mixture
(100 ml) was added. Next, the mixture was placed under orbital agitation
at 300rpm for 48 h, at room temperature and in darkness.
The liquid phase was then separated from the solid by vacuum
filtration through a glass filter and a 45m Millipore (Billerica,
MA) polyvinylidene fluoride (PVDF) membrane filter. The filtrate
was placed on a 100 ml amber glass volumetric flask and the volume
was completed to 100 ml with ethanol/water solvent. Samples
were taken before concentration step to assess the TPC, antioxidant
capacity and iron(II) chelating ability (ICA). Finally, the liquid
was concentrated in a vacuum rotary evaporator (Büchi, Flawil,
Switzerland) at 65 ◦C aided with a nitrogen stream, until dryness.
The dry residue obtained was weighed and redissolved in 50 ml of
water, separated in smaller portions and reserved in an ultra high
deep freezer at −80 ◦C in amber recipients until further analysis.