Grape seed procyanidins extraction

Grape seeds were removed by hand from grapes, lyophilized for 2 days and stored at -20°C. The frozen seeds were finally ground in a ball grinder. An ASE 350 Accelerated Solvent Extraction System (Dionex Corporation. Sunnyvale. CA) was used as previously [24] to extract the tannins from the ground seeds. The ground grape seed and skin (~10 g) were submitted to eight solid/liquid consecutive extractions with acetone/water (80:20, v/v) as solvent systems (40 mL of the corresponding solvent system). The ASE experimental variables were pressure (1500 psi), temperature (40°C), static time (4 min), and preheat time (5 min), by using an N₂ flush to prevent oxidation during extraction. The volume of all collection tubes was combined after extraction and then evaporated under reduced pressure. The obtained solid residue was re-dissolved in 30 mL of water and lyophilized.

The extract was solubilized in 250 mL of water/ethanol (95:5, v/v) and extracted three times with chloroform (v = 250 mL) to remove lipophilic material. Then the aqueous phase was extracted three times with ethyl acetate (v = 250 mL) to obtain two distinctive fractions [25]. The organic fraction was concentrated and lyophilized to obtain a dry powder. A crude oligomeric PAs extract was obtained.