Composition of BM extract and its antioxidative activity
Red grapes (Vitis Vinifera L.) seeds, Burgund Mare variety, (BM)
were obtained from Recas vineyard, Romania. 1 g of well grounded
fresh seeds was treated with 20 ml solvent (water/ethanol, 50/50,
v/v) and the mixture was boiled on a water bath for 30 min. After
cooling, the product was filtered, centrifuged and the volume was
adjusted to 20 ml. The total polyphenol (TP) content was determined
with the Folin–Ciocalteu method and the polyphenol
concentration was expressed as mg/l gallic acid solution, used as
standard for calibration [13]. Total catechin content was evaluated
using a method with vanilin HCl [14], the results being
expressed as g EC/L (EC-epicatechin equivalents). Procyanidins
were assessed by the spectrophotometric method of Lebreton
[15] and expressed as gLC/L (LC-leucocyanidin). Anthocyans were
determined using the method of differentiated pH [16]. Antioxidant
activity was assessed with the stable free radical DPPH scavenging
assay and expressed by IC50 (the concentration of the
substrate needed to reduce 50% of the initial DPPH activity) [17].
High-performance liquid chromatography (HPLC) analysis coupled
with mass spectrometry (MS) was used to determine the
polyphenolic composition of the extract on a Shimadzu HPLC (Shimadzu
Corporation, Japan) with diode array detector (DAD) and
mass spectrometry (MS) detectors, using electronspray ionization
(ESI) in negative ionization mode (LC-DAD-ESI(-)-MS). A LiChrosorb
RP-C18 column (250 46 mm, 5 lm) was used for the chromatographic
separation at 26 C. The eluent system was
ultrapure water with HCOOH at pH 3 (A) and methanol (B) at
10:90, v/v ratio.