Vitis vinifera L. cv. Shiraz
(syn. Syrah) grape berries grown during the 2001 harvest
season on the Waite Campus at the University of Adelaide
were used as the source material for proanthocyanidins.
Samples were collected at three specific times during berry
development: three weeks after fruit set, ve´raison (the commencement
of fruit ripening), and commercial maturity. The
sample collected at ve´raison was divided into unpigmented and
pigmented berries. All samples (5 kg) consisted of randomly
collected fruit clusters within the same vineyard block.
Briefly, proanthocyanidin purification was carried out as
follows. Isolated skins were extracted in 2:1 acetone/water.
Afterward, acetone was removed under reduced pressure, and
the aqueous portion was extracted with chloroform, freezedried
to a powder, and purified by adsorption chromatography
using Toyopearl TSK HW 40-F size exclusion media (Sigma;
Castle Hill, NSW, Australia). Adsorbed proanthocyanidins
were rinsed (3 column volumes) with 1:1 methanol/water
containing 0.1% v/v trifluoroacetic acid to remove low molecular
weight contaminants. The sample collected at commercial
maturity was rinsed with an additional 7 column volumes of
this solvent in an attempt to remove apparent anthocyanin
impurities. After rinsing the samples, proanthocyanidins were
eluted with 2:1 acetone/water with 0.1% v/v trifluoroacetic acid
added. The acetone was evaporated, and the aqueous residue
was freeze-dried to a powder. With the exception of the
chloroform extraction, proanthocyanidin isolation and purification
has been previously described (19).