534 C. Santos-Buelga and V. de Freitas
polymerization, are likely to be related with the free concentrations of phenolic sub-
strates, since part of them is involved in the copigmentation slower reaction rates and
evolution might be expected in wines with greater copigmentation (Boulton 2001).
On the other hand, some authors have suggested that the copigmentation could act
as a first stage in the formation of new pigments and determine both the type of
compounds formed and their levels, thereby affecting the color of aged red wines
(Brouillard et al. 2003; Brouillard and Dangles 1994). Likewise, copigmentation
could also influence the extraction of pigments from the grape and their retention in
the must during winemaking (Boulton 2001). Some of these points will be revised
below.
Different types of copigmentation can be distinguished:intermolecular copig-
mentationwhen anthocyanins associate with other molecules,self-associationwhen
it involves anthocyanins themselves, andintramolecular copigmentationwhen the
anthocyanin chromophore interacts with other residues of its own molecule. This
latter type of copigmentation is restricted to anthocyanins which are acylated by
phenolic acids linked to the anthocyanidin through a suitable spacer, so that it allows
the molecule to fold in such a way that the aromatic acyl group(s) can interact
with the flavylium nucleus and protect it from hydration (Dangles et al. 1993).
Vitis viniferaanthocyanins have only one sugar moiety and at most one hydroxycin-
namoyl residue (i.e.,p-coumaric acid or less frequently caffeic acid). Although the
possibility of some intramolecular copigmentation cannot be ruled out, they are not
as flexible as more complex flower anthocyanins having at least two sugar molecules
between the anthocyanidin and the acyl moiety allowing a suitable folding of the
structure. Furthermore, the contents of acylated anthocyanins are reduced in most
grape varieties and are even worse represented in red wines. In these circumstances,
it does not seem that the intramolecular copigmentation can constitute an important
mechanism for the enhancement of the color in red wines, although acylated antho-
cyanins may be more involved in intermolecular copigmentation than non-acylated
ones (Boulton 2001).
A particular case of copigmentation is self-association, consisting of a positive
deviation from Beer’s law that occurs onincreasing the concentration of antho-
cyanins in the medium. Although it is accepted that this phenomenon contributes
to the color expression in flowers (Hoshino 1991; Hoshino et al. 1981), its partic-
ipation in the color of red wine is matter of discussion. Self-association at acidic
pH was found to occur between the flavylium cation itself and with itsZ-chalcone
(Houbiers et al. 1998). The interaction withZ-chalcone forms cannot be expected
to explain self-association in red wines, given the low existing concentrations of
these form that should exist in wine. However, it might be possible the interaction
among flavylium forms of different anthocyanins that can act as cofactors for each
other. Somers and Evans (1979) considered that self-association could be responsi-
ble for much of the non-Beer’s law behaviour in red wines, whereas Boulton (2001),
based on studies carried out by circular dichroism, concluded that self-association
was not much relevant to the enhancement of color in young red wines, but inter-
molecular copigmentation between anthocyanins and different phenolic compounds
would be mainly responsible for the nonlinear color deviations observed. Assays