Biology Reference
In-Depth Information
gallic acid cannot be oxidatively coupled into ellagic acid, and it was
concluded that “
ellagic acid has been formed in nature by
dehydrogenation of gallic acid-esters
(
e.g.
,
depsidic tannins
)” (Erdtman,
1935). Similar conclusions were drawn 20 years later by Schmidt and
Mayer (1956), stating that “
it is much more likely that hexaoxydiphenic
acid is formed in situ by dehydrogenation of two gallic acids bound to
sugar in suitable positions
” (both quotations are translated from the
original text written in German). This view that the
hexahydroxydiphenoyl (HHDP,
5
) groups of ellagitannins originated
from C-C coupling of neighboring galloyl residues of a depside
precursor was corroborated and refined later in several laboratories, with
particular emphasis on the role of PGG (
3
) as a general precursor of
these compounds (Haslam, 1982, Hatano
et al.
, 1986).
Numerous attempts to unravel the mechanism of such an oxidative
C-C coupling have been carried out for decades, either using chemical
oxidants (O
2
, Fe
3+
) or by relying on
in vitro
studies with fungal or plant
enzymes (phenolase, laccase, peroxidase) that utilize O
2
or H
2
O
2
as
substrates. Free ellagic acid (
6
), the spontaneously formed dilactone of
HHDP units (
5
), was occasionally obtained in experiments with gallic
acid (
1
), methyl gallate, β-glucogallin (
2
), 3,6-digalloylglucopyranose or
pentagalloylglucopyranose (
3
). A true ellagitannin, however,
characterized by a glucose-bound HHDP group (
5
), was never found (see
references in Gross, 1999). Also in my laboratory, only negative results
were encountered in intensive efforts on this question, employing soluble
and microsomal enzyme preparations from various plants and testing
many oxidants and oxidoreductase cofactors. It was finally concluded
that inadequate analytical techniques presented a major problem, being
caused by a combination of extremely low enzyme reaction rates and the
possible formation of numerous similar products and by-products that
could hardly be discriminated even with the best available HPLC
techniques. Another concern was the possible contamination of samples
with
in vivo
formed ellagitannins.
The necessity of a radically altered approach to these questions was
evident. In this connection, it was our idea to reduce the number of
possible reaction products to be identified to a minimum, preferentially
to only one variable parameter. As summarized in Fig. 3.8, such a
