Research Article

1808
H 3CO
H 3 CO
H
HO
OH
HO O
OH
IV
8
5
8
5
O
O
5
HO
4
OH
OH
OH
8
O
O
O
OCH 3
4
OCH 3
OCH 3
OCH 3
I
OH
O
O
www.soci.org D Dobberstein, M Bunzel
OH
OH
V
O
HO
OH
8
O
O
OCH 3
5
HO
OCH 3
H
O
H 3 CO
OCH 3
5
OH
HO O OH
OH
OH
Figure 5. Structures of the isolated and identified trimers 8-O-4/8-5-dehydrotriferulic acid (I), 8-8(aryltetralin)/8-O-4-dehydrotriferulic acid (II), 8-O-4/8-
O-4-dehydrotriferulic acid (III), and 5-5/8-O-4-dehydrotriferulic acid (IV). Compound V, 8-5/5-5-dehydrotriferulic acid, was only tentatively identified in
corn stover.
5-dehydrotriferulic acid. 11 However, due to solubility issues and
low sample amounts the final characterization by NMR was not
successful leaving this assignment tentative.
5-5/8-O-4-Dehydrotriferulic acid (Fig. 5, IV) was successfully
isolated from fraction S tri 13. UV spectrum, mass spectrum, proton
and carbon NMR data of the isolated compound perfectly match
those of 5-5/8-O-4-dehydrotriferulic acid. 14 Four doublets with
16 Hz coupling constants in the proton spectrum indicate two
intact trans-cinnamate side chains, a singlet at 7.40 ppm is
indicative for one 8-coupled side chain, and the fact that only one
doublet-of-doublets is present shows that two guaiacyl units are
linked in 5-position (demonstrated by the missing 8 Hz coupling
for the protons in the 6 position).
To date, seven dehydrotriferulates were isolated. 36 As corn
bran is (1) an excellent source for ferulates in general, 24,37 (2) only
slightly lignified, 38 and (3) obtained from the seeds which are
protected from light by the husk suppressing the formation
of cyclobutane dimers, it was chosen as ideal starting material
searching for higher radically formed ferulate oligomers. As
recently reviewed, 36 the 5-5/8-O-4-trimer was also identified
by HPLC-UV and comparison with a standard compound in
other cereal grains (wheat bran, 39 rye bran 40,41 and wild rice 41 ).
However, the identification of ferulate trimers from the vegetative
plant organs of grasses has not been performed yet. As this
material shows such a complex composition of hydroxycinnamate
derivatives, simple identification of these trimers by HPLC-UV or
HPLC coupled to a single-quadrupole MS could not be readily
achieved. By performing the more laborious but unequivocal
4
OCH 3
5
8
8
4
III
OCH 3
HO
O
H 3CO
O
8
OCH 3
O
4
OH
O
O
II
OH
OH
8
8
O
O
OH
OH
OCH 3
method of isolating these compounds throughout a series
of chromatographic steps, four of the seven known trimers
were isolated, and a fifth trimer was tentatively assigned. This
demonstrates that the formation of ferulate trimers is not limited
to the reproductive organs of the grasses but also contributes
to the formation of networks in the cell walls of vegetative
organs. Quantitative aspects are hard to judge from these
studies. The isolation or synthesis of higher milligram quantities
of ferulate trimers and higher oligomers and the development
and full validation of an HPLC/UPLC–triple-quadrupole MS or
HPLC/UPLC-quadrupole-ion trap MS methodology is required to
gain quantitative information about these compounds in different
plant tissues.
CONCLUSION
By isolation of ferulate dehydrotrimers, we demonstrated that
higher ferulates potentially contribute to the formation of
networks in the cell walls of corn stover. Although only four
trimers were unambiguously identified it can be assumed that
more trimers and even higher oligomers contribute to the
structure of the cell walls of corn stover. The complexity of the
alkaline hydrolyzate, however, prevented us from isolating more
compounds in sufficient purity to unambiguously identify their
structures. As quantification of ferulate trimers is yet not possible
the significance of these compounds for the formation of cell
wall cross-links in corn stover and for the reduction of forage
digestibility cannot be judged at this time. In addition to the
www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1802–1810