Determination of free diferulic, disinapic and dicoumaric acids in plants and foods

Article abstract of DOI:10.1016/j.foodchem.2014.08.131

Hydroxycinnamates are common phenolic compounds of plants and plant foods, often found in substantial quantities. Due to their high in vitro antioxidant activity they can easily be oxidized under oxidative conditions. In this study, we found that in vitro

Full text of DOI:10.1016/j.foodchem.2014.08.131

Food Chemistry 171 (2015) 280–286
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Determination of free diferulic, disinapic and dicoumaric acids
in plants and foods
a,b,
a,b
b
b
a,b
⇑
ˇ
ˇ
ˇ
Jirí Grúz
, Jirí Pospíšil , Hana Kozubíková , Tomáš Pospíšil , Karel Dolezal
,
Mirko Bunzel c, Miroslav Strnad a,b
a Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacky University,
Šlechtitelu˚ 11, 783 71 Olomouc, Czech Republic
b Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University and Institute of
Experimental Botany, Academy of Sciences of Czech Republic, Šlechtitelu˚ 11, 783 71 Olomouc-Holice, Czech Republic
c Department of Food Chemistry and Phytochemistry, Karlsruhe Institute of Technology (KIT), Adenauerring 20A, 76131 Karlsruhe, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2014
Received in revised form 25 August 2014
Accepted 30 August 2014
Available online 8 September 2014
Hydroxycinnamates are common phenolic compounds of plants and plant foods, often found in substan-
tial quantities. Due to their high in vitro antioxidant activity they can easily be oxidized under oxidative
conditions. In this study, we found that in vitro oxidation of coumaric, ferulic and sinapic acids resulted
mainly in dimeric compounds. We hypothesized that these dimers are present in plants and plant foods
not only in their bound form but also as free acids that can be extracted from non-hydrolyzed samples. By
applying sensitive UHPLC–MS/MS method, we were able to identify and quantify four free hydroxycin-
namic acid dimers for the first time, namely 8-80-disinapic, 8-50-diferulic, 8-O-40-diferulic and
8-30-dicoumaric acids, in wheat sprouts, Chinese cabbage, millet sprouts, light beer and parsley. Concen-
Keywords:
Hydroxycinnamic and dicinnamic acids
Phenylpropanoids
Oxidative coupling
Food
trations of dicinnamates in plant tissues ranged from 0.05 to 2.8
ratio ranged from 2 to 850.
l
g gꢀ1 DW and the monomer:dimer
Ó 2014 Elsevier Ltd. All rights reserved.
Plant
Liquid chromatography
Mass spectrometry
1. Introduction
in the formation of radical cations or quinones that are subse-
quently coupled with other molecules. The products have been
suggested to be insoluble polymers (e.g., lignin) or soluble low
molecular weight compounds, such as oligomeric proanthocyani-
dins, lignans and some dimeric alkaloids (Costa et al., 2008). The
in vitro oxidation and subsequent oligomerization of other highly
abundant phenolics, such as quercetin, resveratrol, hydroxycin-
namic and hydroxybenzoic acids, have been studied several times,
but the oxidation products have not yet been unambiguously
identified in plants or have only been identified after release from
their insoluble form (Bunzel, 2010; Chervyakovsky et al., 2008;
Cichewicz, Kouzi, & Hamann, 2000; Liu, Wan, Huang, & Kong,
2007; Mouterde, Flourat, Cannet, Ducrot, & Allais, 2013; Rouau
et al., 2003).
Hydroxycinnamic acids, the products of PAL enzyme, have been
demonstrated to protect plant cell against free radicals and oxida-
tive damage (Tamagnone et al., 1998; Yamasaki, Sakihama, &
Ikehara, 1997). During the elimination of oxidants, hydroxycin-
namic acids as electron donors are oxidized and the majority is
irreversibly modified although they can partially be regenerated
Undesirable oxidation and browning of plant food diminishes
its quality and could also affect its safety (Friedman, 1996). The
oxidation of phenolic compounds is poorly understood at the
molecular level and the effects of oxidized phenolic compounds
on human health are unknown. Hydroxycinnamic acids, the early
metabolites of the phenylpropanoid pathway, are present in sub-
stantial quantities in all land plants (Emiliani, Fondi, Fani, &
Gribaldo, 2009). Their high in vitro antioxidant activity suggests
that they are easily oxidized under oxidative conditions. Besides
direct oxidation by oxidants and oxidizing radicals, phenolics are
commonly oxidized by a few enzymes, including polyphenol oxi-
dase (PPO), peroxidase and laccase (Weng & Chapple, 2010;
Yoruk & Marshall, 2003). Whereas PPO oxidizes phenols by adding
molecular oxygen to their molecule, class III peroxidases and lac-
cases mediate the one electron oxidation of phenolics, resulting
⇑
Corresponding author at: Laboratory of Growth Regulators, Palacky´ University
& Institute of Experimental Botany ASCR, Šlechtitelu˚ 11, CZ-783 71 Olomouc, Czech
Republic. Tel.: +420 585632173.
via the ascorbate and glutathione cycles (Sgherri, Cosi,
&
Navari-Izzo, 2003). In general, hydroxycinnamic acids are found
E-mail address: jiri.gruz@upol.cz (J. Grúz).
http://dx.doi.org/10.1016/j.foodchem.2014.08.131
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

J. Grúz et al. / Food Chemistry 171 (2015) 280–286
281
both attached to cell wall and as soluble compounds in food
plants. However their oligomers have only been determined after
saponification, acid hydrolysis or cinnamoyl esterase treatment
(Bunzel, 2010; Faulds, Sancho, & Bartolome, 2002). In the present
study, we report that diferulic, disinapic and dicoumaric acids
are low molecular weight oxidation products present in common
plants and foods as free compounds.
was placed in close proximity to a tungsten light and irradiated for
3 days. 1H NMR spectra indicated >98% conversion of (E)-17c to
(Z)-17c. 1H NMR (500 MHz, acetone-d6) d = 3.83 (s, 6H), 3.86 (s,
6H), 4.11 (dd, J = 1.9, 5.0 Hz, 1H), 5.75 (d, J = 5.1 Hz, 1H), 6.77 (s,
2H), 7.19 (d, J = 2.0 Hz, 1H), 7.43 (broad s, 1H), 7.61 (s, 2H), 7.96
(broad s, 1H), 11.2–13.5 (broad s, 1H). To synthesize diacid 14c, a
solution of dilactone 16c (11 mg, 0.02 mmol, 1.0 equiv) in DMSO
(1 mL) was added to 0.1 M phosphate buffer (9 mL, pH 7.4) and
the resulting mixture was stirred at RT for 3 days. The water and
remaining DMSO were removed by sublimation under high vac-
uum and the residue was suspended in EtOAc (50 mL). The organic
layer was dried over Na2SO4, filtered, and the solvents were
removed under reduced pressure, yielding the diacid 14c (46%).
1H NMR (500 MHz, acetone-d6) d [ppm] = 3.76 (s, 12H), 7.01 (s,
4H), 7.63 (broad s, 2H), 7.85 (s, 2H), 11.5–13.3 (broad s, 2H); 13C
NMR (125 MHz, acetone-d6) d [ppm] = 56.5, 108.8, 126.4, 126.6,
138.8, 142.6, 148.5, 168.6. To synthesize thomasidioic acid 15c, a
solution of sinapic acid 1c (500 mg, 2.23 mmol) in water
(110 mL, 0.02 M) was gently stirred in an open flask at RT. The
pH of the solution was adjusted to 7.5–8.0 using 0.5 M aq. NaOH.
Air was bubbled through the solution at RT and the resulting mix-
ture was stirred in the dark for 72 h. The whole mixture was then
extracted with CH2Cl2 (3 ꢁ 150 mL). Combined organic layers were
washed with brine (50 mL), dried over Na2SO4, filtered and the sol-
vents removed under reduced pressure to yield 378 mg (76%) of
2. Materials and methods
2.1. Chemical synthesis
Sinapic, ferulic and 4-coumaric acids were purchased from
Sigma–Aldrich Fine Chemicals (St. Louis, MO, USA). 8-O-40-dehy-
drodiferulic acid 12b was isolated earlier (Bunzel, Funk,
&
Steinhart, 2004). 8,50-dehydrodiferulic 10b and 8,30-dehydrodi-p-
coumaric 10a acids were prepared according to published proto-
cols (Ralph, Quideau, Grabber,
& Hatfield, 1994; Torres &
Rosazza, 2001). Proton and carbon chemical shifts (s) are reported
in ppm downfield from internal reference of residual (CHD2)-
CO(CD3) peak in (CD3)2CO (for 1H-NMR; calibrated to 2.05 ppm)
and the carbonyl peak in (CD3)2CO (for 13C-NMR; calibrated to
206.26 ppm). To synthesize bis-lactone 16c, a solution of sinapic
acid 1c (1.0 g, 4.46 mmol, 1.0 equiv) in EtOH (5 mL) was added to
a solution of dry FeCl3 (1.59 g, 9.81 mmol, 2.2 equiv) in EtOH
(12 mL) at RT under vigorous stirring. The reaction was stirred
for 2 h, followed by removal of the organic solvents under reduced
pressure. The residue was suspended in water (20 mL) and
extracted with EtOAc (3 ꢁ 50 mL). Combined organic layers were
washed with brine, dried over Na2SO4, filtered, and the solvents
were removed under reduced pressure. The residue was purified
by flash column chromatography on silica gel (CH2Cl2:
MeOH = 50:1–>20:1), yielding 665 mg (67%) of slightly yellow
thomasidioic acid 15c. 1H NMR (500 MHz, acetone-d6)
d
[ppm] = 3.40 (d, J = 1.5 Hz, 1H), 3.61 (s, 3H), 3.69 (s, 6H), 3.89 (s,
1H), 5.05 (broad s, 1H), 6.35 (s, 2H), 6.91 (s, 1H), 7.68 (s, 1H),
8.01 (broad s, 1H), 8.07 (broad s, 1H), 11.5–13.3 (broad s, 2H);
13C NMR (125 MHz, acetone-d6) d [ppm] = 40.3, 47.3, 56.55,
56.62, 60.4, 106.2, 108.8, 124.07, 124.11, 125.0, 134.7, 1357,
138.3, 142.6, 146.4, 148.45, 148.51, 168.6, 173.4. Spectral data of
the isolated acid 15c were in agreement with those previously
reported (Ahmed, Lehrer, & Stevenso, 1973).
crystals. Mp = 232–234 °C; 1H NMR (500 MHz, acetone-d6)
[ppm] = 3.79 (s, 12H), 4.07 (d, J = 0.5 Hz, 2H), 5.71 (s, 2H), 6.69 (s,
4H), 7.47 (broad s, 2H); 13C NMR (125 MHz, acetone-d6)
d
2.2. Oxidation by HRP
d
[ppm] = 176.7, 149.7, 138.2, 130.5, 104.9, 84.0, 57.4, 49.7. To syn-
thesize lactone (E)-17c, a solution of dilactone 16c (150 mg,
0.33 mmol, 1.0 equiv) in THF (1 mL) was cooled to 0 °C and 1.0 M
aq. sol. of NaOH (10 mL) was added. The reaction mixture was stir-
red at 0 °C for 15 min and then acidified to pH 4 (2.0 M aq. HCl).
The mixture was extracted with EtOAc (3 ꢁ 25 mL) and the result-
ing organic layers were combined, dried over Na2SO4, filtered and
the solvents removed under reduced pressure. The residue was
purified by flash column chromatography on silica gel (CH2Cl2:
MeOH = 20:1–>10:1), yielding 69 mg (46%) of yellow crystals.
Mp = decomp; 1H NMR (500 MHz, acetone-d6) d [ppm] = 3.75 (s,
6H), 3.81 (s, 6H), 4.28 (m, 1H), 5.69 (d, J = 2.4 Hz, 1H), 6.63 (s,
2H), 7.02 (s, 2H), 7.33 (broad s, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.86
(broad s, 1H), 11.2–13.5 (broad s, 1H); 13C NMR (75 MHz, CDCl3)
d [ppm] = 174.5, 171.4, 147.4, 147.2, 141.9, 137.7, 135.2, 130.2,
124.4, 118.1, 107.7, 101.9, 80.5, 56.4, 56.3, 53.5. To synthesize lac-
Sinapic, ferulic and 4-coumaric acids (1 mM) were oxidized in
35% acetone (in distilled water) by adding hydrogen peroxide (final
concentration of 0.5%) and horseradish peroxidases Type II and IV
(5–250 l
g mLꢀ1; Sigma–Aldrich Fine Chemicals, St. Louis, MO,
USA). The reaction was monitored by UPLC–ESI-MS (full scan
mode) at various time points (5, 15, 30, 60 and 240 min) after
diluting 5 lL of the reaction mixture in 145 lL 10% MeOH.
2.3. Plant and food material
Wheat (Triticum aestivum) and millet (Panicum miliaceum)
sprouts were grown in vermiculite at room temperature and
shoots were collected after 4 days. Parsley, Chinese cabbage, rad-
ish, cranberries, rice, rice bread, carrot, light beer (lager) and white
wine were purchased from a grocery store (Olomouc, Czech
Republic).
tone (Z)-17c, a solution of (E)-17c (ꢂ1.5 mg) in acetone-d6 (550
lL)
Table 1
Free dicinnamic acids quantified in plant-derived samples. Concentrations are given as mean SD (n = 3).
Compound
MRM
Plant material
Concentration (
l
g gꢀ1 DW)
Monomer/dimer ratio
8-80-Disinapic acid (14c)
401.0 > 357.0
Wheat sprouts
Chinese cabbage
Millet sprouts
Light beer
2.30 0.10
1.94 0.21
0.05 0.01
2.78 0.26a
0.15 0.01
2.1
9.9
80.0
852.6
133.0
8-50-Diferulic acid (10b)
8-O-40-diferulic acid (12b)
8-30-Dicoumaric acid (10a)
385.1 > 341.2
385.1 > 193.1
325.1 > 281.2
Parsley
a
ng mLꢀ1
.

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J. Grúz et al. / Food Chemistry 171 (2015) 280–286
Fig. 1. Overview of possible products formed by single-electron oxidation of hydroxycinnamic acids (1a-c). The arrangement of atoms in optically active molecules is shown
in a relative configuration.
2.4. Sample preparation
standards. Test tubes containing finely ground plant material were
sonicated for 15 min, vortexed for 30 min and then subjected
to centrifugation for 10 min at 20,000g and 7 °C (Avanti 30,
Beckman, Krefeld, Germany). The supernatant was directly
loaded onto an SPE cartridge (Spe-ed C18, 1 g/6 mL, Applied
Separations), which was dried with nitrogen and eluted with
100% methanol. The eluent was removed using a vacuum rotary
500 mg of lyophilized samples (with the exception of the beer
and wine samples, which were directly used for SPE after the
addition of the internal standards) were homogenized in 5 mL
aqueous solution of the internal standards with or without
sodium azide (0.1 mM) using an oscillation ball mill (MM 301,
Retsch, Haan, Germany) in 5 mL aqueous solution of internal
standards with or without sodium azide (0.1 mM). Deuterated
salicylic (3,4,5,6-D4) and 4-hydroxybenzoic (2,3,5,6-D4) acids
evaporator, re-dissolved in 10% MeOH, filtered through a 0.45 lm
Nylon membrane microfilter (Alltech, Breda, Netherlands) and
analyzed by UPLC–MS/MS. All samples were analyzed in
triplicate.
(0.3 lM in the extraction solvent) were used as internal

J. Grúz et al. / Food Chemistry 171 (2015) 280–286
283
2.5. UPLC–MS/MS
The UPLC–MS/MS instrumentation and conditions were similar
to those described earlier (Gruz, Novak, & Strnad, 2008). Briefly,
analyses were performed using an ACQUITY Ultra Performance
LC™ system (Waters, Milford, MA, USA) coupled to both a PDA
2996 photo diode array detector (Waters, Milford, MA, USA) and
a Micromass Quattro micro™ API benchtop triple quadrupole mass
spectrometer (Waters MS Technologies, Manchester, UK) equipped
with an electrospray ionization (ESI) source operating in negative
mode. Samples were injected onto a reversed phase column (BEH
C8, 1.7
lm, 2.1 ꢁ 150 mm, Waters, Milford, MA) maintained at
30 °C and eluted by acetonitrile-based mobile phase. The effluent
was introduced into a PDA detector (scanning range 210–600 nm,
resolution 1.2 nm) and subsequently into an electrospray source
(source block temperature 100 °C, desolvation temperature
350 °C, capillary voltage 2.5 kV, cone voltage 25 V). Argon was used
as collision gas (collision energy 16 eV) and nitrogen as desolvation
gas (500 L hꢀ1). MRM transitions used for quantification of dimers
Fig. 2. Time course of the most abundant products (disinapates 14c and 16c)
detected in reaction mixture of sinapic acid, hydrogen peroxide and HRP.
Normalization of each curve was performed by dividing data points by their mean.
a
Fig. 3. Product ion spectra of selected dicinnamic acids detected in reaction mixtures with H2O2 and horseradish peroxidase (ions given in bold were used in MRM mode).

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J. Grúz et al. / Food Chemistry 171 (2015) 280–286
are listed in Table 1. The quantification of both monomers and
dimers was based on deuterium labeled internal standards of 4-
hydroxybenzoic and salicylic acids to compensate for losses during
the extraction procedure and reduce matrix effects that can sup-
press the ionization efficiency. Analyte concentrations were calcu-
lated by using the ratio of analyte:internal standard.
3.2. Chemical synthesis and stability of dimers
Stereo- and regio-isomers of disinapic acid, compounds 14–17c,
were synthesized in mg amounts from bislactone dimer 16c, which
was the primary product of chemical oxidation of sinapic acid 1c.
Depending on the pH of the reaction media and reaction time,
the bislactone 16c was transformed in the absence of oxygen into
diacids 14c and 17c (Fig. 4). It is interesting to note that we
observed the formation of 2–5 different dimers during in vitro oxi-
dation, but typically only one isomer was detected in plants. This
might be caused by both enzymatic and chemical factors. Plant
enzymes are usually more specific than HRP and can work together
with other stereospecific proteins, such as dirigent proteins, to
form specific stereoisomers (Davin & Lewis, 2000). The presence
of only one isoform can also be explained by chemical means.
We found that the ratio of the different 8,80-coupled disinapic acid
dimers (diacid 14c, monolactone 17c and dilactone 16c) strongly
depended on the pH of the aqueous media, as well as on the pres-
ence/absence of oxygen. Since phenolic acids are mostly located in
the vacuole and apolast, compartments with generally low pH,
dimer formation and stability may be affected by this factor. The
experiments with bislactone 16c demonstrated that under acidic
pH conditions, the stability of the open form, diacid 14c, was mark-
edly improved. Accordingly, 14c was the only detected disinapate
in plant samples. These findings suggested that pH adjustments
or usage of buffers during the extraction procedures can accelerate
the transformation of natural isoforms of phenolic dimers.
3. Results and discussion
3.1. Oxidation of phenolic acids by HRP
Hydroxycinnamic acids 1a-c (Fig. 1) were oxidized in vitro by
HRP, and the reactions were monitored at several time points by
UPLC-PDA–MS operating in the full scan mode. The main products
under the described conditions were dimers with molecular
weights of (2 ꢁ MWcinnamate ꢀ 2H). Typically, not only one dimeric
compound but various stereo and regioisomers were formed.
As minor products, several other oxidation products, such as
oxodimers (2 ꢁ MWcinnamte ꢀ 2H + O) and dehydrodimers (2 ꢁ
MWcinnamate ꢀ 4H), were also detected. Although the cinnamic
acids reacted quickly with hydrogen peroxide in the presence of
HRP, they were stable for several hours when only hydrogen
peroxide was added. An unexpected time course was observed for
the reaction of sinapic acid, i.e., its re-appearance in the reaction
mixture after 60 min (Fig. 2). By adding synthesized disinapates
to the reaction mixture, we found that this increase was not due
to their breakdown but more likely due to the transformation of
other products undetectable by our system (e.g., hydrolysable
oligomers or stable radicals). This phenomenon was not observed
at lower concentrations of hydrogen peroxide (<0.1%).
To search for oxidation products in food sources, we developed
a sensitive MRM-based UPLC–MS/MS method based on analyzing
collision induced fragmentation spectra of major peaks (ca 25 com-
pounds) present in the reaction mixtures of HRP induced dimeriza-
tion of hydroxycinnamic acids (Fig. 3). Pseudo-molecular ions
[MꢀH]ꢀ were used as parent ions, with the exception of disinapic
acid 14c, which exhibited an abundant [MꢀHꢀCOO]ꢀ ion with m/z
401 due to in-source fragmentation.
3.3. Determination of oxidation products in foods
The initial screening of food products, including parsley, wheat
sprouts, millet sprouts, radish, cranberries, rice, rice bread, carrot,
Chinese cabbage, beer and wine, was performed on extracts used
later for SPE. As a result, a few tentative identifications were made
based on the MS fragmentation, UV–VIS spectra and retention
times of the products identified in the in vitro reaction mixture
with HRP. To further improve the UPLC–MS/MS detection, we per-
formed SPE pre-concentration of all samples to remove interfering
compounds and to increase the signal-to-noise ratio. However, we
Fig. 4. Conversion of 8-80-disinapic acid into different stereo- and regio-isomers under specified conditions.

J. Grúz et al. / Food Chemistry 171 (2015) 280–286
285
found that evaporation from aqueous solutions of up to 80%
organic solvent resulted in substantial losses of some dicinna-
mates, especially 8-30-dicoumaric acid 10a. Therefore, we mini-
mized the sample processing by using water extraction, which
allowed direct application onto the SPE column, thus avoiding
problematic evaporation of the primary extract. The retention of
diacids was always checked in parallel experiments. After loading
the primary extract, we dried the SPE cartridge with nitrogen to
remove any remaining water and subsequently eluted analytes
with 100% MeOH. Due to low solubility of diacids in water, we ver-
ified that all of them were quantitatively redissolved in 10% MeOH
methods (e.g., grinding, steaming, roasting), which cause cell
membrane disruption and exposure to oxidative conditions.
Homogenizing plant tissues can therefore result in mixing the for-
merly separated components and subsequent formation of extrac-
tion artifacts. To prevent overestimation of hydroxycinnamic acid
dimers, we freeze-dried all solid samples to remove hydrogen per-
oxide, a substrate of class III peroxidases. We also tested how addi-
tion of sodium azide, an inhibitor of both laccase and peroxidase
activities, affects the concentrations of hydroxycinnamic acid
dimers (Johannes & Majcherczyk, 2000; Tuisel, Grover, Lancaster,
Bumpus, & Aust, 1991). We found only a minor decrease in the
concentration of disinapate (ranging from 20 to 30%) and no signif-
icant decrease in the concentrations of dicoumaric and diferulic
acids extracted with sodium azide, demonstrating that these
dimers are mostly formed in plants before extraction. Besides class
III peroxidases and laccases, phenolics are also oxidized by PPO,
which is commonly associated with food browning. However,
PPO incorporates molecular oxygen into the substrate molecule
and is therefore unlikely to be directly involved in the formation
of the described dicinnamates.
when the final concentration was 60.5 lM (highest concentration
allowed). The pre-concentrated extracts dissolved in 10% MeOH
were stable for at least 24 h at 4 °C and for weeks at ꢀ80 °C. The
signal-to-noise ratio was increased by approximately 6ꢁ times
and was sufficient to clearly identify dimers of sinapic, ferulic
and coumaric acids in wheat sprouts, Chinese cabbage, millet
sprouts, light beer and parsley (Fig. 5). In contrast, we were not
able to detect any dicinnamates in radish, cranberries, rice, rice
bread, carrot and white wine samples.
The oxidation of hydroxycinnamates seems to be mainly medi-
ated by redox enzymes, such as peroxidases, laccases and PPO,
which are compartmentalized in the plant cell (Costa et al., 2008;
Strack, Pieroth, Scharf, & Sharma, 1985). Compartmentalization
can be destroyed during food processing using harsh processing
Mostly unprocessed plant foods, such as wheat sprouts and
parsley, where the production of dicinnamates can mainly be
attributed to enzymatic activity in planta, were investigated in this
study. As one exception, we also analyzed light beer, which is a
highly processed end-product formulated to be stable for months.
Fig. 5. MRM chromatograms of free dicinnamic acids identified in food and plant samples.

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J. Grúz et al. / Food Chemistry 171 (2015) 280–286
Beer was found to contain 8-O-40-diferulic acid 12b, possibly orig-
inating from either the barley sprouts or formed during processing,
such as mashing and wort boiling, where dicinnamates can be
formed by enzymatic oxidation, chemical oxidation and/or may
be released from their insoluble form (cell wall bound). Whereas
8-O-40-diferulic acid 12b was present in beer, 8,50-diferulic 10b
was found in millet sprouts, suggesting different mechanisms (or
enzyme specificity) by which these dimers are produced. It is also
unlikely that the different structural forms were a consequence of
the extraction procedure, providing further evidence of the extrac-
tion procedure validity. The concentrations of 12b and 10b were
more than 1000ꢁ times lower than the reported concentrations
of bound diferulates released after saponification of barley
References
Ahmed, R., Lehrer, M.,
& Stevenso, R. (1973). Synthesis of thomasic acid.
Tetrahedron, 29, 3753–3759.
Allerdings, E., Ralph, J., Schatz, P. F., Gniechwitz, D., Steinhart, H., & Bunzel, M.
(2005). Isolation and structural identification of diarabinosyl 8-O-4-
dehydrodiferulate from maize bran insoluble fibre. Phytochemistry, 66,
113–124.
Bunzel, M. (2010). Chemistry and occurrence of hydroxycinnamate oligomers.
Phytochemistry Reviews, 9, 47–64.
Bunzel, M., Allerdings, E., Ralph, J.,
& Steinhart, H. (2008a). Cross-linking of
arabinoxylans via 8-8-coupled diferulates as demonstrated by isolation and
identification of diarabinosyl 8-8 (cyclic)-dehydrodiferulate from maize bran.
Journal of Cereal Science, 47, 29–40.
Bunzel, M., Funk, C.,
& Steinhart, H. (2004). Semipreparative isolation of
dehydrodiferulic and dehydrotriferulic acids as standard substances from
maize bran. Journal of Separation Science, 27, 1080–1086.
Bunzel, M., Heuermann, B., Kim, H., & Ralph, J. (2008b). Peroxidase-catalyzed
oligomerization of ferulic acid esters. Journal of Agricultural and Food Chemistry,
56, 10368–10375.
Bunzel, M., Ralph, J., Marita, J. M., Hatfield, R. D., & Steinhart, H. (2001). Diferulates
as structural components in soluble and insoluble cereal dietary fibre. Journal of
the Science of Food and Agriculture, 81, 653–660.
Chervyakovsky, E. M., Bolibrukh, D. A., Kurovskii, D. L., Gilep, A. A., Vlasova, T. M.,
Kurchenko, V. P., et al. (2008). Oligomeric oxidation products of the flavonoid
quercetin. Chemistry of Natural Compounds, 44, 427–431.
Cichewicz, R. H., Kouzi, S. A., & Hamann, M. T. (2000). Dimerization of resveratrol by
the grapevine pathogen Botrytis cinerea. Journal of Natural Products, 63, 29–33.
Costa, M. M. R., Hilliou, F., Duarte, P., Pereira, L. G., Almeida, I., Leech, M., et al.
(2008). Molecular cloning and characterization of a vacuolar class III peroxidase
involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant
Physiology, 146, 403–417.
Davin, L. B., & Lewis, N. G. (2000). Dirigent proteins and dirigent sites explain the
mystery of specificity of radical precursor coupling in lignan and lignin
biosynthesis. Plant Physiology, 123, 453–461.
) ) samples (Bunzel, Ralph,
(0.7 mg gꢀ1 and millet (2.6 mg gꢀ1
Marita, Hatfield, & Steinhart, 2001). The most abundant free dicin-
namic acids in the studied samples was 8-80-disinapic acid, which
was found in both wheat sprouts and Chinese cabbage. The chro-
matogram of an extract from Chinese cabbage also showed another
peak with the same transition (Fig. 5), but its retention time of
9.40 min did not match with any of the studied disinapate isoforms
(14c, 15c, 16c or 17c). This might be explained by in-source frag-
mentation of unknown disinapate derivatives, such as glucosides
or esters, resulting in pseudo MS3 spectra. Unfortunately, we were
not able to gather clean full/daughter scan MS spectra due to the
low intensity of this unknown compound. Although the concentra-
tion of free disinapic acid was relatively high, it was lower than
that of bound disinapates released after saponification from rice
l
g gꢀ1 (Qiu, Liu, & Beta,
Emiliani, G., Fondi, M., Fani, R., & Gribaldo, S. (2009). A horizontal gene transfer at
the origin of phenylpropanoid metabolism: A key adaptation of plants to land.
Biology Direct, 4, 12.
samples and ranging between 5 and 19
2010).
In general, all dimers were found in lower quantities than their
corresponding monomers (between 2 and 850 times). The high
ratio of monomer/dimer may be due to several reasons. One is
the incorporation of dimers into the cell wall, which contains high
quantities of dehydrodiferulic acids attached to arabinoxylans
(Allerdings et al., 2005; Bunzel, Allerdings, Ralph, & Steinhart,
2008). In addition, we found that dimers were chemically less sta-
ble than simple cinnamates, and thus were degraded faster. It was
also demonstrated that dehydrodihydroxycinnamates can be fur-
ther oxidized to trimers and higher oligomers that are difficult to
detect by conventional LC methods, and thus would also remain
undetected by our method (Bunzel, Heuermann, Kim, & Ralph,
2008; Mouterde et al., 2013; Rouau et al., 2003).
Faulds, C. B., Sancho, A. I., & Bartolome, B. (2002). Mono- and dimeric ferulic acid
release from brewer’s spent grain by fungal feruloyl esterases. Applied
Microbiology and Biotechnology, 60, 489–493.
Friedman, M. (1996). Food browning and its prevention: An overview. Journal of
Agricultural and Food Chemistry, 44, 631–653.
Gruz, J., Novak, O., & Strnad, M. (2008). Rapid analysis of phenolic acids in beverages
by UPLC–MS/MS. Food Chemistry, 111, 789–794.
Johannes, C., & Majcherczyk, A. (2000). Laccase activity tests and laccase inhibitors.
Journal of Biotechnology, 78, 193–199.
Liu, H. L., Wan, X., Huang, X. F., & Kong, L. Y. (2007). Biotransformation of sinapic
acid catalyzed by Momordica charantia peroxidase. Journal of Agricultural and
Food Chemistry, 55, 1003–1008.
Mouterde, L. M. M., Flourat, A. L., Cannet, M. M. M., Ducrot, P. H., & Allais, F. (2013).
Chemoenzymatic total synthesis of
a
naturally occurring (5-50)/
(80-O-400) dehydrotrimer of ferulic acid. European Journal of Organic Chemistry,
173–179.
Qiu, Y., Liu, Q., & Beta, T. (2010). Antioxidant properties of commercial wild rice and
analysis of soluble and insoluble phenolic acids. Food Chemistry, 121, 140–147.
Ralph, J., Quideau, S., Grabber, J. H., & Hatfield, R. D. (1994). Identification and
synthesis of new ferulic acid dehyrodimers present in grass cell-walls. Journal of
the Chemical Society-Perkin Transactions, 1, 3485–3498.
4. Conclusion
In the present study, we demonstrated that hydroxycinnamic
acid dimers are present as free and soluble compounds in common
foods and plants. The dimers are products of the oxidation of
hydroxycinnamic acids which are mostly considered as health-
promoting compounds with antioxidant properties. It is however
unclear whether they remain beneficial or become detrimental
after fulfilling their antioxidative role, which results in their oxida-
tion and dimer formation. As oxidation products are known to be
present in common foods, further work is needed to study their
biological activities.
Rouau, X., Cheynier, V., Surget, A., Gloux, D., Barron, C., Meudec, E., et al. (2003). A
dehydrotrimer of ferulic acid from maize bran. Phytochemistry, 63, 899–903.
Sgherri, C., Cosi, E., & Navari-Izzo, F. (2003). Phenols and antioxidative status of
Raphanus sativus grown in copper excess. Physiologia Plantarum, 118, 21–28.
Strack, D., Pieroth, M., Scharf, H.,
& Sharma, V. (1985). Tissue distribution of
phenylpropanoid metabolism in cotyledons of Raphanus sativus L. Planta, 164,
507–511.
Tamagnone, L., Merida, A., Stacey, N., Plaskitt, K., Parr, A., Chang, C. F., et al. (1998).
Inhibition of phenolic acid metabolism results in precocious cell death and
altered cell morphology in leaves of transgenic tobacco plants. Plant Cell, 10,
1801–1816.
Torres, J., & Rosazza, J. P. N. (2001). Microbial transformations of p-coumaric acid by
Bacillus megaterium and Curvularia lunata. Journal of Natural Products, 64,
1408–1414.
Tuisel, H., Grover, T. A., Lancaster, J. R., Bumpus, J. A., & Aust, S. D. (1991). Inhibition
of lignin peroxidase-H2 by sodium azide. Archives of Biochemistry and Biophysics,
288, 456–462.
Weng, J. K., & Chapple, C. (2010). The origin and evolution of lignin biosynthesis.
New Phytologist, 187, 273–285.
Yamasaki, H., Sakihama, Y., & Ikehara, N. (1997). Flavonoid-peroxidase reaction as a
detoxification mechanism of plant cells against H2O2. Plant Physiology, 115,
1405–1412.
Yoruk, R., & Marshall, M. R. (2003). Physicochemical properties and function of plant
polyphenol oxidase: A review. Journal of Food Biochemistry, 27, 361–422.
Acknowledgements
We would like to thank Ing. Michaela Šubrtová for excellent
technical assistance and Sees-Editing, U.K. for editing the manu-
script. This research was supported by project GACR 503/12/P166
and a Czech Ministry of Education grant from the National Program
for Sustainability I (LO1204). J.G. and K.D. also acknowledge the
support of the Operational Program Education for Competitiveness
– European Social Fund (project CZ.1.07/2.3.00/20.0165).