Plant phenolics affect oxidation of tryptophan

Article abstract of DOI:10.1021/jf800708t

The effect of berry phenolics such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), black currant (Ribes nigrum), and cranberry (Vaccinium oxycoccus) and byproducts of deoiling processes rich in phenolics such as rapese

Full text of DOI:10.1021/jf800708t

7472 J. Agric. Food Chem. 2008, 56, 7472–7481
Plant Phenolics Affect Oxidation of Tryptophan
HANNA SALMINEN* AND MARINA HEINONEN
Department of Applied Chemistry and Microbiology, Division of Food Chemistry,
P.O. Box 27, 00014 University of Helsinki, Finland.
The effect of berry phenolics such as anthocyanins, ellagitannins, and proanthocyanidins from
raspberry (Rubus idaeus), black currant (Ribes nigrum), and cranberry (Vaccinium oxycoccus) and
byproducts of deoiling processes rich in phenolics such as rapeseed (Brassica rapa L.), camelina
(Camelina sativa), and soy (Glycine max L.) as well as scots pine bark (Pinus sylvestris) was
investigated in an H2O2-oxidized tryptophan (Trp) solution. The oxidation of Trp was analyzed with
high-performance liquid chromatography using both fluorescence and diode array detection of Trp
and its oxidation products. Mechanisms of antioxidative action of the phenolic compounds toward
the oxidation of Trp were different as the pattern of Trp oxidation products varied with different phenolic
compounds. The antioxidant protection toward oxidation of Trp was best provided with pine bark
phenolics, black currant anthocyanins, and camelina meal phenolics as well as cranberry
proanthocyanidins.
KEYWORDS: Tryptophan; protein oxidation; HPLC; antioxidants; phenolic compounds; berries
INTRODUCTION
and/or at high temperatures, for example, during broiling/
grilling of meat products, exhibit mutagenic and carcinogenic
activities (5). Enhanced Trp degradation is observed during
pregnancy and in a variety of diseases and disorders, for
example, infectious diseases, autoimmune diseases, and
neuropsychiatric and neurological disorders such as Alzhe-
imer’s and Parkinson’s diseases as well as dementia (6).
The oxidation of proteins and peptides occurs during
processing and storage of food and under physiological
conditions leading to altered physicochemical and functional
properties and may even result in toxic products (1). The
oxidation of proteins contributes to changes in food structure,
decreases in protein solubility, color changes (browning
reactions), and changes in nutritive value (2, 3). The nature
and extent of reactions involved in food processing depend
on the composition of food and food processing conditions,
such as temperature, pH, the presence of oxygen, the
application of chemicals, fermentation, and irradiation (4, 5).
Processing-induced changes leading to denaturation of pro-
teins may improve the digestibility, especially of plant
proteins containing antinutritional compounds, or impair the
digestibility and biological availability by destruction of
essential amino acids, conversion of amino acids into
nonmetabolizable derivatives, and intra- or intermolecular
cross-linking. In addition, the Maillard reaction initiated by
reaction between amines and carbonyl compounds at elevated
temperatures also has a great impact on the organoleptic and
nutritional properties of proteins (4). The essential amino acid
tryptophan (Trp) is exceptional in its diversity of biological
functions such as a precursor for a series of metabolic
reactions. Therefore, its stability in processed foods is a major
concern as the oxidation products of Trp are biologically
active (5). In addition to Trp-derived oxidation products,
kynurenines (Kyn), Trp-derived nitroso compounds, and the
carbolines formed in the presence of carbonyl compounds
In relation to potential health effects, phenolic bioavail-
ability and phenolic-mediated cell effects may involve the
interactions of phenolic compounds with specific biological
targets, mainly proteins, and possible participation of phenolic
compounds in the regulation of gene expression for specific
proteins (7). The interaction of tannins (oligomeric procya-
nidins) with salivary proteins developing sensation of
astringency is currently the sole binding process with clear
in vivo biological significance (8).
The mechanisms of inhibiting oxidation by phenolic com-
pounds, as food components or potential drugs, are related to
their reducing ability (antioxidant properties by electron or
H-atom donation), their ability to interact with proteins, and
the formation of complexes between protein molecules. These
flavonoid-protein or protein-protein complexations may pro-
vide protection to the protein against oxidative degradation. In
addition, flavonoid-protein interactions can modify the redox
properties of flavonoids that underline their antioxidant, and
eventually their pro-oxidant, activity (7).
The molecular interactions responsible for phenolic-protein
complexation can be divided into van der Waals (dispersion)
interactions and electrostatic interactions. Flavonoid oxidation
by autoxidation, radical scavenging, or enzymatic oxidation
leads to the formation of flavonoid oxidation products. Being
both electrophilic and oxidizing, the flavonoid oxidation products
* To whom correspondence should be addressed. Tel: +358 9 191
58285. Fax: +358 9 191 58475. E-mail: hanna.salminen@helsinki.fi.
10.1021/jf800708t CCC: $40.75  2008 American Chemical Society
Published on Web 07/23/2008

Phenolics Affect Oxidation of Tryptophan
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7473
Figure 1. Formation of Trp-derived oxidation products. See panel A for abbreviations.
Camelina (Camelina satiVa) meal was obtained from Raisio Ltd.
(Finland). Soy flour (from soybean Glycine max L.) (Soyolk) was from
Cereform Ltd. (Northampton, England), and soy meal (Risetti) was
from Risetti Ltd. (Finland). Protein, fatty acid, and tocopherol composi-
tions as well as isoflavone and lignan contents of the oilseed materials
have been reported in a previous study by Salminen et al. (14). Scots
pine (Pinus sylVestris L.) bark drink was obtained by extraction with
water so that it contained 30% pine bark and phloem (Ravintorengas
Ltd., Siikainen, Finland). All berries (raspberry, black currant, and
cranberry) were purchased from a market place. Samples were packed
immediately into a vacuum and stored in a freezer at -20 °C until
use.
Caffeic acid, chlorogenic acid, cyanidin-3-glucoside, delphinidin-
3-glucoside, ellagic acid, ferulic acid, gallic acid, daidzein, genistein,
procyanidin B1, rutin, sinapic acid, and taxifolin were from Extrasyn-
the`se (Genay, France). Catechin, quercetin, L-Trp, 3-hydroxy-tryp-
tophan, 5-hydroxy-L-tryptophan, L-Kyn, Tra, and kynurenic acid (KynA)
were obtained from Sigma. Vinylsyringol was synthesized as described
by Rein et al. (16). Oia was synthesized as described by Simat et al.
(17). Catalase (EC 1.11.1.6) from bovine liver (1824 units/mg) was
from Sigma. Sodium tetraborate decahydrate, potassium iodide, and
acetic acid were from Riedel-de Hae¨n (Germany). Trifluoroacetic acid
(TFA) was from Sigma. All solvents were high-performance liquid
chromatography (HPLC) grade and purchased from Rathburn Chemicals
Ltd. (Walkerburn, Scotland). A MilliQ water purification system was
used (Millipore, Bedford, MA). Chemicals used were of analytical
purity.
Isolation of Plant Phenolics. The extractions of camelina meal, soy
meal, and soy flour and the extraction of rapeseed meal were performed
according to methods of Salminen et al. (14) and Vuorela et al. (15)
with some modifications, respectively. Plant material (0.8 g) and 20
mL of 80% methanol (70% ethanol for rapeseed) was put in a centrifuge
tube that was shaken in a water bath (75 °C) for 60 min. The clear
phenolic extract was collected after centrifugation (6000 rpm, 15 min).
Berry samples were freeze-dried prior to analysis and stored at -20
°C until use. Extraction and isolation of berry anthocyanins and
raspberry ellagitannins were carried out as described by Ka¨hko¨nen et
al. (18). The berry anthocyanin fractions were further purified by
preparative HPLC as described by Ka¨hko¨nen et al. (18). A method by
Ma¨a¨tta¨-Riihinen et al. (19) was followed to isolate cranberry proan-
thocyanidins. Berry isolates were freeze-dried and stored at -20 °C.
Total Phenolics and Phenolic Profiles. The amount of total
polyphenols was measured colorimetrically according to the Folin-
Ciocalteau procedure (20). Phenolic extract (0.2 mL) was evaporated
to dryness. After that, 0.2 mL of methanol/water (1:2), 1 mL of
Folin-Ciocalteau reagent (1:10), and 0.8 mL of sodium dicarbonate
(aryloxyl radicals, quinones, and quinonoid compounds) may
react with nucleophilic or oxidizable amino acid residues,
thereby irreversibly modifying the protein by oxidation or
covalent bonds (7).
The oxidative attack on Trp residues occurs first on the
pyrrole ring and second on the phenyl moiety (9). Therefore,
Trp is oxidized (Figure 1) with the formation of primary
oxidation products such as diastereomers A and B of 3a-
hydroxypyrroloindole-2-carboxylic acid (PIC), ꢀ-oxindolylala-
nine (Oia) (10), and dioxindolylalanine (DiOia) (11), respec-
tively, as the pyrrole ring remains uncleaved. The phenyl moiety
of Trp is susceptible to oxidation forming 5-hydroxy-tryptophan
(5-OH-Trp), and the alanyl moiety undergoes changes forming,
for example, tryptamine (Tra) and indole derivatives. Cleavage
of the pyrrole ring of Trp leads to the formation of N-
formylkynurenine (NFK), Kyn, and 3-hydroxykynurenine (3-
OH-Kyn) (9). In mammalian cells, Trp is degraded primarily
by a complex enzymatic cascade known as the Kyn pathway
(12).
To our knowledge, the role of plant phenolics on the
oxidation of proteins and amino acids is not well-established.
In addition, the mechanisms of phenolic-protein interactions
are still incompletely understood in part due to lack of specific
methods for measuring oxidation products of proteins. To
control and prevent oxidation, the oxidation mechanisms and
protein-phenolic interactions must be elucidated. The objec-
tive of this study was to investigate the effects of plant
phenolics on the oxidation of Trp and the pattern of Trp-
derived oxidation products. All plant materials selected were
found antioxidatively active in our previous studies (13–15),
and these materials included byproducts of deoiling processes
rich in phenolics, such as rapeseed, camelina and soy meal, soy
flour, pine bark, and berry phenolics from raspberry, black
currant, and cranberry. The main phenolic compounds found
in the selected plant materials are comprised of anthocyanins,
hydroxycinnamic acids, hydroxybenzoic acids, catechins, pro-
cyanidins, flavonols, and isoflavones (Figure 2).
MATERIALS AND METHODS
Materials. The rapeseed (Brassica rapa L.) meal used was the
residue of a rapeseed deoiling process in which the oil was expelled
from the seeds at an elevated temperature by Mildola Ltd. (Finland).

7474 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Salminen and Heinonen
Figure 2. Structures of phenolic compounds.
Table 1. Phenolic Composition of Berry Isolates (mg/g, Mean ( SD)a
B(OH)3 + H2O2 T (HO)3BOOH- + H+
(1)
black
(HO)3BOOH- + H2O2 T (HOO)2B(OH)2- + H2O
B(OH)3 + H2O2 T (HO)2BOOH + H2O
(2)
(3)
concentration
(mg/g)
raspberry
raspberry
currant
cranberry
anthocyanins ellagitannins anthocyanins proanthocyanidins
anthocyaninsb
ellagitanninsc
534 ( 25
ND
18 ( 0
314 ( 44
ND
ND
554
ND
ND
ND
369 ( 44 ND
It has been observed that at pH ∼4.6 only one major boron-containing
species, B(OH)3, is present in 0.1 M solution with 1.0 M H2O2. In
addition, at lower pH levels (acid or neutral), boric acid has no effect
on the photodegradation of H2O2 (24). In acidic solutions, peroxoborates
have not been detected due to their low concentrations. Therefore, under
the experimental conditions used in this study, the amount of perox-
oborates formed may be negligible. It has also been shown that borate
and boric acid have insignificant rates of reaction with hydroxyl radicals
as compared with hydrogen peroxide and thus cannot act as free radical
scavengers in the decomposition of hydrogen peroxide (25).
proanthocyanidinsd ND
ND
ND
ND
ellagic acidc
flavanolse
ND
ND
ND
16 ( 2
ND
ND
12 ( 1
ND
hydroxybenzoic
acidsf
hydroxycinnamic ND
ND
ND
ND
acids and
derivativesg
flavonolsh
ND
8 ( 1
ND
1
a ND, not detected/concentration under detection limit. b Cyanidin 3-glucoside
as standard. c Ellagic acid as standard. d Procyanidin B1 as standard. e Catechin
as as standard. f Gallic acid as standard. g Chlorogenic acid as standard. h Rutin
as standard.
The concentrations of added phenolic compounds (presented as molar
equivalent concentrations) were based on the total phenolic content in
the cases of soy and pine bark drink and on the total hydroxycinnamic
acid derivatives in the cases of rapeseed and camelina meal. The amount
of raspberry and black currant anthocyanin isolates, raspberry ellagi-
tannin isolate, and cranberry proanthocyanidin isolate was calculated
as cyanidin-3-glucoside, ellagic acid, and procyanidin B1 equivalents,
respectively. All tested berry isolates and other plant materials were
dissolved in methanol, ethanol (rapeseed), or water (pine bark).
Aliquots of plant phenolics were pipetted into 4 mL screw-top vials,
and the solvent was evaporated with nitrogen. In control sample, no
antioxidant was added. Small magnets were placed into vials. The Trp
solution (2 mL) and 40 µL (0.19 M) of H2O2 (30%) were then added
into the vials resulting in final Trp concentration of 317 µg/mL. Every
hour during the incubation, an aliquot of 40 µL of H2O2 was added to
samples to keep the catalyst abundant. Samples were agitated on a
vortex mixer after additions of H2O2, and the mixtures in sealed vials
were stirred during incubation. After incubation, the reaction was
stopped by 30 min of treatment with 100 µL of catalase (about 300
units). The absence of H2O2 was subsequently checked by potassium
iodide test. After acidification with 164 µL of 2 M acetic acid, the
sample was filtered (0.45 µm GPH) prior to HPLC analysis.
solution (7.5%) were added. After 30 min, the total phenolic content
was measured at 765 nm by Perkin-Elmer λ25 UV-vis spectropho-
tometer (Norwalk, CT). Gallic acid was used as a standard compound.
The results are expressed as gallic acid equivalents (GAE), µg/g of
plant material. The HPLC analysis of phenolics was performed
according to the method outlined by Koski et al. (21) for phenolic acids
and their derivatives, and by Ka¨hko¨nen et al. (22) for other phenolic
compounds. Catechin, chlorogenic acid, cyanidin-3-glucoside, ellagic
acid, gallic acid, procyanidin B1, rutin, sinapic acid, and vinylsyringol
were used as standard compounds.
Oxidation of Trp. A 2 mM Trp model solution using 0.1 M borate
(pH 6.3) in the presence of selected extracts of phenolic compounds
(at concentrations of 10, 50, and 100 µM) was oxidized with hydrogen
peroxide (H2O2) added hourly (final concentration of 1.05 M) for 6 h
at 37 °C in dark. The addition of H2O2 (1.05 M) resulted in a final pH
of 4.6. Oxidation was performed by modifying a method outlined by
Simat and Steinhart (9). H2O2 was used as an oxidant since it is an
oxidant in many biological molecules, and it is a member of the reactive
oxygen species (23). Borate/boric acid buffers have been reported to
catalyze hydrogen peroxide oxidation reactions leading to the formation
of peroxoborates, which may accelerate some reactions. The pH of
borate buffer solution decreased as H2O2 was added due to the rapid
equilibria in the formation of monoperoxoborate (eq 1) and diperox-
iborate (eq 2). However, at lower pH, peroxoboric acid was formed
(eq 3) (24).
Analysis of Trp Oxidation Products with HPLC. Trp and its
oxidation products were determined using an analytical HPLC method
combined with diode array and fluorescence detection outlined by Simat
and Steinhart (9) with some modifications. The HPLC system (Waters,
Milford, MA) consisted of 717 plus autosampler, 515 and 510 pumps
with a pump control module, a column oven with a temperature control
module, a PDA 996 diode array detector, and a HP 1046 scanning
fluorescence detector (Hewlett-Packard, Palo Alto, CA). The separation

Phenolics Affect Oxidation of Tryptophan
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7475
Table 2. Phenolic Composition of Plant Extracts (µg/g, Mean ( SD)a
concentration (µg/g)
rapeseed meal
camelina meal
soy meal
soy flour
pine bark drink (µg/mL)
total phenolicsb
6730 ( 290
ND
ND
8030 ( 140
1800 ( 180
140 ( 20
64 ( 7
ND
3940 ( 110
2110 ( 410
ND
3020 ( 160
650 ( 40
30 ( 3
2770 ( 65
760 ( 10
ND
2650 ( 50
790 ( 70
ND
3400 ( 250
80 ( 10
3 ( 0
flavanolsc
hydroxybenzoic acidsb
hydroxycinnamic acids and derivativesd
sinapinee
ND
ND
ND
sinapic acide
vinylsyringolf
ND
2150 ( 70
flavonolsg
ND
ND
ND
a ND, not detected/concentration under detection limit. b Gallic acid as standard. c Catechin as standard. d Chlorogenic acid as standard. e Sinapic acid as standard.
f Vinylsyringol as standard. g Rutin as standard.
Figure 3. Separation by RP-HPLC of Trp-derived oxidation products of H2O2-oxidized Trp (solid line) and with added camelina meal phenolics at 10 µM
(dotted line) after 6 h of oxidation. (a) PDA 260 nm. (b) Fluorescence detection, 0-13 min: Ex, 230 nm; Em, 342 nm; 13-46 min: Ex, 280 nm; Em,
335 nm. The Trp concentration of the control sample (without added phenolics) before oxidation was 317 µg/mL and after oxidation was 158 µg/mL.
Peaks: 1, PIC A; 2, DiOia A; 3, DiOia B; 4, PIC B; 5, Kyn; 6, NFK; 7, Oia; 8, 5-OH-Trp; 9, Tra; and 10, Trp. See panel A for abbreviations.
of oxidation products was carried out on a Nova-Pak C18 column (3.9
mm × 150 mm, 4 µm) equipped with a Spherisorb S5 ODS2 precolumn
(20 mm × 2 mm, 5 µm) (Waters, Milford, MA). The mobile phase
consisted of 0.1% (v/v) TFA in water (solvent A), methanol (solvent
B), and acetonitrile (solvent C). The elution conditions were as follows:
linear gradient from 95% A and 5% B to 86% A and 14% B, 0-10
min; to 46% A, 14% B and 40% C, 10-40 min; to 26% A, 14% B,
and 60% C, 40-42 min; to 95% A and 5% B, 42-46 min. The flow
rate was 1.0 mL/min, and the column oven temperature was 35 °C.
The injection volume was 20 µL. The Trp oxidation products were
confirmed by spectral identification and retention times. The Trp
oxidation product standards were dissolved in 0.1 M borate (pH 6.3)
to a concentration of approximately 0.5 mg/25 mL except for Trp to 6
mg/25 mL.
Trp oxidation products by HPLC was made using Trp, Tra, Kyn, Oia,
and 5-OH-Trp as external standards. The quantifications for NFK, PIC
(A/B), and DiOia (A/B) were calculated as equivalents to 5-OH-Trp
as external standard since these standards were not commercially
available. The results are given as the mean values of triplicate
analyses.
Statistical Analysis. Statistical analysis of multivariance (ANOVA)
was performed using Statgraphics Plus (STCC Inc., Rockville, MD).
Differences at p < 0.05 were considered to be significant.
RESULTS AND DISCUSSION
Phenolic Profiles of Berry and Plant Extracts. The phenolic
composition of berry isolates is shown in Table 1. Raspberry
anthocyanin isolate consisted of purely anthocyanins. The main
phenolics in black currant isolate were anthocyanins with a
modest amount of flavanols present. Raspberry ellagitannin
A photodiode array (PDA) detector wavelength of 260 nm was used
to monitor oxidation products. The fluorescence program was modified
as follows: from Ex 230 nm and Em 342 nm, 0-13 min; to Ex 280
and Em 335, 13-46 min. The quantitative determination of Trp and

7476 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Salminen and Heinonen
Table 3. Concentrations of Trp-Derived Oxidation Products (after 6 h of
Oxidation with H2O2) in a Trp Model Solution (µg/mL, Mean ( SD, n ) 36)a
Trp-derived oxidation product
concentration (µg/mL)
Trp
Oia
NFK
Tra
DiOia B
PIC B
Kyn
DiOia A
5-OH-Trp
PIC A
157.7 ( 13.5
12.6 ( 0.9
4.9 ( 0.6
3.2 ( 0.3
2.1 ( 0.2
1.2 ( 0.1
1.3 ( 0.2
1.1 ( 0.1
0.7 ( 0.2
0.6 ( 0.0
a See panel A for abbreviations.
tion of Trp-derived products has been observed (9) reporting a
higher Trp loss (68%). However, it was reported that the amount
of degradation products related to Trp content was approxi-
mately 11 and 13% with Trp loss of 50 and 68%, respectively
(9). Thus, under strong H2O2 treatment, Trp is oxidized to such
an extent (50%) that secondary oxidation products (∼20%) are
efficiently formed. The major differences were the temperature
and pH used; instead of pH 8.3 and a temperature of 40 °C, pH
6.3 and a temperature of 37 °C were used. Degradation patterns
of Trp and rate of oxidation depend on the oxidative conditions
suchasincubationtime,theamountofoxidant,andtemperature(5,9).
Oxidation of Trp did not reach its end point since Trp loss could
have been more substantial by extending the incubation time.
It has been also observed that under strong oxidative conditions
(1.05 M H2O2) PIC A is decreased, and the amount of Kyn,
NFK, and DiOia is increased (9). This is due to the fact that
Trp oxidation products (PIC A, Oia, DiOia, Kyn, and 5-OH-
Trp) are even more susceptible to oxidation than Trp is itself.
In addition, the oxidation rate for Trp oxidation products is more
rapid (9).
It still remains unclear as to what other compounds Trp is
degraded into as 80-90% of the degradation products related
to Trp loss have not been explicated. However, the formation
of Trp oxidation products, NFK and Kyn, has been shown to
correlate with a loss of Trp due to oxidation (26). Oxidative
modification of Trp residues to Kyn type products may also
induce covalent aggregation in proteins (27), and oxidized milk
proteins have been observed to polymerize (26). In addition,
oxidation of myofibrillar proteins at high H2O2 and FeCl3
concentrations has been reported to cross-link mainly from
disulfide bonds, but also, dityrosine and ditryptophan bonds may
be involved (28).
Effect of Plant Phenolics toward Oxidation of Trp. The
loss of Trp affected by different phenolic compounds was
30-50% after 6 h of oxidation (Table 4). However, as only
5-20% of the determined oxidation products depending on the
Trp loss could be elucidated, the appearance of less polar UV
and fluorescent compounds was detected (Figure 3) after Trp
was eluted. However, the formation of the putative Trp-dimers,
Trp-phenolic, or phenolic-phenolic adducts was not substanti-
ated. Labieniec et al. (29) showed that phenols can react with
proteins and modify the formation of CO groups.
Figure 4. UV-vis spectra of Trp and its oxidation products from PDA
detector of the HPLC. See panel A for abbreviations.
isolate consisted mainly of ellagitannins and ellagic acid with
minor amounts of anthocyanins and flavonols. Cranberry
proanthocyanidin isolate contained mainly proanthocyanidins
with minor amounts of anthocyanins and flavonols. The phenolic
composition of plant extracts is presented in Table 2. Camelina
meal extract was predominated by hydroxycinnamic acids,
flavanols, and flavonols, whereas rapeseed meal extract was
predominated by hydroxycinnamic acids. Soy meal and flour
consisted mainly of flavanols. The main phenolics in pine bark
drink were flavanols with hydroxybenzoic acids.
Oxidation of Trp. Nine Trp-derived oxidation products
detectable with the modified HPLC method were identified by
PDA and fluorescence (FL) detection (Figure 3). Separation
of Trp and its oxidation products was achieved within 16 min
by using the selected column. DiOia and PIC each gave two
peaks, and Oia gave a double peak, referring to their diaster-
eomers. Some peaks remained unidentified. The identification
of Trp and its oxidation products was confirmed by their spectral
properties (Figure 4). The loss of Trp in the model system was
approximately 50% after 6 h of oxidation with H2O2. Only
∼20% of the determined Trp loss in free Trp could be elucidated
by the determined Trp oxidation compounds (Table 3). Oia and
NFK were shown to be the main oxidation products of Trp,
followed by Tra, DiOia B, PIC B, Kyn, DiOia A, 5-OH-Trp,
and PIC A. A corresponding oxidation susceptibility of forma-
After 6 h of oxidation, PIC A and B, the primary oxidation
products of Trp, exhibited higher concentrations levels with most
of the phenolic compounds than with Trp control regardless of
the amount of Trp loss (Table 5). However, at this point of
oxidation, the formation of PIC has been observed to be
decreasing (9). Therefore, this may be due to the fact that Trp
oxidation is slowed down by the phenolic compounds, but
further oxidation of PIC itself proceeds. Thus, the further

Phenolics Affect Oxidation of Tryptophan
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7477
Table 4. Inhibition of Trp Loss (%) with Phenolics after 6 h of Oxidation with H2O2 Determined by HPLCa
inhibition of Trp loss (%) after oxidation
50 µM
10 µM
100 µM
plant phenolics
black currant as
raspberry as
cranberry pro
raspberry etb
pine bark
camelina meal
rapeseed meal
soy meal
4.1 ( 8.5 c
-11.1 ( 10.0 d
20.7 ( 5.9 ab
41.9 ( 3.7 a
6.0 ( 1.2 d
3.0 ( 9.1 f
2.7 ( 9.9 f
33.3 ( 4.3 ab
16.6 ( 6.7 cd
29.8 ( 6.4 b
10.4 ( 14.8 cd
8.4 ( 5.8 cd
17.8 ( 2.5 c
13.8 ( 7.0 cd
27.0 ( 2.1 b
11.5 ( 2.9 de
35.8 ( 0.7 a
5.8 ( 1.4 ef
22.9 ( 0.7 bc
6.9 ( 2.7 ef
15.2 ( 2.6 cd
20.0 ( 9.3 b
31.8 ( 1.5 a
10.1 ( 8.9 bc
12.5 ( 3.3 bc
7.9 ( 0.9 c
soy flour
phenolic compounds
cyanidin-3-glucoside
delphinidin-3-glucoside
caffeic acid
catechin
chlorogenic acid
ellagic acid
ferulic acid
procyanidin B1
quercetin
sinapic acid
taxifolin
15.2 ( 1.4 cde
32.2 ( 3.9 a
23.8 ( 0.9 b
8.8 ( 5.8 fg
24.4 ( 1.4 c
30.6 ( 5.5 b
35.0 ( 1.8 b
18.7 ( 3.1 de
23.3 ( 1.6 cd
46.3 ( 1.6 a
22.0 ( 3.8 cd
-3.5 ( 4.8 g
21.9 ( 1.9 cd
24.5 ( 0.8 c
3.9 ( 2.8 f
30.1 ( 3.3 d
37.9 ( 3.5 bc
38.2 ( 0.5 b
25.3 ( 2.2 de
30.2 ( 5.5 cd
50.6 ( 1.9 a
23.0 ( 3.7 de
-4.8 ( 6.9 g
26.1 ( 5.2 de
27.1 ( 0.7 d
8.2 ( 0.2 ef
8.6 ( 3.0 fg
17.9 ( 3.4 cd
17.0 ( 3.6 cd
-11.1 ( 7.1 h
6.4 ( 3.1 g
19.9 ( 1.3 bc
-5.4 ( 3.9 efg
-19.0 ( 3.5 i
13.2 ( 1.9 def
13.3 ( 1.7 def
vinylsyringol
daidzein
genistein
-9.0 ( 4.2 h
16.5 ( 0.5 ef
15.5 ( 1.2 ef
-18.5 ( 11.0 h
14.7 ( 2.2 f
11.7 ( 1.5 f
a Trp loss (%) of the control after oxidation was 50.3 ( 4.2 (n ) 36). Values in the same column at the same concentration within plant phenolics and within phenolic
compounds followed by different letters are significantly different (p < 0.05) (percent inhibition, mean ( SD, n ) 3). b Raspberry ellagitannin isolate concentrations at 50
and 100 µM correspond to 58 and 115 µM, respectively. Abbreviations: As, anthocyanins; pro, proanthocyanidins; and et, ellagitannins.
oxidation of primary oxidation products PIC A, Oia, and DiOia
yielding Kyn and NFK, DiOia and Kyn, and Kyn, respectively
(9), results in higher concentrations of Kyn, NFK, and DiOia.
This phenomenon is observed with results of raspberry and black
currant anthocyanins (Table 5). However, the formation of
DiOia A and B with following oxidation susceptibility to PIC
A/B and Oia is retarded with most of the plant phenolics and
phenolic reference compounds (Tables 5 and 6). Therefore, the
faster oxidation of primary oxidation products may thus lead
to an increased formation of secondary oxidation products. That
explains why some phenolic compounds inhibited the formation
of primary oxidation products poorly or at a moderate level
while they were excellent antioxidants toward the formation of
secondary oxidation products.
Berry Phenolics. Black currant anthocyanins (50 µM) ex-
hibited the best inhibition (42%) among plant phenolics against
Trp loss as compared to the inhibition levels of pine bark
phenolics (100 µM) of 36%, cranberry procyanidins of (50 µM)
of 33%, and camelina meal phenolics (10 µM) of 32% (Table
4). However, black currant anthocyanins at other concentrations
(10 and 100 µM) were poor in inhibiting Trp loss. Even though
the amount of Trp loss in the presence of black currant
anthocyanins at 100 µM did not differ from the control sample,
the pattern of the Trp-derived oxidation products was similar
to that at black currant anthocyanin concentration level of 50
µM (Table 5). Black currant anthocyanins were able to inhibit
almost totally Tra, indicating that the oxidation of alanyl moiety
of Trp is highly affected by black currant anthocyanins. In
addition, black currant anthocyanin isolate (100 µM) was an
excellent inhibitor of DiOia B. NFK as the second most
important oxidation product formed as well as Kyn and DiOia
A were inhibited approximately 70% by black currant isolate.
The inhibition of Oia was about 40% by black currant.
Raspberry anthocyanins in all concentrations showed no dif-
ference in Trp loss as compared to oxidized Trp (Table 4). The
antioxidant effect of different berry anthocyanins reflects their
anthocyanin composition (Table 1). Raspberry consists mainly
of the 3-sophorisides (59%), 3-glucosides (16%), and 3-gluco-
sylrutinosides (16%) of cyanidin with minor amounts of
pelargonidin (4%) with different 3-glucosyl substituents, while
black currant contains four major anthocyanins: the 3-glucosides
and 3-rutinosides of cyanidin (7 and 38%) and delphinidin (16
and 39%) (30). Cyanidin-3-glucoside and delphinidin-3-gluco-
side were able to inhibit the Trp loss by 30% (Table 4). Effects
of cyanidin-3-glucoside and delphinidin-3-glucoside (although
with less effect) (Table 6) showed a consistency in the pattern
of oxidation products formed with black currant and raspberry
anthocyanins. Therefore, according to these results, it seems
that cyanidin- and delphinidin-3-glucosides present are the main
compounds responsible for the antioxidant activities in black
currant isolates. It has been reported (31) that black currant
consists also of procyanidins (43%), and prodelphinidins (57%)
with low molecular weight (LMW) (1-10 µg/g) and insoluble
high molecular weight (HMW) (100 µg/g) proanthocyanidins,
which may also contribute to the antioxidant activity. Prodel-
phinidins in the form of trimeric gallocatechins (31) and flavan-
3-ols such as catechin (8 µg/g) and epicatechin (11 µg/g) have
been identified from black currant (32). This suggests that the
amount of 3.7% of flavanols in black currant isolate (Table 1)
may also affect the oxidation of Trp, which is in accordance
with the results that catechin as a reference compound inhibited
the Trp oxidation (Tables 4 and 6).
Cranberry proanthocyanidins (50 and 100 µM) exhibited
approximately 30% inhibition toward Trp loss (Table 4).
However, Trp with cranberry proanthocyanidins showed a
higher susceptibility to oxidation as compared to pine bark
(Table 5) although the inhibition of Trp oxidation was similar
(30%) (Table 4). Cranberry procyanidins were less efficient in
retarding oxidation of Oia and DiOia A as they still yielded
DiOia and Kyn, respectively (Table 5). Although procyanidin

7478 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Salminen and Heinonen
Table 5. Inhibitions of Trp Oxidation Products (after 6 h of Oxidation) by Plant Phenolics in a Trp Model Solution (Percent Inhibition, Mean ( SD)a
concentration
(µM) of
extracts
PIC A
DiOia A
DiOia B
PIC B
5-OH-Trp
Kyn
NFK
Oia
Tra
raspberry anthocyanins
-4.3 ( 4.7 d
10
50
100
-25.1 ( 17.1 bc -37.7 ( 23.4 e -0.8 ( 2.7 e
-46.6 ( 17.0 d 15.0 ( 3.9 e 10.4 ( 6.0 f
-4.2 ( 27.1 e 65.5 ( 13.2 c 26.0 ( 4.5 c
63.5 ( 0.9 c 100.0 ( 0.0 a 25.6 ( 0.6 f
8.3 ( 6.9 e
85.0 ( 2.4 c
93.3 ( 0.2 e
-56.7 ( 2.6 ab
-76.6 ( 6.7 c
3.5 ( 7.8 e
50.5 ( 0.3 d -162.9 ( 0.0 f
41.1 ( 1.1 e
98.0 ( 0.3 b
31.0 ( 9.8 a
black currant anthocyanins
-21.1 ( 1.0 e
10
50
100
-6.8 ( 1.4 a
-23.0 ( 2.0 ab
-81.9 ( 1.4 c
16.0 ( 4.6 d
63.4 ( 5.2 bc
68.5 ( 0.7 b
31.2 ( 2.8 c
78.0 ( 5.4 b
95.2 ( 0.3 c
-13.4 ( 14.9 c 37.8 ( 2.4 d 22.9 ( 9.0 e
48.1 ( 7.4 d
50.5 ( 3.3 a
71.0 ( 6.3 bc 71.9 ( 1.4 c 40.1 ( 4.2 b 100.0 ( 0.0 a
-6.1 ( 10.2 b
71.5 ( 1.1 b
73.2 ( 4.6 c 41.1 ( 0.9 d
97.8 ( 0.5 b
cranberry proanthocyanidins
38.6 ( 10.2 b
10
50
100
-14.5 ( 2.6 ab
-29.5 ( 13.6 ab
-21.3 ( 6.2 b
19.5 ( 0.5 cd
18.6 ( 13.0 d
22.4 ( 0.6 b
56.7 ( 14.0 c 24.5 ( 2.6 d 36.2 ( 7.3 b
41.7 ( 3.1 d 39.0 ( 5.4 f
12.7 ( 11.7 e 8.4 ( 4.6 f
56.3 ( 10.5 cd 70.8 ( 8.7 c
44.9 ( 2.9 a
74.7 ( 2.8 e
47.6 ( 1.8 d
58.8 ( 3.4 f
31.9 ( 3.5 a
34.1 ( 2.3 e 100.0 ( 0.0 a
raspberry ellagitannins
-21.2 ( 16.8 c
-6.4 ( 0.6 b
57.5
115
-5.4 ( 4.0 a
-341.3 ( 2.3 f
14.7 ( 16.6 e 97.6 ( 1.5 a
36.4 ( 2.2 f 89.3 ( 0.3 d
93.0 ( 2.0 a 100.0 ( 0.0 a 51.1 ( 8.2 a
87.0 ( 0.4 a 100.0 ( 0.0 a 22.7 ( 0.4 g
99.0 ( 0.2 ab
pine bark
50.1 ( 1.6 a
10
50
100
-36.4 ( 3.5 c
-30.2 ( 1.5 ab
1.5 ( 0.4 a
68.3 ( 0.2 ab 93.2 ( 0.3 a
80.0 ( 3.0 a
81.2 ( 0.7 ab 50.4 ( 2.0 ab 100.0 ( 0.0 a
73.5 ( 0.5 b
61.6 ( 0.1 c
97.9 ( 0.1 a
100.0 ( 0.0 a
39.7 ( 1.9 a
80.3 ( 1.1 ab 85.8 ( 0.5 b 55.4 ( 3.8 a 100.0 ( 0.0 a
35.5 ( 1.4 a
64.6 ( 1.8 c
79.9 ( 0.3 b 51.6 ( 0.8 b 100.0 ( 0.0 a
camelina meal
10
50
100
-78.6 ( 1.8 d
-79.4 ( 4.6 c
-170.2 ( 4.1 d
71.7 ( 0.2 a
41.9 ( 0.2 d
29.8 ( 1.1 g
95.2 ( 0.3 a
96.1 ( 0.5 a
24.1 ( 0.6 c
-767.1 ( 71.0 e
86.1 ( 1.0 a
92.1 ( 0.5 a
90.9 ( 0.0 a
87.7 ( 0.5 a 53.4 ( 0.4 a 98.8 ( 0.3 ab
83.7 ( 0.5 b 52.5 ( 7.0 a 100.0 ( 0.0 a
73.8 ( 0.9 c 55.1 ( 1.3 a 100.0 ( 0.0 a
-86.9 ( 20.6 d
95.5 ( 0.1 c -143.8 ( 0.6 d
rapeseed meal
10
50
100
-36.4 ( 9.3 c
-93.0 ( 1.1 bc
-210.6 ( 11.4 e
54.9 ( 1.2 b
47.7 ( 1.4 d
15.8 ( 1.5 h
79.9 ( 1.2 b
84.2 ( 0.1 b
72.5 ( 0.2 e
36.2 ( 1.8 b
11.1 ( 1.1 b
-224.5 ( 17.8 c
74.0 ( 1.0 a
72.0 ( 0.9 c 45.5 ( 2.8 bc 89.7 ( 0.6 c
-577.8 ( 123.5 d 79.1 ( 1.5 ab 73.0 ( 0.8 c 51.4 ( 2.6 a
97.2 ( 0.4 bc
97.3 ( 0.2 b
-44.8 ( 1.5 c -1077.1 ( 131.2 f
71.9 ( 2.1 b
62.9 ( 0.7 d 44.1 ( 0.5 c
soy meal
-2.7 ( 5.8 d
10
50
100
-251.1 ( 10.9 e
-825.8 ( 15.2 d -101.3 ( 2.6 f
-1817.5 ( 20.2 h -110.3 ( 4.1 i
35.3 ( 0.7 c
95.2 ( 0.2 a
100.0 ( 0.0 a
74.1 ( 0.3 a
80.7 ( 1.1 ab 41.5 ( 2.0 cd 94.2 ( 1.2 abc
-98.7 ( 1.6 d
35.1 ( 5.2 d 100.0 ( 0.0 a 26.0 ( 1.7 c
98.2 ( 1.8 ab
96.6 ( 0.4 c
100.0 ( 0.0 a -188.8 ( 3.0 e
36.6 ( 5.4 e
59.1 ( 1.7 de 10.3 ( 1.7 h
soy flour
-8.4 ( 2.1 d
100.0 ( 0.0 a 100.0 ( 0.0 a -141.7 ( 1.2 e
100.0 ( 0.0 a 100.0 ( 0.0 a -262.3 ( 1.6 f
10
50
100
-298.9 ( 3.9 f
-1150.2 ( 2.0 e
-1418.8 ( 11.6 g
32.9 ( 0.4 c
90.9 ( 0.1 a
-99.0 ( 12.2 b
-81.5 ( 12.2 ab 59.4 ( 3.6 c
31.9 ( 117.9 a 24.9 ( 1.9 f
72.4 ( 4.0 a
75.9 ( 0.6 bc 37.1 ( 0.6 d
65.4 ( 2.7 c 16.2 ( 3.1 d
57.8 ( 2.6 e -6.5 ( 2.6 i
92.5 ( 0.3 bc
95.7 ( 0.7 c
95.4 ( 0.3 d
a Values in the same column at the same concentration followed by different letters are significantly different (p < 0.05). See panel A for abbreviations.
B1 used as a reference compound was effective in inhibiting
the formation of Trp oxidation products (Table 6), it showed
no effect in inhibiting Trp loss as compared to control (Table
4). Cranberry procyanidin fractions have been found to be
effective antioxidants in DPPH test and toward lipid oxidation
such as inhibiting the oxidation of methyl linoleate emulsion
and LDL (33). Cranberry, blueberry, and grape seed extracts
alone and in combinations showed antioxidant activity assayed
by using a DPPH radical inhibition test (34). In addition,
cranberry juice powder and its synergies with ellagic and
rosmarinic acids have been shown to reduce oxidative stress
and mediate antioxidant enzyme responses in porcine muscle
tissue induced by H2O2 (35).
Raspberry ellagitannin isolate provided only a weak protection
against the oxidation of Trp (Table 4). Among plant phenolics
such as pine bark drink and soy meal (10 and 50 µM), cranberry
proanthocyanidins (10 and 100 µM), and rapeseed (100 µM)
with the same Trp loss (40%), raspberry ellagitannins had a
more pronounced effect on oxidation products such as DiOia
A and B, Kyn, NFK, and Tra. In contrast, rapeseed meal, pine
bark, and soy meal affected the oxidation of Oia about the same
as raspberry ellagitannins. According to Ka¨hko¨nen et al. (36),
the main compounds in ellagitannin fraction consist of mixture
of monomers (MW ∼ 936 g/mol), dimers (sanguiin H6), trimers
(lambertianin C), and polymers. Raspberry is reported to contain
minor amounts of flavonols such as 3-glucosides and 3-glucu-
ronides of quercetin (19), which is accordance with our results
(Table 1). Ellagic acid and raspberry ellagitannins have been
attributed with antioxidative properties (37). Ellagic acid with
increasing concentration exhibited the best activity against
oxidation of Trp (Table 4) by decreasing the Trp loss by
50%.
Oilseed Processing Byproducts. Camelina meal phenolics
inhibited the oxidation of Trp by 32% already at a concentration
of 10 µM in comparison to rapeseed meal phenolics with 23%
inhibition at 100 µM (Table 4 and Figure 3). Camelina meal
phenolics at 50 and 100 µM exhibited a similar rate of Trp
oxidation with oxidized Trp (control), whereas the amount of
Trp-derived oxidation products was clearly decreased as com-
pared to less oxidized Trp with camelina meal phenolics at 10
µM (Table 5 and Figure 3). In general, camelina meal phenolics
showed more pronounced effects in retarding the oxidation of
Oia and Kyn as compared to other plant phenolics. The
inhibition of Kyn may be due to the fact that oxidation of Oia
was slowed down, thus yielding less Kyn. The phenolic
composition of camelina meal extract was predominated by
flavanols, hydroxycinnamic acids, and flavonols, while rapeseed
meal phenolics were mainly hydroxycinnamic acids (Table 2),
which is accordance with results reported earlier (14, 15).
Consequently, flavanols and flavonols, such as quercetin glu-
cosides, contribute to the antioxidant activity of camelina meal,
which was also concluded in an earlier study (14). With regard
to the reference compounds, catechin and quercetin provided a
protection toward Trp and Trp oxidation compounds (Table 4).

Phenolics Affect Oxidation of Tryptophan
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7479
Table 6. Inhibitions of Trp Oxidation Products (after 6 h of Oxidation) by Phenolic Compounds in a Trp Model Solution (Percent Inhibition, Mean ( SD)a
concentration
(µM) of
extracts
PIC A
DiOia A
DiOia B
PIC B
5-OH-Trp
Kyn
NFK
Oia
Tra
cyanidin-3-glucoside
10
50
100
-2.6 ( 1.2 a
-15.9 ( 1.7 b
-69.0 ( 36.8 c
7.3 ( 1.2 ab
43.4 ( 0.8 c
49.8 ( 4.5 c
5.5 ( 1.1 g
58.0 ( 0.8 e
78.2 ( 0.6 e
13.0 ( 1.7 a
22.2 ( 0.5 bc
42.3 ( 0.1 cd
12.8 ( 5.5 bc
53.4 ( 2.1 c
70.1 ( 4.8 c
4.8 ( 1.9 cd 34.8 ( 0.2 ab
31.3 ( 1.1 cd 79.6 ( 0.9 c
40.7 ( 2.2 d
92.9 ( 1.6 bc
delphinidin-3-glucoside
10
50
100
-39.8 ( 0.9 i
-20.9 ( 1.5 c
-134.7 ( 10.4 f
-53.3 ( 25.6 g -10.7 ( 0.9 i
-50.6 ( 25.2 cd -4.5 ( 5.6 b
-60.9 ( 2.2 c -30.9 ( 3.3 f
8.9 ( 0.8 bc
1.7 ( 0.8 d
15.5 ( 0.4 cdef
39.0 ( 1.4 f
74.5 ( 1.4 e
-6.2 ( 5.1 h
23.5 ( 11.2 e
16.8 ( 2.7 j
37.9 ( 4.4 i
26.7 ( 3.9 ef 11.7 ( 1.1 fg
-84.0 ( 1.5 de
7.5 ( 6.5 i
47.5 ( 2.1 d
23.5 ( 0.5 f
caffeic acid
10
50
100
-21.4 ( 0.7 g
-136.3 ( 3.4 l
-175.6 ( 5.7 g
10.0 ( 0.2 a
23.1 ( 0.8 e
23.1 ( 0.3 e
22.8 ( 1.0 cd
61.7 ( 0.1 de
75.2 ( 0.2 f
9.0 ( 1.9 d
-12.3 ( 20.3 bc
33.0 ( 13.9 b
44.9 ( 3.0 bc
16.4 ( 7.4 bc
51.8 ( 2.4 c
64.8 ( 2.1 c
8.3 ( 1.6 bc 36.0 ( 0.2 ab
43.6 ( 1.4 ab 92.0 ( 0.3 a
55.6 ( 1.8 bc 96.4 ( 0.1 ab
33.7 ( 0.5 c
35.4 ( 0.4 ab
catechin
10
50
100
-0.8 ( 0.3 a -12.7 ( 13.6 de 21.2 ( 0.2 cd
-0.9 ( 0.1 e
27.0 ( 0.6 d
36.9 ( 0.1 ab
-20.9 ( 11.7 bcd 17.4 ( 0.1 abc 8.6 ( 3.4 bc 25.8 ( 0.5 bc
26.2 ( 0.5 bc 51.6 ( 5.4 c 32.7 ( 1.8 cd 81.2 ( 0.2 c
23.3 ( 8.3 efgh 65.1 ( 3.8 c 50.0 ( 4.5 c 93.2 ( 0.0 bc
-60.5 ( 1.9 g
33.7 ( 0.7 d
54.8 ( 1.5 c
64.4 ( 0.6 d
81.3 ( 0.8 d
-120.0 ( 3.3 ef
chlorogenic acid
10
50
100
-11.8 ( 0.5 de
-49.7 ( 2.4 f
-91.1 ( 2.8 cd
7.8 ( 3.5 ab
47.2 ( 0.6 b
67.9 ( 7.5 b
23.9 ( 0.4 c
63.7 ( 0.3 d
86.4 ( 1.7 c
25.6 ( 1.2 b
33.4 ( 0.0 c
-26.5 ( 30.1 bc -6.1 ( 10.5 bc
16.1 ( 1.6 bc
49.4 ( 2.2 c
69.6 ( 5.7 c
9.1 ( 4.5 bc 20.7 ( 8.4 cd
37.1 ( 2.6 bc 83.8 ( 0.2 bc
61.9 ( 4.7 ab 94.4 ( 3.3 bc
-64.9 ( 8.6 c
9.0 ( 6.5 cde
41.5 ( 0.9 ab -83.3 ( 24.3 de 14.9 ( 3.0 hi
ferulic acid
10
50
100
-40.4 ( 1.6 i
-91.1 ( 0.9 i
-28.4 ( 1.7 f
-18.8 ( 2.8 j
13.5 ( 0.2 ef
26.6 ( 7.9 gh
30.4 ( 1.1 j
8.9 ( 0.6 d
-51.7 ( 0.4 cd -11.3 ( 10.9 bc -2.7 ( 37.2 c
-59.6 ( 11.8 c
5.0 ( 5.7 cd 38.8 ( 2.5 a
23.2 ( 1.7 e
11.6 ( 11.6 cde 38.7 ( 11.0 d 17.5 ( 6.2 ef
82.4 ( 3.6 c
90.4 ( 0.6 c
-101.3 ( 1.5 de -27.7 ( 1.3 fg
23.4 ( 3.0 bc -53.8 ( 27.4 cd 13.9 ( 18.7 fghi 47.1 ( 6.7 d
21.5 ( 0.3 f
ellagic acid
-2.9 ( 1.1 efg -40.9 ( 5.1 cd -21.2 ( 11.5 bcd 27.6 ( 2.2 ab
10
50
100
-7.9 ( 1.3 c
-110.7 ( 3.0 j
-183.3 ( 3.5 g
-5.7 ( 3.9 bcd -2.6 ( 3.6 h
5.0 ( 2.4 cd
51.2 ( 3.0 a
7.5 ( 2.5 f
53.9 ( 1.0 a
82.6 ( 0.3 a
82.7 ( 0.3 a
99.0 ( 0.0 a
11.6 ( 1.4 f
-81.2 ( 4.3 c
61.1 ( 4.9 a
94.5 ( 1.0 a
100.0 ( 0.0 a
100.0 ( 0.0 a
93.2 ( 1.2 a
13.8 ( 1.3 bc -94.1 ( 2.0 e
63.3 ( 1.2 a 100.0 ( 0.0 a
procyanidin B1
10
50
100
-33.2 ( 1.0 h
-132.0 ( 0.0 k
-120.2 ( 1.7 ef
-6.7 ( 2.5 cde 61.0 ( 4.7 a
49.6 ( 0.5 a
50.2 ( 1.1 a
59.7 ( 0.7 a
90.0 ( 10.5 a -49.8 ( 14.1 e
27.7 ( 11.1 a -1.7 ( 21.6 e
37.8 ( 10.5 a 26.4 ( 6.2 a
41.8 ( 5.0 a
25.6 ( 1.1 e
72.7 ( 0.1 b
76.7 ( 0.2 b
89.4 ( 0.0 b
63.3 ( 5.9 b
81.5 ( 1.6 b
44.3 ( 4.7 ab 87.5 ( 0.7 b
60.7 ( 2.4 ab 96.6 ( 0.2 ab
31.1 ( 1.3 a
58.0 ( 3.1 b
sinapic acid
10
50
100
-9.7 ( 0.5 cd -19.4 ( 1.3 ef
-2.4 ( 0.4 h
29.6 ( 1.7 g
44.5 ( 0.5 h
-2.3 ( 1.3 ef
35.9 ( 0.8 b
-3.1 ( 11.8 b -22.8 ( 3.6 cd
4.0 ( 12.1 ab 15.5 ( 2.0 cde
10.1 ( 2.1 bc
25.9 ( 2.1 f
45.1 ( 3.1 d
4.2 ( 4.2 cd 12.4 ( 2.6 def
15.3 ( 2.1 efg 61.6 ( 1.9 d
-30.7 ( 1.8 d
-40.5 ( 1.8 b
-0.5 ( 0.7 g
25.5 ( 0.5 e
38.6 ( 0.4 ab -14.4 ( 35.6 b
29.1 ( 4.4 de
21.7 ( 1.4 f
83.0 ( 0.3 d
taxifolin
10
50
100
-12.0 ( 1.8 e -11.4 ( 1.0 de
-14.9 ( 3.2 ab -7.4 ( 1.6 h
30.0 ( 1.2 b
37.4 ( 5.1 f
64.8 ( 0.3 g
-4.7 ( 0.2 g
12.4 ( 0.2 f
-24.5 ( 28.4 bc -36.1 ( 2.8 de
12.5 ( 1.8 bc
6.0 ( 0.6 bcd 6.6 ( 0.3 f
-29.3 ( 9.5 bc
20.3 ( 10.2 bcd 28.1 ( 4.1 ef 11.1 ( 0.4 fg
46.4 ( 1.3 e
82.1 ( 2.7 d
-85.9 ( 1.8 cd
19.8 ( 0.7 e
-0.8 ( 0.5 cd -27.7 ( 5.6 bc
9.6 ( 0.5 ghi
42.4 ( 2.2 d
23.2 ( 0.7 f
vinylsyringol
19.7 ( 1.1 c
10
50
100
-16.9 ( 2.5 f
-35.8 ( 2.1 e
-13.4 ( 2.3 a
9.7 ( 1.0 a
18.1 ( 0.4 f
27.1 ( 11.7 e
33.7 ( 9.0 b
60.9 ( 4.8 de
76.6 ( 3.1 ef
-11.5 ( 9.8 bc
4.6 ( 13.0 de
23.3 ( 8.1 ab 12.3 ( 7.8 b
34.9 ( 1.5 de 17.5 ( 5.7 ef
14.6 ( 4.8 def
45.8 ( 5.2 e
5.0 ( 1.1 g
18.2 ( 6.1 bc
22.6 ( 14.0 efg 50.1 ( 10.5 d 30.8 ( 11.7 e 74.4 ( 6.1 e
quercetin
10
50
100
-5.5 ( 2.4 b
-65.3 ( 1.3 h
4.6 ( 0.1 abc 17.7 ( 2.9 de
-3.3 ( 0.9 fg -69.2 ( 27.4 d -6.2 ( 12.4 bc
71.1 ( 0.3 c -331.9 ( 1.1 j -155.9 ( 92.4 d 35.4 ( 16.6 b
12.1 ( 20.9 bc 3.2 ( 4.2 cd 18.6 ( 21.3 cde
58.0 ( 2.7 bc 24.8 ( 18.8 de 83.2 ( 2.7 bc
41.4 ( 2.4 c
-97.8 ( 32.3 de 40.0 ( 1.7 d
79.2 ( 0.2 de -1131.7 ( 57.0 e -306.0 ( 47.2 f
daidzein
-60.4 ( 5.5 d -23.1 ( 10.0 cd -1.0 ( 0.8 c -0.1 ( 0.8 d
30.1 ( 5.0 de
67.7 ( 5.3 c
42.9 ( 2.9 d
93.2 ( 0.4 bc
10
50
100
-13.9 ( 0.5 e -13.1 ( 0.8 de
-23.3 ( 0.7 c -14.8 ( 1.9 i
-14.3 ( 0.7 a -32.4 ( 0.8 g
3.2 ( 2.6 g
18.9 ( 0.4 ij
14.6 ( 0.4 l
-8.2 ( 2.2 h
-7.4 ( 0.6 h
8.0 ( 4.0 ef
18.6 ( 1.2 h
15.8 ( 6.8 g
-49.8 ( 4.7 c -24.9 ( 6.0 f
20.4 ( 3.4 f
6.1 ( 2.1 g
-2.7 ( 1.3 cd -89.1 ( 12.4 de -51.4 ( 5.8 j
12.7 ( 7.0 f -0.1 ( 2.3 h
genistein
10
50
100
-8.8 ( 1.6 c -20.0 ( 1.6 ef
-12.1 ( 0.7 a -26.2 ( 0.4 k
-19.0 ( 1.0 ab -20.8 ( 0.5 f
7.9 ( 1.8 fg
22.5 ( 0.6 hi
27.0 ( 1.5 k
-2.0 ( 0.9 ef
-28.5 ( 1.9 i
-5.8 ( 1.1 b -21.9 ( 2.0 bcd -33.6 ( 1.5 d
5.1 ( 1.5 cd 14.6 ( 3.4 def
17.8 ( 9.0 ab -30.9 ( 7.6 f
22.9 ( 12.3 f
7.0 ( 3.1 g
8.9 ( 4.4 g
34.4 ( 5.8 g
43.8 ( 3.2 f
-20.3 ( 0.5 d -106.9 ( 6.4 e
27.2 ( 10.4 def 25.4 ( 13.3 e
a Values in the same column at the same concentration followed by different letters are significantly different (p < 0.05). See panel A for abbreviations.
Camelina meal has been shown to inhibit protein carbonyls in
cooked pork meat patties with similar antioxidant activity as
rapeseed meal (14). The pattern of Trp oxidation products
formed and inhibited with rapeseed meal phenolics was similar
to camelina meal phenolics; however, the antioxidant effect was
more pronounced with camelina meal (Table 5). Rapeseed
phenolics have been reported to show moderate radical scaveng-
ing activity (DPPH test) (37) and antioxidant activity in cooked
pork meat patties (protein carbonyls) (14, 15) as well as in
liposomes (37). Among hydroxycinnamic acids, caffeic and
chlorogenic acids showed a more profound antioxidant effect
than ferulic and sinapic acids toward the oxidation of Trp (Table
4). In this regard, the differences in activities may be due to
the different structures of hydroxycinnamic acids. Caffeic and
chlorogenic acids with only hydroxyl groups are more prone to
interaction with Trp than ferulic and sinapic acids or sinapine
with one or more methoxy groups. However, as camelina,
rapeseed, and soy meal as well as pine bark used in this study

7480 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Salminen and Heinonen
ABBREVIATIONS USED
were rich in diverse phenolics, the pure phenolic compounds
used as reference compounds may not explicate the exact
activity of a certain compound. In addition, the antioxidant
activity of certain compound(s) contributing to activity is
difficult to demonstrate since there may also be synergistic
effects between the different phenolics present in the extracts.
Trp, tryptophan; NFK, N-formylkynurenine; Kyn, kynurenine;
3-OH-Kyn, 3-hydroxykynurenine; KynA, kynurenic acid; 5-OH-
Trp, 5-hydroxy-tryptophan; Tra, tryptamine; PIC A/B, 3a-
hydroxypyrroloindole-2-carboxylic acid; Oia A/B, oxindolyla-
lanine; DiOia A/B, dioxindolylalanine.
Soy meal and soy flour consist mainly of flavanols (Table
2) with isoflavones and lignans dominating (16). Soy meal and
soy flour phenolics (10 µM) provided more protection toward
the formation of Trp oxidation compounds than at higher
concentrations (Table 5), even though there were only minor
differences in Trp loss (Table 4). However, soy meal phenolics
at 50 and 100 µM were able to inhibit DiOia B but not DiOia
A. This may be due to stereoselective reaction. Soy flour
phenolics, however, was able to inhibit both diastereomers of
DiOia. Isoflavones, daidzein and genistein, as such acted mostly
as weak antioxidants toward Trp oxidation. In addition, genistein
and daidzein were incapable of affecting oxidation of Oia,
thereby yielding more DiOia A/B and Kyn (Table 6). In
previous study (14), soy meal and soy flour were effective in
inhibiting protein carbonyls only in combination with rosemary
extract due to their synergistic interactions. It has been reported
that in foods where phenolic compounds are present together
in interactions with other (bioactive) ingredients, one phenolic
could synergistically improve the chances of the other phenolics
to function effectively (37).
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Received for review March 7, 2008. Revised manuscript received May
30, 2008. Accepted June 7, 2008.
JF800708T