New series of avenanthramides in oat seed

Article abstract of DOI:10.1080/09168451.2014.946390

Avenanthramides are characteristic constituents of oat seeds. We analyzed the methanol extract of oat seeds by HPLC and detected three compounds 1, 2, and 3 eluted at retention times similar to avenanthramides. The three compounds were purified by column

Full text of DOI:10.1080/09168451.2014.946390

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New series of avenanthramides in oat seed
Atsushi Ishiharaa, Kana Kojimaa, Takeshi Fujitaa, Yuya Yamamotoa & Hiromitsu Nakajimaa
a Faculty of Agriculture, Tottori University, Tottori, Japan
Published online: 13 Aug 2014.
To cite this article: Atsushi Ishihara, Kana Kojima, Takeshi Fujita, Yuya Yamamoto & Hiromitsu Nakajima (2014): New
series of avenanthramides in oat seed, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.946390
To link to this article: http://dx.doi.org/10.1080/09168451.2014.946390
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Bioscience, Biotechnology, and Biochemistry, 2014
New series of avenanthramides in oat seed
Atsushi Ishihara*, Kana Kojima, Takeshi Fujita, Yuya Yamamoto and Hiromitsu Nakajima
Faculty of Agriculture, Tottori University, Tottori, Japan
Received April 19, 2014; accepted June 14, 2014
http://dx.doi.org/10.1080/09168451.2014.946390
Avenanthramides are characteristic constituents of
oat seeds. We analyzed the methanol extract of oat
seeds by HPLC and detected three compounds 1, 2,
and 3 eluted at retention times similar to avenanthra-
mides. The three compounds were purified by col-
umn chromatography and HPLC. Spectroscopic
analyses of 1, 2, and 3 suggested that they are amides
of 4,5-dihydroxyanthranilic acid with caffeic, p-cou-
maric, and ferulic acids, respectively. Their identities
were confirmed by comparing spectra and chromato-
graphic behavior with compounds synthesized from
4,5-dihydroxyanthranilic acid and N-hyrdroxysuccin-
imide esters of hydroxycinnamic acids. LC-MS/MS
analysis with multiple reaction monitoring showed
that the amounts of 1, 2, and 3 were 16.5–26.9% of
corresponding avenanthamides with 5-hydroxyanth-
ranilic acid. Compounds 1, 2, and 3 showed stronger
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scav-
enging activity than the corresponding avenanthra-
mides with 5-hydroxyanthranilic acid, indicating the
involvement of 4,5-dihydroxyanthranilic acid moiety
in the scavenging of DPPH radicals.
gens3−5) and by treatment with elicitors.6) In addition,
avenanthramides have been shown to be incorporated
into cell walls upon pathogen infection.7) The incorpo-
ration of avenanthramides is considered to reinforce
cell walls against cell wall-degrading enzymes secreted
by pathogens. It has been demonstrated that the hy-
droxyanthranilate hydroxycinnamoyl-CoA hydroxycin-
namoyltransferase (HHT) activity that catalyzes the
final condensation reaction of avenanthramide synthesis
is induced by elicitation.8,9) Enzyme activity is also
detected in the oat seed extract, indicating the involve-
ment of the same enzyme in the synthesis of avenan-
thramides in seeds.10) The genes encoding HHTs have
been cloned by Yang et al.11)
Oats are usually consumed as whole grains and con-
tain the highest levels of protein and fat of any of the
cereal grains.12) In ancient Rome, oat flour was topi-
cally applied to treat various dermatologic condi-
tions.13) Recently, various bioactivities have been found
in avenanthramides. Liu et al. demonstrated that aven-
anthramides are anti-inflammatory and anti-atherogenic
by showing that they inhibit the adhesion of monocyte
cells to aortic endothelial cell monolayers, the expres-
sion of adhesion molecules, and the production of pro-
inflammatory cytokines.14) Sur et al. indicated that
avenanthramides exhibit anti-inflammatory and anti-
itching activity of the skin.15) They showed that topical
application of avenanthramides mitigated inflammation
in murine models of contact hypersensitivity and neuro-
genic inflammation and reduced pruritogen-induced
scratching in a murine itch model. In addition, it is
interesting that avenanthramides are very similar in
their chemical structure to tranilast (N-3,4-dimethoxy-
cinnamoylanthranilic acid, Rizaben®), which is an anti-
allergy drug used to treat asthma, autoimmune diseases,
and atopic and fibrotic pathologies.16,17)
Key words: Avena sativa; avenanthramide; anti-oxidant;
phytoalexin
Plants have acquired biosynthetic pathways leading
to various secondary metabolites in the relationships of
the plants with environmental factors during the evolu-
tionary process. Secondary metabolites have unique
structures, some of which are useful to human life.
Avenanthramides are secondary metabolites that have
been found in oat (Avena sativa) groats and hulls.1)
Avenanthramides are hydroxycinnamic acid amides
with hydroxyanthranilic acids (Fig. 1). Collins
described that at least 20 different phenolic conjugates
with anthranilic acid derivatives are present in oats,2)
but their chemical structures have not been fully identi-
fied. The major avenanthramides in oat groats are aven-
The anti-oxidant activity of avenanthramides has
also been investigated. Peterson et al. measured anti-
oxidant activity using two in vitro systems: inhibition
of β-carotene bleaching and reaction with the free
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). They
indicated the order of activity was avenanthramide
C > B > A in both assay systems.18) Fagerlund et al.
included synthesized analogs of avenanthramides in
the analysis of the anti-oxidant activity of avenanthra-
mides. They determined the activity by DPPH assay
anthramides
A
(N-p-coumaroyl-5-hydroxyanthranilic
acid), B (N-feruloyl-5-hydroxyanthranilic acid), and C
(N-caffeoyl-5-hydroxyanthranilic acid).
Avenanthramides play a role as phytoalexins in oat
leaves because they exhibit anti-fungal activity and
their synthesis is induced by infection with patho-
*Corresponding author. Email: aishihara@muses.tottori-u.ac.jp
© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
A. Ishihara et al.
Fig. 1. Chemical structures of avenanthramides.
and by the linoleic acid hydroperoxide assay. In the
scavenging DPPH radical, avenanthramides with feu-
loyl-, sinapoyl, and caffeoyl groups showed high
activity.19) In addition, they found that avenanthra-
mides with the 4-methoxy-5-hydroxyanthranilic moi-
ety showed greater activity than avenanthramides
bearing anthranilic and 5-hydroxyanthranilic moieties.
Thus, the 4-methoxy-5-hydroxyanthranilic moiety is
involved in the radical scavenging reaction. In the
linoleic acid assay, avenanthramides with one or
more hydroxyl groups inhibited linoleic acid oxida-
tion. However, the structure-activity relationship
revealed by this system is not simple, and the order
of activity was not explained in a rational way.
Packed Column 5C18-AR-II, 20 × 250 mm; Nacalai
Tesque, Kyoto, Japan) at a flow rate of 7.0 mL/min and
a column temperature of 40 °C. Unless stated otherwise,
a two-solvent system was used for HPLC to generate
the mobile phase: solvent A, H2O-TFA (100:0.1); and
solvent B, MeCN Analytical HPLC was performed
using an ODS column (Cosmosil Packed Column 5C18-
AR-II, 4.6 × 150 mm; Nacalai Tesque) at a flow rate of
0.8 mL/min and a column temperature of 40 °C. The
same solvents were used as for preparative HPLC. A
linear gradient 5–60% B/(A + B) within 60 min was
applied.
Isolation of avenanthramides.
Oat (Avena sativa
In oat groats, multiple uncharacterized avenanthra-
mides are present. Even in the identified avenanthra-
mides, usually only avenanthramides A, B, and C have
been the target of the analysis. The uncharacterized
avenanthramides did not attract attention probably
because their amounts were small in comparison with
known avenanthramides. However, the uncharacterized
avenanthramides may have stronger or distinct activity.
From this viewpoint, we searched for uncharacterized
avenanthramides in oat seed and identified avenanthra-
mides containing 4,5-dihydroxyanthranilic acid.
Because they have an additional hydroxyl group that
may contribute to anti-oxidant activity, we measured
anti-oxidant activity using the DPPH radical scavenging
assay. In addition, we developed a multiple reaction
monitoring (MRM) method of liquid chromatography-
tandem mass spectrometry (LC-MS/MS), and deter-
mined their contents in oat groats.
L. cv. Shokan 1) seeds harvested from an experimen-
tal farm of the Faculty of Agriculture, Tottori Univer-
sity, Japan, in July 2010 and 2011 were used for
extraction of avenanthramides. Oat seeds (515 g)
without removal of hulls were powdered using a
food processor. The powder was extracted three times
with 4 L MeOH. The extracts were combined and
evaporated. The residue was suspended in a mixture
of 250 mL H2O and 50 mL MeOH. The suspension
was washed with hexane three times to remove
lipids, and was passed through an ODS column
(Cosmosil 75C18-PREP, 100 g; Nacalai Tesque) equili-
brated with MeOH H2O AcOH (80:10:1). The
column was washed with 600 mL of the same sol-
vent. The column through fraction and MeOH H2O
AcOH (80:10:1) fraction were combined and evapo-
rated. The obtained residue was dissolved in MeOH
H2O–AcOH (5:90:1) and applied to an ODS column
(Cosmosil 75C18-PREP, 100 g; Nacalai Tesque) equili-
brated with MeOH H2O AcOH (5:95:1). The column
was eluted with 750 mL each of a mixture of MeOH
H2O AcOH (5:95:1, 20:80:1, 40:60:1, and 80:20:1)
and MeOH. Avenanthramides were eluted in the
MeOH H2O AcOH (60:40:1) fraction. The fraction
was concentrated to dryness, and 0.70 g extract was
obtained. This extract was applied to preparative
HPLC with an ODS column (Wakosil-II, 5C18HG
Prep, 20 × 250 mm; Wako Pure Chemical Industries).
HPLC conditions were as follows: solvents: 0.1%
TFA in H2O (A); acetonitrile (B); gradient: 28–35%
B/(A + B) within 40 min; flow rate: 7 mL/min;
column temperature: 40 °C; and detection: 340 nm.
Peaks 1 and 3 were eluted at 12.2 and 17.3 min,
respectively.
Materials and methods
General experimental procedures.
1H and 13C
NMR spectra were recorded on a Bruker Avance II 600
MHz spectrometer (Bruker, Billerica, MA, USA).
Chemical shifts were referenced to CD3OD (δH 3.31)
and (CD3)2SO (δH 2.50, δC 39.5). Positive and negative
ESI MS were obtained using a Waters Quattro Micro
mass spectrometer combined with a Waters PDA UPLC
system equipped with an ODS column (Acquity UPLC
BEH C18, 1.7 μm, 2.2 × 50 mm; Waters, Milford, MA,
USA). The HRMS were obtained with an Exactive mass
spectrometer (Thermo Fisher Scientific, Waltham, MA,
USA). Preparative HPLC was performed using an ODS
column (Wakosil-II, 5C18HG Prep, 20 × 250 mm; Wako
Pure Chemical Industries, Osaka, Japan; Cosmosil

New avenanthramides in oat seed
3
Since the fraction corresponding to peak 1 con-
tained impurities, the fraction was further subjected
to HPLC purification with a small column (Mightysil
RP-18 GP, 150 × 4.6 mm (3 μm); Kanto Kagaku,
Tokyo, Japan). HPLC conditions were as follows:
solvents: 0.1% TFA (A), MeCN (B); elution: 20%
A/(A + B); flow rate: 0.6 mL/min, column tempera-
ture: 40 °C, and detection 340 nm. The fraction cor-
responding to peak 1 (Rt. 9.6 min) was concentrated
and subjected to a small ODS column (Cosmosil
75C18-PREP) to remove TFA. The column was
washed with H2O and eluted with MeOH H2O (8:2)
to give compound 1.
4,5-Dihydroxyanthranilic acid. 4,5-dihydroxyanth-
ranilic acid was prepared from 4,5-dimethoxyanthranilic
acid according to Breuer et al.22) 2-Amino-4,5-dimeth-
oxybenzoic acid (0.99 g, 5.0 mmol) was mixed with
hydrobromic acid (25 mL) and the mixture was
refluxed for 3 h, and then evaporated in vacuo. After
washing with ether, this material was dissolved in a
small amount of ice water. The pH of the solution was
adjusted to 4 with saturated NaHCO3 solution, precipi-
tating 4,5-dihydroxyanthranilate.
Dark violet solid (0.50 g, 3.0 mmol, yield 60%). ¹H
NMR (600 MHz, (CD3)2SO): δH (ppm) 9.42 (brs, 1H,
OH), 8.23 (brs, 1H, OH), 8.02 (brs, 3H, NH3), 7.07
(s, 1H, H-6), 6.13 (s, 1H, H-3). 13C NMR (150 MHz,
(CD3)2SO): δC (ppm) 169.5 (COOH), 152.4 (C-4),
147.2 (C-2), 135.9 (C-5), 116.7 (C-6), 102.5 (C-1),
101.1 (C-3).
1
1 (1.0 mg). H NMR (600 MHz, CD3OD): δH (ppm)
8.23 (s, 1H, H-3), 7.51 (d, 1H, J = 15.6 Hz, H-7′), 7.50
(s, 1H, H-6), 7.07 (d, 1H, J = 2.0 Hz, H-2′), 6.98 (dd,
1H, J = 2.0 Hz and 8.1 Hz, H-6′), 6.79 (d, 1H, J = 8.1
Hz, H-5′), 6.46 (d, 1H, J = 15.6 Hz, H-8′). HR-ESI-MS:
m/z 332.0764 ([M + H]+) (m/z 332.0765 calcd. for
C16H14O7N).
N-Caffeoyl-4,5-dihydroxyanthranilic acid.
4,5-Di-
hydroxyanthranilic acid (43.3 mg, 0.26 mmol) was dis-
solved in distilled H2O (5 mL). The pH of the solution
was adjusted to 8.0 by adding NaHCO3 (12.5 mg, 0.15
mmol). Caffeic acid N-hydroxysuccinimide ester (12.5
mg, 0.049 mmol) was dissolved in 5 mL acetone and
was added to the solution of 4,5-dihydroxyanthranilic
acid. After standing for 24 h, acetone was removed by
evaporation. The pH of the solution was neutralized by
adding AcOH. The mixture was applied to an ODS
column equilibrated with MeOH H2O AcOH (5:95:1).
The column was eluted with 450 mL methanol
H2O-AcOH (5:95:1, 60:40:1) and MeOH. The MeOH-
H2O-AcOH (60:40:1) fraction was concentrated and
subjected to preparative HPLC. HPLC conditions were
the same as for isolation of 1 from oat grain extract.
Yellowish black solid (4.0 mg, 0.012 mmol, 24.5%).
1H NMR (600 MHz, CD3OD): δH (ppm) 8.23 (s, 1H,
H-3), 7.50 (d, 1H, J = 15.6, H-7′), 7.50 (s, 1H, H-6),
7.07 (d, 1H, J = 2.0 Hz, H-2′), 6.98 (dd, 1H, J = 2.0
and 8.2 Hz, H-6′), 6.79 (d, 1H, J = 8.2 Hz, H-5′), 6.45
(d, 1H, J = 15.6 Hz, H-8′). ¹H NMR (600 MHz,
(CD3)2SO): δH (ppm) 13.01 (brs, 1H, NH), 11.28 (brs,
1H, COOH), 10.07 (brs, 1H, OH), 9.52 (brs, 1H, OH),
9.14 (brs, 1H, OH), 9.11 (brs, 1H, OH), 8.19 (s, 1H,
H-3), 7.39 (d, 1H, J = 15.8 Hz, H-7′), 7.37 (s, 1H,
H-6), 7.07 (s, 1H, H-2′), 6.98 (d, 1H, J = 7.9 Hz, 1H,
H-6′), 6.76 (d, 1H, J = 7.9 Hz, 1H, H-5′), 6.42 (d, 1H,
J = 15.8 Hz, 1H, H-8′). 13C NMR (150 MHz,
(CD3)2SO): δC (ppm) 169.6 (C-7), 163.5 (C-9′), 150.9
(C-4), 147.8 (C-4′), 145.6 (C-3′), 141.1 (C-7′), 140.4
(C-5), 135.5 (C-2), 126.0 (C-1′), 121.0 (C-6′), 118.9
(C-8′), 117.1 (C-6), 115.7 (C-5′), 114.4 (C-2′), 107.3
(C-3), 106.8 (C-1).
The fraction corresponding to peak 3 also contained
impurities. The fraction was subjected to preparative
HPLC with a small column (Mightysil RP-18 GP,
150 × 4.6 mm (3 μm)). Peak 3 was eluted at 21.4 min.
HPLC conditions were as follows: solvents: 0.1% TFA
(A), MeCN (B); elution: 25% B/(A + B); flow rate: 0.6
mL/min, column temperature: 40 °C, and detection:
340 nm.
3 (2.5 mg). ¹H NMR (600 MHz, CD3OD): δH (ppm)
8.24 (s, 1H, H-3), 7.57 (d, 1H, J = 15.5 Hz, H-7′), 7.51
(s, 1H, H-6), 7.24 (d, 1H, J = 1.9 Hz, H-2′), 7.10 (dd,
1H, J = 1.9 Hz and 8.2 Hz, H-6′), 6.83 (d, 1H, J = 8.2
Hz, H-5′), 6.55 (d, 1H, J = 15.5 Hz, H-8′), 3.93 (s, 3H).
HR-ESI-MS: m/z 346.0921 ([M + H]+) (m/z 346.0921
calcd. for C17H16O7N).
Oat seeds (150 g) were extracted with 1 L of MeOH
three times, washed with hexane, and fractionated
with an ODS column in a similar way to the isolation
of 1 and 3. The MeOH-H2O-AcOH (60:40:1) fraction
was subjected to preparative HPLC with an ODS col-
umn (Cosmosil Packed Column 5C18-ARII, 10 × 250
mm) with flow rate of 3 mL/min . The conditions for
preparative HPLC other than the flow rate were the
same as for the isolation of 1 and 3. The compound
corresponding to peak 2 was eluted at 9.0 min. The
fraction was concentrated to a small volume, and TFA
was removed using a small ODS column to give
compound 2.
1
2 (1.0 mg). H NMR (600 MHz, CD3OD): δH (ppm)
8.22 (s, 1H, H-3), 7.56 (d, 1H, J = 15.7 Hz, H-7′), 7.51
(s, 1H, H-6), 7.48 (d, 2H, J = 8.5 Hz, H-2′, 6′), 6.82 (d,
2H, J = 8.5 Hz, H-3′, 5′), 6.51 (d, 1H, J = 15.7 Hz, H-
8′). HR-ESI-MS: m/z 316.0814 ([M + H]+) (m/z
316.0821 calcd. for C16H14O7 N).
N-p-Coumaroy-4,5-dihydroxyanthranilic acid. N-p-
Coumaroy-4,5-dihydroxyanthranilic acid was synthe-
sized from 4,5-dihydroxyanthranilic acid (76.5 mg, 0.45
mmol) and p-coumaric acid N-hydroxysuccinimide ester
(361.5 mg, 1.53 mmol) in a similar way to the synthesis
of N-caffeoyl-4,5-dihydroxyanthranilic acid. Preparative
HPLC gave N-coumaroyl-4,5-dihydroxyanthranilic acid.
HPLC conditions were the same as for isolation of 2
from oat seed extract.
Chemicals. N-hydroxysuccinimide esters of p-cou-
maric, ferulic, and caffeic acids were prepared by
transesterification, according to the method described
by Stöckigt and Zenk.20) Syntheses of avenanthramides
A, B, C, and D (N-4-hydroxycinnamoyl-5-hydroxyanth-
ranilic acid) and L (N-[5-(4-hydroxyphenyl)-penta-2, 4-
dienoyl]-5-hydroxyanthranilic acid) are described else-
where.1,21)

4
A. Ishihara et al.
Pale brown solid (53.5 mg, 0.17 mmol, yield 38%).
equivalent of each avenanthramide was calculated. In
this study, Trolox equivalent means Trolox amount
(moles) that shows the same DPPH radical-scavenging
activity with 1 mol of each avenanthramide.
1H NMR (600 MHz, CD3OD): δH (ppm) 8.22 (s, 1H,
H-3), 7.56 (d, 1H, J = 15.6 Hz, H-7′), 7.51 (s, 1H, H-
6), 7.48 (d, 2H, J = 8.6 Hz, H-2′, 6′), 6.82 (d, 2H,
J = 8.6 Hz, H-3′, 5′), 6.51 (d, 1H, J = 15.6 Hz, H-8′). ¹H
NMR (600 MHz, (CD3)2SO): δH (ppm) 13.01 (brs, 1H,
NH), 11.29 (brs, 1H, COOH), 10.08 (brs, 1H, OH),
9.95 (brs, 1H, OH), 9.11 (brs, 1H, OH), 8.22 (s, 1H,
H-3), 7.55 (d, 2H, J = 8.6 Hz, H-2′, 6′), 7.48 (d, 1H,
J = 15.5 Hz, H-7′), 7.38 (s, 1H, H-6), 6.80 (d, 1H,
J = 8.6 Hz, 1H, H-3′, 5′), 6.54 (d, 1H, J = 15.5 Hz, 1H,
H-8′). 13C NMR (150 MHz, (CD3)2SO): δC (ppm)
169.6 (C-7), 163.6 (C-9′), 159.3 (C-4′), 150.9 (C-4),
140.8 (C-7′), 140.4 (C-5), 135.6 (C-2), 129.9 (C-2′, 6′),
125.5 (C-1′), 119.0 (C-8′), 117.1 (C-6), 115.7 (C-3′,
5′), 107.3 (C-3), 106.8 (C-1).
Determination of avenanthramide contents in groats
by MRM of LC-MS/MS analysis. Ten oat groats were
homogenized with a mortar and pestle to a powder.
The powder was extracted with 20 volumes of MeOH
for 24 h. After filtration (Millex-LH, 0.45 μm;
Millipore, Billerica, MA, USA), the extract was sub-
jected to MRM of LC-MS/MS (Waters UPLC coupled
with Quattro Micro). The amounts of avenanthramides
in groats of oat cultivar Shokan 1 were calculated on
the basis of calibration curve generated by analysis of
external standards. UPLC conditions were as follows:
column: Acquity UPLC BEH C18 1.7 μm (2.1 × 100
mm; Waters); solvents: A: 0.1% HCOOH in H2O and
B: 0.1% HCOOH in MeCN; gradient: 15–55%
B/ (A + B) within 10 min. MRM conditions were
optimized with synthesized compounds (Table S1).
N-Feruloyl-4,5-dihydroxyanthranilic acid. N-Feru-
loyl-4,5-dihydroxyanthranilic acid was synthesized
from 4,5-dihydroxyanthranilic acid (37.2 mg, 0.22
mmol) and ferulic acid N-hydroxysuccinimide (205.6
mg, 0.77 mmol) by a procedure similar to the synthesis
of N-caffeoyl-4,5-dihydroxyanthranilic acid. The com-
pound was purified by preparative HPLC. HPLC condi-
tions were the same as for the isolation of 3.
Results and discussion
Identification of avenanthramides
Yellow solid (23.4 mg, 0.068 mmol, 31%) ¹H NMR
(600 MHz, CD3OD): δH (ppm) 8.24 (s, 1H, H-3), 7.56
(d, 1H, J = 15.5 Hz, H-7′), 7.51 (s, 1H, H-6), 7.24 (d,
1H, J = 1.7 Hz, H-2′), 7.10 (dd, 1H, J = 1.7 Hz and 8.2
Hz, H-6′), 6.83 (d, 1H, J = 8.2 Hz, H-5′), 6.55 (d, 1H,
J = 15.5 Hz, H-8′), 3.93 (s, 3H). ¹H NMR (600 MHz,
(CD3)2SO): δH (ppm) 12.97 (brs, 1H, NH), 11.27 (brs,
1H, COOH), 10.08 (brs, 1H, OH), 9.54 (brs, 1H, OH),
9.12 (brs, 1H, OH), 8.23 (s, 1H, H-3), 7.47 (d, 1H,
J = 15.5 Hz, H-7′), 7.38 (s, 1H, H-6), 7.32 (s, 1H, H-
2′), 7.11 (d, 1H, J = 8.0 Hz, 1H, H-6′), 6.78 (d, 1H, J =
8.0 Hz, 1H, H-5′), 6.63 (d, 1H, J = 15.5 Hz, 1H, H-8′),
3.83 (s, 3H, OCH3). 13C NMR (150 MHz, (CD3)2SO):
δC (ppm) 169.5 (C-7), 163.7 (C-9′), 150.9 (C-4), 148.8
(C-4′), 147.9 (C-3′), 141.2 (C-7′), 140.4 (C-5), 135.6
(C-2), 126.1 (C-1′), 122.6 (C-6′), 119.3 (C-8′), 117.1
(C-6), 115.6 (C-5′), 111.2 (C-2′), 107.4 (C-3), 106.7
(C-1), 55.7 (OCH3).
To find new avenanthramides, we analyzed a MeOH
extract of oat seeds by HPLC (Fig. 2(A)). Detected
peaks 1 and 3 were collected, concentrated, and
subjected to LC-PDA-MS analysis. For a small-scale
isolation of peak 2, a different gradient program with a
larger column was applied (Fig. 2(B)). The respective
positive ESI mass spectra of peaks 1, 2, and 3 showed
ions at m/z 332, 316, and 346 as [M + H]+ ions and
ions at m/z 163, 147, and 177 as fragment ions
(Fig. 3). In negative ESI mass spectra, peaks 1, 2, and
3 showed ions at m/z 330, 314, and 344 as [M − H]−,
suggesting that their molecular masses are 331, 315,
and 345, respectively. Because we recorded ESI mass
spectra in MS scan mode, fragment ions are considered
to be generated in ionization process. Ions at m/z 163,
147, and 177 are characteristic fragment ions of com-
pounds bearing caffeoyl, p-coumaroyl, and feruloyl
moieties. The subtraction of masses of the fragment
ions from molecular masses was commonly 168. This
suggested that the remaining part of the molecules is
dihydroxyanthranilic acid moiety, with a mass of 168,
because, in avenanthramides, fragment ions are gener-
ated by the cleavage of the amide bond between
anthranilate and hydroxycinnamoyl moieties.
DPPH assay. Anti-oxidant activity of avenanthra-
mides was evaluated by the DPPH assay according to
the method described by Oki et al. with modifica-
tions.23) Trolox (Wako Pure Chemical Industries) solu-
tions (50, 100, 150, and 200 μM) in 80% EtOH were
added to a mixture of 300 μL of 400 μM 1,1-diphenyl-
2-picrylhydrazyl (DPPH; Wako Pure Chemical Indus-
tries) in ethanol, 300 μL of 200 mM MES buffer
(pH6.0), and 200 μL of 20% EtOH, and vortexed.
After 30 min incubation, the absorbance at 520 nm was
measured with a Hitachi U-2001 spectrophotometer. In
the same way, avenanthramide solutions in 80% EtOH
at different concentrations were added to a mixture
containing DPPH, and absorbance at 520 nm was mea-
sured 30 min after adding avenanthramide. From the
slope of the graph generated by plotting absorbance
values as a function of concentration, the Trolox
Then, we performed large-scale extraction of oat
seeds. The obtained MeOH extract was fractionated by
ODS column chromatography, and the 60% MeOH
fraction was subjected to preparative HPLC. Finally,
we purified compounds 1, 2, and 3, which corre-
sponded to peaks 1, 2, and 3.
The molecular formula of 1 was revealed to be
C16H13O7N on the basis of HR-ESI-Orbitrap MS. This
is compatible with the idea that 1 is avenanthramide
composed of dihydroxyanthranilic acid and caffeic
1
acid. H NMR spectrum exhibited signals correspond-
ing to a trans double bond (δH 7.57 and 6.55) and to a
1,2,4-trisubstituted benzene ring (δH 7.07, 7.04, and

New avenanthramides in oat seed
5
Fig. 2. HPLC analysis of avenanthramides in oat groats (A) and chromatogram for the isolation of 2 (B).
Notes: MeOH extract of oat groats was subjected to analytical HPLC. HPLC conditions were described in the part of general experimental proce-
dures in materials and methods section. Sixty percent MeOH fraction obtained by ODS column chromatography of oat groat extract was subjected
to preparative HPLC. HPLC conditions were described in the part of isolation of avenanthramides in materials and methods section.
Fig. 3. Mass and absorption spectra of peak 1, 2, and 3.
Notes: Positive (A, D, and G) and negative (B, E, and H) ESI mass spectra, and absorption spectra (C, F, and I) of peak 1, 2, and 3.
6.79). These signals were assigned to a caffeoyl moiety.
The remaining signals were two singlets (δH 8.23, and
7.50), suggesting the presence of 4,5-dihydroxyanthra-
nilic acid.
This compound was synthesized from 4,5-dihydroxy-
anthranilic acid and caffeic acid. First, 4,5-dihydroxy-
anthranilic acid was prepared by demethylation of 4,5-
dimethoxyanthranilic acid by refluxing with HBr. Then,
we tried direct condensation with 4,5-dihydroxyanthra-
nilic acid and caffeic acid by dicyclohexylcarbodiimide
(DCC) in various solvents. However, the formation of
target compound was not detected by LC-MS analysis
of the reaction mixture. Thus, we employed the con-
densation reaction with activated caffeic acid with 4,5-
dihydroxyanthranilic acid. Caffeic acid was converted
to N-hydroxysuccinimide ester by the condensation
reaction with N-hydroxysuccinimide using DCC. Then,
N-hydroxysuccinimide ester of caffeic acid was reacted

6
A. Ishihara et al.
with 4,5-dihydroxyanthranilic acid under alkaline con-
ditions. The 1H NMR spectrum and chromatographic
behavior of 1 and the synthesized compound were
identical, indicating that 1 is N-caffeoyl-4,5-dihydroxy-
anthranilic acid.
N-benzoyl-4-hydroxyanthranilic acid was indicated to
be formed by the hydroxylation of N-benzoylanthranilic
acid on the basis of the detection of hydroxylase activ-
ity.29) Similar hydroxylation of anthranilate moiety may
form 4- and 5-hydroxyanthranilic and 4,5-dihydroxy-
anthranilic acids in oats. In addition, it is plausible that
the biosynthetic routes of avenanthramides may consist
of a metabolic grid. To resolve this issue, extensive
feeding studies with labeled avenanthramides are
needed.
The molecular formula of 2 was revealed to be
C16H13O6N on the basis of HR-ESI-Orbitrap MS. The
molecular formula agreed with that of N-p-cou-
marroyldihydroxyanthranilic acid. 1H NMR showed
signals corresponding to a trans double bond (δH 7.56
and 6.51), and a 1,4-disubstituted benzene ring (δH
7.48 and 6.82). These signals were assigned to the
p-coumaroyl moiety. The spectrum also showed two
singlets (δH 8.22 and 7.51) corresponding to
4,5-dihydroxyanthranilic acid. The identity was con-
firmed by the synthesis of the compound from
4,5-dihydroxyanthranilic acid and p-coumaric acid in a
similar way to the synthesis of N-caffeoyl-4,5-dihy-
Determination of avenanthramide contents in oat
groats
Conditions for MRM of avenanthramides were opti-
mized with synthetic compounds (Table S1). Avenan-
thramides were detected by MRM of LC-MS/MS
analysis, as shown in Fig. 4. The amounts of avenan-
thramides in groats of oat cultivar Shokan 1 were cal-
culated on the basis of calibration curve generated by
analysis of external standards. The results are summa-
rized in Table 1. The amounts of avenanthramides with
4,5-dihydroxyanthranilic acid were 26.9–16.5% of cor-
responding avenanthramides with 5-hydroxyanthranilic
acid. In this experiment we extracted 10 groats
together, and 9 samples were analyzed. The results of
analysis still showed a large variation in the avenan-
thramide contents, indicating that the amounts largely
differ among groats.
1
droxy anthranilic acid. The H NMR and mass spectra
and chromatographic behavior of 2 were identical to
those of synthesized N-p-coumaroyl-4,5-dihydroxyanth-
ranilic acid. Blaakmeer et al. synthesized this com-
pound and reported its oviposition deterrent activity for
1
Pieris brassicae.24) The H NMR data was identical to
that reported by Blaakmeer et al.24)
The molecular formula of 3 was determined to be
C17H15O6N on the basis of HR-ESI-Orbitrap MS.
The molecular formula suggested that 3 is N-fer-
uloyldihydroxyanthranilic acid. Supporting this, 1H
NMR showed signals corresponding to a trans double
bond (δH 7.56 and 6.51), a 1,3,4-substituted benzene
ring (δH 7.24, 7.10 and 6.83), and a methoxy group
(δH 3.83). These signals were assigned to a feruloyl
moiety. The spectrum also showed two singlets (δH
8.22 and 7.51) corresponding to 4,5-dihydroxyanthra-
nilic acid part. N-Feruloyl-4,5-dihydroxyanthranilic
acid was synthesized from 4,5-dihydroxyanthranilic
acid and ferulic acid in a similar way to the synthesis
of 1 and 2. Their identical 1H NMR and ESI-mass
spectra, and the same chromatographic behavior on
reversed phase HPLC indicated that 3 is N-feruloyl-
4,5-dihydroxyanthranilic acid.
Major avenanthramides in oat seeds are those com-
posed of 5-hydroxyanthranilic acid. Avenanthramides
with anthranilic, 4-hydroxyanthranilic, and 4-meth-
oxyl-5-hydroxyanthranilic acids have also been identi-
fied. In addition, Wise described the detection of 2
as avenanthramide 5p by ESI-MS.25) The same
author also showed a chromatogram of avenanthra-
mides in oat grain and indicated the peak of 3, but
no spectroscopic data were reported.26) Collins also
described the presence of avenanthramides with
4,5-dihydroxyanthranilic acid, but without showing
any spectroscopic data.27)
DPPH
radical-scavenging
activity
of
avenanthramides
We evaluated anti-oxidant activity by the DPPH
method according to Oki et al.23) Trolox equivalent of
the DPPH radical-scavenging activity of avenanthra-
mides is summarized in Table 2. Avenanthramide
composed of 4,5-dihydroxyanthranilic acid scavenged
DPPH radical more effective than the corresponding
avenanthramides composed of 5-hydroxyanthranilic
acid. In the comparison of the activity of avenanthra-
mides with 5-hydroxyanthranilic acid, the radical-scav-
enging activity of avenanthramides was dependent on
the hydroxycinnamoyl moiety; the order of activity was
caffeoyl > feruloyl > p-coumaroyl. This tendency was
also observed for avenanthramides with 4,5-dihydroxy-
anthranilic acid, although the difference in the activities
of avenanthramides with p-coumaroyl and feruloyl
moiety was small.
Radical-scavenging activity of avenanthramides was
evaluated by DPPH assay by Peterson et al.,
Fagerlund et al., and Bratt et al.18,19,30) The order of
activity in our assay for avenanthramide A, B, and C
was the same as their results. As demonstrated for
hydroxycinnamic acids, ortho substitution with an
electron-donating methoxy group in the ferulic acid
moiety is considered to increase its activity by stabi-
lizing the phenoxy radical.31−33) On the other hand,
the presence of a second hydroxyl group in the ortho
position of the 4-hydroxy group in p-coumaric acid is
known to increase radical-scavenging activity by
additional resonance stabilization and o-quinone for-
mation.33−35) The small Trolox equivalent of avenan-
thramide D indicates that the contribution of the
Because of the presence of avenanthramide with 4-
methoxyl-5-hydroxyanthranilic acid,28) the occurrence
of avenanthramides with 4,5-dihydroxyanthranilic acid
is not surprising. Avenanthramides were shown to be
synthesized by the condensation of anthranilic acids
and hydroxycinnamoyl-CoAs.8,9) Enzyme HHT
accepted anthranilic acid and 4- and 5-hydroxyanthrani-
lic acids as substrates, but not 3-hydroxyanthranilic
acid. Thus, the enzyme may accept 4,5-dihydroxyanth-
ranilic acid. On the other hand, in carnation,

New avenanthramides in oat seed
7
Fig. 4. Analysis of avenanthramides in oat groats by MRM of LC-MS/MS analysis.
Notes: Compound 1 (A), avenanthramide C (B), compounds 2 (C) and 3 (D), and avenanthramides A (E), B (F), L (G), and D (H) were
extracted from oat groats and analyzed by LC-MS/MS.
Table 1. Amounts of avenanthramides in oat groats. Ten groats
were extracted and analyzed. The values are the average of nine repli-
cates with SD.
avenanthramide D. The introduction of another
hydroxyl group at 4-position of anthranilate further
Avenanthramides
Amount (nmol/gFW)
increased activity. This occurred for the same reason
as the increase in radical-scavenging activity in the
introduction of the ortho hydroxyl group in the caf-
feoyl moiety. Moreover, the relatively large Trolox
equivalent value of 2 indicates that the activity of 2
is mainly attributable to 4,5-dihydroxyanthranilic acid
in the molecule. Similarly, the enhancement of DPPH
radical-scavenging activity by introducing a methoxy
group at 4-position on the anthranilic moiety in aven-
anthramides was reported by Fagerlund et al.19) Con-
sidering the high radical-scavenging activity of 1–3
and their relatively larger amounts, it is likely that
they significantly contribute to the anti-oxidant activ-
ity of oat seed extract.
A
B
C
1
2
3
101.7 34.4
73.9 20.8
173.8 60.8
46.8 16.4
16.8 5.5
18.7 7.3
65.2 25.2
5.4 1.8
L
D
hydroxyl group of p-coumaroyl moiety to DPPH
radical scavenging is marginal.
The 5-hydroxyl group on anthranilic acid was
shown to also contribute to the radical-scavenging
activity of avenanthramides, because the activity of
avenanthramide A is substantially higher than that of
Avenanthramides have been shown to have bioactivi-
ties mainly in relation to the anti-inflammatory pro-
cess.14,36,37) It would be of interest to assay the activity
of newly identified avenanthramides in various experi-
mental systems.

8
A. Ishihara et al.
Table 2. Trolox equivalent of avenanthramides determined by DPPH assay. Values indicate Trolox amounts (moles) that show the same DPPH
radical-scavenging activity with 1 mol of each avenanthramide. Abbreviated names and compound numbers were shown in parentheses. Values are
averages of 3 replicates with SD.
Hydroxycinnamoyl moiety of avenanthramides
Anthranilate moiety of avenanthramides
p-Coumaroyl
Feruloyl
Caffeoyl
5-Hydroxyanthranilate
4,5-Dihydroxyanthranilate
Anthranilate
0.38 0.03 (Av. A)
0.76 0.03 (2)
0.018 0.005 (Av. D)
0.66 0.05 (Av. B)
1.06 0.09 (Av. C)
0.84 0.04 (3)
2.08 0.22 (1)
a
a
–
–
aNot determined.
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Supplemental material
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2014.946390.
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This study was supported by the Foundation for
Dietary Scientific Research and by the Adaptable
and Seamless Technology Transfer Program (A-step)
through Target-driven R&D, Japan Science and Tech-
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