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R E S E A R C H Open Access
Avenanthramide supplementation attenuates
exercise-induced inflammation in
postmenopausal women
Ryan Koenig
1
, Jonathan R Dickman
1
, Chounghun Kang
2
, Tianou Zhang
2
, Yi-Fang Chu
3
and Li Li Ji
1,2*
Abstract
During aging, chronic systemic inflammation increases in prevalence and antioxidant balance shifts in favor of
oxidant generation. Avenanthramide (AVA) is a group of oat phenolics that have shown anti-inflammatory and
antioxidant capability. The present study investigated whether dietary supplementation of avenanthramides (AVA)
in oats would increase antioxidant protection and reduce inflammation after a bout of downhill walking (DW) in
postmenopausal women. Women at age of 50–80 years (N = 16) were randomly divided into two groups in a
double-blinded fashion, receiving two cookies made of oat flour providing 9.2 mg AVA or 0.4 mg AVA (control, C)
each day for 8 weeks. Before and after the dietary regimen, each group of subjects walked downhill on a treadmill
(−9% grade) for 4 bouts of 15 minutes at a speed of 4.0 km/h with 5 minutes rest between sessions. Blood samples
were collected at rest, 24 h post-DW, and 48 h post-DW pre- and post-supplementation. Both DW sessions
increased plasma creatine kinase activity (P < 0.05). Before supplementation, in vitro neutrophil respiratory burst
(NRB) activity was increased at 24 h post-DW (P < 0.05) and C-reactive protein (CRP) was increased 48 h post-DW
(P < 0.05). AVA supplementation decreased DW-induced NRB at 24 h (P < 0.05) and CRP level 48 h (P < 0.05). Plasma
interleukin (IL)-1βconcentration and mononuclear cell nuclear factor (NF) κB binding were suppressed at rest and
during post-DW period in AVA but not C group (P < 0.05). Plasma total antioxidant capacity (P < 0.05) and
erythrocyte superoxide dismutase activity were increased in AVA vs. C (P < 0.05), whereas glutathione redox status
was elevated 48 h post-DW but not affected by AVA. Thus, chronic AVA supplementation decreased systemic and
DW-induced inflammation and increased blood-borne antioxidant defense in postmenopausal women.
Introduction
The skeletal muscle of aged individuals decreases muscle
mass, force generation and metabolic functions known as
sarcopenia. Recent research points to a strong link be-
tween aging and inflammation [1,2]. So many diseases
have been identified to have an etiological origin of in-
flammation that the term “inflammaging”has been coined
[3-6]. Therefore, developing strategies to prevent and re-
duce inflammation in the aging population has become a
priority in gerontological research in recent years.
The increase in inflammation during aging has been
linked to increased nuclear factor (NF) κB binding to
DNA in many organs and tissues, as well as several types
of blood borne cells [7]. NFκB is sensitive to oxidative
stress and a variety of other stimuli and is responsible
for the regulation of the transcription of a variety of
gene targets, including pro-inflammatory cytokines such
as interleukin (IL)-1, 6 and tumor necrosis factor (TNF)-
α[8]. Aged rats and mice displayed increased nuclear
NFκB binding activity in several major organs studied,
whereas no increase in cytoplasmic NFκB was observed
[9]. IL-1 and 6 gene expression in the T cells of older hu-
man subjects was elevated compared with younger
counterparts with or without NFκB induction, indicating
aging is associated with immune dysregulation resulting
in a pro-inflammatory state [10].
In women the phenomenon of menopause results in a
lack of production of estrogen, which adds complexity
to the aging milieu. Estrogens function as antioxidants,
* Correspondence: lllji@umn.edu
1
Department of Kinesiology, University of Wisconsin–Madison, Madison,
WI 53706, USA
2
Laboratory of Physiological Hygiene and Exercise Science, University of
Minnesota, 1900 University Avenue, Minneapolis, MN 55455, USA
Full list of author information is available at the end of the article
© 2014 Koenig et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Koenig et al. Nutrition Journal 2014, 13:21
http://www.nutritionj.com/content/13/1/21
and their absence in postmenopausal women could con-
tribute to an increased susceptibility to oxidative stress
[11]. Estrogen was recently shown to up-regulate anti-
oxidant enzymes via mitogen activated protein kinase
(MAPK) and NFκB pathways [12]. In addition estrogens
may function to stabilize cell membranes and to regulate
cell signaling through the binding to estrogen receptors
[13]. These mechanisms are thought to provide import-
ant protection from muscle damage to women following
a bout of unaccustomed exercise. Indeed, postmeno-
pausal women exhibited increased serum creatine kinase
(CK) and lactate dehydrogenase (LDH) as well as in-
creased mRNA expression of pro-inflammatory cyto-
kines following strenuous eccentric exercise compared
to their counterparts with hormone therapy [14].
Downhill walking is a muscular activity that involves
lengthening or eccentric contraction (EC) and breaks
weaken myofibrils and activate proteases and lipases,
followed by immunological responses such as infiltration
of neutrophils, free radical generation and expression of
pro-inflammatory cytokines and chemokines [15]. NFκB
activation escalates the process and provokes systemic
inflammation that could have broad health outcomes
such as muscle pain, chronic inflammation (rheuma-
toid), leading to underperformance and fear of participa-
tion in exercise and sports. However, recent research
have shown pharmacological treatments of EC-induced
inflammation such as NSAID might interrupt normal
healing process and large doses supplementation of anti-
oxidants of pharmaceutical source can be more detri-
mental than beneficial as it interferes with intrinsic
adaptive responses and sometimes takes away the benefit
of exercise [16,17]. Thus, seeking phytochemicals dem-
onstrating antioxidant and anti-inflammatory properties
for daily dietary supplements is desirable.
Oat (Avena sativa), although consumed in consider-
ably lower quantities worldwide than wheat and rice, has
a highly edible quality and contains high antioxidants
such as tocopherols, tocotrienols, and flavonoids [18]. In
addition, oat contains a unique group of approximately
40 different types of avenanthramides (AVA) that consist
of an anthranilic acid derivative and a hydroxycinnamic
acid derivative linked by a pseudo- peptide bond [19]. Of
all the AVA that have been identified, three stand out
due to their abundance and have been labeled as AVA-
A, −B, and –C, which differ by a single moiety on the
hydroxycinnamic acid ring. All three AVA of interest
showed antioxidant activity with AVA-C being the most
potent [20]. Additional studies performed have shown
that AVA have the anti-inflammatory and anti-
atherogenic effects of decreasing monocyte adhesion to
human aortic endothelial cells (HAEC), as well as their
expression of adhesion molecules and pro-inflammatory
cytokines [21]. AVA-C displayed further antiatherogenic
potential by inhibiting vascular smooth muscle cell
(SMC) proliferation and enhancing nitric oxide produc-
tion in both SMC and HAEC in parallel with the up-
regulation of mRNA expression of endothelial nitric
oxide synthase [22]. These effects were shown to be de-
rived from decreased NFκB activity [23].
The antioxidant, anti-inflammatory, and NFκB inhibi-
tory properties of AVA make it a candidate for supple-
mentation in the cause of decreasing inflammation and
muscle damage in post-menopausal women. Thus, the
present study was designed to test the anti-inflammatory
and antioxidant capability of AVA in postmenopausal
women. We hypothesize that AVA supplementation
would increase plasma antioxidant defense, inhibit
NFκB-DNA binding in the mononuclear cells and de-
crease DW-induced systemic inflammation.
Materials and methods
A. Subjects
Older women aged 50–80 years were recruited from the
Madison, Wisconsin, community or from faculty and
staff of the University of Wisconsin-Madison. The re-
cruitment procedure and study conducts were approved
by the Health Science Institutional Review Board for
Human Subjects of UW-Madison. The subjects were
randomly assigned to one of two groups (N = 8 per
group) receiving a high dose of AVA supplementation in
the diet, or receiving a low dose of AVA present in nor-
mal oats, serving as Control. Other than this difference,
the groups were treated exactly the same in a double-
blind fashion.
All participants gave informed consent before enrol-
ling in the study. They also completed a Health History
Survey to ensure that they were eligible for the study
and healthy enough to exercise. Criteria for exclusion
from the study were smoking or other tobacco use,
drinking alcohol in excess of 5 drinks per week, use of
dietary antioxidants, blood pressure medication, non-
steroidal anti-inflammatory drugs (NSAIDs) and antico-
agulants or antidiabetic or hypoglycemic drugs.
B. Dietary supplementation
Because the goal of the study is to study the biological
efficacy of AVA, we employed a dietary regimen wherein
both groups of subjects were supplemented with oats,
which differed only in AVA concentration but processed
identical nutritional and antioxidant contents (see
below). Both dietary groups of subjects received cookies
made with oat samples with standardized AVA concen-
tration provided courtesy of Ceapro Inc. (Edmonton,
AB, Canada). AVA concentrations were verified by high-
performance liquid chromatography (HPLC) in our la-
boratory. The high-AVA oat flour contained 190 mg/kg,
and the Control group flour contained 8 mg/kg, the
Koenig et al. Nutrition Journal 2014, 13:21 Page 2 of 11
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lowest AVA concentration among all oat lines available.
The recipe for each type of cookie was identical except
in the type of flour used. Each cookie contained 30 g
flour (high- or low-AVA), 5.91 mL unsweetened apple
sauce (Surefine), 12.32 mL artificial sweetener (Natra-
taste Gold), 0.616 mL baking soda, and 0.0308 mL table
salt. They were baked in a low temperature oven (121°C)
for 15 minutes to ensure that AVA was not broken down
or produced by the oat during the process. AVA concen-
tration in the high-AVA cookies was 4.6 mg/cookie
(9.2 mg/day), and it was 0.2 mg/cookie (0.4 mg/day) in
the control cookies. Each cookie provided 125 kilocalo-
ries (250 kcal/day) regardless of group.
Supplementation began on the evening of the second
study visit following the third blood draw and ended on
the evening of the third study visit following the fifth
blood draw (see below). Subjects were furnished with
cookies and instructed to consume two per day: one in
the morning with breakfast and one in the evening with
dinner.
C. Study visits
A total of six visits were required for each subject follow-
ing recruitment and consenting. There were three pre-
supplementation visits and three post-supplementation
visits identical to the pre-supplementation visits. The first
visit of each trio consisted of completion of the Inter-
national Physical Activity Questionnaire (IPAQ) and
health history questionnaire, downhill walking (DW), and
a blood draw. The second and third visit occurred 24 hours
after the first and consisted of a single blood draw. The
pre-supplementation and post-supplementation visits
were separated by 8 weeks of dietary supplementation
regimen as described above.
D. Downhill walking
DW was performed on a treadmill in the UW Biodynamics
Laboratory. Each of the 2 DW sessions consisted of 4 bouts
of 15 minutes of treadmill walking separated by 3 sessions
of 5 minutes of quiet rest. The treadmill grade was set
at −9% and the speed to 4.0 km/h. Heart rate was re-
corded every 5 minutes using a heart rate monitor.
E. Blood sample collection and preparation
Mixed venous blood was drawn from an antecubital vein
into 4 EDTA-coated Vacutainer tubes (7 mL each, Fisher
Scientific). Whole blood was placed on ice and then im-
mediately centrifuged at 500 × gat 4 degrees C for use in
the glutathione assay (see below) or gently pipetted over
two layers (3 ml of each) of density gradient (Histopaque
and Ficoll-Paque) for isolation of blood cells. After centri-
fugation at 500 × gfor 30 minutes at 20°C, plasma was re-
moved by aspiration and frozen at −80°C. A band of
monocytes was then removed by aspiration, washed with
phosphate buffered saline (PBS), and frozen at −80°C. Next,
the remaining fluid (not packed erythrocytes) was removed
and washed with ice cold PBS to attain neutrophils. Any
erythrocytes contaminating the sample were lysed with the
addition of nanopure water. After gentle inversion, tonicity
was restored by the addition of 3% NaCl. After centrifuga-
tion at 900 × gfor 5 minutes at 4°C, the neutrophil pellet
was resuspended in Hank’s balanced salt solution (HBSS)
and the cells counted by microscope and hemacytometer
and diluted to 1.5 × 10
6
cells/mL for immediate analysis of
respiratory burst (see below). Packed erythrocytes were re-
moved and stored immediately at −80°C.
F. Biological measurements
1. ELISA
Enzyme-linked immunosorbent assay (ELISA) kits
(eBioscience, Read-Set-Go! ELISA, San Diego, CA) were
used to test for the plasma concentrations of interleukin
(IL)-1β, IL-6, tumor necrosis factor (TNF)-α,andC-
reactive protein (CRP) per manufacturer’s instruction. All
samples were measured in duplicate using 96-well plates
coated with capture antibody. Following sample addition,
detection antibody, avidin horse radish peroxidase, and
enzyme substrate were added in succession with each step
separated by room-temperature incubation and thorough
washing with a PBS-Tween 20 wash buffer. Absorption at
525 nm was measured on a plate reader (Spectra MAx
340, Molecular Devices) and used to determine plasma
concentration from a standard curve generated using re-
combinant standards provided by the manufacturer.
NFκB binding to DNA was measured by ELISA in nuclear
extracts of mononuclear cells. The assay principle is as
above; however, only p65 bound to DNA was detected. Nu-
clear extraction was conducted according to manufacturer’s
instructions (Millipore Nuclear Extraction Kit). Manufac-
turer’s instructions were followed for the ELISA process,
which utilized an antibody against p65 (eBioscience
InstantOne ELISA). Samples were scanned using a
luminometer (Turner Biosystems #2030-000).
2. HPLC
a. Plasma glutathione Glutathione concentrations were
measured by HPLC based on the method described by Ji
and Fu [24]. Both GSH and GSSG were detected, and the
ratio of GSH:GSSG calculated. This assay was performed
on plasma separated from a blood sample that was kept
on ice and centrifuged at 4°C immediately upon being
drawn. 250 μL of plasma was transferred to a tube con-
taining 10 μL of 0.4 mmol/L iodoacetic acid and ex-
cess sodium bicarbonate. After incubation at room
temperature for 1 hour, 2 μL of 2,4-dinitrofluorobenzene
(Sanger’s reagent, Sigma Chemical, St. Louis, MO) was
added, and the samples were kept in the dark for 28 hours
before the HPLC detection. Concentrations of GSH and
Koenig et al. Nutrition Journal 2014, 13:21 Page 3 of 11
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GSSG were determined using a Shimadzu UV–VIS de-
tector at 365 nm wavelength and quantified with standard
curves generated using GSH and GSSG standards.
b. Avenanthramide concentration Cookie AVA concen-
trations were measured using HPLC. The method of Chen
et al. [25] was modified for use on homogenized cookies.
To 200 μl of sample, 20 μl of vitamin C-EDTA was added.
Then 500 μl of 100% acetonitrile (ACN) was added to the
tubes. After 5 minutes, the samples were centrifuged at
15000 × gfor 5 min. The supernatant, which contained
the AVA, was removed, and the solvent was evaporated by
motorized vacuum pump (Fisher Scientific) at a pressure
of approximately 200 mm Hg for approximately 5 minutes.
The residue was reconstituted in 200 μlofHPLCaqueous
solvent. Again the samples were centrifuged at 15000 × g
for 5 min. The supernatant, which contained the AVA,
was removed, and the solvent was evaporated by motor-
ized vacuum pump (Fisher Scientific) at a pressure of ap-
proximately 200 mm Hg for approximately 5 minutes.
The residue was reconstituted in 200 μLofHPLCaqueous
solvent. Again the samples were centrifuged at 15000 × g
for 5 min. All samples were analyzed for AVA concentra-
tion with a procedure based on Milbury [26] on a dual
pump Shimadzu HPLC system with a UV–VIS spectro-
photometric detector, a Supelco C18 column with inline
guard column. Absorption at 330 nm was tracked by
Shimadzu EZStart 7.2.1 software.
3. Neutrophil respiratory burst (NRB)
Neutrophils diluted in HBSS to 1.5 × 10
6
cells/mL were
assayed for respiratory burst activity by luminometer
using a procedure according to Benbarek et al. [27] with
modifications. Neutrophils were incubated with luminol
(Sigma, MO) for 5 minutes at 37°C in a shaking water
bath. Three concentrations of phorbol myristate acetate
(PMA) were used to evaluate the effects of maximal,
moderate, and minimal stimulation of the cells. The
highest concentration was 160 μmol/L, the middle con-
centration was 16 μmol/L, and the low concentration
was 1.6 μmol/L. A total of 1 × 10
6
neutrophils were used
in each trial. The respiratory burst chemiluminescence
was tracked for 30 minutes by luminometer (Turner
Biosystems) with 10 measurements in one second of in-
dividual samples every 2.5 minutes. A cell-free blank
containing equal volume of HBSS received luminol and
maximal PMA concentration in order to measure
chemiluminescence not associated with cellular activity.
The mean of 10 measurements at each time point was
calculated and the time course of the respiratory burst
plotted. Area under curve was calculated by the trapez-
oidal rule and used as a measure of total respiratory
burst activity.
4. Spectrophotometric assays
a. Plasma total antioxidant capacity Plasma total
antioxidant capacity (TAC) was measured by spectro-
photometer by monitoring the attenuation of 2,2’-azinobis-
3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation at
734 nm according to Re et al. [28]. A solution of 7 mmol/L
ABTS and 2.45 mmol/L aluminum potassium sulfate (APS)
was made immediately before the conducting of the assay
and kept in the dark. An aliquot of 100 μLplasmawas
added to a final volume of 1 mL with ABTS/APS solution.
The cuvette was mixed by inversion and then incubated at
37°C for 5 minutes. The cuvettes were then read against
a Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox-
ylic acid) standard curve using a spectrophotometer
(Shimadzu UV160).
b. Plasma creatine kinase Plasma ceatine kinase (CK)
activity was measured as a marker of eccentric muscle
damage according to the procedure of Tanzer and Gil-
varg [29]. The CK reaction was coupled to NADH con-
version to NAD by lactate dehydrogenase (LDH), which,
along with pyruvate kinase (PK), phosphoenol pyruvate
(PEP), and NADH, were present in the reaction mixture.
The decrease in NADH concentration was tracked using
a spectrophotometer (Shimadzu UV160).
c. Erythrocyte superoxide dismutase Erythrocyte super-
oxide dismutase (SOD) activity was measured by spec-
trophotometrically by tracking the decrease in auto-
oxidation of epinephrine to adrenochrome according to
Sun and Zigman [30]. Epinephrine autoxidation rate was
measured at 320 nm for 3 minutes in the presence of an
aliquot of erythrocyte lysate. The slope of the linear por-
tion of the absorption graph was used to determine SOD
activity by determining the percent inhibition of epi-
nephrine autoxidation via comparison to the blank.
Activity was normalized to hemoglobin concentration.
d. Erythrocyte glutathione peroxidase Erythrocyte gluta-
thione peroxidase (GPx) activity was measured by moni-
toring the change in NADPH concentration in a system
with excess GSH and glutathione reductase (GR) in
the presence of H
2
O
2
[24]. Activity was normalized to
hemoglobin concentration.
Erythrocyte hemoglobin was measured using Drabkin’s
reagent (potassium ferricyanide and potassium cyanide
in sodium bicarbonate), which binds hemoglobin to
cause a shift in maximal absorbance, which can be mea-
sured by the spectrophotometer [31].
G. Statistical analysis
Data were shown as mean ± SEM and analyzed using the
Planned Comparison method. A three-way repeated mea-
sures ANOVA was conducted using R (version 2.14.1)
Koenig et al. Nutrition Journal 2014, 13:21 Page 4 of 11
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statistical software. The three main factors are (a) post- vs.
pre-AVA supplementation, (b) timing with respect to DW
test (rest vs. 24 h post-DW vs. 48 h post-DW), and (c)
high-AVA vs. Control supplementation. The standard
error of estimate of the ANOVA was used to complete a
priori planned comparisons. Significance for each com-
parison was set at P< 0.00455, which is the quotient of
0.05 divided across the 11 comparisons.
Results
A. Participant data
The age, height, weight, and body mass index (BMI) of
the study participants are displayed in Table 1. There
were no significant differences between groups for any
of the parameters measured. Body weight and BMI were
unchanged following dietary supplementation.
B. Muscle damage caused by DW
Plasma CK activity was significantly elevated 24 h after
DW both before and after the dietary supplementation
regimen (P< 0.05; Figure 1). CK activity was not differ-
ent 48 h after DW compared to resting levels in both
groups. No difference was observed between AVA and
control across all groups.
C. Inflammatory markers
In the current study inflammatory responses to DW by
older women subjects were assessed by several bio-
markers including neutrophil respiratory burst (NRB)
activity in vitro, plasma CRP concentration and pro-
inflammatory cytokine levels. NRB activity increased sig-
nificantly 24 h after DW before supplementation (P<
0.05; Figure 2). This post -DW elevation of NRB activity
maintained in Control group after 8 week dietary regi-
men, but was abolished in the high-AVA supplemented
group (P< 0.05).
Plasma level of CRP was not different between rest and
24 h post-DW either before or after the 8 week supple-
mentation, but increased significantly 48 h after DW prior
to supplementation (P< 0.05; Figure 3). Following supple-
mentation, plasma CRP level was elevated 48 h post-DW
only in Control group but not in AVA group (P<0.05).
Plasma IL-1βconcentration was not altered by DW
prior to dietary supplementation (Figure 4). Following 8
wk of high-AVA supplementation, IL-1βlevel at rest and
24 h post-DW was decreased by nearly 50% in the high-
AVA group compared to their Control counterparts (P<
0.05). This difference vanished at 48 h post-DW.
We measured plasma concentration of two other pro-
inflammatory cytokines, TNF-αand IL-6. TNF-αlevels
were not affected by DW before or after dietary supple-
mentation regimen, but showed a strong trend to de-
crease (0.05 < P < 0.1) after supplementation (data not
shown). IL-6 levels were not affected by DW or AVA
supplementation (data not shown).
DW did not significantly affect monocyte NFκB bind-
ing activity before dietary supplementation (Figure 5).
After supplementation NFκB binding was lower in AVA
vs. Control at rest as well as 24 and 48 h post-DW (P<
0.05).
D. Antioxidant defense
Plasma TAC did not change significantly in response to
DW either before or after dietary oat supplementation,
however dietary supplementation regimen resulted in a
significant increase in TAC regardless of exercise status
or dietary AVA concentration (P< 0.05; Figure 6).
Erythrocyte SOD activity was unchanged with DW be-
fore the dietary supplementation regimen (Table 2). Fol-
lowing supplementation, SOD activity was significantly
greater in high AVA compared to Control group 48 h after
DW (P< 0.05, interaction). Erythrocyte GPx activity was
not altered by DW but showed a trend toward a lower
level in high-AVA vs. Control at Rest (P < 0.1, interaction).
Plasma GSH concentration was not significantly altered
by DW or dietary supplementation (Table 2). Plasma
GSSG concentration increased significantly (P< 0.05) 24 h
after DW and returned to baseline levels at 48 h both be-
fore and after supplementation. Change in GSSG was not
affected by AVA content in the diet. Plasma GSH:GSSG
ratio did not change at 24 h but was significantly increased
48 h post-DW vs. Rest both before and after dietary sup-
plementation (P< 0.05). No difference between AVA and
Control groups was observed.
Discussion
In the present study, DW resulted in a significant,
though modest, increase in CK activity after 24 h indi-
cating that the DW protocol was sufficient to elicit
muscle damage among older womon subjects. The re-
peated eccentric contractions of DW might have caused
sarcomere stretching and membrane damage, leading to
Table 1 Characteristics of postmenopausal participants
Pre-Supplementation Post-Supplementation
Age (y) Height (m) Body Weight (kg) BMI Body Weight (kg) BMI
Control 60.125 ± 2.20 1.48 ± 0.025 57.50 ± 2.42 26.40 ± 1.59 57.65 ± 2.53 26.42 ± 1.61
AVA 59.000 ± 2.25 1.45 ± 0.051 60.45 ± 2.66 29.01 ± 1.27 59.91 ± 2.69 28.74 ± 1.39
Note: Data are mean ±SEM. AVA, avananthramide. BMI, body mass index defined by the ratio of body weight (kg) divided by height (meter)
2
.
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the escape of sarcoplasmic constituents such as CK to
the circulation. AVA supplementation did not affect the
extent of muscle damage due to DW, as the CK re-
sponse was unchanged after supplementation.
Previous studies have shown that a single bout of un-
accustomed eccentric exercise can lead to a protective
effect whereupon a second bout of eccentric exercise
may result in less muscle damage [32-34]. This effect
was not observed in this study possibly because the
8 week period between DW sessions was a sufficiently
long washout period and the DW protocol was mild
providing relatively small stimulus to the muscles in-
volved. Furthermore, postmenopausal women lack estro-
gen, which may provide membrane stability by
intercalating among plasma membrane phospholipids.
Therefore, the skeletal muscle of postmenopausal
women may be more prone to DW-induced damage
compared to younger subjects [13].
Figure 1 Plasma CK activity in postmenopausal women in response to downhill walking (DW). Data are mean ± SEM (N = 8). * P< 0.05,
24 h post-DW vs. Rest.
Figure 2 Neutrophil respiratory burst activity in postmenopausal women in response to DW. Data are mean ± SEM (N = 8), normalized to
Pre-Supplementation Rest value. * P< 0.05, 24 h vs. Rest. § P< 0.05, AVA vs. Control in 24 h post-DW.
Koenig et al. Nutrition Journal 2014, 13:21 Page 6 of 11
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AVA supplementation reduced plasma inflammatory
markers
The local response to eccentric contraction-induced
muscle damage is the triggering of inflammation [35]. A
major part of the muscle repair process is the arrival and
infiltration of neutrophils at the site of damage followed
by phagocytosis. During this process, NADPH oxidase
activity increases in a respiratory burst to convert O
2
to
superoxide radical and cause oxidative damage [36]. In
the current study we did not obtain muscle biopsy sam-
ples to test this scenario, however, concomitant with the
increase in CK 24 h post-DW, NRB increased signifi-
cantly in circulating neutrophils (Figure 2). This finding
suggests that DW-associated muscle damage might have
stimulated either receptor binding or NADPH oxidase
activity, or both of the circulating neutrophils.
After 8 weeks of dietary oat supplementation, DW
triggered a NRB level at 24 h in the Control group, simi-
lar to the response seen prior to the dietary regimen.
However, high-AVA supplementation resulted in a pro-
tection against this response. In a previous study, Brickson
et al. [37] showed in a rabbit muscle stretch injury
Figure 3 Plasma C-reactive protein (CRP) concentrations in postmenopausal women in response to DW. Data are mean ± SEM (N = 8).
*P< 0.05, 48 h post-DW vs. Rest. § P < 0.05, AVA vs. Control in 48 h post-DW.
Figure 4 Plasma interleukin (IL)-1βconcentrations in young women in response to DW. Data are mean ± SEM (N = 8). § P< 0.05, AVA vs.
Control in Rest and 24 h post-DW.
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model that M1/70 antibody could block the iC3b do-
main of neutrophil receptors for respiratory burst with-
out affecting its adhesive effect. Previous work
conducted in HAEC demonstrated that AVA was able to
inhibit both adhesion and inflammatory cytokine pro-
ductions [38]. Our data were the first time to show that
AVA could suppress neutrophil respiratory burst activity
in human in response to physical stress and thus sup-
port the notion that AVA is a potent anti-inflammatory
agent.
A key step for the signal transduction of mononuclear
cells is NFκB activation [39,40]. Peripheral blood mono-
nuclear cells isolated from patients with certain inflam-
matory diseases and pathogenesis showed increased
Figure 5 Mononuclear cell NFκB binding activity in postmenopausal women in response to DW. Data are mean ± SEM (N = 8), normalized
to Pre-Supplementation Rest value. § P< 0.05, AVA vs. Control.
Figure 6 Plasma total antioxidant capacity (TAC) in postmenopausal women. Data are mean ± SEM (N = 8). + P< 0.05, Post- vs. Pre-
supplementation regardless of time or AVA treatment.
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NFκB binding. Because previous studies showed that
AVA could block NFκB signaling in vitro [38,41], we
measured NFκB binding to DNA in the nuclear extracts
of mononuclear cells ex vivo. We found that older
women receiving 8 weeks of high-AVA diet showed sig-
nificantly reduced NFκB binding in mononuclear cells
compared to those who received low-AVA control diet
both at rest and during the two day post-DW recovery
period (Figure 5). These effects may be of particular im-
portance in postmenopausal women, who suffer from in-
creased risk of inflammation.
DW appeared to elicit a whole-body inflammatory re-
sponse among the older women as indicated by elevated
CRP levels in the plasma 48 h post-DW, a reliable
marker of systemic inflammation [42] Furthermore, rest-
ing CRP levels in older women were twice as high com-
pared to a study we conducted with young women at
18–25 year of age [43]. However, 8 weeks of high-AVA
supplementation completely abolished CRP response to
DW seen in the control diet group at 48 h. Inflammation
is a double-edged sword. While at young age inflamma-
tion in response to heavy muscle contraction especially
eccentric contraction is viewed as a necessary process
for muscle to recover from injury, among aged human
subjects muscle growth and repair appear to be inhibited
at high level of inflammation [11]. Furthermore, systemic
inflammation has been shown to disrupt local inflamma-
tory responses that are responsible for muscle repair
[35]. The repercussions of a lifetime of repeated inflam-
matory response are thought to be experienced in the
aged individuals as the result of a process known as
“inflammaging”[6].
AVA supplementation suppressed pro- inflammatory
cytokine production
Inflammation is associated with increased pro-inflammatory
cytokine production, which could occur in both activated
leukocytes and injured muscle cells [35]. In the current
study, DW did not significantly increase plasma levels of
three major pro-inflammatory cytokines among older
women, presumably because the DW protocol was not
strenuous enough. However, plasma IL-1βconcentration
was decreased at rest and 24 h post-DW among subjects re-
ceiving high-AVA supplementation, whereas TNF-αlevel
showed a trend of reduction. It is known that plasma IL-1β
is associated with increased adhesion molecule expression,
hypothalamic modulation of body temperature, and hyper-
algesia [44]. By reducing IL-1β, AVA might have decreased
leukocyte invasion and, together with the decrease in neutro-
phil respiratory burst, protect skeletal muscle from elevated
inflammatory status seen among older women [45]. Reduced
plasma TNF-αmight also play a role in attenuating NFκBac-
tivation and NRB in response to DW. Guo et.al [38] reported
that the inhibitory effect of AVA on the expression of pro-
inflammatory cytokines was mediated through NFκBactiva-
tion in HAEC. Our finding that AVA could suppress NFκB
activation in human plasma mononuclear cells provided fur-
ther evidence that AVA may inhibit inflammatory responses
through a common pathway of NFκB.
AVA had no adverse effect on blood antioxidant and
redox status
Besides AVA, oats contain a variety of phytochemicals
such as phytic acid, polyphenols, flavonoids, and tocols
that function as antioxidants [18]. Thus, oat supplemen-
tation, regardless of AVA concentration, significantly in-
creased plasma TAC indicating an overall antioxidant
protection. There has been a concern, however, about a
high dose of dietary antioxidant supplementation that
may cause some adverse effects such as interference with
intrinsic antioxidant systems and preventing exercise-
induced metabolic benefit [17]. In older women receiv-
ing 8-weeks of high-AVA supplementation, erythrocyte
SOD activity showed no change, whereas GPx activity
showed a modest but significant reduction (Table 2).
Plasma GSH content and GSH:GSSG ratio were normal
Table 2 Erythrocyte antioxidant enzyme activity and plasma glutathione status
SOD GPX GSH GSSG GSH:GSSG
C AVA C AVA C AVA C AVA C AVA
Pre-
Supplementation
Rest 408 ± 20.6 410 ± 19.9 440 ± 18.9 452 ± 16.2 5.90 ± 0.19 5.90 ± 0.10 1.10 ± 0.09 1.10 ± 0.13 5.36 ± 0.20 5.36 ± 0.19
24 h
Post-DW
393 ± 34.6 436 ± 10.0 399 ± 19.7 445 ± 19.3 6.57 ± 0.19 6.26 ± 0.12 1.15 ± 0.08 1.20 ± 0.10* 5.71 ± 0.21 5.20 ± 0.04
48 h
Post-DW
383 ± 15.3 403 ± 20.3 406 ± 14.1 436 ± 50.2 6.44 ± 0.21 7.05 ± 0.12 1.07 ± 0.10 1.14 ± 0.13 6.03 ± 0.22* 6.18 ± 0.17*
Post-
Supplementation
Rest 374 ± 38.7 419 ± 21.1 419 ± 14.1 319 ± 17.9¥ 5.76 ± 0.17 6.46 ± 0.20 1.07 ± 0.07 1.16 ± 0.12 5.41 ± 0.15 5.59 ± 0.13
24 h
Post-DW
353 ± 23.6 362 ± 60.1 376 ± 11.9 312 ± 24.6¥ 6.00 ± 0.15 6.65 ± 0.18 1.19 ± 0.05* 1.19 ± 0.13 5.03 ± 0.17 5.57 ± 0.10
48 h
Post-DW
389 ± 17.5 449 ± 6.5§ 397 ± 11.9 336 ± 61.9 6.27 ± 0.08 6.48 ± 0.15 1.02 ± 0.07 1.10 ± 0.06 6.16 ± 0.16* 5.89 ± 0.17
Note: Data are mean ± SEM (N = 8). SOD, superoxidae dismutase. Gpx, glutathione peroxidase. GSH, reduced glutathione; GSSG, glutathione disulfide. DW, downhill
walking (see text for details). * P < 0.05, 24 or 48 h Post-DW vs. Rest. § P < 0.05, Post-supplementation vs. Presupplementation. ¥ 0.05 < P < 0.1, AVA vs. Control.
Koenig et al. Nutrition Journal 2014, 13:21 Page 9 of 11
http://www.nutritionj.com/content/13/1/21
both at rest and in response to DW. These data indicate
that despite the clear benefit of anti-inflammatory ef-
fects, high-AVA supplementation did not cause major
adverse effects on the endogenous antioxidant system.
The modest drop in GPx activity in red blood cells
should not raise a concern as the GPx in vivo activity
has a large margin of protection due to its relatively low
Km (~1 mM).
Conclusions
High levels of dietary AVA significantly decreased sys-
temic inflammatory response of the older women to
downhill walking as indicated by lowered neutrophil re-
spiratory burst activity and plasma CRP concentration.
AVA supplementation attenuated plasma IL-1βlevels
and suppressed mononuclear cell NFκB activation.
These effects did not adversely affect the endogenous
antioxidant system. Thus, dietary supplementation of
AVA at the given dose appeared to be a useful dietary
supplement in reducing inflammation after demanding
physical exercise.
Abbreviations
AVA: avenanthramides; BMI: body mass index; CK: ceatine kinase;
CRP: C-reactive protein; DW: downhill walking; GSH: glutathione;
GSSG: glutathione disulfide; GPx: glutathione peroxidase; IL: interleukin; (NF)
κB: nuclear factor-kappaB; NRB: neutrophil respiratory burst; ROS: reactive
oxygen species; SOD: superoxide dismutase; TAC: total antioxidant capacity;
TNF-α: tumor necrosis factor-α.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
RK and LLJ designed research; RK, JRD and CK conducted research; RK
analyzed data; RK, CK, YC, and LLJ wrote the paper; TOZ formatted the
paper; LLJ had primary responsibility for final content. All authors read and
approved the final manuscript.
Acknowledgments
This research was supported by a grant from the University of Wisconsin
Foundation.
Author details
1
Department of Kinesiology, University of Wisconsin–Madison, Madison,
WI 53706, USA.
2
Laboratory of Physiological Hygiene and Exercise Science,
University of Minnesota, 1900 University Avenue, Minneapolis, MN 55455,
USA.
3
Quaker Oats Center of Excellence, PepsiCo Nutrition, 617 W Main
Street, Barrington, IL 60010, USA.
Received: 1 November 2013 Accepted: 4 March 2014
Published: 19 March 2014
References
1. Peake J, Della Gatta P, Cameron-Smith D: Aging and its effects on
inflammation in skeletal muscle at rest and following exercise-induced
muscle injury. Am J Physiol Regul Integr Comp Physiol 2010, 298:R1485–R1495.
2. Ferrucci L, Corsi A, Lauretani F, Bandinelli S, Bartali B, Taub DD, Guralnik JM,
Longo DL: The origins of age-related proinflammatory state. Blood 2005,
105:2294–2299.
3. Toth MJ, Ades PA, Tischler MD, Tracy RP, LeWinter MM: Immune activation
is associated with reduced skeletal muscle mass and physical function in
chronic heart failure. Int J Cardiol 2006, 109:179–187.
4. BalA,UnluE,BaharG,AydogE,EksiogluE,YorganciogluR:
Comparison of serum IL-1β,sIL-2R,IL-6,andTNF-αlevels with
disease activity parameters in ankylosing spondylitis. Clin Rheumatol
2007, 26:211–215.
5. Cesari M, Kritchevsky SB, Baumgartner RN, Atkinson HH, Penninx BW,
Lenchik L, Palla SL, Ambrosius WT, Tracy RP, Pahor M: Sarcopenia, obesity,
and inflammation–results from the Trial of Angiotensin Converting
Enzyme Inhibition and Novel Cardiovascular Risk Factors study. Am J Clin
Nutr 2005, 82:428–434.
6. Franceschi C, Capri M, Monti D, Giunta S, Olivieri F, Sevini F, Panourgia MP,
Invidia L, Celani L, Scurti M, Cevenini E, Castellani GC, Salvioli S:
Inflammaging and anti-inflammaging: a systemic perspective on aging
and longevity emerged from studies in humans. Mech Ageing Dev 2007,
128:92–105.
7. Hinojosa E, Boyd AR, Orihuela CJ: Age-associated inflammation and Toll-
like receptor dysfunction prime the lung for pneumococcal pneumonia.
J Infect Dis 2009, 200:546–554.
8. Schreck R, Albermann K, Bauerle PA: Nuclear factor kappa B: an oxidative-
stress responsive transcription factor of eukaryotic cells (a review). Free
Radic Res Commun 1992, 17:221–237.
9. Helenius M, Hanninen M, Lehtinen SK, Salminen A: Changes associated
with aging and replicative senescence in the regulation of transcription
factor nuclear factor-kB. Biochem J 1996, 318:603–608.
10. Bektas A, Zhang Y, Wood WH 3rd, Becker KG, Madara K, Ferrucci L, Sen R:
Age-associated alterations in inducible gene transcription in human
CD4+ T lymphocytes. Aging 2013, 5:18–36.
11. Gomez CR, Plackett TP, Kovacs EJ: Aging and estrogen: modulation of
inflammatory responses after injury. Exp Gerontol 2007, 42:451–456.
12. Vina J, Sastre J, Pallardo FV, Gambini J, Borras C: Role of mitochondrial
oxidative stress to explain the different longevity between genders:
protective effect of estrogens. Free Radic Res 2006, 40:1359–1365.
13. Enns DL, Tiidus PM: The influence of estrogen on skeletal muscle: sex
matters. Sports Med 2010, 40:40–58.
14. Dieli-Conwright CM, Spektor TM, Rice JC, Schroeder ET: Hormone therapy
attenuates exercise-induced skeletal muscle damage in postmenopausal
women. J Appl Physiol 2009, 107:853–858.
15. Liao P, Zhou J, Ji LL, Zhang Y: Eccentric contraction induces inflammatory
responses in rat skeletal muscle: role of tumor necrosis factor-alpha. Am
J Physiol Regul Integr Comp Physiol. 2010, 298:R599–607.
16. Ristow M, Zarse K, Oberbach A, Klöting N, Birringer M, Kiehntopf M,
Stumvoll M, Kahn CR, Blüher M: Antioxidants prevent health-promoting
effects of physical exercise in humans. Proc Natl Acad Sci USA 2009,
106:8665–70.
17. Gomez-Cabrera MC, Domenech E, Romagnoli M, Arduini A, Borras C,
Pallardo FV, Sastre J, Viña J: Oral administration of vitamin C decreases
muscle mitochondrial biogenesis and hampers training-induced
adaptations in endurance performance. Am J Clin Nutr 2008, 87:142–9.
18. Peterson DM: Oat antioxidants. J Cereal Sci 2001, 33:115–129.
19. Collins FW: Oat phenolics: Avenanthramides, novel substituted
N-cinnamoylanthranilate alkaloids from oat groats and hulls. J Agric Food
Chem 1989, 37:60–66.
20. Peterson DM, Hahn MJ, Emmons CL: Oat avenanthramides exhibit
antioxidant activities in vitro. Food Chem 2002, 79:473–478.
21. Liu L, Zubik L, Collins FW, Marko M, Meydani M: The antiatherogenic
potential of oat phenolic compounds. Atherosclerosis 2004, 175:39–49.
22. Nie L, Wise ML, Peterson DM, Meydani M: Avenanthramide, a polyphenol
from oats, inhibits vascular smooth muscle cell proliferation and
enhances nitric oxide production. Atherosclerosis 2006, 186:260–266.
23. Nie L, Wise ML, Peterson DM, Meydani M: Mechanism by which
avenanthramide-c, a polyphenol of oats, blocks cell cycle progression in
vascular smooth muscle cells. Free Radic Biol Med 2006, 41:702–08.
24. Ji LL, Fu R: Responses of glutathione system and antioxidant enzymes to
exhaustive exercise and hydroperoxide. J Appl Physiol 1992, 72:549–554.
25. Chen CY, Milbury PE, Kwak HK, Collins FW, Samuel P, Blumberg JB:
Avenanthramides and phenolic acids from oats are bioavailable and act
synergistically with vitamin C to enhance hamster and human LDL
resistance to oxidation. J Nutr 2004, 143:1459–1466.
26. Milbury P: Analysis of complex mixtures of flavonoids and polyphenols
by high-performance liquid chromatography electrochemical detection
methods. Methods Enzymol 2001, 335:15–26.
27. Benbarek H, Deby-Dupont G, Deby C, Caudron I, Mathy-Hartert M, Lamy M,
Serteyn D: Experimental model for the study by chemiluminescence of
the activation of isolated equine leucocytes. Res Vet Sci 1996, 61:59–64.
Koenig et al. Nutrition Journal 2014, 13:21 Page 10 of 11
http://www.nutritionj.com/content/13/1/21
28. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C:
Antioxidant activity applying an improved ABTS radical cation
decolorization assay. Free Radic Biol Med 1999, 26:1231–1237.
29. Tanzer ML, Gilvarg C: Creatine and creatine kinase measurement. J Biol
Chem 1959, 234:3201–3204.
30. Sun M, Zigman S: An improved spectrophotometric assay for superoxide
dismutase based on epinephrine autoxidation. Anal Biochem 1978,
90:81–89.
31. Ji LL, Gomez-Cabrera M-C, Vina J: Exercise and hormesis: activation of
cellular antioxidant pathway. Ann NY Acad Sci 2006, 1067:425–435.
32. Clarkson PM, Nosaka K, Braun B: Muscle function after exercise-induced
muscle damage and rapid adaptation. Med Sci Sports Exerc 1992,
24:512–520.
33. Faulkner JA, Brooks SV, Opiteck JA: Injury to skeletal muscle fibers during
contractions: conditions of occurrence and prevention. Phys Ther 1993,
73:911–921.
34. Ebbeling CB, Clarkson PM: Exercise-induced muscle damage and
adaptation. Sports Med 1989, 7:207–234.
35. Proske U, Allen TJ: Damage to skeletal muscle from eccentric exercise.
Exerc Sport Sci Rev 2005, 333:98–104.
36. Ji LL, Gomez-Cabrera M-C, Vina J: Role of antioxidants in muscle health
and pathology. Infectious disorders special issue. Infect Disord Drug Targets
2009, 9:428–444.
37. Brickson S, Hollander J, Corr DT, Ji LL, Best TM: Oxidant formation and
immune response following muscle stretch injury in rabbit skeletal
muscle. Med Sci Sports Exer 2001, 33:2010–2015.
38. Guo W, Wise ML, Collins FW, Meydani M: Avenanthramides, polyphenols
from oats, inhibit IL-1β-induced NF-κB activation in endothelial cells.
Free Radic Biol Med 2008, 44:415–29.
39. Ichiyama T, Nishikawa M, Yoshitomi T, Hasegawa S, Matsubara T, Hayashi T,
Furukawa S: Clarithromycin inhibits NF-κB activation in human peripheral
blood mononuclear cells and pulmonary epithelial cells. Antimicrob
Agents Chemother 2001, 45:44–47.
40. Tsai WJ, Yang SC, Huang YL, Chen CC, Chuang KA, Kuo YC: 4-Hydroxy-17-
methylincisterol from Agaricus blazei decreased cytokine production and
cell proliferation in human peripheral blood mononuclear cells via
inhibition of NF-AT and NF-κB activation. Evid Based Complement Alternat
Med 2013:435916. doi: 10.1155/2013/435916.
41. Sur R, Nigam A, Grote D, Liebel F, Southall MD: Avenanthramides,
polyphenols from oats, exhibit anti-inflammatory and anti-itch activity.
Arch Dermatol Res 2008, 300:569–574.
42. Ridker PM, Hennekens CH, Buring JE, Rifai N: C-reactive protein and other
markers of inflammation in the prediction of cardiovascular disease in
women. N Engl J Med 2000, 342:836–43.
43. Koenig R: Avenathranmide Supplementation Attenuate Eccentric Exercise-
induced Inflammation and Oxidative Stress in Young Women. Koenig R. Ph.D.
dissertation: University of Wisconsin-Madison; 2011.
44. Morgan MM, Clayton CC, Heinricher MM: Dissociation of hyperalgesia
from fever following intracerbroventricular administration of
interleukin-1beta in the rat. Brain Res 2004, 1022:96–100.
45. Chedraui P, Jaramillo W, Pérez-López FR, Escobar GS, Morocho N, Hidalgo L:
Pro-inflammatory cytokine levels in postmenopausal women with the
metabolic syndrome. Gynecol Endocrinol 2011, 27:685–91.
doi:10.1186/1475-2891-13-21
Cite this article as: Koenig et al.:Avenanthramide supplementation
attenuates exercise-induced inflammation in postmenopausal women.
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