Avenanthramides and Phenolic Acids from Oats Are Bioavailable and Act
Synergistically with Vitamin C to Enhance Hamster and Human LDL
Resistance to Oxidation
Chung-Yen Chen, Paul E. Milbury, Ho-Kyung Kwak, F. William Collins,* Priscilla Samuel,
and Jeffrey B. Blumberg
Antioxidants Research Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research
Center on Aging, Tufts University, Boston, MA; *Eastern Cereal and Oilseed Research Centre, Agriculture
and Agri-Food Canada, Ottawa, Canada; and
John Stuart Research Laboratories, The Quaker Oats
Company, Barrington, IL
ABSTRACT The intake of phenolic acids and related polyphenolic compounds has been inversely associated with
the risk of heart disease, but limited information is available about their bioavailability or mechanisms of action.
Polyphenolics, principally avenanthramides, and simple phenolic acids in oat bran phenol-rich powder were
dissolved in HCl:H
O:methanol (1:19:80) and characterized by HPLC with electrochemical detection. The bioavail-
ability of these oat phenolics was examined in BioF1B hamsters. Hamsters were gavaged with saline containing
0.25 g oat bran phenol-rich powder (40
mol phenolics), and blood was collected between 20 and 120 min. Peak
plasma concentrations of avenanthramides A and B, p-coumaric, p-hydroxybenzoic, vanillic, ferulic, sinapic, and
syringic acids appeared at 40 min. Although absorbed oat phenolics did not enhance ex vivo resistance of LDL to
-induced oxidation, in vitro addition of ascorbic acid synergistically extended the lag time of the 60-min
sample from 137 to 216 min (P ⱕ 0.05), unmasking the bioactivity of the oat phenolics from the oral dose. The
antioxidant capability of oat phenolics to protect human LDL against oxidation induced by 10
also determined in vitro. Oat phenolics from 0.52 to 1.95
mol/L increased the lag time to LDL oxidation in a
dose-dependent manner (P ⱕ 0.0001). Combining the oat phenolics with 5
mol/L ascorbic acid extended the lag
time in a synergistic fashion (P ⱕ 0.005). Thus, oat phenolics, including avenanthramides, are bioavailable in
hamsters and interact synergistically with vitamin C to protect LDL during oxidation. J. Nutr. 134: 1459 –1466,
Studies showing an inverse association between the intake
of polyphenolic compounds, particularly ﬂavonoids from fruits
and vegetables, and cardiovascular disease risk suggest that a
beneﬁcial effect may be observed from other foods containing
these compounds (1–3). For example, polyphenolics have
been identiﬁed in several grains, including wheat, rice, corn,
and oats (4). These phytochemicals have a range of biological
activities, including antiatherosclerotic, anti-inﬂammatory,
and antioxidant effects (5). Similar to their actions in other
foods, simple phenolic acids and polyphenolic compounds
from oats (referred to here as oat phenolics) may serve as
potent antioxidants via scavenging reactive oxygen and nitro-
gen species and/or by chelating transition minerals both in
plants and in those animals that consume them (6).
Because most phenolics are located in the bran layer of
grains (7), oats (Avena sativa L.), which are normally con-
sumed as whole-grain cereal, could be a signiﬁcant dietary
source of these compounds (8). Several oat phenolics have
been identiﬁed, including ferulic acid, caffeic acid, p-hydroxy-
benzoic acid, p-hydroxyphenylacetic acid, vanillic acid, proto-
catechuic acid, syringic acid, p-coumaric acid, sinapic acid,
tricin, apigenin, luteolin, kaempferol, and quercetin (9,10).
These oat phenolics are present as free or simple soluble esters
and, to a greater extent, as complex insoluble esters with
polysaccharides, proteins, or cell wall constituents (6,8). In
addition, Collins (11) isolated and characterized a group of
cinnamoylanthranilate alkaloid oat polyphenols, called ave-
nanthramides, which appear to be unique to oats.
The antioxidant capacity of oat phenolics was demon-
strated via in vitro studies (12–15). However, few studies have
explored the in vivo activity of oat phenolics. Hulless (“na-
Presented in part at Experimental Biology 02, April 2002, New Orleans, LA
[Chen, C.-Y., Milbury, P, O’Leary, J., Collins, F. W. & Blumberg, J. (2002)
Synergy between oat polyphenolics and
-tocopherol in prevention of LDL oxi-
dation. FASEB J. 16: A1106 (abs.)].
Supported by the U.S. Department of Agriculture (USDA) Agricultural Re-
search Service under Cooperative Agreement No. 58 –1950-00; the Agriculture
and Agri-Food Canada Matching Investment Initiative Program agreement No.
A01989, ECORC contribution No. 03–330; and The Quaker Oats Company. The
contents of this publication do not necessarily reﬂect the views or policies of the
USDA nor does mention of trade names, commercial products, or organizations
imply endorsement by the U.S. government.
To whom correspondence should be addressed.
0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences.
Manuscript received 9 December 2003. Initial review completed 29 January 2004. Revision accepted 1 March 2004.
ked”) oats fed to cows resulted in a greater stability of their
milk against oxidative degradation (16). Similarly, carcasses of
broiler chickens fed oats or hulless oats had a lower content of
lipid peroxidation products (17,18). However, the antioxidant
capacity of serum was not affected in people consuming an oat
milk product (19).
To date, no studies have explored directly the bioavailabil-
ity of oat phenolics and their subsequent effect on antioxidant
activity. Therefore, we conducted this study with the following
goals: 1) to measure the bioavailability of oat phenolics using
a hamster model; 2) to determine the in vivo effect of absorbed
oat phenolics on the antioxidant capacity of hamsters; and 3)
to test in vitro the effect of oat phenolics on the resistance of
human LDL to oxidation and its potential interactions with
vitamin C in this system.
METHODS AND MATERIALS
Chemicals and reagents. The following reagents were obtained
from Sigma Chemical: copper sulfate,
-tocopherol, sodium chloride,
p-hydroxybenzoic acid, syringic acid, p-coumaric acid, vanillin,
vanillic acid, ferulic acid, sinapic acid, sodium phosphate monobasic,
sodium phosphate dibasic, Folin Ciocalteu’s phenol reagent, and
-glucuronidase type H-2 (containing sulfatase). All organic sol-
vents, glacial acetic acid, ascorbic acid, and potassium bromide were
purchased from Fisher Scientiﬁc. Food-grade ascorbic acid was from
Mallinckrodt, and lithium hydroxide was from Fluka.
Production of oat bran phenol-rich powder. Oat bran was col-
lected from hulless oats passed 3 times through a Satake Rice Ma-
chine (type RMB, Satake Engineering). The ﬁnal weight removed
was 20% of the original hulless oats. The oat bran was extracted twice
with ethanol:water (80:20, v:v) for2hat35°C with continuous
agitation. The extraction slurry was centrifuged at 1250 ⫻ g (Invert-
ing Filter Centrifuge, Model HF-600.1, Heinkel Filtering Systems, 5
mm-bag) to provide the supernatant. Food-grade ascorbic acid was
added to the supernatant as a processing aid preservative, but was
removed during late processing. The supernatant was vacuum con-
centrated (Alfa-Laval, Model 6 ⫻ 2) at 35– 40°C to a thick oat bran
extract and then lyophilized (Virtis Model 50-SRC-6, Virtis) to an
oat bran phenol-rich powder and stored at ⫺20°C until use.
Measurement of phenolics from oat bran phenol-rich powder.
Oat phenolics in oat bran phenol-rich powder were dissolved in
O:methanol (1:19:80). After centrifugation at 11,000 ⫻ g for
10 min, an aliquot of the supernatant was dried under puriﬁed
nitrogen. The residue was reconstituted with the aqueous mobile
phase, and the oat phenolics proﬁle was characterized by HPLC
equipped with electrochemical detection (ECD)
according to Mil-
bury (20). The quantity of individual oat phenolics was calculated
according to concentration curves constructed with authenticated
phenolic acid standards and with pure avenanthramide A and B.
Phenolic esters were not determined in this study. The total phenolic
content of the oat bran powder was also determined using the
Folin-Ciocalteu reaction against a gallic acid standard curve and
expressed as molar equivalents of gallic acid (21).
Animals. BioF1B strain Golden Syrian Hamsters (n ⫽ 30; Bio-
Breeders), 1 y old, mean body weight 156.7 ⫾ 12.7 g, were housed in
cages with a 10-h:14-h light:dark cycle. Hamsters were used due to
the similarity of their lipoprotein metabolism to that of humans (22).
To increase lipoprotein formation for subsequent collection, hamsters
consumed ad libitum a nonpuriﬁed diet (Harlan) enriched with 10 g
coconut oil and 0.5 g cholesterol/100 g diet for 2 wk before the acute
oat phenolics feeding experiments (23).
After overnight food deprivation, 30 hamsters were randomly
assigned on the basis of their body weight into 6 time point groups:
0, 20, 40, 60, 80, and 120 min. A slurry with 250 mg oat bran
phenol-rich powder containing 40
mol phenolics (6.8 mg) was
delivered in 1.0 mL of 0.154 mol/L saline via stomach gavage to
hamsters anesthetized with Aerrane (Baxter). The same volume of
saline was given to hamsters in the baseline control group. The
estimated daily polyphenolic intake for a 70-kg body person is 14
mg/kg (24). We chose a dose of 45 mg/kg body weight (40
phenolics/per hamster) because rodents consume 5– 6 times more
food-based energy than humans on a body weight basis (25). Blood
samples from each hamster were collected into tubes containing
EDTA via orbital bleeding at selected time points. Plasma samples
were collected after whole blood was centrifuged at 1000 ⫻ g for 15
min at 4°C. Two aliquots of plasma were stored at ⫺80°C for
determination of oat phenolics and antioxidant capacity; the remain-
der was used immediately for analysis of LDL oxidation. This study
was approved by the Animal Care and Use Committee of the Jean
Mayer USDA Human Nutrition Research Center on Aging at Tufts
Analysis of plasma oat phenolics. Oat phenolics in plasma were
measured via HPLC-ECD (20). Brieﬂy, 20
L vitamin C-EDTA
(1.136 mmol ascorbic acid plus 3.42
mol EDTA in 1 mL of 0.4
) and 20
L glucuronidase were added to 200
plasma, and the mixture was incubated at 37°C for 45 min. Oat
phenolics were extracted with acetonitrile; the 500-
was removed after centrifugation at 14,000 ⫻ g for 5 min, dried under
puriﬁed nitrogen, and reconstituted in 100
L of the aqueous HPLC
mobile phase. After centrifugation at 14,000 ⫻ g for 5 min, the 50-
supernatant was injected into the HPLC for analysis of oat phenolics.
Quantiﬁcation was accomplished using authenticated standards that
were spiked into human plasma and processed through the extraction
procedure. An internal standard was not used in this study to calcu-
late the recovery rate or for quantiﬁcation; rather, spiked authenti-
cated standards were used in constructing standard curves that ac-
count for extraction losses. We observed an 80% recovery rate for the
internal standard (2⬘,3⬘,4⬘-trihydroxyacetophenone), which has char-
acteristics similar but not identical to the oat phenolic compounds of
interest; the recovery rate did not always parallel the recovery rates of
authenticated oat phenolic standards during the extraction procedure
(data not shown).
Ex vivo antioxidant capacity of oat phenolics. Absorbed oat
phenolics were tested ex vivo to characterize their antioxidant effect
on the resistance of hamster LDL to Cu
-induced oxidation accord
ing to a slight modiﬁcation of the method described by Esterbauer et
al. (26). Brieﬂy, LDL was separated from the plasma according to
Chung et al. (27) using a Beckman NVT-90 rotor in a Beckman
L8-mol/L centrifuge. Salt and EDTA were removed from the sample
using a PD-10 column (Amershan Pharmacia Biotech). LDL protein
was determined using a BCA protein assay kit (Pierce). Because LDL
content in hamster plasma is less than that found in human plasma,
91 nmol/L LDL was oxidized by 5
with or without the
mol/L ascorbic acid in a total volume of 1.0 mL phos-
phate buffer (pH 7.4). Formation of conjugated dienes was monitored
by absorbance at 234 nm at 37°C over 6 h using a Shimadzu UV1601
spectrophotometer equipped with a 6-position automated sample
changer. The results of the LDL oxidation were expressed as lag time
(deﬁned as the intercept at the abscissa in the diene-time plot) (28).
The total antioxidant capacity of the plasma was measured with the
oxygen radical absorbance capacity (ORAC) assay according to a
slight modiﬁcation of the method described by Huang et al. (29).
Synergistic relationship of oat phenolics and vitamin C in the in
vitro human LDL oxidation. Because the amount of LDL available
from hamsters is limited, human LDL was used to conﬁrm the
observed synergistic relation between oat phenolics and vitamin C.
An added beneﬁt of using human LDL in this assay is the extension
of the results in an animal model to future applications for clinical
evaluations. Venous blood was obtained at 1400 h from nonfasting
healthy adult Caucasian women (n ⫽ 6), 28 – 64 y old, with a mean
body weight of 63 ⫾ 15 kg, and plasma immediately separated after
centrifugation as described above. LDL samples from the ﬁrst 3
subjects were used to assess the dose-response relation of oat pheno-
lics and from the last 3 subjects for experiments on the interaction
between oat phenolics and vitamin C. All LDL experiments were
performed on 3 subjects in duplicate. The kinetics of LDL oxidation
Abbreviations used: C
, maximal concentration; ECD, electrochemical
detector; ORAC, oxygen radical absorbance capacity; pca, perchloric acid–
treated; RT, retention time; TE, Trolox equivalent; T
, time to maximal concen
CHEN ET AL.1460
were monitored after the addition of 10
nmol/L LDL protein in a total volume of 1.0 mL phosphate buffer
(pH, 7.4) and the formation of conjugated dienes monitored as
described above. An aliquot of oat phenolics in acidiﬁed methanol
was dried under nitrogen and redissolved in an equal volume of
phosphate buffer (pH 7.4) for testing in the assay. The lowest con-
centration of oat phenolics (0.52
mol/L) used in the in vitro LDL
oxidation experiment was selected because it consistently extended
the lag time. Additional concentrations of oat phenolics were se-
lected to reﬂect the concentrations observed in the plasma from the
hamster study described above. Oat phenolics were incubated with
182 nmol/L LDL at 37°C for 30 min before initiation of oxidation.
When used in the assay, ascorbic acid was dissolved in PBS and added
to the reaction immediately before initiation of oxidation. The effect
of oat phenolics and ascorbic acid on the resistance of LDL against
oxidation was expressed as the lag time increase compared with the
lag time of LDL without the addition of oat phenolics or vitamin C.
Statistics. All results are reported as means ⫾ SD. The Tukey-
Kramer honestly signiﬁcant difference (HSD) test was used after
signiﬁcant differences were obtained by one-way ANOVA in exper-
iments on plasma phenolics in hamsters, ex vivo and in vitro hamster
LDL oxidation, and in vitro human LDL oxidation. When variance
was unequal, Hartley’s test (30) was used and data (including that for
ferulic and sinapic acids) were square root–transformed before
ANOVA. A paired t test was performed to determine the signiﬁcance
of the synergy between oat phenolics and vitamin C in human LDL
oxidation by comparing the observed lag time during their coincu-
bation with the expected (calculated) sums of values observed for oat
phenolics and vitamin C treatments alone. Differences with P ⱕ 0.05
were considered signiﬁcant. The JMP IN 4 statistical software pack-
age (SAS Institute) was used to perform all statistical analyses.
The total polyphenolic content in the oat bran phenol-rich
powder was 162
mol gallic acid equivalents/g as determined
by the Folin-Ciocalteu method. As revealed by a typical
HPLC-ECD chromatogram, there were ⬃30 peaks with de-
tectable redox potential (Fig. 1). We identiﬁed and quantiﬁed
9 phenolics in oat bran phenol-rich powder (in descending
order of concentration) as: avenanthramide A (2.50
avenanthramide B (1.97
mol/g), vanillin (2.40
p-coumaric acid (1.28
mol/g), ferulic acid (0.64
vanillic acid (0.53
mol/g), syringic acid (0.39
napic acid (0.25
mol/g), and p-hydroxybenzoic acid (0.03
mol/g). Although oats are rich in vitamin E (8,31), none was
detectable by our HPLC method in this oat bran phenol-rich
powder because most of tocopherols and tocotrienols are lo-
cated in the germ and endosperm (31), both of which were
eliminated by abrasion milling.
Although there were numerous compounds in oat bran
phenol-rich powder, avenanthramide A and B, vanillic acid,
syringic acid, p-coumaric acid, ferulic acid, sinapic acid, and
p-hydroxybenzoic acid (not shown in the chromatogram) were
bioavailable in hamsters (Figs. 2 and 3). In addition, 2 un-
known compounds in plasma were noted at a retention time
(RT) of 17.80 and 30.95 min. Comparing the ratio observed of
oat phenolic concentrations in oat bran phenol-rich powder
and plasma, the compounds syringic, ferulic, and p-hydroxy-
FIGURE 1 HPLC-ECD proﬁle of phenolic acids and avenan-
thramides in oat bran phenol-rich powder identiﬁed by HPLC-ECD.
Labeled peaks are: (1) p-hydroxybenzoic acid, (2) vanillic acid, (3) sy-
ringic acid, (4) p-coumaric acid, (5) vanillin, (6) ferulic acid, (7) sinapic
acid, (8) avenanthramide A, (9) avenanthramide B.
FIGURE 2 HPLC-ECD chromatographs of hamster plasma sam-
ples obtained 40 min after administration of 0.25 g oat bran phenol-rich
powder in saline, containing 40
mol phenolics (gallic acid equivalents)
and immediately after gavage with saline (baseline). (A) The 420-mV
ECD trace. (B) The 560-mV ECD trace. Labeled peaks are: (a) 17.80-min
RT compound, (2) vanillic acid, (3) syringic acid, (4) p-coumaric acid, (b)
30.95-min RT compound, (6) ferulic acid, (7) sinapic acid.
BIOAVAILABILITY OF ANTIOXIDANT OAT PHENOLICS 1461
benzoic acids possessed a similar bioavailability, whereas p-
coumaric acid had at least 10% greater bioavailability (Table
1). Among identiﬁed oat phenolics, avenanthramides were
found at the highest concentration in the oat bran phenol-rich
powder but the lowest concentration in hamster plasma.
On the basis of their pharmacokinetic proﬁle, the maxi-
mum plasma concentrations (C
vanillic acid, sinapic acid, syringic acid, ferulic acid, and
p-coumaric acid ranged from 0.10 to 1.55
mol/L (Fig. 4). The
for avenanthramide A and B was 0.04 and 0.03
respectively (Fig. 4). The C
of these oat phenolics and the
compound identiﬁed at 30.95 min RT was reached at 40 min
). In contrast, the compound identiﬁed at 17.80 min RT
at 80 min. At 120 min, the plasma concentrations
of these 10 compounds did not differ from the baseline refer-
Absorbed oat phenolics did not change the resistance of
hamster LDL collected at 40 and 60 min against Cu
oxidation (Fig. 5). However, after 5
mol/L ascorbic acid was
added to the assay mixture, LDL collected at 60 min had a
58% longer lag time than that collected at baseline (216 and
137 min, respectively; P ⱕ 0.05). The ORAC assay for total
antioxidant capacity, expressed as
mol/L Trolox equivalent
(TE), was measured in plasma (ORAC
) and protein-pre
cipitated, perchloric acid–treated plasma (ORAC
sorbed oat phenolics did not change the ORAC
⫾ 1243 and 7079 ⫾ 777
mol/L TE) or ORAC
⫾ 123 and 1081 ⫾ 171
mol/L TE) in samples collected
at baseline and 40 min, respectively.
The antioxidant activity of oat phenolics in vitro was
apparent through a dose-dependent increase in the resistance
of human LDL against Cu
-induced oxidation (P ⱕ 0.0001)
(Fig. 6). The lowest concentration of oat phenolics tested for
this effect was 0.52
mol/L gallic acid equivalents which
resulted in a lag time 9.6 ⫾ 1.7 min greater than that of the
control absent oat phenolics. Oat phenolics doses of 0.78, 1.3,
mol/L further extended the lag time by 12.8 ⫾ 2.1,
21.8 ⫾ 2.4, and 37.5 ⫾ 3.3 min, respectively. The addition of
ascorbic acid alone at 2.5 and 5.0
mol/L increased the lag
time by 11.8 ⫾ 2.6 and 46.3 ⫾ 3.5 min, respectively (Fig. 7).
A 1-fold synergy (i.e., an observed value twice the expected
value from additive calculation) was observed with oat phe-
nolics and the 5.0
mol/L ascorbic acid dose, but no such
interaction was found with the 2.5
mol/L dose (P ⱕ 0.005).
In addition to their protein and micronutrient content,
whole grains contain an array of phytochemicals that may
contribute substantially to the total intake of dietary antioxi-
dants. Although typically consumed in lower quantities than
grains such as rice and wheat, oats are normally consumed as
a whole-grain cereal; thus, the antioxidant-rich portion of the
grain is retained. Among other potential health beneﬁts, these
constituents may contribute to the reduction in risk of cardio-
vascular disease associated with whole-grain intake as found in
several observational studies (32–35). Interestingly, the anti-
oxidant capacity of oats had been recognized many years ago
with their use as additives in food and beverage products to
preserve their quality (36,37).
Using HPLC-ECD to analyze phenolics of oat bran phenol-
rich powder, we identiﬁed (in descending order of concentra-
tion) avenanthramide A and B, vanillin, ferulic acid, p-cou-
maric acid, vanillic acid, syringic acid, sinapic acid, and
p-hydroxybenzoic acid. These results are consistent with those
of Peterson et al. (9) and Daniels and Martin (10). However,
caffeic acid, protocatechuic acid, tricin, apigenin, luteolin,
FIGURE 3 HPLC-ECD chromatograph of avenanthramide A (peak
8) and B (peak 9) in hamster plasma obtained immediately after a
gavage with saline (baseline, lower trace) and 40 min after administra-
tion of 0.25 g oat bran phenol-rich powder in saline, containing 40
phenolics (gallic acid equivalents) (upper trace).
Relative bioavailability of 8 phenolics in hamsters fed 40
mol total phenolics of oat bran phenol-rich powder
p-Coumaric acid 0.32 1.55 ⫾ 0.91 4.84 ⫾ 2.84 100
Sinapic acid 0.06 0.26 ⫾ 0.38 4.30 ⫾ 6.30 89.5
Syringic acid 0.10 0.38 ⫾ 0.25 3.80 ⫾ 2.50 78.5
p-Hydroxybenzoic acid 0.03 0.10 ⫾ 0.04 3.33 ⫾ 1.33 68.8
Ferulic acid 0.50 1.20 ⫾ 1.08 2.40 ⫾ 2.60 49.5
Vanillic acid 0.13 0.15 ⫾ 0.05 1.20 ⫾ 0.38 23.8
Avenanthramide A 0.63 0.04 ⫾ 0.03 0.06 ⫾ 0.05 1.3
Avenanthramide B 0.49 0.03 ⫾ 0.02 0.06 ⫾ 0.04 1.3
Oral dose is the absolute amount of each phenolic compound fed to each hamster.
Values are means ⫾ SD, n ⫽ 5.
The ratio of plasma C
/oral dose for p-coumaric acid was arbitrarily set at 100.
CHEN ET AL.1462
kaempferol, and quercetin were not found in the oat bran
phenol-rich powder by our HPLC-ECD method. We identiﬁed
2 of the 6 reported oat avenanthramides (11,15) by HPLC-
ECD using authenticated standards. Our chromatographic re-
sults suggest that there are numerous other antioxidant phy-
tochemicals in oat bran that remain to be identiﬁed and fully
characterized, such as phenolic esters (9). The absence of free
caffeic acid and some other oat phenolics from our material, in
contrast to reports by others (9,10), is likely due to the
different methods employed to isolate these compounds, in-
cluding factors such as extraction solvent, heating, and ester-
ase activity. As noted, the oat bran phenol-rich powder em-
ployed in our studies contained no vitamin E or other tocols as
detected by HPLC and reported by Peterson (31); thus, they
are not a source of the antioxidant activity noted in our
Oat phenolics from the oat bran phenol-rich powder were
found to be bioavailable in hamsters. Ji et al. (38) recently
reported that the dietary administration of a synthetic ave-
nanthramide had an antioxidant effect in selected tissues of
exercised rats. Vanillic, p-hydroxybenzoic, sinapic, ferulic, and
p-coumaric acids from other food sources were found previ-
ously to be bioavailable (39–42), but our results appear to be
the ﬁrst to identify syringic acid and avenanthramides in
plasma and suggest their bioactivity. The T
of the phenolic
acids and avenanthramides in hamsters were reached at 40
min and essentially eliminated by 120 min. p-Coumaric acid
was the most bioavailable among the identiﬁed oat phenolics.
In contrast, although the polyphenolic avenanthramides had
the greatest concentration in the oat bran phenol-rich powder,
their apparent relative bioavailability was only 5% of the least
bioavailable phenolic acid (vanillic acid). Although p-cou-
maric acid is the most bioavailable phenolic acid, the apparent
relative bioavailabilities among phenolics might be inﬂuenced
FIGURE 4 Time course of oat
phenolic compounds in the plasma of
hamsters administered 0.25 g oat bran
phenol-rich powder in saline contain-
mol phenolics (gallic acid
equivalents). Values are mean ⫾ SD, n
⫽ 5. In panels I and J,
C is the area
under the ECD trace with time. Means
in each panel without a common letter
differ, P ⱕ 0.05.
BIOAVAILABILITY OF ANTIOXIDANT OAT PHENOLICS 1463
by the distribution and/or biotransformation of phenolic acids
in the hamsters. For example, vanillin was the richest phenolic
acid in oat bran phenol-rich powder, but none was observed in
plasma, possibly due to its conversion to vanillic acid in vivo
(43). Because the concentrations of the oat phenolics were
measured only in plasma, it is not possible to determine from
this study to what extent these compounds were distributed to
other tissues. The C
of the unidentiﬁed 17.80- and 30.95-
min RT compounds was achieved at 80 and 40 min, respec-
tively. The 17.80-min RT compound may be a metabolite
because its T
was substantially delayed relative to other oat
phenolics, and it was not present in baseline plasma. In addi-
tion to hepatic metabolism, the biotransformation of polyphe-
nolics by colonic microﬂora was demonstrated (44– 46). In
contrast, the 30.95-min RT compound was likely produced
endogenously because it was present, albeit at a lower concen-
tration, in the baseline plasma. The identiﬁcation of these
compounds could not be achieved without authenticated stan-
dards by our HPLC-ECD method; therefore, an effort is un-
derway to identify these and other oat phenolic metabolites
In vitro studies of ferulic, syringic, and other phenolic acids
clearly reveal the capacity of these compounds to bind to LDL
and increase its resistance against oxidation (47,48). We eval-
uated the potential antioxidant activity of the oat phenolics
using an ex vivo hamster LDL oxidation model and found no
apparent change in the lag time after induction by Cu
lack of an effect might be due to an inadequate concentration
of the oat phenolics in the plasma or to their biotransforma-
tion [hepatic phase 2 enzymes have been shown to reduce the
antioxidant capacity of polyphenolics relative to their parent
compounds (49,50)]. Although these results appear in contrast
to our in vitro results with oat phenolics in human LDL, it is
important to note that the ex vivo assay reﬂects the action of
only those bioavailable oat phenolics that remain associated
with the LDL through its isolation process.
Despite no apparent change in the resistance of LDL to
oxidation ex vivo, the oat phenolics had a subtle action on the
lipoprotein that was indicated by their interaction with vita-
min C. When ascorbic acid was added in vitro, an increase in
the lag time was observed compared with its respective con-
trol. This increase was synergistic in nature, i.e., the lag time
was greater than the calculated additive effect of the antioxi-
dants, although the mechanism for such an interaction has yet
to be elucidated. This synergistic relationship is consistent
with that reported between isoﬂavones and vitamin C on LDL
in vitro (51). Interestingly, the synergy appears only in LDL
collected at 60 min rather than at 40 min, the T
of most of
the oat phenolics. This time difference in action may be due to
an equilibration period between peak plasma and LDL con-
FIGURE 5 Lag time to Cu
-induced oxidation of hamster LDL
obtained at 0, 40, and 60 min after administration of 0.25 g oat bran
phenol-rich powder in saline, containing 40
mol phenolics (gallic acid
equivalents) without (A) or with (B)5
mol/L ascorbic acid added in
vitro. Values are means ⫾ SD, n ⫽ 5. Means in the same category
without a common letter differ, P ⱕ 0.05.
FIGURE 6 Effect of oat phenolics on increased lag time to Cu
induced oxidation of human LDL in vitro;182
mol/L LDL was oxidized
with addition of oat phenolics. Lag time of control
(no added oat phenolics) was 49.3 ⫾ 3.7 min. Values are means ⫾ SD,
n ⫽ 3. Means without a common letter differ, P ⱕ 0.0001.
FIGURE 7 The synergistic effect of oat phenolics and vitamin C
on the increased lag time of human LDL oxidation in vitro;182
LDL was oxidized by 10
with the addition of oat phenolics,
vitamin C, or oat phenolics and vitamin C combined. Values are means
⫾ SD, n ⫽ 3. Means without a common letter differ, P ⱕ 0.005. Lag time
of control (no added oat phenolics or ascorbic acid) (A) was 45.5 ⫾ 0.7
min and (B) 47.7 ⫾ 1.5 min. Open bar (oat phenolics) and hatched bar
(ascorbic acid) are stacked to illustrate expected values by calculation
of the additive effect of individual ingredients. Solid bar represents the
observed effect of oat phenolics ⫹ ascorbic acid. The percentage value
above the solid bar indicates the observed synergistic increase of
combined antioxidants over expected calculated values of the individ-
CHEN ET AL.1464
centrations or the duration necessary for the oat phenolics to
bind and remodel LDL conformation.
The total antioxidant activity of plasma, assessed with the
ORAC assay, was not affected by absorbed oat phenolics in
hamsters. Although high doses of some ﬂavonoids were shown
to increase ORAC values (52), this assay may not be sufﬁ-
ciently sensitive to detect the changes obtained in this study
against the background antioxidant activity contributed by
protein, urate, and other redox constituents in plasma as
suggested by Ninfali and Aluigi (53).
In addition to the hamster model, we examined the inter-
action between the oat phenolics and vitamin C on human
LDL in vitro. Oat phenolics increased the resistance of human
LDL to oxidation in a dose-dependent fashion within concen-
trations that were achieved in hamsters. Whether these con-
centrations can be achieved and maintained in humans is not
known. A synergy between the oat phenolics and ascorbic acid
was evident at selected doses of the vitamin. These results are
consistent with the observation of a synergy between isoﬂa-
vones and ascorbic acid as reported by Hwang et al. (51) who
found as much as a 5-fold increase in lag time over the
calculated effect. We also noted a synergy between vitamin E
and phenolic compounds from almond bran (54). The mech-
anism(s) for this interaction has not been established, al-
though a regeneration of vitamins C and E by polyphenolics
was proposed as contributing to this effect (51,55). Hwang et
al. (51) also suggested that polyphenolics may stabilize the
LDL particle structure via a dynamic interaction with its
apoprotein-B domain. Further, as suggested by the absence of
an effect with our low vitamin C concentration (2.5
ascorbic acid may also contribute to a synergy via its inhibition
of the decomposition of lipid peroxides and/or prevention of
binding to LDL.
We thank Jennifer O’Leary and Ting-Huang Li for their excellent
technical assistance and Mark Andon for his valuable comments on
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