Absorption of hydroxycinnamates in humans after high-bran cereal consumption

Article abstract of DOI:10.1021/jf0302299

Hydroxycinnamic acids are a group of phenolic compounds that exhibit a wide range of in vitro chemoprotective and antioxidant properties. Cereals containing a high proportion of the bran layers are rich in ester-linked hydroxycinnamic acids, such as ferul

Full text of DOI:10.1021/jf0302299

6050 J. Agric. Food Chem. 2003, 51, 6050−6055
Absorption of Hydroxycinnamates in Humans after High-Bran
Cereal Consumption
SANDRA M. KERN,†,§ RICHARD N. BENNETT,† FRED A. MELLON,†
PAUL ANTHONY KROON,† AND MARIA-TERESA GARCIA-CONESA*,†
Institute of Food Research, Nutrition Division, Norwich Research Park,
Colney, Norwich NR4 7UA, United Kingdom, and Institute of Phytopathology, Department of Applied
Biochemistry, Technical University of Munich, 85350 Freising-Weihenstephan, Germany
Hydroxycinnamic acids are a group of phenolic compounds that exhibit a wide range of in vitro
chemoprotective and antioxidant properties. Cereals containing a high proportion of the bran layers
are rich in ester-linked hydroxycinnamic acids, such as ferulic and diferulic acids. The present work
investigated the absorption in humans of hydroxycinnamic acids from high-bran breakfast cereal
(wheat). Plasma and urine samples from six volunteers were collected before and after cereal
consumption and analyzed for total hydroxycinnamic acids content after â-glucuronidase/sulfatase
treatment both by HPLC-DAD and by LC-MS (SIM monitoring). High-bran cereal administration
resulted in increased plasma ferulic and sinapic acid concentrations (maximum levels detected of
∼200 and ∼40 nM, respectively) with absorption peaks between 1 and 3 h. Increases of ∼4-fold in
ferulic acid and ∼5-fold in feruloylglycine were detected in 24-h urine after consumption of the cereal.
Most of the ferulic acid detected in urine and plasma was present as conjugates (feruloylglycine
and/or glucuronides). Diferulic acids were undetectable. The data show that ferulic and sinapic acids
are taken up in humans from dietary high bran wheat but that absorption is limited and may originate
only from the free and soluble portions present in the cereal.
KEYWORDS: Hydroxycinnamic acids; diferulic acids; bran; cereals; absorption; humans
INTRODUCTION
Epidemiological studies suggest a link between the consump-
tion of whole grain products and the prevention of chronic
diseases such as coronary heart disease (1) and certain forms
of cancer (2). The exact mechanisms linking whole grains to
disease prevention are not known but may include gastrointes-
tinal effects and antioxidant protection, which can be attributed
to various nutrients of the whole grain cereals (bran plus germ)
such as fiber, vitamins, minerals, or phenolic compounds (3).
Cereal bran contains significant quantities of phenolic com-
pounds, mainly hydroxycinnamic acids (Figure 1), that are
present mainly ester-linked to polymers in the plant cell wall
(4, 5). Thus, regular consumption of cereal bran and bran-
enriched products would result in the ingestion of significant
amounts of these plant components, in particular, ester-linked
ferulic and diferulic acids (Figure 2). For example, an intake
of 20 g of wheat bran would provide approximately 100 mg of
total ferulate, 2 mg of sinapate, 3 mg of p-coumarate, and 20
mg of diferulates (4).
Hydroxycinnamic acids and diferulic acids possess significant
antioxidant properties and chemoprotective effects as shown by
Figure 1. Chemical structures of some dietary hydroxycinnamates.
* Corresponding author [telephone +44 (0)1603 255051; fax +44 (0)-
1603 255038; e-mail maria.conesa@bbsrc.ac.uk].
† Institute of Food Research.
multiple in vitro assays and animal studies (6-11), and it is
suspected that they may contribute to some of the beneficial
effects derived from the consumption of cereal bran. However,
§ Technical University of Munich.
10.1021/jf0302299 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/20/2003

Absorption of Hydroxycinnamates in Humans from Cereals
J. Agric. Food Chem., Vol. 51, No. 20, 2003 6051
Figure 2. Structures of the main diferulates present in cereal bran.
to establish their possible in vivo effects, it is essential to
determine whether these compounds are absorbed from the diet
and which forms are found circulating in plasma and may reach
specific tissues. Esterified hydroxycinnamates and diferulates
cannot be absorbed as part of complex molecules, but esterases
present in the small intestine (12, 13) and in the colon of humans
(14, 15) are able to cleave the ester bonds and may release a
proportion of the hydroxycinnamic acids and diferulic acids free
into the lumen, which can then be absorbed. It has been shown
in humans that some hydroxycinnamic acids (mostly ferulic acid
and caffeic acid) can be absorbed from various sources such as
tomatoes (16), beer (17), bark extract (18), herbal extract (19),
and prunes (20) and that the main circulating metabolites were
glucuronide and sulfate conjugates (17-19, 22).
Our knowledge about hydroxycinnamate absorption from the
diet and their metabolism in humans is incomplete, one major
unresolved point consisting of the absorption of the bound
compounds (ester-linked to complex polymers of the plant cell
wall). The aim of this study was to investigate the bioavailability
of hydroxycinnamates and diferulates from high-bran wheat (rich
in the ester-linked forms of these compounds) and to determine
if some of the covalently bound forms of these phenolics are
absorbed.
Figure 3. Scheme showing the study design.
pH 4.0 with HCl, and extracted with ethyl acetate (3× vol). The pooled
organic phases were dried by rotary evaporation to obtain the solid
glycine conjugate as free acid. The final product was analyzed by
HPLC-DAD, and the percentage of unreacted ferulic acid was <5%.
The molecular mass of the conjugate was confirmed by LC-MS using
ESI+ mode ([M + H]+ ) 252).
Study Design. This study was approved by the Norwich District
Research Ethics Committee and was performed under their guidelines.
Six healthy volunteers, three males and three females (mean age )
32, range ) 26-39; mean body mass index, 25.7 kg/m2, range ) 21.7-
30.3), were recruited from the Norwich Research Park by advertisement.
Volunteers were excluded from the study if they were suffering from
diagnosed acute or chronic illness or taking medication, giving blood
as a donor within a month (before or after) the study, outside the
reference range of blood parameters, known to have a history of
gastrointestinal disease, exhibiting altered gut behavior (constipation
or diarrhea), pregnant, vegetarian, vegan, or known to have a specific
food allergy to all bran or cereal bran. The six volunteers underwent a
2 day low polyphenol diet prior to the study day. The diet specifically
avoided bran cereals, whole grain products, seeds and nuts, all fruits
and vegetables, herbs and spices, and other products such as ready meals
as well as specific beverages (black, green, or herbal tea; coffee; fruit
juice; red wine; cider; or beer). Volunteers were allowed to eat peeled
potatoes, white bread, white pasta, white rice, meat, fish, eggs, cheese,
unflavored yogurt, salt, and pepper. Water, semiskimmed, or skimmed
milk and white wine were the only beverages allowed. The study
protocol and the collection of samples are depicted in Figure 3. On
the study day and immediately before eating the test meal a cannula
was inserted into an appropriate vein and a sample of blood (30 mL)
MATERIALS AND METHODS
Chemicals. trans-Ferulic acid (FA), trans-p-coumaric acid (p-CA),
trans-sinapic acid (SA), vanillin, ascorbic acid, pure â-glucuronidase
from Escherichia coli (EC 3.2.1.31, type IX-A), and sulfatase from
Helix pomatia (EC 3.1.6.1, type H-1) were purchased from Sigma
Chemical Co. (Poole, Dorset, U.K.). o-Coumaric acid was obtained
from Avocado Research Chemicals Ltd. (Heysham, Lancashire, U.K.).
The free diferulic acids (diFA) 8-5-diFA (open ring), 5-5-diFA, 8-O-
4-diFA, and 8-5-benzofuran-diFA were prepared as described elsewhere
(12). All other chemicals were of analytical or HPLC grade purity.
Ultrapure water (18 Ω) was used throughout the study.
Synthesis of Feruloylglycine. The glycine conjugate of ferulic acid
was prepared using a method to synthesize glycine-conjugated bile acids
(21) as follows: 0.25 mmol of ferulic acid was dissolved in 1.0 mL of
dry dimethylformamide and 0.5 mmol of glycine methyl ester hydro-
chloride, 0.3 mmol of diethylcyanophosphonate and 0.4 mL of
triethylamine were added successively, and the mixture was incubated
for 20 h at room temperature. The product of the reaction was extracted
with ethyl acetate (3× vol), and the dried residue was refluxed in 5
mL of 5% methanolic KOH (1 M) for 30 min at 100 °C. The methanolic
solution was then applied to a preconditioned C-18 cartridge (Strata
X, Phenomenex), and the unbound fraction was collected, acidified to

6052 J. Agric. Food Chem., Vol. 51, No. 20, 2003
Kern et al.
was taken as a baseline control. Volunteers were then asked to consume
100 g of a commercial breakfast cereal (85% wheat bran) with skimmed
milk. The time the volunteer finished his/her meal was recorded as t
) 0 (maximum ) 20 min). Blood samples (30 mL each) were taken
by cannulation at 1, 3, 6, 9, and 12 h after the test meal. Low-polyphenol
meals and water were served during the study day. Volunteers were
asked to collect urine for 24 h on the day prior to the study day and
throughout the study day. The following morning the volunteers
returned for giving a 24-h blood sample. The venous blood samples
were immediately centrifuged at 1500g for 10 min in 10 mL lithium-
heparin tubes (Sarstedt Ltd., Leicester, U.K.); plasma was collected,
placed on dry ice, and stored at -20 °C until analysis. The 24-h urine
samples were collected in plastic bottles containing 2 g of ascorbic
acid and were also stored at -20 °C.
Water Extractable Hydroxycinnamates in Cereals. A water
soluble fraction containing free hydroxycinnamic acids and low
molecular weight feruloylated material was prepared by extracting the
finely ground high-bran cereal (5.0 g) with 20 mL of water for 30 min
at 50 °C with constant agitation (200 rpm) under nitrogen in the dark.
The solubilized fraction was centrifuged at 13845g for 10 min. The
remaining pellet was re-extracted once more, and the supernatants were
combined (final volume ) 33 mL). A 10 mL aliquot of the resulting
supernatant was spiked with 4 µg of o-coumaric acid as internal
standard, acidified to pH <2.0 with HCl, and extracted with ethyl
acetate (3× vol). The combined organic phases were dried by rotary
evaporation at 40 °C and dissolved in 800 µL of 50% methanol,
followed by centrifugation at 15000g for 5 min and filtering (0.2 µm)
prior to analysis of free hydroxycinnamic acids by HPLC-DAD.
HPLC Analysis. Hydroxycinnamic acids and diferulic acids were
analyzed by reverse-phase HPLC-DAD with a constant flow rate of 1
mL/min. The chromatographic conditions have been described else-
where (12). Eluent was monitored at 325 and 280 nm, and sample
identification was established by comparing retention time and absorp-
tion spectra to reference standards. Hydroxycinnamic acids and diFAs
present in the cereal and feruloylglycine in the urine samples were
quantitated using the response factor determined for authentic synthetic
compounds.
Liquid Chromatography)Mass Spectrometry (LC-MS). Positive
ion electrospray LC-MS measurements were obtained using a Micro-
mass Quattro II triple quadrupole mass spectrometer (Micromass,
Manchester, U.K.) coupled to a Jasco PU-1585 triple-pump HPLC with
an AS-1559 cooled autoinjector, a CO-1560 column oven, and a UV
detector [Jasco (U.K.) Ltd., Great Dunmow, U.K.]. The HPLC column
temperature was maintained at 25 °C and the autoinjector at 4 °C. Eluent
flow (1 mL/min) was split between the mass spectrometer and the UV
detector in a ratio of 1:5 using an ASI 600 fixed ratio splitter valve
(Presearch, Hitchin, U.K.). Approximately 800 µL of the flow split,
monitored using a Humonics Optiflow 1000 flow meter (Sigma-Aldrich,
Dorset, U.K.), was diverted to the UV detector to carry out detection
at 325 nm. HPLC conditions were described above. All mass spectra
were performed using a Micromass Z-spray ion source with the
following tuning parameters: the electrospray capillary voltage was
set to 3.5 kV and the cone voltage to 22 V; the source block temperature
was 120 °C and the desolvation temperature 350 °C; nitrogen was used
as the drying and nebulizing gas at flows of 500 and 15 L/h,
respectively; full-scan spectra were obtained in positive ion mode
between m/z 50 and 1000 with a scan rate of 1.5 s. Identification and
quantitation of hydroxycinnamic acids in plasma and urine samples
was based on retention time and SIM monitoring observing ions at
m/z 195 [M + H]+ and 177 [M - H2O + H]+ for ferulic acid, m/z 225
[M + H]+ and 207 [M - H2O + H]+ for sinapic acid, m/z 165 [M +
H]+ and 147 [M - H2O + H]+ for o-coumaric acid, and m/z 387 [M
+ H]+, 369 [M - H2O + H]+, 389 [M + 2H + H]+, 371 [M - H2O
+ 2H + H]+, and 409 [M + Na]+ for diferulic acids. Instrument control
and data acquisition and processing were performed using Micromass
MassLynx version 3.4 data system and software.
Total Esterified Hydroxycinnamates in Cereals. Total esterified
hydroxycinnamates in the cereal and in the water-soluble fraction were
determined after alkaline hydrolysis following a protocol described
previously (12). Samples (200 mg of ground cereal or 10 mL of the
water-soluble fraction) were incubated with NaOH (1 M final concen-
tration) for 18 h at room temperature under N2 and in the absence of
light. o-Coumaric acid (4 µg) was added as internal standard, and the
mixture was acidified to pH <2.0 with HCl and extracted with ethyl
acetate (3× vol). The combined organic phases were dried by rotary
evaporation at 40 °C and dissolved in 800 µL of 50% methanol,
followed by centrifugation at 15000g for 5 min and filtering (0.2 µm)
prior to analysis of total hydroxycinnamic acids and diferulic acids by
HPLC-DAD.
Calibration curves were prepared by injecting samples of standard
solutions in 50% methanol for the concentration ranges of 10 nM-5
µM for ferulic and sinapic acid and 500 nM-25 µM for o-coumaric
acid and then plotting the peak areas of selected ions monitored against
concentration. The limit of detection for ferulic acid and sinapic acid
was 2.5 pmol as injected (which corresponds to ∼4.5 nM concentration
in plasma and ∼10 nM in urine). For the diferulic acids the limit of
detection as injected was 5 pmol (approximate plasma concentration
of 10 nM and urine concentration of 25 nM). A peak was identified as
positive by retention time and area increase of at least two monitored
ions above a signal-to-noise level of 3.
Plasma Sample Preparation. Plasma samples (7 mL) were spiked
with 0.5 µg of o-coumaric acid as internal standard and processed using
a slightly modified version of a method described previously (12).
Methanol (20 mL) was added to the plasma sample, and the mixture
was vortexed for 30 s and centrifuged at 1000g for 10 min at 4 °C.
The pellet was washed twice with 20 mL of methanol and centrifuged
for 10 min at 4 °C at 2000g and 2500g, respectively. The pooled
methanol phases were dried by rotary evaporation at 40 °C and
dissolved in 10 mL of PBS buffer. After acidification (pH 6.5, 0.5 M
HCl), samples were incubated with â-glucuronidase (7.5 × 104 units/
L) and sulfatase (1.5 × 104 units/L) for 1 h at 37 °C. This mixture was
further acidified with HCl to pH <2 and then extracted with ethyl
acetate (3× vol), and the organic phase was rotary evaporated to
dryness. The residue was taken up in 600 µL of 50% methanol,
centrifuged for 5 min at 15000g, and filtered through a 0.2 µm syringe
filter prior to analysis by LC-MS and HPLC-DAD.
RESULTS
Composition of the Cereal. In this study high-bran breakfast
cereal (wheat) was used as a dietary source of hydroxycinnamic
acids because of its high content of these phenolic compounds
(∼325 mg/100 g of fresh weight), particularly ferulic acid and
diferulic acids. Table 1 shows the content (milligrams per 100
g of fresh weight) of free and total (free plus ester-linked) water
soluble hydroxycinnamic acids as well as total hydroxycinnamic
acids (soluble plus insoluble) of a commercially available high-
bran breakfast cereal. The most abundant hydroxycinnamic acid
detected was trans-ferulic acid followed by sinapic acid (about
80% and 6%, respectively, of the total quantified phenolics).
Only a small proportion of trans-ferulic acid was solubilized
in the water extract (∼7% of the total compound), whereas just
over half of the total sinapic acid was found in the water soluble
fraction. Small amounts of the free soluble acids were also
detected. cis-Ferulic acid, p-coumaric acid, and vanillin were
present only in low quantities. Approximately 9% of the
Urine Sample Preparation. From the 24-h urine samples an aliquot
of 40 mL was mixed with 4 mL of PBS buffer (×10) and the pH was
adjusted to 6.5 with 1 M NaOH. The sample solution was incubated
with â-glucuronidase (7.5 × 104 units/L) and sulfatase (1.5 × 104 units/
L) for 1 h at 37 °C. After the addition of o-coumaric acid (13 µg) as
internal standard, the pH was adjusted to 2.0 with HCl, and the sample
mixture was applied onto a preconditioned C-18 SPE column (TechElut,
1000 mg/6 mL) with a flow rate of 1 mL/min (19). The SPE cartridges
were equilibrated with 10 mL of methanol followed by 10 mL of 5
mM HCl. The samples were eluted with 4 mL of methanol. An aliquot
of this methanolic fraction was diluted with water (1:1), centrifuged
for 5 min at 15000g, and filtered through a 0.2 µm syringe filter prior
to analysis by LC-MS.

Absorption of Hydroxycinnamates in Humans from Cereals
J. Agric. Food Chem., Vol. 51, No. 20, 2003 6053
minor compounds with the same retention time and spectral
properties similar to those of ferulic acid.
Table 1. Hydroxycinnamate and Diferulate Composition of the
High-Bran Cereal As Consumed (Milligrams per 100 g of Fresh
Weight)a
To determine the type of ferulic conjugates in human plasma,
plasma samples from two volunteers (at time points 1 and 3 h)
were treated according to the following procedures: (i) no
enzyme treatment (to detect free ferulic acid); (ii) â-glucu-
ronidase treatment (the â-glucuronidase enzyme used in this
study is pure (22); and (iii) â-glucuronidase plus sulfatase
treatment. One hour after consumption of the cereal, the
concentration of free ferulic acid detected in untreated plasma
was 56 nM (mean value of two volunteers). After treatment
with â-glucuronidase, the levels of free ferulic acid rose to 190
nM. Treatment with a combination of the two enzymes showed
no substantial increase in ferulic acid (199 nM). Similar results
were observed 3 h after consumption of the cereal (31 nM of
ferulic acid in untreated plasma, 172 nM after treatment with
â-glucuronidase, and 177 nM after treatment with the mixture
of enzymes). These data indicate that ferulic acid detected in
human plasma after the consumption of high-bran cereal was
present mostly as a glucuronide conjugate.
total (soluble and
water soluble
free and
insoluble)
free and
ester-linked
free
ester-linked
vanillin
1.35 ± 0.12
0.14 ± 0.06
0.19 ± 0.04
2.45 ± 0.64
0.12 ± 0.16
1.39 ± 0.02
0.47 ± 0.11
9.00 ± 1.41
18.54 ± 4.40
0.84 ± 0.71
2.72 ± 0.40
5.44 ± 0.45
19.60 ± 1.73
259.10 ± 15.63
9.22 ± 4.96
4.94 ± 0.18
6.43 ± 0.24
14.46 ± 1.92
2.85 ± 0.75
trans-p-coumaric acid
trans-sinapic acid
trans-ferulic acid
cis-ferulic acid
8-5-diFA (open form)
5-5-diFA
8-O-4-diFA
8-5-benzofuran-diFA
a Values are means ± SD of two independent analyses (n ) 2).
Trace levels of sinapic acid were also detected and identified
(retention time, LC-MS, SIM mode) in the plasma of four
volunteers with the highest levels of total free compound 1 h
after consumption of the cereal. Although quantitation of sinapic
acid was problematic due to the presence of minor interfering
peaks, we were able to estimate maximum plasma values of
∼10-40 nM. Sinapic acid was not detected in any plasma
samples taken at 3 h. We were not able to detect any of the
selected ions for the diferulic acids in the plasma samples after
consumption of the high-bran cereal at any of the time points
examined. We cannot completely exclude that traces of these
compounds could be present in the samples at concentrations
below the detection limit of our method (plasma concentration
of ∼10 nM) or that different metabolites with molecular masses
different from those selected by us were present in the plasma.
Figure 4. Concentration of free ferulic acid in plasma immediately prior
to and for 24 h following consumption of 100 g of high-bran cereal for six
volunteers (plasma samples treated with â-glucuronidase plus sulfa-
tase): volunteers A ([), B (9), C (2), D (×), E (b), and F (/). Time
zero was taken as the point at which consumption of the dose cereal
was completed.
Urinary Excretion of Hydroxycinnamic Acids. Free ferulic
acid, sinapic acid, and a glycine conjugate of ferulic acid were
detected after enzyme treatment, in the 24-h urine collected from
all subjects, both before and after consumption of high-bran
cereal (as confirmed by LC-MS). Total excreted amounts in 24
h (after â-glucuronidase plus sulfatase treatment) are shown in
Table 2. On average, the amounts of ferulic acid (3.17 ( 0.67
µmol) and feruloylglycine (5.33 ( 2.94 µmol) excreted in the
24-h urine collected after a 2 day low-polyphenol diet increased
∼4-fold (12.26 ( 3.22 µmol) and ∼5-fold (28.99 ( 15.92
µmol), respectively, after the cereal consumption. Two of the
volunteers showed increases of feruloylglycine of up to ∼10-
fold. Low but measurable quantities of total sinapic acid were
detected in 24-h urine after the low-polyphenol diet, and only
a small increase was observed for most volunteers after the
consumption of the cereal. For subject D, sinapic acid decreased
slightly on the study day.
quantified total phenolics comprise diferulic acids. 8-O-4-diFA
was the most abundant diferulate (50% of total diferulates)
followed by the 8-5-diFA (open form plus benzofuran, 27%),
whereas the 5-5-dimer accounted for 22% of the diferulates.
No diferulic acids were detected in the water extract obtained
from the cereal, indicating that these dimeric phenolics were
mainly present as ester-linked compounds in the insoluble
fraction of the cereal.
Plasma Levels of Hydroxycinnamic Acids after Cereal
Consumption. The major hydroxycinnamic acid detected in
human plasma after the consumption of the high-bran cereal
was ferulic acid. Figure 4 shows the plasma levels of total free
ferulic acid (after glucuronidase plus sulfatase treatment)
measured before and after consumption of 100 g of a high-
bran breakfast cereal in six volunteers. For most volunteers the
plasma concentration of ferulic acid reached a maximum (150-
210 nM) between 1 and 3 h after ingestion of the cereal and
decreased rapidly between 3 and 6 h and then more slowly until
24 h. For one subject (volunteer D) the maximum peak of
absorption was, however, detected at 6 h after the consumption
of the cereal. At times 0 and 24 h, plasma concentrations
between 5 and 30 nM were measured. The presence in plasma
of ferulic acid before the test meal may originate from trace
quantities of this compound present in the diet of the volunteers.
We cannot discard, however, the presence in human plasma of
Urine samples from two volunteers were treated with â-glu-
curonidase or â-glucuronidase plus sulfatase according to the
procedures described above. Treatment with pure â-glucu-
ronidase raised the levels of free ferulic acid in urine from 2.2
to 9.7 µmol/24-h urine. Additional treatment with sulfatase
yielded a slight (but not significant) increase of free ferulic acid
up to 14.4 µmol/24-h urine. Our results indicate that the major
ferulic conjugates detected in the urine were feruloyl glycine
(∼70% of the total ferulic acid excreted) followed by glucu-
ronide derivatives. A small proportion of sulfate derivatives may
also be present.

6054 J. Agric. Food Chem., Vol. 51, No. 20, 2003
Kern et al.
Table 2. Total Amounts of Ferulic Acid, Feruloylglycine, and Sinapic Acid Excreted in Urine before and after Consumption of High-Bran Cereal
(Micromoles per 24 h)
ferulic acid
sinapic acid
free + glucuronides + sulfates
prestudy day study day
feruloylglycine
prestudy day study day
26.81
free + glucuronides + sulfates
volunteer
prestudy day
study day
A
3.19
10.45
8.94
1.24
1.88
B
4.40
15.09
3.20
11.42
1.20
2.74
C
2.60
16.07
3.78
22.61
0.54
2.78
D
2.80
7.77
2.03
24.68
2.62
1.48
E
2.69
13.79
5.19
58.89
0.78
2.94
F
3.32
10.39
8.83
29.55
1.96
2.63
mean ± SD
3.17 ± 0.67
12.26 ± 3.22
5.33 ± 2.94
28.99 ± 15.92
1.39 ± 0.77
2.41 ± 0.59
Table 3. Intake, Subsequent Excretion, and Recovery of Ferulic Acid and Sinapic Acid in Urine of All Six Volunteers after Consumption of High-Bran
Cereal
urine excretiona (% intake)
total urine excretionb
(mg/24 h)
intake (mg)
acid
total
water soluble
free
total
water soluble
ferulic
259.10
18.54
9.00
2.45
8.10 ± 3.34
[5.15−14.63]c
0.54 ± 0.13
[0.33−0.66]
3.13 ± 1.29
[1.99−5.65]
2.76 ± 0.67
[1.69−3.36]
43.68 ± 18.02
[27.77−78.92]
6.00 ± 1.46
sinapic
19.60
0.19
[3.69−7.32]
a Total excreted (mg) × 100/intake (mg). Values are means ± SD (n ) 6). b Values are means ± SD (n ) 6). c Range of data for all six volunteers.
DISCUSSION
levels of hydroxycinnamic acids found in plasma 6 h after
consumption of the cereal are indicative of little or no absorption
from the large intestine. If hydroxycinnamates from cereals are
released in the colon, they are more likely to be further
metabolized by the microflora (25, 26) or excreted via feces
(14).
Cereal bran and bran-enriched products are the most important
dietary sources of cell wall bound hydroxycinnamates and
diferulates (4, 5). Although there are considerable data concern-
ing the composition of plants, nothing is known of the phenolic
composition of cereal-derived food products (e.g., breakfast
cereals, baked products) that are consumed by humans. In this
paper we report for the first time the composition in hydroxy-
cinnamates and diferulates of a commercial high-bran breakfast
cereal and show that the consumption of a 40 g portion of this
product provides ∼130 mg of these phenolic antioxidants. We
have also shown that only a small proportion of these phenolics,
specifically ferulic acid and sinapic acid, are detected in the
plasma and urine of volunteers after consumption of the cereal.
As shown in Table 3, the percentage of each of these two
phenolics recovered in the urine in relation to the total amount
ingested is ∼3%. The amounts of ferulic and sinapic acid
excreted in urine are greater than the amount of total free acids
present in the test meal (approximately 2.5 and 0.2 mg,
respectively; Table 1), indicating that some de-esterification
must have taken place. It has been reported that esterases with
the ability to cleave ferulate from a model substrate are present
in cell free extracts from duodenum, jejunum, and ileum human
mucosa (13). In the present study, maximum absorption of
ferulic acid takes place between 1 and 3 h after the test meal,
suggesting that absorption of this compound occurs primarily
in the small intestine (23). Thus, it is likely that cleavage, release,
and absorption of ferulic and sinapic acids from the high-bran
breakfast cereal take place mainly in the small intestine and
from the soluble compounds present in the test meal (the urinary
total ferulic and sinapic acids account for approximately 44 and
6% of the respective total water soluble compounds in the cereal;
Table 3). Further release of hydroxycinnamic acids from both
the soluble and insoluble portions of the high-bran cereal is
possible in the large intestine due to the action of bacterial
enzymes (12-15). However, it has been shown that bioavail-
ability of ferulic acid from a complex cereal matrix in rats is
severely reduced probably due to limited access of the digestive
enzymes to the phenolic substrate (24). In this study, the low
Free diferulic acids can be absorbed from the gut in rats (12),
but there are no reports on the absorption of these compounds
in humans from a dietary source. In vitro assays have shown
that human colonic esterases (mainly luminal and probably
derived from the microflora) are capable of releasing diferulic
acids from cereal brans (12) and, thus, it is possible that a small
proportion of these cereal components may have been released
into the lumen. In this study, none of the diferulic acids were
detected in the plasma or urine samples from any volunteer,
indicating that absorption of the ester-linked dimers, if any, is
very restricted (plasma and urine levels were below the detection
limits, ∼10 and ∼25 nM, respectively). Instead, released dimers
may be further metabolized by colonic bacteria as it has been
shown that diferulic acids can undergo hydrogenation reactions
and opening of the hydrofuran rings by human fecal micro-
organisms (27). Among the selected ions for the detection of
the diferulic compounds we included the mass corresponding
to a monohydrogenated form of the dimer (389 [M + 2H +
H]+, 371 [M - H2O + 2H + H]+, but we were not able to
detect it either in plasma or in urine. Overall, it is likely that
the bulk of the diferulic acids remains covalently bound and is
excreted in the feces.
It is important to consider the circulating levels of hydroxy-
cinnamics and conjugates after the cereal intake in relation to
their putative in vivo protective effects against cardiovascular
diseases or cancer. For example, oxidation of lipids or low-
density lipoproteins (LDL) is important in the pathogenesis of
atherosclerosis, and recent in vitro studies show that hydroxy-
cinnamic and diferulic acids can partially inhibit the oxidation
process at micromolar concentrations (∼1-100 µM) (7, 28).
Also, the anticarcinogenic properties of ferulic acid have been
reported in rats using milligram doses of the free compound
(10, 11). In this study the maximum levels of hydroxycinnamic

Absorption of Hydroxycinnamates in Humans from Cereals
J. Agric. Food Chem., Vol. 51, No. 20, 2003 6055
(12) Andreasen, M. F.; Kroon, P. A.; Williamson, G.; Garcia-Conesa,
M. T. Intestinal release and uptake of phenolic antioxidant
diferulic acids. Free Radical Biol. Med. 2001, 31, 304-314.
(13) Andreasen, M. F.; Kroon, P. A.; Williamson, G.; Garcia-Conesa,
M. T. Esterase activity able to hydrolyze dietary antioxidant
hydroxycinnamates is distributed along the intestine of mammals.
J. Agric. Food Chem. 2001, 49, 5679-5684.
acids found in plasma after the consumption of the cereal at
any time point were ∼200 nM for ferulic acid, ∼40 nM for
sinapic acid, and <10 nM for diferulic acids, considerably lower
than the concentrations required to induce responses in vitro.
Other studies looking at absorption in humans of dietary
hydroxycinnamic acids have also reported plasma concentrations
in the nanomolar range (20, 26). Thus, in vitro and animal
studies looking at chemoprotective and antioxidant effects of
dietary hydroxycinnamic acids need to be carried out using more
physiologically relevant concentrations of these compounds and
their metabolic conjugates. Additionally, and given the likely
presence of a proportion of free hydroxycinnamic acids in the
lumen, studies looking at the biological effects of these
compounds on intestinal epithelia cells are worth considering.
In conclusion, we have shown that (1) ferulic acid and sinapic
acid are the major hydroxycinnamic acids taken up in humans
after the consumption of a high-bran cereal (maximum levels
reached in plasma in the nanomolar range), with absorption
occurring mostly from the small intestine, and (2) covalently
bound diferulic acids either are not absorbed or are absorbed
only in very small amounts (<10 nM in plasma), indicating
that the bulk of ester-linked dimeric compounds are excreted
in feces or further metabolized by colonic microflora.
(14) Buchanan, C. J.; Wallace, G.; Fry, S. C. In ViVo release of 14C-
labelled phenolic groups from intact dietary spinach cell walls
during passage through the rat intestine. J. Sci. Food Agric. 1996,
71, 459-469.
(15) Kroon, P. A.; Faulds, C. B.; Ryden, P.; Robertson, J. A.;
Williamson, G. Release of covalently bound ferulic acid from
fiber in human colon. J. Agric. Food Chem. 1997, 45, 661-
667.
(16) Bourne, L. C.; Rice-Evans, C. Bioavailability of ferulic acid.
Biochem. Biophys. Res. Commun. 1998, 253, 222-227.
(17) Bourne, L.; Paganga, G.; Baxter, D.; Hughes, P.; Rice-Evans,
C. Absorption of ferulic acid from low-alcohol beer. Free Radical
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(18) Virgil, F.; Paganga, G.; Bourne, L.; Rimbach, G.; Natella, F.;
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ACKNOWLEDGMENT
We thank all of the volunteers who took part in this study and
Aliceon Blair, Lesley Maloney, and Yvonne Clements in the
Human Nutrition Unit at the Institute of Food Research. We
also thank John Eagles for mass spectrometry analysis.
(20) Cremin, P.; Kasim-Karakas, S.; Waterhouse, A. L. LC/ES-MS
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(21) Momose, T.; Tsubaki, T.; Iida, T.; Nambara, T. An improved
synthesis of taurine- and glycine-conjugated bile acids. Lipids
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Received for review March 28, 2003. Revised manuscript received July
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