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Bioavailability of Anthocyanins from Purple
Carrot Juice: Effects of Acylation and Plant Matrix
Craig S. Charron, Anne C. Kurilich, Beverly A. Clevidence, Philipp W. Simon,
Dawn J. Harrison, Steven J. Britz, David J. Baer, and Janet A. Novotny
J. Agric. Food Chem., 2009, 57 (4), 1226-1230• DOI: 10.1021/jf802988s • Publication Date (Web): 23 January 2009
Downloaded from http://pubs.acs.org on March 5, 2009
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Bioavailability of Anthocyanins from Purple Carrot
Juice: Effects of Acylation and Plant Matrix
CRAIG S. CHARRON,†ANNE C. KURILICH,†,‡BEVERLY A. CLEVIDENCE,†
PHILIPP W. SIMON,§DAWN J. HARRISON,†STEVEN J. BRITZ,†DAVID J. BAER,†
AND JANET A. NOVOTNY*,†
Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of
Agriculture, Beltsville, Maryland 20705, and Vegetable Crop Research Unit, Agricultural Research
Service, U.S. Department of Agriculture, Department of Horticulture, University of Wisconsin,
Madison, Wisconsin 53706
Absorption of cyanidin-based anthocyanins is not fully understood with respect to dose or anthocyanin
structure. In feeding studies using whole foods, nonacylated anthocyanins are more bioavailable
than their acylated counterparts, but the extent to which plant matrix determines relative bioavailability
of anthocyanins is unknown. Using juice of purple carrots to circumvent matrix effects, a feeding trial
was conducted to determine relative bioavailability of acylated and nonacylated anthocyanins and to
assess dose-response effects. Appearance of anthocyanins in plasma was measured in 10 healthy
adults for 8 h following consumption of purple carrot juice. Each subject consumed 50, 150, and 250
mL of juice containing 76 µmol (65 mg), 228 µmol (194 mg), and 380 µmol (323 mg) of total
anthocyanins, respectively. Acylated anthocyanins comprised 76% of total anthocyanins in the juice,
yet their bioavailability was found to be significantly less than that of nonacylated anthocyanins. Peak
plasma concentrations of nonacylated anthocyanins were 4-fold higher than that for acylated
anthocyanins. Absorption efficiency declined across the doses administered. Because the treatments
were consumed as juice, it could be discerned that the difference in bioavailability of acylated versus
nonacylated anthocyanins was not primarily caused by interactions with the plant matrix.
KEYWORDS: Anthocyanin; bioavailability; absorption; carrot (Daucus carota)
Anthocyanins are flavonoids found in fruits, vegetables,
leaves, flowers, and grains. The presence of these polyphenols
imparts bright colors of red, blue, and purple. In plants,
anthocyanins offer photoprotection, scavenging of free radicals,
and attraction of animals for pollination and seed dispersal (1, 2).
Anthocyanins play a role in industry by offering a replacement
for some synthetic food colorants. As dietary constituents,
anthocyanins possess a variety of health benefits, including
reduced risk of cardiovascular disease (3-6), decreased risk of
cancer (4, 7-13), protection against age-related neurodegen-
An important factor in the ability of a dietary component to
provide health benefits is bioavailability. Anthocyanins appear
to have low bioavailability, because recovery of anthocyanins
in biological samples after volunteers have consumed antho-
cyanin-rich foods and extracts has been low (19-25). Antho-
cyanin structure is one factor that appears to affect bioavail-
ability. Anthocyanins in nature are derivatives of six common
backbone structures that are glycosylated, and the glycosylations
can form linkages with aromatic acids, aliphatic acids, and
methyl ester derivatives (26). Both glycosylation and acylation
appear to affect bioavailability. A study using Caco-2 human
intestinal cell monolayers showed that cyanidin 3-glucoside and
peonidin 3-glucoside had higher transport efficiencies than
cyanidin 3-galactoside and peonidin 3-galactoside, respectively,
indicating the higher bioavailability of glucose-based antho-
cyanins (27). This lends support to the proposition that antho-
cyanin absorption may be mediated by the sodium-dependent
glucose transporter, which is involved in the transport of the
flavonoid quercetin (28), or the organic anion membrane carrier
bilitranslocase (29), because the efficiency of carrier proteins
in anthocyanin transport would likely be related to anthocyanin
structure. In the same study, it was found that the presence of
free hydroxyl groups as opposed to methoxyl groups on the
aglycone was generally associated with decreased anthocyanin
bioavailability (27). Studies have also suggested that acylation
of anthocyanins can significantly affect anthocyanin absorption.
Nonacylated anthocyanins from steamed red cabbage were found
to be 4-fold more bioavailable than acylated anthocyanins (30),
* Corresponding author [telephone (301) 504-8263; fax (301) 504-
9098; e-mail Janet.Novotny@ars.usda.gov].
†Beltsville Human Nutrition Research Center.
‡Present address: Quaker/Tropicana/Gatorade, Barrington, IL 60010.
§Vegetable Crop Research Unit.
J. Agric. Food Chem. 2009, 57, 1226–1230
10.1021/jf802988s This article not subject to U.S. Copyright. Published 2009 by the American Chemical Society
Published on Web 01/23/2009
and nonacylated anthocyanins from purple carrots were found
to be 11-14-fold more bioavailable than acylated ones (31).
Both of those studies were conducted with whole foods. Thus,
the effects of anthocyanin localization in the plant matrix could
not be isolated from the effects of anthocyanin structure. Plant
matrix is an important factor in bioavailability of other phyto-
nutrients. For example, ?-carotene from orange fruits is
substantially more bioavailable than ?-carotene from green, leafy
vegetables (32). Therefore, we conducted a study of bioavail-
ability of acylated and nonacylated anthocyanins from purple
carrot juice, an anthocyanin-rich vehicle that contains both
acylated and nonacylated derivatives and which would not be
vulnerable to interference by plant matrix issues. In addition,
treatments were administered at three different dose levels to
further elucidate anthocyanin dose-response.
MATERIALS AND METHODS
Chemicals and Materials. High-performance liquid chromatography
(HPLC) grade ethyl acetate, methanol, and water were purchased from
Fisher Scientific (Norcross, GA). Reagent grade formic acid and
trifluoroacetic acid (TFA) were purchased from Sigma Chemical Co.
(St. Louis, MO). Cyanidin 3-galactoside and malvidin 3-galactoside
were purchased from Indofine Chemical Co. (Somerville, NJ). Sep-
Pak Vac RC (500 mg) C18 cartridges for solid-phase extraction (SPE)
were obtained from Waters Corp. (Milford, MA).
Subjects and Study Design. The study protocol was approved by
the Johns Hopkins University Institutional Review Board, and written
informed consent was obtained from each study subject. The 10 subjects
were healthy, nonsmoking volunteers (5 males, 5 females) averaging
38 ( 15 years old and 70.0 ( 13 kg in body weight. Individuals with
active disease (peripheral vascular disease, degenerative kidney disease,
degenerative liver disease, cancer, acid reflux disease, or endocrine
disorders) that may interfere with the study were excluded. Individuals
with malabsorptive disorders or history of bariatric surgery were also
excluded. Subjects’ dietary history showed typical intake of three meals
Three purple carrot juice treatments were administered to subjects
in a crossover experimental design. All subjects received each of the
treatments, and the treatment periods were separated by 4-week breaks.
Subjects were randomly assigned to one of two groups, and each group
had a different treatment order. Treatments consisted of 50, 150, and
250 mL of purple carrot juice and were served to fasting subjects. Blood
was collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, and 8 h. Subjects
were provided with an anthocyanin-free diet during the treatment day
and for the prior 2 days. During the treatment day, a snack, lunch, and
dinner were provided at 2, 4, and 10 h after consumption of the purple
carrot juice, respectively. Caffeine consumption was prohibited on the
treatment day and for 1 day prior to the treatment day. Vitamins and
supplements were prohibited throughout the study.
Preparation of Purple Carrot Juice. The purple carrots were U.S.
Department of Agriculture inbred B217 and were grown at the
University of California Desert Research and Education Center in
Holtville, CA. After the carrots had been washed, the carrot tissue was
juiced in a model JE900 commercial juicer (Breville, Torrance, CA)
and remaining pulp was discarded. Four batches of carrot juice were
produced. Each batch was pasteurized at 82 °C, cooled in cold tap
water, and frozen at -20 °C. Two days prior to use, the frozen carrot
juice was thawed in a refrigerator at 4 °C.
Analysis of Anthocyanins in Purple Carrot Juice. Triplicate
samples from each of the four batches were analyzed. Each sample
was prepared by diluting 1 mL of purple carrot juice with 20 mL of
water and then diluting a 0.5 mL aliquot of this mixture with 5 mL of
methanol/10% aqueous formic acid (1:9 v/v). After this final dilution,
50 µL samples were injected onto a liquid chromatograph-mass
spectrometer for anthocyanin identification and quantification.
Blood Collection and Preparation. Blood was collected into
vacutainers containing EDTA and centrifuged at 2560g for 10 min.
Plasma aliquots of 2.2 mL were combined with 1.3 mL of 0.44 M
aqueous TFA in cryovials and stored at -80 °C. Anthocyanins were
extracted on SPE columns as previously described (31). After extraction
mass spectrometer for anthocyanin identification and quantification.
HPLC-DAD-MS Analysis of Anthocyanins. Anthocyanins were
analyzed on an LC-MS system composed of an Agilent (Agilent
Technologies, Palo Alto, CA) series 1100 LC with a 250 mm × 4.6
mm i.d., 5 µm, Zorbax SB-C18 column (Agilent), G1315A diode array
detector (DAD), and G1946A mass spectrometer (MS). The LC-MS
conditions and solvent system were as previously described (31).
Selected ion monitoring was used to identify individual anthocyanins
and to search for cyanidin and anthocyanin glucuronides or sulfates.
Calculations and Statistics. Malvidin 3-galactoside was used as
an internal standard to account for extraction losses, which averaged
47 ( 5% (SEM). A standard curve was created using cyanidin
3-galactoside to calculate molar concentrations of individual antho-
cyanins expressed as cyanidin 3-galactoside equivalents. Gram con-
centrations of individual anthocyanins were calculated using their
respective molecular weights.
The percent recovery was calculated by dividing the peak plasma
anthocyanin quantity (expressed in micromoles) by the quantity
(micromoles) of anthocyanins in the dose. The peak plasma quantity
was determined by multiplying the plasma anthocyanin concentration
by the estimated plasma volume (45 mL of plasma/kg of body mass).
The data were tested for normality (using the Kolmogorov-Smirov
test) and equal variance (using the Levene median test), and then a
one-way repeated measures analysis of variance was used to compare
plasma responses among treatments (P < 0.05). The Holm-Sidak
method was used for pairwise multiple comparisons between treatments.
The percent recoveries of acylated and nonacylated anthocyanins for
each treatment were compared by t test (P < 0.05). SigmaStat software,
version 3.11 (SPSS Inc., Chicago, IL), was used for these statistical
analyses. The area under the plasma concentration time curve (AUC)
was calculated by the trapezoidal method using Microsoft Excel 2003 v.
RESULTS AND DISCUSSION
Five anthocyanins were identified in the purple carrot juice.
These anthocyanins were previously detected in purple carrots
in studies using mass spectrometry and nuclear magnetic
resonance spectroscopy (31, 33). These anthocyanins consist
of a cyanidin aglycone to which a glycosidic residue is attached
at the 3-position of the cyanidin: cyanidin 3-(2′′-xylose-6′′-
glucose-galactoside), cyanidin 3-(2′′-xylose-galactoside), cya-
nidin 3-(2′′-xylose-6′′-sinapoyl-glucose-galactoside), cyanidin
3-(2′′-xylose-6′′-feruloyl-glucose-galactoside), and cyanidin 3-(2′′-
xylose-6′′-(4-coumaroyl)glucose-galactoside) (Figure 1). Cya-
nidin 3-(2′′-xylose-6-glucose-galactoside) and cyanidin 3-(2′′-
xylose-galactoside) are nonacylated anthocyanins. Cyanidin
3-(2′′-xylose-6′′-sinapoyl-glucose-galactoside), cyanidin 3-(2′′-
xylose-6′′-feruloyl-glucose-galactoside), and cyanidin 3-(2′′-
cyanidin 3-(2′′-xylose-6-glucose-galactoside) by acylation with
sinapic acid, ferulic acid, and p-coumaric acid, respectively. All
anthocyanins except cyanidin 3-(2′′-xylose-6′′-(4-coumaroyl)-
glucose-galactoside) were also observed in plasma following
consumption of purple carrot juice.
treatments were 76.1 µmol (64.5 mg) in 50 mL of purple carrot
juice, 228.1 µmol (193.6 mg) in 150 mL of purple carrot juice,
1). This concentration of 152 µmol/100 g (129 mg/100 g) is higher
than that of most vegetables, which have been analyzed whole
rather than as juice and which range from 1.5 mg/100 g for red
leaf lettuce and 6 mg/100 g for red beans to 113 mg/100 g for red
cabbage and 116 mg/100 g for red radish (34). This level is similar
to our previous analysis of purple carrot tap root, which contained
166 mg/100 g (31). By way of reference, a medium carrot weighs
Bioavailability of Anthocyanins fromPurple Carrot JuiceJ. Agric. Food Chem., Vol. 57, No. 4, 2009
61 g, on average, and the juice yield was about half the mass of
the carrot. Acylated anthocyanins comprised 76% of total antho-
24%. This distribution is similar to our previous findings for whole
purple carrots, in which 86% of anthocyanins were acylated (31).
The mean concentration of total anthocyanins in plasma
following consumption of purple carrot juice is represented in
Figure 2. Anthocyanins were detected at the first time point
(0.5 h) for each treatment, with the most rapid accumulation of
anthocyanins in blood occurring between 0 and 2 h, and
anthocyanins were still detectable at the final time point (8 h).
The maximum mean concentrations of total anthocyanins were
measured at 2 h for the 50- and 250-mL treatments and at 1 h
for the 150-mL treatment.
Plasma total anthocyanin response is represented by peak
concentration, percent recovered at peak, and area under the
plasma concentration-time curve (AUC) (Table 2). The peak
total anthocyanin concentration increased significantly with
increasing dose consumed. The peak concentration was 2.5
nmol/L when 50 mL of purple carrot juice was ingested and
was 1.6-fold higher for the 150-mL dose level (i.e., 3-fold higher
dose) and 2.8-fold higher for the 250-mL dose level (i.e., 5-fold
higher dose). Thus, on a percentage basis, the increase in peak
concentration was lower than the increase in dose level.
Although the plasma AUCs at the 150- and 250-mL dose levels
were significantly higher than at the 50-mL dose level, the
percent recovered at peak did not vary significantly with dose
level, probably due to high coefficients of variation. The similar
values for AUC at the 150- and 250-mL dose levels suggest
that anthocyanin absorption mechanisms began to saturate
somewhere between these two levels of purple carrot juice
Previous studies of anthocyanin dose response have had
mixed results with respect to saturation. A study of strawberry
anthocyanins showed no change in absorption efficiency over
three dose levels ranging from 15 to 60 µmol (35). A study of
red cabbage anthocyanins showed that absorption of anthocya-
nins increased with increasing dose, but with decreasing
absorption efficiency, over doses ranging from 138 to 414 µmol
(30). A study of whole purple carrot anthocyanins demonstrated
no increase in total anthocyanins absorbed when dose was
doubled, suggesting saturation of absorption at doses over the
range of 357-714 µmol (31). Considering these studies together,
absorption efficiency was most greatly affected by dose at the
higher dose ranges. The anthocyanin dose range for this study
(76-380 µmol) was chosen to be below the dose range of our
Figure 1. Chemical structures of anthocyanins detectedinpurplecarrot
juice. Structures are represented as previously reported (33).
Table 1. Acylated and Nonacylated Anthocyanin Content of Treatmentsa
µmol per treatment
aValues are expressed as means ( SEM.
consumption of 50, 150, or 250 mL of purple carrot juice. Error bars
represent ( SEM(n ) 10).
Mean plasma total anthocyanin concentration following
Table 2. Plasma Total Anthocyanin Responsea
%recovered at peak
aValuesareexpressedasmeans( SEM. Meansfollowedbydifferent letters
withinacolumnaresignificantlydifferent, P<0.05. Notethat Figure2showsthe
mean of anthocyanin concentrations at each time point and this table shows the
meanpeakconcentrationfor total anthocyanins. Becausethepeakvaluesdidnot
occurat thesametimeforall subjects, thevaluesinthistabledonot matchthose
in Figure 2.
J. Agric. Food Chem., Vol. 57, No. 4, 2009 Charron et al.
previous study of anthocyanin bioavailability from whole purple
carrots because absorption of anthocyanins appeared to be
saturated through the dose range used for the previous study
(357-714 µmol) (31). We also chose the highest dose level of
this study (380 µmol) to be close to the lowest dose level of
the previous study (357 µmol) (31) so that the two studies would
together provide a wide dose range tested for a clearer picture
of where saturation might occur. Thus, the 250-mL dose of juice
provided a similar dose of anthocyanins as the 250-g dose of
carrot sticks in the previous study (250 g of cooked purple carrot
sticks delivered 357 µmol of anthocyanins and 250 mL of juice
provided 380 µmol of anthocyanins) (31). Taken together, the
studies suggest that absorption of cyanidin-based anthocyanins
saturates between dose levels of approximately 250 and 350
Because the anthocyanin level in the 250-mL dose of juice
provided in this study was not different from the anthocyanin
level in the 250-g dose of whole purple carrots used in the
previous study (31), comparison of the anthocyanin response
after ingestion of these treatments provides information about
the overall role of the plant matrix in affecting anthocyanin
absorption. Percent recovery of anthocyanins in the juice at peak
plasma concentration was approximately double that for the
carrot sticks, but the AUCs were similar [32.0 ( 5.0 nmol h/L
for juice vs 26.6 ( 3.5 nmol h/L for whole carrots (31), mean
( SEM, difference not statistically significant]. These results
suggest that the total anthocyanins absorbed were similar for
carrot juice and whole carrots providing equivalent amounts of
anthocyanins, but anthocyanin absorption from juice was more
rapid. The time required for digestive processes to liberate
anthocyanins from the plant matrix may account for the slower
rate of anthocyanin absorption from whole carrots.
The peak concentrations of nonacylated anthocyanins in-
creased significantly with increasing dose size. In contrast, for
acylated anthocyanins, the peak concentrations at the 150- and
250-mL dose levels did not differ from one another, but were
higher than the peak concentration corresponding to the 50-
mL dose level (Table 3). (Note that Figure 2 shows the mean
of anthocyanin concentrations at each time point and Table 3
shows the mean peak concentrations for acylated and nonacy-
lated anthocyanins. Because the peak values did not occur at
the same time for all subjects, the values in Table 3 do not
match those in Figure 2. In addition, the peak concentration
for acylated anthocyanins did not occur simultaneously with
the peak concentration for nonacylated anthocyanins. Thus,
summing the peak values for acylated and nonacylated antho-
cyanins from Table 3 does not yield the peak value for total
anthocyanins in Table 2.) The percentages recovered at peak
for nonacylated anthocyanins were 2.9-, 3.1-, and 4.7-fold higher
than those of acylated anthocyanins for the 50-, 150-, and 250-
mL doses, respectively. This difference in recovery is similar
to that found in a study of red cabbage anthocyanins (30).
Acylated anthocyanins from carrot sticks were also found to
be significantly less bioavailable than nonacylated anthocyanins
(31). The previous studies comparing absorption of acylated
and nonacylated anthocyanins used whole foods, in which the
anthocyanins would have been compartmentalized in intact plant
cell vacuoles. Thus, it could not be determined if the reduction
in bioavailability was strictly related to anthocyanin structure
or if the more important factor was the association of acylated
versus nonacylated anthocyanins with the plant matrix. In this
study, we processed the carrots through juicing, thus removing
the anthocyanins from the plant matrix and insoluble fiber.
Therefore, differences in bioavailability of different anthocyanin
forms can be attributed primarily to chemical structure.
In conclusion, nonacylated anthocyanins are more bioavail-
able than acylated anthocyanins from juiced carrots, a finding
consistent with our previous observation from whole carrots and
red cabbage. Because this study was performed using juiced
carrots, thus having the plant matrix removed, the findings
demonstrate that the lower relative bioavailability of acylated
anthocyanins compared to nonacylated counterparts is not
primarily related to differential hindrance of acylated antho-
cyanins within the plant matrix. In addition, absorption efficiency
decreased over increasing anthocyanin dose. These findings
related to dose and acylation should be considered in the
development of dietary guidance related to anthocyanin intake.
Adjustments should be included in dietary guidance so that
delivery of desired levels of anthocyanins can be achieved.
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Received for review September 24, 2008. Revised manuscript received
December 19, 2008. Accepted December 19, 2008. This work was
supported by a USDA CSREES IFAFS Grant 2000-4258.
J. Agric. Food Chem., Vol. 57, No. 4, 2009 Charron et al.