Original Communication

Urinary Excretion of Anthocyanins

Following Consumption of
Strawberry and Red Grape Juice
Yana Cahyana1,2, Michael H. Gordon1, and Trevor M. Gibson3
1 Hugh Sinclair Unit of Human Nutrition and Institute of Cardiovascular and Metabolic Research,
Department of Food and Nutritional Sciences, The University of Reading, Whiteknights, UK
2 Food Technology Department, Faculty of Agricultural Industrial Technology, Universitas Padjadjaran, West Java, Indonesia
3 The Chemical Analysis Facility (CAF), Harborne Building, The University of Reading, Whiteknights, UK

Received: May 6, 2015; Accepted: April 12, 2016

Abstract: The excretion of anthocyanins in urine following consumption of a mixed juice prepared from strawberry and red grape juice was
investigated in a human intervention study. Unmetabolised anthocyanins, and glucuronide derivatives have been detected, with pelargonidin-3
glucoside metabolised into pelargonidin monoglucuronide and pelargonidin-3-glucoside monoglucuronide. The mass of urinary anthocyanins
excreted in 24 h following consumption of the mixed juice (containing 21.93 mg of anthocyanins) was 144 nmol consisting of 106 nmol of
anthocyanins derived from strawberries (0.32% of ingested dose) and 38 nmol derived from red grapes (0.22% of ingested dose). A higher
proportion of delphinidin–3-glucoside was excreted in the unmetabolised form than less polar anthocyanins (at p  0.05). Excretion of
anthocyanins peaked between 2 and 4 h following consumption of the juice. The proportion of the ingested anthocyanins excreted was not
significantly different for strawberry and red grape anthocyanins despite the differences in the structures.

Keywords: anthocyanins, metabolites, urinary excretion, bioavailability

Introduction memory and cognitive function [3–8]. Amongst the promis-

Anthocyanins in plants occur as glycosides, formed by
O-glycosylation of anthocyanidins. The sugar moieties in
monoglycosides are mainly linked at the C3 position and
in diglycosides at the C3, C5 positions, with glucose, galac-
tose, rhamnose, xylose, arabinose and fructose being the
most commonly occurring sugars.
An early study reported intake of anthocyanins in the
United States was as high as 185–215 mg/day/person [1],
but a more recent evaluation has reported a much lower
mass of anthocyanins consumed at about 12.5 mg/day/
person [2]. Cyanidin and its derivatives represented 45%
of the daily anthocyanin intake, followed by delphinidin
and malvidin reaching as much as 21% and 15% respec-
tively [2]. In wine-drinking countries, wine may be an
important source of anthocyanin intake, thus malvidin-
based anthocyanins may be more important in these
countries than in others.
Anthocyanins are believed to exert beneficial effects
on health including reducing the risk of cardiovascular dis-
ease, exerting anti-cancer, or anti-inflammatory effects,
modulation of the immune response, as well as boosting
ing health benefits of anthocyanin consumption, evidence
for the association of consumption of anthocyanin-rich
fruits and vegetables with reduced risk of cardiovascular
disease is increasingly well documented [9].
The effects of anthocyanins on health are limited by their
bioavailability. Bioavailability refers to the fraction of an
ingested compound that reaches the systemic circulation
and the specific sites in which it can exert its biological
action [10]. In order to be bioavailable, a compound should
be bioaccessable, referring to the availability of an ingested
compound for absorption in the gastrointestinal tract. Once
the compound has been absorbed, it is transported for tissue
distribution and then exerts its bioactivity [11].
Bioavailability is a pivotal parameter for a compound in
order to deliver its significant effect in a biological system.
Understanding the bioavailability permits the determina-
tion of an effective dose for the compound to have an effect
which along with information about their metabolism is key
to identifying the most likely compounds to have biological
activity in vivo.
Studies on anthocyanin absorption and metabolism have
demonstrated that anthocyanins were absorbed into the

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https://doi.org/10.1024/0300-9831/a000546
2 Y. Cahyana et al., Urinary Excretion of Anthocyanins
systemic system and were present as both metabolised and
unmetabolised molecules, circulating at low concentrations
[12, 13]. Other studies have shown that metabolites of
anthocyanins include glucuronide, sulfate and methylated
derivatives. Studies of anthocyanin absorption following
dietary interventions with rats [14], or human subjects
[13, 15–17] have been reported. The information on bioavail-
ability is therefore important in order to assess the benefi-
cial effects of anthocyanins.
Although anthocyanin bioavailability has been previ-
ously reported, factors affecting the bioavailability such as
different anthocyanin structures, or the presence of other
food components such as sugar, has been rarely found in
the literature. The mechanisms of absorption and metabo-
lism of anthoyanins also remain obscure. The glucuronida-
tion pathway for instance is a subject of debate as to whether
it involves UDP-glucuronosyltransferase or UDP-glucose.
The mechanism by which anthocyanin glucosides are
absorbed needs to be clarified since it may involve either
the glucose transporter pathway or passive diffusion.
In this study we investigated the bioavailability of antho-
cyanins with various structures from strawberry and red
grape juices. In order to get a more precise comparison of
the effect of anthocyanin structure on excretion, the straw-
berry and red grape juices were consumed as a mixed juice,
but the individual juices were also consumed separately to
aid identification of the origin of the excreted anthocyanins.
The metabolites present in the urine help to reveal the
mechanism of absorption of anthocyanins as well as that
of the glucuronidation pathway. We also determined sugar
content and compared it with the sugar content in other
studies to elucidate whether or not the sugar content affects
anthocyanin bioavailability.
It has been reported that anthocyanin metabolites may
result from bacterial action in the colon. Berry anthocyanins
have been found to be metabolised into homovanillic and
vanillic acids [18]. Protocatechuic acid has been found to
be the major metabolite following cyanidin-glucoside con-
sumption [19]. The metabolites resulting from bacterial
action in the colon were not taken into account in this study
due to the difficulties of measurement. Still this current
human intervention study provided important information
not only on bioavailability per se but also on the effect of
anthocyanin structure, and improved understanding of
the metabolism and the absorption of the anthocyanins,
despite its limitations.
Materials and methods
Reagents and materials
Acetonitrile, methanol, formic acid, water (HPLC grade),
anthocyanin standards, β glucuronidase and sodium
Int J Vitam Nutr Res (2019), xx (xx–xx), xx–xx
hydroxide were purchased from Sigma Chemical
Company, Poole, Dorset, UK. Concentrate of strawberry
was purchased from Cobell, Exeter, UK and red grape
concentrate from Homebrew shop, Aldershot, UK.
Subjects and study design
The study protocol was given an ethical approval to proceed
by the University of Reading’s Research Ethics Committee.
All participants gave informed consent before taking part in
the dietary intervention study. Six healthy volunteers
comprising 3 men and 3 women, aged 35 ± 2 y with an aver-
age body mass index of 24.6 ± 2.0 kg/m2, were recruited to
participate in this study. Those who were suffering from dis-
ease, on a weight reducing dietary regimen or taking any
dietary supplements were excluded from the study. Those
who were in vigorous exercise, pregnant and lactating were
also excluded. All subjects were asked not to consume fruits
and vegetables containing anthocyanins for 2 days prior to
and on the day of the experiment. The list of fruits and
vegetables was provided and given to the volunteers.
Following overnight fasting, the volunteers consumed a
mixed juice prepared from strawberry and red grape con-
centrates containing 55 g of strawberry and 140 g of red
grape juice concentrate. Blank urine was collected prior to
juice consumption followed by urine collection at 2, 4, 6,
8, and 24 h after consumption. The volunteers were free
to drink water during the experiment. The urine was imme-
diately acidified with 4% formic acid following collection
and analysed by HPLC.
The volunteers were also divided randomly into 2 groups
to consume either the strawberry or red grape juice concen-
trate 2 weeks later for similar analysis of urinary metabolites.
Isolation of anthocyanins
Anthocyanins were extracted from acidified urine using a
solid-phase extraction (SPE) C-18 cartridge (Bond Elute,
200 mg, 3 mL) which was preconditioned with 6 mL of
1% formic acid in methanol, followed by 6 mL of 1% aque-
ous formic acid. Acidified urine was loaded onto the precon-
ditioned SPE cartridge, and 6 mL of 1% aqueous formic acid
was passed through the cartridge. The anthocyanins
retained in the cartridge were then eluted with acidified
methanol (1% formic acid). The methanolic extract was
dried under nitrogen at room temperature. The residue
was then dissolved in 200 μL solvent (1% formic acid in
methanol: 1% aqueous formic acid in the ratio 1:9), filtered
into a vial and then analysed by HPLC and LC-MS. Rutin
was used as an internal standard for the HPLC analysis, with
detection at 260 nm. The standard curve for urinary antho-
cyanins was prepared by spiking pelargonidin-3-glucoside
into blank urine which was then treated similarly to the
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Y. Cahyana et al., Urinary Excretion of Anthocyanins 3

above process using SPE and analysed by HPLC. The recov-

ery of anthocyanins by SPE extraction was about 93%.
To confirm the presence of glucuronide conjugates
in urine by LC-MS, acidified urine was brought to pH 5 with
NaOH (10 M). The urine was then treated with 30x106 U/L
of β glucuronidase, and incubated at 37°C for 15 min. The
sample was then acidified with formic acid to pH 2 and
the anthocyanins were extracted using SPE as described
above. Control urine without β glucuronidase treatment
was also analysed.

Anthocyanin analysis
Anthocyanin analysis was performed using an Agilent 1200
series HPLC with quaternary pump and UV-visible diode
array detector. Sample (50 μl) was injected onto a reverse Figure 1. Chromatogram of Anthocyanins in Strawberry (A) and Red
grape (B): 1=delphinidin-3-glucoside, 2=cyanidin-3-glucoside, 3=
phase Nova-pak@C18 column (4 μm, 4.6 x 250 mm)
petunidin-3-glucoside, 4= pelargonidin-3-glucoside, 5= unidentified,
(Waters, Elstree, UK) equipped with a guard column. 6=pelargonidin-3-rutinoside, 7=peonidin-3-glucoside, 8=malvidin-
The mobile phases were 1% aqueous formic acid as 3-glucoside, 9=peonidin-3-glucoside pyruvic acid, 10=unidentified
solvent A and acetonitrile as solvent B. The flow rate was 11=malvidin-3-glucoside pyruvic acid, 12=pelargonidin-
3-malonylglucoside.
1 mL/min with the column temperature of 35°C. Elution
of compounds was carried out using a gradient comprising
standard curve for quantification. The detected compounds
100% solvent A at 0 min; solvent A changed to 81% and B
which did not match the standards were analysed by LCMS
19% at 19 min, 68% solvent A and 32% solvent B at 45 min,
to determine the m/z value, from which they could be iden-
then 0% solvent A and 100% solvent B at 52 min. Solvent B
tified. The methods used in this study allowed identification
was maintained at 100% for 5 min, and then returned to
of 5 anthocyanins in strawberry concentrate and eight
100% solvent A and 0% solvent B at 60 min. Wavelengths
anthocyanins in red grape juice (Figure 1). Strawberry juice
of 280 and 520 nm were chosen to detect the anthocyanins.
(Figure 1A) contained cyanidin-3-glucoside, pelargonidin-
Detection by LC-MS was carried out with the same
3-glucoside, pelargonidin-3-rutinoside, pelargonidin-
column and mobile phase system as in HPLC analysis.
3-malonylglucoside representing 3%, 65.4%, 20.5% and
The LC-MS system consisted of an Agilent 1100 HPLC
3.9% of the detected peaks respectively. The unknown
and a Bruker MicrotofQ II high resolution time of flight
components corresponded to about 7.2% of the detected
(TOF) mass spectrometer. The analysis was in positive
peaks. Red grape juice (Figure 1B) contained anthocyanins
ion mode at 180°C with electrospray ionisation. The nebu-
corresponding to the aglycones delphinidin, cyanidin, petu-
lizer pressure was 1 bar N2, and the flow rate of N2 gas was
nidin, peonidin and malvidin with peak areas of 2.5%, 5.7%,
8 L/min. The collision voltage was 10 eV, collision RF
10%, 9.1% and 24.9% respectively. It also contained 2 com-
210 Vpp, capillary voltage 4000 V. Data was collected
pounds with m/z of 531 and 561. These compounds were
in high resolution mode.
also glucosides, as indicated by the peaks corresponding
to loss of 162 Da by fragmentation in the mass spectrum.
Statistical analysis These two compounds had the molecular formulae
C25H23O13 and C26H25O14 respectively indicating that the
PASW statistics 17 was used to analyse the data. A Student’s
compounds were peonidin-3-O-glucoside pyruvic acid
t-test was used to detect significant differences (p < 0.05).
and malvidin-3-O-glucoside pyruvic acid (vitisin A), which
contributed about 8.8% and 26.1% of the detected peaks.
The composition of anthocyanins in strawberry and red
Results grape juice is presented in Table 1.

Anthocyanins in juice
Analysis of the anthocyanins in strawberry and red grape
juice concentrates has been carried out using HPLC and
LCMS with the aid of anthocyanin standards, and
pelargonidin-3-glucoside was used as a standard in the
Urinary anthocyanins
The compounds present in strawberry juice included
pelargonidin-3-glucoside as well as pelargonidin-3-rutino-
side which were detected in urine after consumption of

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4 Y. Cahyana et al., Urinary Excretion of Anthocyanins

Table 1. Anthocyanin composition in dose of mixed strawberry and

red grape juice consumed by volunteers, containing 33.5 and 17 μmol
of strawberry and red grape anthocyanins respectively
Anthocyanins Strawberry Red grape
(μmol) (μmol)
Pelargonidin-3-glucoside 21.9 ± 0.8
Pelargonidin-3-rutinoside 6.9 ± 0.4
Pelargonidin-3-malonylglucoside 1.3 ± 0.2
Cyanidin-3-glucoside 1 ± 0.2 1 ± 0.1
Delphinidin-3-glucoside 0.5 ± 0.1
Peonidin-3-glucoside 1.6 ± 0.1
Petunidin-3-glucoside 1.7 ± 0.2
Malvidin-3-glucoside 4.2 ± 0.3
Peonidin-3-glucoside pyruvic acid 1.5 ± 0.1
Malvidin-3-glucoside pyruvic acid 4.4 ± 0.1 Figure 2. Chromatogram of urinary anthocyanins from strawberry (A)
and Red grape (B) 1 = delphinidin-3-glucoside; 2 = cyanidin-3-gluco-
Unknown compounds 2.4 ± 0.4 2.1 ± 0.2
side; 3= petunidin-3-glucoside; 4 = pelargonidin-3-glucoside; 6=
pelargonidin-3-rutinoside; 10 = unidentified; 11 = malvidin-3-glucoside
pyruvic acid; 13 =unidentified; a,c,d,e,f,g,h,i=unidentified metabolites;
b=pelargonidin monoglucuronide.
the juice (Figure 2A). New peaks labelled as peaks a, b, c, d, e
which were absent in the blank urine appeared in the urine
following consumption of strawberry juice concentrate.
Peak b was identified as pelargonidin monoglucuronide
characterised by LC-MS with m/z 447/271. The presence
of a fragment with loss of 176 Da indicated that the
compound was a glucuronide. Pelargonidin monoglu-
curonide coeluted with pelargonidin-3-glucoside. The other
new peaks were unidentified. Pelargonidin-3-glucoside
monoglucuronide characterised by LC-MS with m/z of
609/271 was also detected but it was present at too low
concentration to be quantified by HPLC.
Several anthocyanins from red grape juice including del-
phinidin-3-glucoside, cyanidin-3-glucoside and malvidin-
3-glucoside pyruvic acid were detected in urine following
consumption of red grape juice concentrate (Figure 2B).
Figure 3. Chromatogram of strawberry urinary anthocyanins without
New peaks appeared in the urine labelled as peaks f, g, h, treatment (A), urine sample treated with glucuronidase (B).
and i in the figure but these peaks were unidentified. These
peaks were absent in the blank urine. No evidence for the
presence of glucuronide conjugates in the urine was found a concentration of 0.47% of that in the original juice, was
after consumption of red grape juice. significantly higher than that of cyanidin-3-glucoside which
In order to confirm the presence of glucuronides in the was 0.07% of the concentration in the original juice indicat-
urine following strawberry juice consumption, the urine ing that the additional hydroxyl substituent facilitated the
was treated with glucuronidase, incubated at pH 5, 37°C excretion of unmetabolised anthocyanins.
for 15 min. The chromatograms of untreated urine and urine The excretion of unmetabolised petunidin-3-glucoside at
treated with glucuronidase were determined (Figure 3). The 0.08% of the concentration in the juice consumed was
area of peaks a, b, c in untreated urine (Figure 3A) decreased similar to that of cyanidin-3-glucoside and was significantly
following treatment with glucuronidase (Figure 3B). This less than that of delphinidin-3-glucoside. The excretion of
demonstrated that compounds a, b, c were glucuronides. malvidin-3-glucoside pyruvic acid at 0.076% of the
The effect of anthocyanin structure on uptake and excre- corresponding ingested compound was similar to that of
tion was studied by determining the peak areas of urinary petunidin-3-glucoside and cyanidin-3-glucoside.
anthocyanins following consumption of red grape juice The total excretion of strawberry and red grape antho-
since several anthocyanins with different structures were cyanins has been calculated from the urinary anthoyanins
detected in the urine (Figure 4). The chromatogram showed collected at 2, 4, 6, 8 and 24 h. Knowing the strawberry
that the excretion of delphinidin-3-glucoside, which was at and red grape anthocyanin intake which was 14.54 and

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Y. Cahyana et al., Urinary Excretion of Anthocyanins 5

Figure 4. Effect of structure on the excretion of anthocyanins in red

grapes. Dp= delphinidin, Cy=cyanidin, Pet=petunidin, glc=glucoside.
* significant at p  0.05. The measurement was conducted triplicate.

7.39 mg respectively, the percentage of the urinary antho- Figure 5. Excretion of urinary anthocyanins as a function of time
cyanins can be calculated. The mean excretion of straw- following juice consumption presented as percentage of ingested
berry anthocyanins excreted by volunteers during 24 h anthocyanin dose. ACNs=anthocyanins, STRW=strawberry, RG=red
grape. The measurement was conducted triplicate.
collection was 24611.89 ng. Taking into account the molec-
ular weight of pelargonidin-3-glucoside which was used as a
standard, the total excretion of strawberry anthocyanin in
urine was about 0.17% of the dose consumed which was
equal to about 56.8 nmol. Similar calculation has also been
applied to the urinary anthocyanins from red grape. The
total excretion of strawberry anthocyanins was slightly
lower than the mass of red grape anthocyanins excreted
which was 43.4 nmol corresponding to about 0.25% of the
corresponding dose. The difference in the percent excretion
was however not statistically significant, suggesting that the
anthocyanins of strawberry juice were absorbed to a similar
extent to those of red grape juice. The composition of the
juices is very different with strawberry juice containing
89% of pelargonidin and red grape juice containing 51% Figure 6. Relative excretion of total urinary anthocyanins (ACNs)
of malvidin, so this result indicates that there was no evi- (expressed in %) from mixed juice compared to strawberry and red
grape anthocyanins. ACNs=anthocyanins, STRW=strawberry, RG=red
dence of a significant difference in the absorption of
grape. The measurement was conducted triplicate.
pelargonidin compared to malvidin.
The absorption of anthocyanins from the individual
juices could be affected by other components present in excreted urinary anthocyanins. The excretion of intact
the juices capable of inhibiting or modulating the absorp- urinary anthocyanins reached 84% within 6 h and the
tion, or by other variables. In order to provide a more precise excretion was as much as 95% of the total intact urinary
comparison and to confirm the above result, urinary antho- anthocyanins excreted within 8 h.
cyanins from volunteers who consumed a mixed juice The excretion of anthocyanins in the 24 h following
containing strawberry and red grape anthocyanins was consumption of the mixed juice was about 0.28% of the
analysed (Figure 5). Approximately 144 nmol of antho- initial dose of anthocyanins (Figure 6). The concentration
cyanins following consumption of a mixed juice consisting of urinary anthocyanins derived from strawberry was
of 106 nmol of strawberry anthocyanins and 38 nmol of 0.32% of the ingested strawberry anthocyanins compared
red grape anthocyanins was excreted within 24 h following with 0.22% for the ingested red grape anthocyanins but
the consumption of 50.6 μmol of mixed juice. Most antho- the difference was not statistically significant (p  0.05).
cyanins were excreted between 2–6 h following mixed juice Thus, this result confirmed the finding following consump-
consumption with maximum excretion at 2 and 4 h repre- tion of the individual juices that the absorption and excre-
senting respectively about 30% and 34% of the total tion of strawberry and red grape anthocyanins was similar.

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6 Y. Cahyana et al., Urinary Excretion of Anthocyanins
Discussion
Anthocyanins in juice
Strawberry juice used in this study contained pelargonidin
as the major compound present in the juice. Red grape juice,
however, contained other compounds which were not
present in strawberry juice. In this juice peonidin-3-O-
glucoside pyruvic acid and malvidin-3-O-glucoside pyruvic
acid (vitisin A) were identified along with peonidin-3-O-
glucoside and malvidin-3-O-glucoside.
The presence of vitisin A in high quantity in red grape
juice is uncommon and unexpected. It seems that the juice
has been partly fermented during the processing or storage.
Normal red grape juice is usually characterised by malvidin-
3-glucoside as a major compound. However due to fermen-
tation, pyruvic acid which is an intermediate product of
aerobic glycolysis or anaerobic fermentation could react
with malvidin or peonidin-3-glucoside to form malvidin-
3-glucoside pyruvic acid and peonidin-3-glucoside pyruvic
acid [20]. Vitisin A and the related pyranoanthocyanin
vitisin B have been reported to occur in wine following
fermentation, but vitisin B has also been found in red grape
juice [21].
Urinary anthocyanins
Volunteers consumed about 50.5 μmol of anthocyanins
from mixed juice concentrate containing the anthocyanins
of strawberry and red grape equal to 33.5 μmol and 17 μmol
respectively. When the volunteers consumed one of the
juices, they either consumed 33.5 μmol of strawberry
anthocyanins or 17 μmol of red grape juice concentrate. This
helped distinguish metabolites from each juice, whilst the
mixed juice allowed more accurate quantification of the
relative uptake of different anthocyanins.
Apart from pelargonidin-3-glucoside as well as pelargoni-
din-3-rutinoside, glucuronide forms of pelargonidin consid-
ered as the metabolite products were also detected in urine
following strawberry juice consumption. Meanwhile uri-
nary anthocyanins following red grape juice consumption
did not indicate the presence of any glucuronide forms.
The presence of glucuronides in urine has been reported
in other studies following the consumption of strawberry
containing anthocyanins [14]. At least 3 different glu-
curonide-conjugates of pelargonidin have been reported
with different elution times depending on the position of
the glucuronide substituent [22].
The presence of pelargonidin-3-glucoside monoglu-
curonide as a metabolite helps identification of the
glucuronidation pathway. Another study also found the
same metabolite following strawberry ingestion [14]. Two
possible pathway of glucuronidation have been proposed.
Int J Vitam Nutr Res (2019), xx (xx–xx), xx–xx
The first one involving UDP-glucuronosyltransferase to
conjugate glucuronide derived from glucose or glycogen
[23]. The second one is catalysed by UDP-glucose dehydro-
genase to convert the attached sugar into the glucuronide
form [24]. The presence of pelargonidin-3-glucoside
monoglucuronide suggests that the former pathway was
the most likely to occur. This also demonstrates that glu-
curonidation may take place without deglycosylation of
anthocyanins.
The glucuronidation mechanism is consistent with the
finding of 3 different pelargonidin monoglucuronides
following strawberry ingestion, and different hesperetin
monoglucuronides following drinking hesperetin-
contained beverage [22, 25]. If UDP glucose dehydrogenase
was involved by direct oxidation of the attached sugar, the
presence of only 1 monoglucuronide would be expected.
Thus, the former mechanism involving UDP glucuronosyl-
transferase is the most probable pathway.
In terms of the absorption mechanism, the presence of
the glucoside in the urine indicates that anthocyanin
absorption may involve the glucose transporter pathway
as also suggested by other studies [26–28]. Glucosides,
which are more polar than aglycones, are unlikely to pass
through lipophilic membranes into cells by passive diffu-
sion as estimated from the partition coefficients of other
flavonoids [29, 30]. The anthocyanin glucoside needs to
be cleaved to form aglycones to pass by passive diffusion
across the brush border of intestinal cells. Thus, this finding
suggests that the glucoside is absorbed through another
mechanism probably by the glucose transporter pathway
[31] but not through passive diffusion.
Regarding the structure effect, overall the increase in free
hydroxyl residues in the B ring from 2 to 3 seemed to
increase the excretion of unmetabolised anthocyanins. It
is likely that the less polar anthocyanins require glu-
curonidation or sulfation to increase their solubility for
excretion, whereas delphinidin-3-glucoside is sufficiently
soluble to be excreted without metabolism.
It is interesting to explore the effect of the glucose pres-
ence on the anthocyanin bioavailability. It was reported that
glucose could inhibit the absorption of anthocyanins [32],
suggesting that anthocyanins and glucose compete for the
same channel namely the glucose transporter pathway.
Our In-vitro study (data is under preparation to be pub-
lished) which was in line with the other study [31] has shown
that glucose inhibits uptake of anthocyanins by Caco-2 cells
which supports the concept that anthocyanins are absorbed
into apical membrane cells through a glucose transporter.
Quercetin-3-glucoside has also been reported to be capable
of inhibiting the absorption of anthocyanins [33], and
uptake of this compound also occurs via a glucose trans-
porter [34, 35]. Other flavonoid glucosides including quer-
cetin-3-glucoside and kaempferol-3-glucoside occur in
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Y. Cahyana et al., Urinary Excretion of Anthocyanins
strawberries and red grapes [36, 37] and hence would be
present in the juice and these may influence absorption of
anthocyanins by competition for the glucose transporter.
To elucidate the effect of sugar on the absorption, we
analysed the sugar content of the juice using HPLC.
A refraction index detector and AMINEX column have
been used in this analysis. Retention times were compared
with standards to identify the peaks. The glucose and fruc-
tose content of red grape juice concentrate used in this
study was 238.4 mg/g and 330 mg/g respectively which
was approximately 37% and 23% higher than that of the
strawberry juice. Sucrose was coeluted with these peaks
so the concentrations may be overestimated, but the total
sugar content of the red grape juice was higher than that
of the strawberry juice. This sugar content in the juices
was relatively high as concentrated juices were consumed,
and this may contribute to the relatively low concentration
of anthocyanins detected in the urine in this study com-
pared to that found in other studies ranging from 1.8–2.1%
[15, 22]. Felgine used 200 g deep frozen strawberries and
added 15 g sugar or equivalent to 75 mg sugar/g sample
and achieved the urinary anthocyanin recovery around
1.8% [22], while Carkeet applied 100–400 g blended straw-
berry fruit with addition of non-caloric sweetener to
improve palatability and achieved the anthocyanin recov-
ery in urine around 2% [15].
Lower bioavailability obtained in this study could also be
due to the formation of colonic metabolites of anthocyanins
which were not detected in this study. The presence of
phenolic acids in plasma such as protocatechuic acid has
been reported following cyanidin-3-glucoside administra-
tion [38]. Phenolic acids such as protocatechuic acid (3,4-
dihydroxybenzoic acid), gallic acid (3,4,5-trihydroxyben-
zoic acid), and vanillic acid (4-hydroxy-3-methoxybenzoic
acid) have also been reported to be present in rat plasma
as unconjugated and glucuronidated forms [39]. In this
study in which a mixed juice was consumed, we decided
not to quantify the phenolic acids since the detection would
be complicated and their presence would be impossible to
link to specific precursors. Consequently the bioavailability
estimated in this study may underestimate the real value.
The highest urinary recovery was obtained with an isotope
tracer study reaching ca. 5.4% [40] or 3.3% using radiola-
belled anthocyanins which was still considered poor [41],
particularly when compared to the bioavailability of other
flavonoids such as epicathecin (ca. 22%) [25] or isoflavones
of glycitin and daidzin (45%) [42].
Conclusion
The present study led to the conclusion that the antho-
cyanins have poor bioavailability as shown by the excretion
Ó 2019 Hogrefe
7
of only 0.32% for strawberry anthocyanins. Higher concen-
trations of glucuronides were formed from pelargonidin-
3-glucoside than from other anthocyanins. Urinary antho-
cyanins were mainly excreted in urine 2–4 h after
consumption and 95% of the total anthocyanins detected
in urine were excreted within 8 h following consumption.
Delphinidin-3-glucoside was excreted at higher concentra-
tions as the unmetabolised anthocyanin than cyanidin-
3-glucoside and petunidin-3-glucoside which may be due
to the higher solubility of the anthocyanin. Delphinidin-
3-glucoside was however a minor component in both stud-
ied juices and there was no significant difference in the
proportions of anthocyanins excreted following consump-
tion of strawberry or red grape juice.
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Acknowledgments
This work was supported by financial sponsorship from Directorate
General of Higher Education (DGHE) of Indonesia.
Conflict of Interest
The authors declare no competing financial interest.
Yana Cahyana
Universitas Padjadjaran
Bandung
Indonesia
Tel. +62 22-7768844
y.cahyana@unpad.ac.id
Ó 2019 Hogrefe