Bioavailability of anthocyanins and derivatives

Iva Fernandes
a
, Ana Faria
a,b,c
, Conceica o Calhau
b,d
, Victor de Freitas
a
, Nuno Mateus
a,
*
a
Chemistry Investigation Centre (CIQ), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
Rua do Campo Alegre, 4169-007 Porto, Portugal
b
Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Al. Prof. Herna ni Monteiro, 4200-319 Porto, Portugal
c
Faculty of Nutrition and Food Sciences, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
d
CINTESIS Center for Research in Health Technologies and Information Systems, University of Porto, Al. Prof Herna ni Monteiro,
4200-319 Porto, Portugal
A R T I C L E I N F O
Article history:
Available online xxxx
Keywords:
Absorption
Anthocyanins
Bioavailability
Metabolism
Microbiota
A B S T R A C T
Anthocyanins are naturally occurring compounds widespread in plant-derived foodstuffs
and therefore abundant in human diet. There are evidences regarding the positive associ-
ation of their intake with healthy biological effects displayed in vivo. This review aims to
highlight some aspects regarding anthocyanins bioavailability; these include a short intro-
ductory part of anthocyanin chemistry, stability, occurrence and intake. This rst part is
followed by a more detailed one concerning the main topic of the review that includes
the bioavailability and metabolism of anthocyanins. Special attention is given to the con-
tribution of the gastric mucosa to anthocyanin absorption as the result of the high content
of intact anthocyanins (2025%) detected is plasma few minutes after intake. The contribu-
tion of intestinal tissue and the microbiota impact in anthocyanin absorption and bioactiv-
ity is also highlighted. Despite the biological activities that have been associated with these
compounds, anthocyanins appear to be rapidly absorbed and eliminated, reaching only low
maximal concentrations in plasma and urine. Some possible critical factors that may con-
tribute to this paradox were also explored including the ability of a compound to cross
membranes, the effect of pH, digestive enzymes, biliary acids and microbiota, the lack of
sensitivity of the analytical method, the possible ingestion of pigments (anthocyanin deriv-
atives, especially in the case of red wine) and the inuence of the food matrix. Generally,
the bioavailability of anthocyanins is presumed but whether the effect is due to the native
compounds or other forms, which mechanism are involved or which factors have crucial
impact on bioavailability still remain underexplored.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Anthocyanins are one of the most widespread families of nat-
ural pigments in the plant kingdom, thereby constituting a
part of the world natural heritage. They confer a great diversity
of colours, touching practically all visible spectra, from or-
ange and red through purple and blue hues.
Over the years, the scientic community has been focus-
ing on these amazing molecules trying to understand antho-
cyanins and their properties. Many different chemical
structures from numerous natural sources have been charac-
terized and their physicalchemical properties determined
(Deroles, 2009); their biosynthesis pathways have been eluci-
dated, and new plants with a la carte colours created by
1756-4646/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.05.010
* Corresponding author. Tel.: +351 220402562.
E-mail address: nbmateus@fc.up.pt (N. Mateus).
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genetic engineering (Davies, 2009); their benets for human
health are being discovered, and the applications of anthocy-
anins as colorants or putative bioactives have been exploited
by food, pharmaceutical and cosmetic industries.
2. Anthocyanins chemistry and stability
Attending to their chemical nature, anthocyanins naturallyoc-
cur as glycosides of avylium (2-phenylbenzopyrylium) salts
and are commonly based on six anthocyanidins: pelargonidin
(Pg3glc), cyanidin (Cy3glc), peonidin (Pn3glc), delphinidin
(Dp3glc), petunidin (Pt3glc) and malvidin (Mv3glc) (Fig. 1).
The sugar moieties vary but are usually glucose, rhamnose,
galactose or arabinose (Francis, 1989). The sugar moiety may
be a mono or disaccharide unit, and it may be acylated with
a phenolic or aliphatic acid (Mazza & Miniati, 1993). These
compounds differ in the methoxyl and hydroxyl substitution
patternof the aromatic Bring. Despite the most commonanth-
ocyanidins being just six, there are 539 anthocyanins reported
to be isolated from plants (Andersen & Jordheim, 2005). The
more widespread anthocyanins in fruits are glycosilated in
the 3-OH position (3-O-monoglycosides) and, in less exten-
sion, in both position 3-OH and 5-OH (3,5-O-diglycosides).
Anthocyanins have characteristic physicochemical proper-
ties that confer them its unique colour and stability. They are
highly reactive molecules and thus sensitive to degradation
reactions. Oxygen, temperature, light, enzymes and pH are
among the factors that may affect anthocyanins chemistry
and, consequently, their stability and colour. Anthocyanins
may be degraded through several processes occurring during
their extraction, food processing and storage. The fact that in
aqueous solution they co-exist in pH-dependent equilibrium
between ve species (avylium cation, carbinol base, chal-
cone, quinonoidal base and anionic quinonoidal base) is crit-
ical and deeply related to the colour displayed by
anthocyanins (Fig. 2) (Brouillard & Delaporte, 1977; Brouillard
& Dubois, 1977; Brouillard & Lang, 1990).
3. Occurrence and intake
Polyphenols arise from the secondary metabolism of plants,
being virtually present in all foods and beverages from plant
origin such as vegetables, fruits, cocoa, tea and wine. How-
ever, the daily intake of polyphenols is difcult to estimate
and depend on several factors. Recently, the construction
and application of a database with polyphenols content in
foods has facilitated this task (Neveu et al., 2010).
In 2000, Scalbert and Williamson reported 1 g/day as the
total dietary intake of polyphenols, which were values way
above the ones described for vitamin C or E which are the
classical dietary antioxidants (Scalbert & Williamson, 2000).
Later, Pe rez-Jime nez and colleagues conrmed polyphenol
daily intake of about 1 g/day by using a French cohort and a
phenolic content in foods database. Even so, this value can
be higher due to a lack or insufcient data on food contents
for more complex polyphenols (Perez-Jimenez et al., 2011).
Anthocyanins are part of human diet as they can be found
in red wine, some cereals and root vegetables (aubergines,
beans, cabbage, radishes, onions) but mainly due to its pres-
ence in red fruits such as cherries, strawberries, plums, black-
berries, raspberries, grapes, red currants and black currants.
Anthocyanins are mainly found in skin but can also appear
in the esh, e.g., cherries and strawberries. Usually, the con-
tent in anthocyanins increases during ripening and can reach
values up to 24 g/kg fresh weight in blackcurrants, blackel-
derberry or blackchokeberry (Clifford, 2000; Mazza & Velioglu,
1992; Perez-Jimenez, Neveu, Vos, & Scalbert, 2010). Red wine is
OR
3
O
OH
OH
R
1
R
2
HO
Anthocyanins R
1
R
2
Pg3glc H H
Pn3glc OCH
3
H
Cy3glc OH H
Mv3glc OCH
3
OCH
3
Pt3glc OCH
3
OH
Dp3glc OH OH
Fig. 1 Representation of the general structure of
anthocyanins (avylium form). R
3
is a sugar moiety.
Fig. 2 Schematic representation of the molar fraction of
anthocyanin equilibrium form according to the GI tract pH
adapted from (Nave et al., 2010).
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also a good source of anthocyanins and contains approxi-
mately 200350 mg of anthocyanins per L; in addition, as
the wine ages, these anthocyanins can be converted into
more complex structures such as anthocyanin-pyruvic
acid adducts and vinylpyranoanthocyanin-catechins (Fig. 3)
(Clifford, 2000; Oliveira, de Freitas, Silva, & Mateus, 2007;
Perez-Jimenez et al., 2010; Pissarra et al., 2004; Silberberg
et al., 2006; Sousa et al., 2007).
4. Bioavailability and metabolism of
anthocyanins
Anthocyanins, for consumers that eat berries and drink red
wine on a routine basis, are major dietary components. How-
ever, the key difference compared to the other avonoid gly-
cosides, is that anthocyanins undergo re-arrangements in
response to pH and temperature (Brouillard & Delaporte,
1977) (Fig. 2). Physiological temperatures are highly suitable
both thermodynamically and kinetically for observing the
chalcone tautomer (Brouillard & Delaporte, 1977). The limited
available experimental evidence indicates that in the acidic
conditions that prevail in the gastric compartment anthocya-
nins are in the positively charged avylium form, whilst all
the other dietary avonoids remain neutral.
The difculty in overcoming those analytical problems
may contribute signicantly to the low bioavailability of
anthocyanins, which does not justify all the biological activi-
ties previously associated with the huge consumption of this
avonoid class.
Extensive knowledge of the bioavailability of anthocyanins
is thus essential if their health effects are to be understood
(Fig. 4).
After ingestion, anthocyanins are readily detected in plas-
ma in their parent forms, possibly as a result of their absorp-
tion through the gastric wall (Cao, Muccitelli, Sanchez-
Moreno, & Prior, 2001; Cao & Prior, 1999; Milbury, Cao, Prior,
& Blumberg, 2002; Mulleder, Murkovic, & Pfannhauser, 2002).
It has been only recently that studies were conducted to
determine tissue concentrations of anthocyanins. The stom-
ach exhibited only native anthocyanins, while in other organs
(jejunum, liver, and kidney) native and methylated anthocya-
nins as well as conjugated anthocyanidins (monoglucuro-
nides) were detected (Talave ra et al., 2005).
In another work, pigs were fed with diets supplemented
with blueberries for 4 weeks. Although no anthocyanins were
detected in the plasma or urine of the fasted animals, intact
anthocyanins were detected in the liver, eye, cortex, and cer-
ebellum. The results suggest that anthocyanins can accumu-
Fig. 3 Chemical structures of anthocyanin derivatives: malvidin-3-glucoside pyruvic acid adduct, vinylpyranomalvidin-3-
glucoside-catechin and (+)-catechin-(4,8)-malvidin-3-glucoside.
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late in tissues, including tissues beyond the bloodbrain bar-
rier (Kalt et al., 2008).
In a more recent work, anthocyanin metabolites (methyl-
ated anthocyanins and glucurono-conjugated derivatives)
were identied in various organs (bladder, prostate, testes,
heart and adipose tissue) in rats fed with a blackberry antho-
cyanin-enriched diet for 12 days (Felgines et al., 2009). In that
study, the bladder contained the highest levels of anthocya-
nins, followed by the prostate. Prostate, testes and heart con-
tained native cyanidin 3-glucoside and a small proportion of
cyanidin monoglucuronide. Cyanidin 3-glucoside and methyl-
ated derivatives were present in adipose tissue. Moreover, two
recent works reported the capacity of dietary anthocyanins
from grapes and berries to reach the brain (Passamonti,
Vrhovsek, Vanzo, & Mattivi, 2005; Talave ra et al., 2005).
4.1. Oral cavity absorption of anthocyanins
Studies involving individual anthocyanins revealed that their
amount in plasma is generally 1% of the consumed quanti-
ties, due to limited intestinal absorption, although additional
factors may contribute to the proposed low anthocyanin bio-
availability such as high rates of cellular uptake, metabolism
and excretion (Fig. 5) (Manach, Williamson, Morand, Scalbert,
& Remesy, 2005).
Upstream to gastrointestinal absorption, a variety of bind-
ing processes can take place, namely interaction with food
proteins or with salivary proteins and digestive enzymes
(Matsui et al., 2001; Walle, Browning, Steed, Reed, & Walle,
2005; Wiese, Ga rtner, Rawel, Winterhalter, & Kulling, 2009).
In a recent work with healthy volunteers, black raspberry
anthocyanins could be detected as their hydrolyzed aglycone
form in the oral cavity, resulting from the activity of b-glyco-
sidase derived both from bacteria and oral epithelial cells
(Mallery et al., 2011). In the same study, parent anthocyanins
and protocatechuic acid, a cyanidin-3-glucoside microbiota
metabolite, were detected in the saliva. Furthermore, saliva
samples revealed the presence of glucuronidated anthocya-
nin conjugates, consistent with intracellular uptake and
phase II conversion of anthocyanins (Mallery et al., 2011).
Still, whether these oral transformation reactions will be
of importance for local effects in the oral epithelium is dif-
cult to assess considering the relatively short residence time
of most foods in the oral cavity.
4.2. Gastric absorption of anthocyanins a new approach
Until the beginning of the twenty rst century the study of
fast kinetics of plasma appearance of anthocyanins in rats
and humans was a challenging eld (Cao & Prior, 1999; Mil-
Fig. 4 Hypothetic pathways of anthocyanins absorption, distribution, metabolism and excretion based on current
information.
4 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x x x x
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bury et al., 2002; Murkovic, Mu lleder, Adam, & Pfannhauser,
2001; Tsuda, Horio, & Osawa, 1999).
In 2003, Passamonti and co-workers performed an in vivo
experiment in rats that suggested the ability of anthocyanins
to cross the gastric mucosa (Passamonti, Vrhovsek, Vanzo, &
Mattivi, 2003).
Later, Talave ra et al., 2003 extended their research to struc-
turally related anthocyanins and demonstrated that anthocy-
anin glycosides were quickly and efciently absorbed in the
stomach (approximately 25%). However their absorption var-
ied greatly according to the anthocyanin structure and they
were rapidly excreted into bile as intact and metabolized
forms.
In 2005, the same authors investigated anthocyanin
metabolism and distribution in different rat organs (stomach,
jejunum, liver, kidney and brain). The only additional infor-
mation of this work concerning the stomach absorption was
its incapacity of metabolizing anthocyanins, since no metab-
olites were detected in this organ.
Moreover, El Mohsen et al. (2006) have analyzed pelargoni-
din gastric absorption in rats having detected the presence of
p-hydroxybenzoic acid in stomach 2 h after ingestion. This re-
sult is not indicative of anthocyanin transformation in gastric
cavity but rather of the instability and degradation of the
anthocyanidin.
The absorption of red orange anthocyanins was studied in
both rat stomach and intestine using in situ models (Felgines
et al., 2006). A high proportion (about 20%) of red orange
anthocyanins was absorbed from the stomach and again no
anthocyanin metabolite was observed in the stomach after
30 min of incubation.
The earliest reference on anthocyanin absorption in stom-
ach is from 2007. In that work the authors examined the gas-
tric absorption of pelargonidin-3-glucoside using rat models.
Once more, a high proportion of pelargonidin-3-glucoside
was found to be rapidly absorbed from the stomach (23%) (Fel-
gines et al., 2007).
Bearing all the above data, it appears that these amazing
compounds could entirely jump the gastric system and
reach the systemic circulation in their native or metabolized
forms, being available to exert their biological activities
approximately 30 min after ingestion. In the meanwhile, all
other avonoid glycoside compounds are still in their journey
towards the circulation. There are some few references con-
cerning the gastric absorption of avonoid aglycones. Querce-
tin, but not quercetin 3-O-glucoside nor quercetin 3-O-
rutinoside, was found to be absorbed in rat stomach (Crespy
et al., 2001). Similarly, the isoavones genistein and daidzein,
but not their glucosides, were also found to be absorbed in the
rat stomach (Piskula, Yamakoshi, & Iwai, 1999). Finally, some
phenolic acids were also found to be absorbed at the gastric
epithelium (Konishi, Zhao, & Shimizu, 2006; Lafay et al.,
2006; Vanzo et al., 2007; Zhao, Egashira, & Sanada, 2004).
The main conclusion common to all the authors is the
high content of anthocyanins in the stomach, but the possible
mechanism of anthocyanin gastric absorption remains
unknown.
On this matter, the bioavailability of cyanidin-3-glucoside
in rats that were fed a red orange extract with or without glu-
cose by gastric intubation was not signicantly affected by
simultaneous ingestion of glucose (Felgines et al., 2008). This
fact may suggest that the glucose transporters are not in-
volved in anthocyanin gastric absorption.
Information regarding the kinetic ux of anthocyanins in
the GI is critical for understanding anthocyanin absorption.
After feeding rats with black raspberry extract by stomach
tube, anthocyanin content in the gastric lumen was found
to decrease linearly during 180 min (He, Wallace, Keatley, Fail-
la, & Giusti, 2009). The estimated time to deplete half of the
anthocyanin content in the gastric lumen of the fasted rat
was approximately 120 min, suggesting that minimal
amounts of anthocyanins would still be present in the stom-
ach after 4 h. In the same work, the authors highlighted an-
other important factor that may contribute to the
underestimated anthocyanin quantication. Anthocyanins
appeared to bind to unidentied protein in the stomach tissue
and thus could not be quantied as free anthocyanins by
HPLC. Such binding may be attributed to nonspecic binding
or perhaps specic binding to some protein transporter.
The organic anion carrier bilitranslocase is expressed in
the stomach (Battiston, Macagno, Passamonti, Micali, & Luigi
Sottocasa, 1999; Nicolin, Grill, Micali, Narducci, & Passamonti,
2005). Its, in vitro, normal transport activity is competitively
inhibited by quinoidal forms of dietary anthocyanins (Fig. 6),
suggesting that bilitranslocase could promote the facilitated
diffusion of anthocyanins (Passamonti, Vrhovsek, & Mattivi,
2002). Nevertheless, it should be noted that those in vitro as-
says performed were conducted at pH 8.0, which is far from
the gastric conditions here no quinoidal forms could be de-
tected (Fig. 2). Therefore, bilitranslocase may be involved in
anthocyanin quinoidal forms absorption in the liver (Fig. 6).
On the other hand, the administration of high amounts of
anthocyanins, far from diet levels, could induce saturation of
this transport and contribute to the lower anthocyanin bio-
availability reported in those particular studies (Talave ra
et al., 2003).
Other transporter candidates may include GLUT1, OAT2,
SMCT1 and SMCT2, since the expression of these transporters
has already been detected in stomach tissue (Eraly, Bush,
Sampogna, Bhatnagar, & Nigam, 2004; Garcia, Brown, Pathak,
& Goldstein, 1995; Yoshikawa et al., 2011).
I
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a
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i
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s
0
1
2
3
4
C
m
a
x

(

M
)
Fig. 5 Maximum plasma concentration of anthocyanins
compared to other avonoid classes adapted from (Manach
et al., 2005).
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The stomach has been widely ignored as a metabolizing
organ although it has been identied as a site of absorption
for different compounds (Crespy et al., 2001; Piskula et al.,
1999). The contribution of the gastric mucosa to the metabo-
lism of anthocyanins should not be ruled out because the
stomach possesses conjugative enzyme activities (UDP-glu-
curonosyltransferase, sulphotransferase, and catechol-O-
methyl transferase) (Harris, Picton, Singh, & Waring, 2000;
Karhunen, Tilgmann, Ulmanen, Julkunen, & Panula, 1994;
Strassburg, Nguyen, Manns, & Tukey, 1998; Strassburg, Oldha-
fer, Manns, & Tukey, 1997). Besides, in vitro studies showed
that some avonoids could be metabolized into glucuronidat-
ed and sulphated metabolites by the gastric wall (De chelotte,
Varrentrapp, Meyer, & Schwenk, 1993; Piskula et al., 1999).
As it can be easily perceived, there is too much ambiguous
information on the literature concerning anthocyanin gastric
absorption.
Working with animal models or human volunteers is obvi-
ously an important vehicle for obtaining new insights on
anthocyanin bioavailability. In the meantime, a gastric cell
barrier model would be extremely useful similarly to what
was gained with the studies with caco-2 cell line that mimics
the intestinal barrier.
The methods available to evaluate the absorption of drugs
at the gastric level make use of isolated gastric epithelial cells,
which are both time and labour consuming.
A critical feature of such a model is that it has to work in
the presence of a reduced pH. A recently published work re-
ported the development of a biologically relevant in vitro mod-
el of moderately differentiated adenocarcinoma stomach
cells (MKN-28) to be used as a gastric barrier model (Fernan-
des, de Freitas, Reis, & Mateus, 2012). In that work, the
absorption and metabolism of anthocyanins through gastric
epithelium cells was evaluated over time in the presence of
proton gradient.
By using this model, it was possible to study different pH
conditions that correspond to the fed and unfed stage, pH
of 3.0 or 5.0, respectively (Dressman et al., 1990; Russell
et al., 1993). It was also possible to conclude that anthocya-
nins could cross the gastric epithelium in a time dependent
manner with no statistically differences in their transport
efciency according to the pH. Attending to the results ob-
tained, a saturable transport for these compounds was thus
proposed.
Considering the previously reported implication of glucose
transporters in the absorption of anthocyanins at the intesti-
nal level (Faria, Pestana, Azevedo et al., 2009) the effect of
anthocyanins on the uptake of
3
H-DG was proposed. Preli-
minary studies on this eld indicated that the anthocyanins
tested did not affected glucose uptake in MKN-28 cell line
(Fig. 7).
4.3. Intestinal absorption of anthocyanins
The anthocyanin fraction that is not absorbed in the stomach
reaches the small intestine. Once anthocyanins enter more
basic conditions in the small intestine the carbinol pseudo-
base is likely to predominate. Unlike avonoids where glyco-
sides are hydrolyzed, anthocyanin glycosides are rapidly and
efciently absorbed in the small intestine (Miyazawa, Nakag-
awa, Kudo, Muraishi, & Someya, 1999; Talave ra et al., 2004;
Tsuda et al., 1999). Furthermore, anthocyanins are quickly
metabolized and appear in the circulation or are excreted into
bile and urine as both intact and metabolized forms (glucu-
ronidated, sulfated or methylated derivatives) (Fig. 4) (Ichi-
yanagi, Shida, Rahman, Hatano, Matsumoto et al., 2005;
Ichiyanagi, Shida, Rahman, Hatano, et al., 2005; Ichiyanagi
et al., 2004; McGhie, Ainge, Barnett, Cooney, & Jensen, 2003;
Miyazawa et al., 1999; Talave ra et al., 2003; Talave ra et al.,
2004).
The potential mechanisms of anthocyanin glycosides
absorption in the small intestine may involve a specic glu-
cose transporter, such as SGLT1, as previously suggested for
other avonoids (Hollman et al., 1999).
A recent work points for the putative involvement of
GLUT2 transporter in anthocyanins absorption at the intesti-
nal level (Faria et al., 2009).
Another possible mechanism may involve the hydrolyza-
tion of anthocyanins by brush border enzymes such as lactase
phloridzin hydrolase, prior to passive diffusion of the agly-
1
2
1
2
Fig. 6 Schematic representation of the in vitro studies conducted to prove the involvement of bilitranslocase in anthocyanin
absorption.
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cone, as already proved for other avonoids (Gee et al., 2000;
Hollman et al., 1999).
Unabsorbed anthocyanins reach the colon where they un-
dergo substantial structural modications. Previous studies
have suggested that this is likely due to the spontaneous deg-
radation under physiological conditions (Woodward, Kroon,
Cassidy, & Kay, 2009) or following microbial metabolism. In
fact, colonic microbiota hydrolyses glycosides into aglycones
and degrades them to simple phenolic acids.
According to Vitaglione and co-workers protocatechuic
acid is the major human metabolite of cyanidin-3-glucoside
in humans (Vitaglione et al., 2007). In particular, proto-
catechuic acid accounts for almost 73% of the ingested antho-
cyanins. This metabolite was detected in plasma 2 h after
orange juice ingestion, indicating that it was possibly formed
through chemical degradation at the physiological conditions
of the systemic circulation or in the intestinal mucosa. This
metabolite is recovered in fecal samples, which suggests that
the gut extensively metabolizes anthocyanins (Riso et al.,
2005).
The metabolism of berry anthocyanins resulting in pheno-
lic acids in humans was recently studied (Nurmi et al., 2009).
The main anthocyanin metabolites detected were homovani-
lic and vanilic acids.
In another fresh study, blueberry anthocyanin absorption
and metabolism in rats was accomplished and the main
metabolite detected in urine was hippuric acid, which may
be produced in liver through a conjugation of glycine with
aromatic phenolic acids (Del Bo` et al., 2009).
Since anthocyanin phenolic acids can be further absorbed
in colon (Williamson & Clifford, 2010) it is possible that they
are additionally metabolized by hepatic cells (Woodward,
Needs, & Kay, 2011). Health benets associated with anthocy-
anin rich foods may also be explained by a slow and continu-
ous release of phenolic compounds through the gut into the
bloodstream.
Despite their low apparent bioavailability, plasma concen-
trations of anthocyanins appear sufcient to induce changes
in signal transduction and gene expression in vivo (DeFuria
et al., 2009; Karlsen et al., 2007) in a manner that suggests
their putative role in physiological functions and health out-
comes. It is known that avonoids may directly interact with
membrane lipids altering membrane physical properties, li-
gandreceptor interactions, modulate signal transduction,
transport and enzyme activity (Verstraeten, Fraga, & Oteiza,
2010).
Some of the human studies investigating anthocyanin bio-
availability were recently revised (Faria, Fernandes, Mateus, &
Calhau, 2013).
The overall analysis of the biokinetic parameters of those
studies has facilitated some main assumptions in what
anthocyanin bioavailability is concerned. The most important
one is that although there is a considerable variability in the
values for the biokinetic parameters, anthocyanins appear
to be rapidly absorbed and eliminated, reaching low maximal
concentrations in plasma and urine.
Several factors including variations in the dose, anthocya-
nin chemical composition in the different sources, food or
beverage matrix or processing, age and gender of the individ-
uals and the analytical methodology used can have a huge ef-
fect on the bioavailability and metabolism of anthocyanins.
5. Microbiota impact on anthocyanins
availability and bioactivity
Microbiota has been considered a metabolizing organ with a
role in human metabolism through endo- and xenobiotic
metabolism, vitamin B12 synthesis, carbohydrate breakdown,
between other important functions.
Besides the obvious role of the gut in normal digestive pro-
cesses, the community of microorganisms in the human GI
tract is now being considered as a microbial organ.
The human gut is composed of a bacterial ecosystem of
around 10
13
10
14
bacterial cells, despite not being fully de-
scribed. Microorganisms living inside humans are estimated
to be more than 10 times human cells, and the microbiome
represent more than 100 times the human genome (Cani &
Delzenne, 2009). It has been described a key metabolic role
for microbiota on diabetes and obesity morbilities (Ley et al.,
2005).
C
o
n
t
r
o
l
C
y
3
g
l
c
M
v
3
g
l
c
C
o
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r
o
l
D
p
3
g
l
c
P
h
l
o
r
i
d
z
i
n

1

m
M
P
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2

m
M M

C
y
t
o
c
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l
a
s
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n
B
5
0
0
50
100
150
*
*
*
3
H
-
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-
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e

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(
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)
Fig. 7 Effect of anthocyanins (100 lM) for 30 min on the uptake of
3
H-DG (100 lM) by MKN-28 cells. MKN-28 cells were
incubated at 37 C with
3
H-DG, for 1 min. MKN-28 cells were preincubated for 30 min with cytochalasin B (50 lM), phloretin
(2 mM) and phloridzin (1 mM) and incubated with
3
H-DG (100 lM), for 1 min. Each value represents the mean SEM (n = 6).
Signicantly different from the respective control (
*
p < 0.05).
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x x x x 7
Please cite this article in press as: Fernandes, I. et al., Bioavailability of anthocyanins and derivatives, Journal of Functional Foods (2014), http://
dx.doi.org/10.1016/j.j.2013.05.010
The chemical forms of anthocyanins ingested in the diet
are not the ones that reach microbiota but instead their
respective metabolites that were excreted in the bile and/or
from the enterohepatic circulation. In colon, anthocyanins
are broadly metabolized by bacteria originating more simple
compounds.
Anthocyanins metabolization includes methylation, sulfa-
tion and conjugation with glucuronic acid but also the break
of glycoside linkages and cleavage of the anthocyanin hetero-
cycle (Goldberg, Yan, & Soleas, 2003; Rechner et al., 2004;
Slimestad, Fossen, & Vagen, 2007). For these reactions to take
place, the involvement of beta-D-glucosidases, beta-D-glucu-
ronidases and alfa-L-rhamnosidases that release aglycones
from their glycoside or glucuronidate forms is necessary
(Aura et al., 2005; Gonthier et al., 2003; Selma, Espin, & To-
mas-Barberan, 2009). Bacteroides could be the genera mostly
involved in these reactions as they express these enzymes.
Because of this and, based on substrate specicity, anthocya-
nins may be responsible for the prebiotic benets associated
with red wine ingestion, in particular for Bacteroides (Que-
ipo-Ortuno et al., 2012). This is a new, but growing, concept,
especially taken into consideration that dysbiosis occurs in
the occidental diet pattern and evidence exists that the Bacte-
roides number needs to grow. Studies comparing microbiota
from lean and obese individuals have shown an increase on
Firmicutes genera and a decrease in Bacteroides (about 50%
reduction) in the obese population (Ley et al., 2005).
The structure of the metabolites produced in colon are not
dependent on sugar moieties but on the structural features of
the polyphenols. As already referred, protocatechuic acid has
been reported as the main metabolite, after anthocyanin con-
sumption (Goldberg et al., 2003; Selma et al., 2009; Slimestad
et al., 2007). A recent work conrmed the degradation of
cyanidin-3-glucoside to protocatechuic acid after incubation
with gut bacteria (Hanske et al., 2013). Curiously, it has been
described that protocatechuic acid improves spatial working
memory (Corona, Vauzour, Hercelin, Williams, & Spencer,
2013).
The individual characteristics of microbiota strongly de-
pend on the dietary habits (Cani & Delzenne, 2011), which
in turn inuence anthocyanins bioavailability.
6. Factors affecting anthocyanins
bioavailability
The basic kinetic concepts used to study drug actions or phar-
macokinetics are usually applied to study or predict the
movement of other substances in the organism, such as tox-
ins, environmental pollutants or even phytochemicals.
Biokinetic or disposition is the term used to describe the
path of a xenobiotic in the body and is dened as the compos-
ite actions of its absorption, distribution, biotransformation
and elimination. The bioactivity of a substance is directly
dependent on its concentration, making the disposition of
any compound a major contributor to its potential bioactivity.
Therefore, because the disposition of a chemical determines
its concentration at the site of action, the concerted action
of absorption, distribution and elimination dictates the po-
tential for biological events to occur.
The major route of entry of phytochemicals, polyphenols
and, in particular, avonoids is by oral ingestion, as they are
consumed as part of a normal diet. The bioavailability of
these xenobiotics can be inuenced by several factors, simi-
larly to what happens with other compounds ingested orally.
All the way through the absorption, distribution, biotransfor-
mation and elimination processes, the movement through
biological membrane is implied. Nevertheless, the ability of
a compound to cross membranes can be determined by its
physicochemical characteristics such as size, lipid/water sol-
ubility or pKa. Usually, larger, hydrophilic or ionic charged
molecules cannot freely cross membranes and a transporter
must be implicated.
Regarding the gastrointestinal (GI) tract several other fac-
tors may also inuence xenobiotics absorption such as pH,
food, digestive enzymes, biliary acids, microbiota and the
motility and permeability of the GI tract. The passage
through GI is extremely important concerning anthocyanins
absorption since these compounds have a complex chemis-
try responsible for their attractive colours but also for their
instability, which will inuence all the biokinetics processes,
and thus, the bioactivity. Its structure is pH-sensitive,
becoming unstable at higher pH (McGhie & Walton, 2007).
The most common methodologies to detect anthocyanins
are centered on their colour in a cationic form and are based
on the total conversion of anthocyanins to this form in
acidic medium. However, the conversion of anthocyanins
to a cation form may not be complete leading to an underes-
timated quantication of these compounds (Fernandes et al.,
2012). In addition, it is usually not considered that anthocy-
anins may be metabolized/biotransformed and, thus, the
conversion back to the cationic form after acidication is
no longer a possibility, contributing to this underestimation.
Further, whether the biological effects attributed to anthocy-
anins are due to their cationic form, their hemiacetal
form or a metabolite of one of these two forms is still
uncertain.
Another important factor affecting anthocyanins bioavail-
ability is their possible ingestion as pigments (anthocyanin
derivatives), especially when considering wine consumption
(Fig. 7). A recent work had already indicated that anthocyanin
pyruvic-acid adducts can rapidly reach rat plasma 15 min
after oral administration of 400 mg/kg bw (21.1 nM kg/lmol
and 28.8 nM kg/lmol, of malvidin-3-glucoside-pyruvic acid
adduct and malvidin-3-glucoside, respectively) (Faria et al.,
2009). The possible absorption of other forms of anthocyanin
derivatives has also been recently explored in a study that
showed that avanolanthocyanin pigments presented a
higher absorption efciency in caco-2 cell model than procy-
anidin B3 (Fernandes, Nave, Gonc alves, de Freitas, & Mateus,
2012). This work showed that not only anthocyanins and av-
anols may cross the caco-2 cell barrier model with similar
efciency, but also dimeric structures containing both antho-
cyanins and avanols, although with a lower efciency than
the respective monomers.
Based on the reported studies, a new eld of interest that
is often overlooked arises: the anthocyanin absorption as
anthocyanin-derived pigments.
Therefore, the overall anthocyanin bioavailability should
result from the contribution of the amount that crosses all
8 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x x x x
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dx.doi.org/10.1016/j.j.2013.05.010
physiological barriers in all their possible forms: native, deg-
radation products, metabolites and anthocyanin derivatives
(Fig. 8).
The availability of phenolic compounds can be also depen-
dent on the food matrix where they are inserted. A more lipo-
philic environment may facilitate avonoids solubilization
and absorption. Additionally, the presence of ethanol can also
be determinant on the extent of anthocyanins absorption
(Faria et al., 2009), promoting their transport across intestinal
epithelia. Another factor to take into consideration is the
interaction between polyphenolic compounds and the other
compounds present during a meal: it is well known the ability
of these compounds to interact with proteins (Bras et al.,
2010; Goncalves, Mateus, & de Freitas, 2010), modifying or
changing their biological function and limiting and/or inter-
fering with both protein and phenolic absorption; moreover,
because the cell does not have specic mechanisms for phen-
olics entry, they use the cell machinery for other substances,
interfering with these molecules absorption, e.g., organic cat-
ions, glucose (Faria, Mateus, de Freitas, & Calhau, 2006; Faria
et al., 2009; Keating, Lemos, Goncalves, & Martel, 2008).
Recently, the frequency of the consumption has also been
a point of discussion since cells long-term exposed to antho-
cyanins demonstrated to be more prone to their transport
(Faria et al., 2009). This point is of specical importance as it
may be the rst step to justify dietary recommendations
and emphasizes the importance of a fruit and vegetable-rich
diet.
Different animal and human studies in the past decade
have related anthocyanin-rich foods with health benecial ef-
fects (Andres-Lacueva et al., 2005; Krikorian et al., 2010). For
this to happen, bioavailability of these compounds is pre-
sumed but whether the effect is due to the native compounds
or its metabolites, which mechanism are involved or which
factors have crucial impact on bioavailability still remains
underexplored.
Fig. 8 Schematic representation of the different anthocyanin forms that could contribute to the net bioavailability and
biological effects of these food components.
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x x x x 9
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dx.doi.org/10.1016/j.j.2013.05.010
7. Conclusion
Overall, anthocyanins are found in diverse food sources, with
berries and red wine as main sources, they are gastric and
intestinally absorbed, but they could also reach the colon
and undergo microbiota metabolization. Content of anthocy-
anins may vary considerably between food sources, environ-
mental conditions, as a direct result of sun exposure,
cultivars, etc. This fact is often neglected in studies, probably
justifying the results variability and the, still existing, gap in
knowledge.
The interest in anthocyanins has been driven primarily by
epidemiological studies that have suggested that diets rich in
these phytochemicals are benecial to human health.
There is a real possibility that some dietary anthocyanins
or derived pigments contribute positively to health and
well-being. Healthy known effects associated with consump-
tion of anthocyanin-rich foods should be attributed to: (i) di-
rect effects of the absorbed parent compounds (or their
metabolites); (ii) indirect effects mediated by non-absorbed
entities that, probably, induce modications on microbiota
environment and, consequently, on human metabolism or
could act at the membrane border inducing signal transduc-
tion pathways.
The health benets associated in epidemiologic studies
with the consumption of anthocyanin-rich foods contradict
the apparent low bioavailability of these compounds.
Nevertheless, the biological activity of absorbed parent
compounds, their metabolites and microbial catabolites and
the potential synergy between them could be the answer to
the anthocyanin paradox bioactivity.
More studies should be carried out in what concerns
anthocyanin or derived pigments transport across biological
membranes. There are studies announcing gastric absorption
and neuroprotective effects of anthocyanin-rich foods, but
there is a gap in the knowledge concerning, for example,
anthocyanin transport across gastric or bloodbrain barrier.
On top of this, it is urgent to know dietary factors able to
modulate anthocyanin bioavailability, helping health profes-
sionals to make dietary recommendations. These recommen-
dations will be relevant for a healthy life but, also, to alert
medical doctors as to possible pharmacological interactions
with anthocyanins.
Acknowledgements
This work was supported by FCT (Fundac a o para a Cie ncia e
Tecnologia) (POCI, FEDER, POPH, QREN) by studentship grants
(SFRH/BPD/75294/2010 and SFRH/BPD/86173/2012) and one
project grant (PTDC/AGR-TEC/2227/2012).
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dx.doi.org/10.1016/j.j.2013.05.010