C H A P T E R
35
Anthocyanins
Britt Burton-Freeman, Amandeep Sandhu and Indika Edirisinghe
INTRODUCTION
Anthocyanins are the natural plant pigments impart-
ing red, blue, and purple colors to fruits, flowers, leaves,
and some vegetables. They not only provide attrac-
tive colors and aesthetic appeal to plants but also play
an important role in pollination and seed dispersal.
Anthocyanins are also used as natural colorants in foods
and beverages.
Anthocyanins are categorized as flavonoids, which is
one of the largest categories of phenolic compounds. The
basic structure of anthocyanins is composed of flavylium
cation (C6-C3-C6), which could be linked to different
sugars or hydroxyl or methoxyl groups resulting in more
than 635 anthocyanins identified so far (He and Giusti,
2010). The most common sugar attached to anthocyanins
is glucose, although rhamnose, xylose, galactose, arabi-
nose, and rutinose have also been reported (Horbowicz
et al., 2008). Anthocyanins can be mono-, di-, or tri-gly-
cosides, depending on the number of sugars attached.
In addition, the sugar residues could also be acylated
by aromatic/aliphatic acids (Böhm, 1994; Giusti et  al.,
1998). The aglycones of anthocyanins are called antho-
cyanidins and a total of 19 anthocyanidins have been
reported, among which the six major ones are cyani-
din, peonidin, delphinidin, pelargonidin, malvidin, and
petunidin (Figure 35.1; Tanaka et  al., 2008). The color
and stability of anthocyanins are affected by their struc-
ture, pH, temperature, light, oxygen, enzymes, inter-
and intramolecular association with other compounds
such as ascorbic acids, metal ions, sugars, and proteins
(Francis, 1989).
Anthocyanins are particularly sensitive to pH exhibit-
ing a reversible change in color from red to blue when
pH changes from acidic to basic. This phenomenon
could be explained by their existence in equilibrium in
aqueous solution with pH between 1 and 8 with at least
five different forms that interconvert via pH-dependent
Nutraceuticals.
DOI: http://dx.doi.org/10.1016/B978-0-12-802147-7.00035-8
equilibrium such as flavylium cation (red), carbinol base
(colorless), chalcone (pale yellow/colorless), quinonoi-
dal base (violet/blue), and anionic quinonoidal base
(blue) (Brouillard and Lang, 1990; Oliveira et al., 2014).
The predominant structural forms of anthocyanins at
different pH levels are shown in Figure 35.2. The high
antioxidant activity of anthocyanins is due to their abil-
ity to scavenge free radicals by donating a hydrogen
atom from a hydroxyl group as well as to support an
unpaired electron (Miguel, 2011). Phenoxyl radicals gen-
erated in this reaction stabilize through resonance and/
or intramolecular hydrogen bonding (Kay, 2006).
The evidence for human health benefits linked to con-
sumption of dietary anthocyanins has been presented
over multiple experimental platforms and disease tar-
gets. Early reports suggested that the antioxidant prop-
erties of these compounds were responsible for their
health promoting effects; however, it is now appreci-
ated that anthocyanins work beyond their antioxidant
properties and likely influence an array of cell signaling
and gene expression pathways. The evolving science has
been a driving force stimulating interest and research
in anthocyanin biological activity, including their bio-
availability in humans, how they are metabolized, and
whether their metabolites or parent compounds are
active in the human body, including whether they pres-
ent a safety risk if consumed in higher amounts, such as
a dietary supplement.
DIETARY SOURCES
OF ANTHOCYANINS
The primary sources of anthocyanins in the human
diet are fruits and vegetables, namely, berries (blueber-
ries, strawberries, blackberries, cranberries, and others),
as well as red-skinned grapes, apples, and pears and
other purple/red-pigmented vegetables such as carrots,
489 © 2012
2016 Elsevier Inc. All rights reserved.
490 35. Anthocyanins
FIGURE 35.1  Structures of main anthocyanidins.
potatoes, radishes, and cabbage to name a few. They are
easily recognized due to their red-blue-purple pigmen-
tation. A few exceptions exist, including red tomatoes,
which are pigmented by lycopene, and beets, which are
pigmented by betalain.
Estimates of anthocyanin intake vary widely within
and between populations, as does the composition of
anthocyanin intake, which is based on food and bever-
age selection. In France, anthocyanin intake is estimated
to be ~57 mg/day (Perez-Jimenez et  al., 2010), and in
Finland an average of 47 mg/day of anthocyanins has
been estimated (Ovaskainen et al., 2008). Wu et al. (2006)
estimated that the daily consumption of anthocyanins in
the United States from fruits, vegetables, and beverages
was ~12.5 mg/day. Of the total anthocyanins consumed
per day, almost 70% was accounted for by fruit intake
(24 g fruit contributing ~8.7 mg anthocyanins). Apples
(red delicious) dominated the percent of fruit intake,
yet apples only contributed 8% of anthocyanin intake.
In contrast, berries comprised only ~13.5% of the fruit
intake, but contributed ~56% of the fruit-derived antho-
cyanins. On average, berries (strawberries, blueberries,
cranberries, raspberries, blackberries) provide approxi-
mately 86 mg anthocyanins per 100 g fruit, whereas the
average anthocyanin content in the most commonly
eaten fruits (apple, banana, grapes, melons) (Murphy
et  al., 2012) is approximately 8 mg per 100 g portion
(Bhagwat et al., 2013).
In addition to fruits and vegetables, anthocyanins are
delivered through the diet as natural colorants (food and
beverages). They are also available as dietary supple-
ments in the form of powders, extracts, capsules, soft
NUTRACEUTICALS
gels, and purified compounds. One of the advantages
of anthocyanin supplement products (and potentially
a disadvantage) is the ability to ingest high doses of
anthocyanins. Compared to delivery in their natural
food matrix, much higher doses can be ingested, pre-
senting potential safety issues. These issues are dis-
cussed in further detail later. A critical consideration
when purchasing anthocyanins as a supplement is
whether their chemistry activity and biological activ-
ity have been maintained. Anthocyanins are relatively
unstable compounds, particularly in high temperatures
and light. They are stable in acidic conditions (pH < 3)
and reduced water activity, such as in a freeze-dried
product. Even so, acidified and freeze-dried powders
should be stored at low temperatures in appropriate
packaging and preferably with little oxygen exposure
for maximum shelf-life (Hua et al., 2010).
ANTHOCYANIN HEALTH BENEFITS
The health benefits of anthocyanins have been stud-
ied in a variety of models ranging from human epi-
demiology and clinical trial intervention to screening
and mechanistic studies in animals and cell culture
models (Lila, 2004; Seeram, 2008; Burton-Freeman,
2010; Blumberg et  al., 2013; Basu et  al., 2014; Joseph
et  al., 2014). As mentioned, berries represent the pri-
mary source of dietary anthocyanins, although other
sources also contribute. With recently populated public
databases assigning flavonoid content to foods, includ-
ing anthocyanin content, epidemiological analyses have
revealed population-based diet–disease relationships
specific to dietary anthocyanins. For example, dietary
anthocyanin intake has been shown to be inversely asso-
ciated with cardiovascular events, including myocardial
infarction and death from coronary heart disease and
cardiovascular disease in multivariate models (Mink
et al., 2007; McCullough et al., 2012; Cassidy et al., 2013).
These results may be explained in part by risk factor
reduction: dietary anthocyanin intake has been reported
to be significantly inversely associated with hyperten-
sion (Cassidy et  al., 2011; Jennings et  al., 2014), type 2
diabetes mellitus (T2DM) (Wedick et  al., 2012), insulin
resistance, and markers of inflammation/C-reactive pro-
tein (Jennings et  al., 2014). Epidemiological data link-
ing dietary anthocyanins in cancer risk reduction are
sparse and to date have shown relatively neutral effects
(Zamora-Ros et al., 2013; Cassidy et al., 2014).
Evidence from randomized clinical trials (RCTs)
shows that dietary sources of anthocyanins, including
strawberries, cranberries, and blueberries, improve ath-
erogenic lipid profiles such as decreasing total and LDL
cholesterol, increasing HDL cholesterol, and improving
lipoprotein particle size patterns (Erlund et  al., 2008;
 Anthocyanin Health Benefits 491

OR3 OR3 OR3

O– OH OH

O O HO O+ HO O
OR2 OR2 OR2
–H+
OR1 OR1 OR1

OH OH O

Ionized Quinonoidal Flavylium cation (red to Quinoidal base (blue)

base (blue) orange color) pH = 1 pH = 4
pH > 8

+H2O, –H+
OR3
OH
OH
HO O
OR2

OR1
OH

Carbinol pseudobase
(colorless/pale yellow)
pH = 5

OR3
OH

HO OH O
OR2

OR1
OH

Chalcone (colorless)
pH = 6

FIGURE 35.2  Anthocyanin structural variation at different pH levels (R1=H or glycoside; R2 and R3=H or methyl group). Source: Adapted
from Miguel (2011).

Lee et al., 2008; Basu et al., 2010a,b, 2014; Zunino et al.,
2012; Novotny et al., 2015). These data are supported by
anthocyanin supplementation studies showing similar
improvements in blood lipid profiles (Qin et al., 2009; Zhu
et al., 2011, 2013; Hassellund et al., 2013). Improvements
in emerging CVD and diabetes risk factors have also
been reported with berry and anthocyanin supplemen-
tation, including inflammation (as reviewed in Joseph
et  al., 2014), glycemic control, and improved insulin
sensitivity (Stull et  al., 2010; Edirisinghe et  al., 2011a,b;
Hidalgo et  al., 2014; Li et  al., 2015). Selected studies
have also indicated blood pressure–lowering effects with
berry supplementation (Erlund et  al., 2008; Basu et  al.,
2010a,b; Dohadwala et  al., 2011), but not with antho-
cyanin supplementation (Hassellund et al., 2012, 2013).
Esophageal and other cancers of the gastrointestinal
tract (GIT) appear sensitive to mitigation by high berry
anthocyanin exposure (Chen et al., 2012); however, as in
all studies using nonpurified anthocyanin preparations,
other components in berries may contribute to the effect,
making it difficult to specify an anthocyanin-specific
anticancer effect in humans. Nonetheless, there is an
impressive preclinical literature of anthocyanins medi-
ating physiological functions related to cancer suppres-
sion, suggesting potential anticancer benefits in humans
that are not entirely recognized yet (Zhang et al., 2005;
Wu et  al., 2007; Brown et  al., 2012; Hou, 2003; Stoner
et al., 2007). Some of these effects are briefly described.
Anthocyanins show inhibitory effects on the growth
of some cancer cells (Kamei et  al., 1995; Koide et  al.,
1997) and also inhibit cell transformation (Hou et  al.,
2004). Pure anthocyanins and anthocyanin-rich extracts

NUTRACEUTICALS
492 35. Anthocyanins
from fruits and vegetables have shown antiproliferative
activity toward multiple cancer cell types in vitro. It is
interesting to note that anthocyanins selectively inhibit
the growth of cancer cells with relatively little or no
effect on the growth of normal cells (Hakimuddin et al.,
2004). Apoptosis plays a key role in the development and
growth regulation of normal cells, and is often dysregu-
lated in cancer cells. Effective chemopreventive agents
are often strong inducers of apoptosis in cancer cells.
Anthocyanin-rich extracts from various fruits/extracts
and several pure anthocyanins and anthocyanidins have
exhibited proapoptotic effects in multiple cell types in
vitro (Kamei et  al., 1995; Koide et  al., 1997). Likewise,
angiogenesis is another important factor in cancer
development, and anthocyanins have been shown to
inhibit angiogenesis in endothelial cells (Bagchi et  al.,
2004) and other cells types (Huang et al., 2006; Rodrigo
et  al., 2006). Anthocyanins have been tested in various
animal models of cancer, including carcinogen-treated
animals and animals with hereditable predisposition to
cancer. Black raspberry powder and fractions therein
have been shown to inhibit esophageal tumorigenesis
in N-nitrosomethylbenzylamine (NMBA)-treated rats
(Kang et  al., 2003) as well as to decrease tumor num-
ber in an azoxymethane (AOM)-induced model of colon
cancer in F344 rats (Harris et al., 2001). Promising results
in the APC(Min) mouse model of intestinal cancer with
tart cherry extract and bilberry have also been observed
(Cooke et al., 2006).
Despite these and other anthocyanin-associated
health effects, anthocyanins and other polyphenols
remain to be fully understood because their bioavail-
ability appears to be relatively limited. With few excep-
tions, such as the case with dietary fiber, it is generally
assumed that to confer health benefits systemically,
compounds (of interest) must be absorbed and avail-
able for use by cells. In a classic bioavailability/pharma-
cokinetic paradigm, the compounds of interest would
be the parent anthocyanin molecule and perhaps one
or two metabolites after phase I and phase II metab-
olism. However, it has become increasingly apparent
and appreciated that polyphenols, including anthocya-
nins, are metabolized to a number of potentially bio-
logically active compounds generated from the human
and gut microbiome (Williamson and Clifford, 2010;
Cardona et al., 2013; Czank et al., 2013; de Ferrars et al.,
2014). Hence, although current literature still suggests
limited bioavailability of anthocyanins, the landscape
is quickly evolving to indicate that our definition and
assessment of bioavailability may require adjustment
to fully describe and understand anthocyanin “total”
bioavailability, including metabolites formed and their
relationship with health-associated effects (efficacy) and
potential safety concerns, together with specific cellular
targets sensitive to each metabolite.
NUTRACEUTICALS
CELLULAR AND MOLECULAR
TARGETS OF ANTHOCYANINS
Anthocyanins, along with many flavonoid com-
pounds, have gained increasing attention over the years
due to their well-established in vitro antioxidant activity
(Rice-Evans et  al., 1996) and potential for health ben-
efits. Because oxidative stress leading to damage and
dysfunction of cells and tissue is considered to be at the
core of aging and chronic diseases, anthocyanins/fla-
vonoids antioxidant activity has been regarded as their
main biological activity in humans. However, this idea
has been challenged in recent years due to low bioavail-
ability of many flavonoids, particularly of anthocyanins
(Manach et al., 2005). Anthocyanin bioavailability is gen-
erally less than 1–2%, although 5% bioavailability has
been reported when delivered as wine (Lapidot et  al.,
1998; McGhie et  al., 2003; Gonzalez-Barrio et  al., 2010;
Banaszewski et  al., 2013). Concentrations of anthocya-
nins and their conjugated metabolites in plasma after
oral ingestion are in the mid–high picomolar (pM) to
low–mid nanomolar (nM) range, well below what is
considered the threshold for radical scavenging activity.
In contrast, classic antioxidant radical scavengers, such
as ascorbic acid and urate, circulate in micromolar (µM)
concentrations. Given these concentration differences
and ranges in which anthocyanins circulate, alternative
mechanisms are more likely to describe their bioactivity.
Recently, research has identified cellular targets where
anthocyanins interact to activate, suppress, receive, and
transmit information, resulting in downstream effects
including gene expression to modulate physiology. The
cascade of responses is presumed to be initiated by
ligand–receptor interactions. Nuclear factor kappa light
chain-enhancer of activated B cells (NF-κB), activator pro-
tein-1 (AP-1), signal transducer and activator of transcrip-
tion-1 (STAT-1), and octamer-binding protein (OCT-1) are
some of the transcription factors that are activated in
cancer cells as well as other cells of the body under
distress and disease conditions. Evidence suggests that
metabolites of anthocyanins can reduce the activation
of these transcription factors (Forester and Waterhouse,
2010; Forester et  al., 2014). NF-κB is a central mediator
of inflammatory responses and, therefore, ROS genera-
tion in cell systems. Hence, attenuation of chronically
activated NF-κB activation can dramatically alter the
inflammatory and oxidative stress status of the system,
which has been linked to multiple chronic, noncommu-
nicable diseases, including the most pressing current
diseases: cardiovascular diseases, diabetes, Alzheimer’s
disease, and some cancers (Hotamisligil, 2006; Lu et al.,
2006; Libby, 2007; Scrivo et  al., 2011). The effects of
anthocyanin-rich foods and products on inflammation
and inflammatory targets have been recently reviewed
(Joseph et al., 2014).
 Anthocyanin Bioavailability
Anthocyanins have been shown to stimulate the
expression of tumor suppressor genes via demethylation
of promoters of cyclin-dependent kinase (CDK) inhibitor
2A (CDKN2A), secreted frizzled-related proteins 2 and
5 (SFRP2, SFRP5), and Wnt inhibitor factor -1 (WIF1)
(Wang et  al., 2013). Inhibitors of CDK activity, specifi-
cally p21 and p27, also appear to be sensitive to anthocy-
anin and anthocyanidin molecules promoting cell cycle
arrest and apoptosis in carcinoma cells.
The ability of anthocyanins to induce antioxidant
and detoxifying enzymes has potential implications in
cancer prevention as well as modifying cellular oxidant
status. Treatment of rat liver clone 9 cells with 50 μM
anthocyanins (Shih et al., 2007) and noncancerous breast
cells with 10–20 μg/mL anthocyanins (Singletary et  al.,
2007) enhanced their antioxidant capacity by activating
glutathione (GSH)-related enzymes, including glutathi-
one reductase (GR), glutathione peroxidase (GPx), and
glutathione S-transferase (GST), as well as the activity of
NAD(P)H:quinone reductase.
Other molecular targets of anthocyanins include pro-
tein kinases, many of which are ATP-binding kinases.
The cellular stress kinases extracellular signal-regu-
lated kinases (ERK), mitogen-activated protein kinases
(MAPK), and c-Jun N-terminal kinases (JNK) have been
shown to be sensitive to anthocyanin treatment, result-
ing in reduced activation and attenuation of downstream
cellular signaling networks associated with cancer, brain
aging, and chronic diseases such as cardiovascular dis-
ease, diabetes, and the processes that underlie them
such as chronic inflammation (Ghosh and Konishi, 2007;
Seeram, 2008; Shukitt-Hale et al., 2008; Blumberg et al.,
2013; Basu et al., 2014; Edirisinghe and Burton-Freeman,
2014; Joseph et al., 2014; Rodriguez-Mateos et al., 2014;
Sehitoglu et al., 2014).
Of particular interest are some of the metabolic traf-
ficking activities shown with anthocyanins. Molecular
targets include enzymes (e.g., AMP-activated protein
kinase (AMPK), phosphatidylinositol-3 (PI3)-kinase,
protein kinase B (Akt)), receptors and transporters (e.g.,
insulin receptors, insulin receptor substrate (IRS)-1, car-
nitine palmitoyl transferase I (CPT1)), and nuclear bind-
ing proteins/transcription factors (i.e., PPARs) involved
in cellular energy metabolism (Tsuda et al., 2004; Tsuda,
2008; Seymour et al., 2011; Tulio et al., 2012; Park et al.,
2015). AMPK appears to be an important target for non-
insulin-mediated anthocyanin effects influencing glu-
cose metabolism (Tsuda, 2008; Kurimoto et  al., 2013).
Anthocyanin-mediated effects via insulin-dependent
pathways appear to involve upregulation of insulin
signaling molecules (Qin and Anderson, 2012) and
mediate phosphorylation patterns along the IRS-1 path-
way (enhanced Tyr, activation; reduced Ser, inhibitory)
enhancing Akt phosphorylation/activation that can be
disrupted under oxidative stress and hyperglycemic and
NUTRACEUTICALS
493
free fatty acid conditions, such as in prediabetes and
diagnosed diabetes (unpublished results, personal com-
munication, and Edirisinghe et al. 2011b).
These targets elicit additional or alternative actions
in other tissues as well. For example, in endothelial
cells, anthocyanin-rich extracts were shown to increase
phosphorylation of the PI3/Akt pathway, leading to
increased eNOS activity (Xu et  al., 2004; Edirisinghe
et  al., 2008, 2011a,b), which is relevant in vasodilation
as demonstrated in various ex vivo aorta models with
anthocyanin or anthocyanin-rich fruit extract treatment
(Andriambeloson et al., 1998; Nakamura et al., 2002; Bell
and Gochenaur, 2006; Edirisinghe et al., 2008). Thrombin
receptor activating peptide (TRAP) and vascular endo-
thelial growth factor (VEGF) have roles in thrombosis
and angiogenesis and may be additional targets for
anthocyanins reducing cancer and atherosclerotic risk
(Rechner and Kroner, 2005; Oak et al., 2006).
It is also well recognized that anthocyanins, like other
polyphenols, interact with drug-metabolizing enzymes,
including cytochrome P450 (CYP450) mono-oxygenases
(phase I) and phase II conjugation enzymes and presum-
ably drug excretion transporters. Interaction of anthocya-
nins with these molecular targets might result in decreased
bioavailability of certain drugs with possible treatment
failure or increased bioavailability with higher probability
of adverse effects and toxicity (Rodriguez-Fragoso et al.,
2011). Based on these interactions and the growing evi-
dence of anthocyanin targets and activity in the human
body, there may be good reason for their low bioavail-
ability; either for safety purposes, because the first line
of mammalian defense is at the level of absorption and
first-pass metabolism, or to ensure their uptake coincides
with cellular machinery to optimize their health benefits.
ANTHOCYANIN BIOAVAILABILITY
Bioavailability is a term that refers to the extent that
a substance reaches systemic circulation unchanged
after a particular route of administration. Intravenous
administration confers 100% bioavailability. However,
anthocyanins are ingested via the oral route; therefore,
bioavailability will be governed by their ability to cross
the epithelial surface of the GIT, pass through the liver,
and appear in systemic circulation. Anthocyanins are
one of the few flavonoids that can be absorbed intact and
can be measured in circulation as the parent compound.
Bioavailability of the parent intact glycoside compounds
is remarkably low and is dependent on aglycone struc-
ture and sugar moiety, but with most reports indicating
much less than 1% (Cao et al., 2001; McGhie et al., 2003;
Wu et al., 2004; Prior and Wu, 2006; Banaszewski et al.,
2013). Nonetheless, an array of anthocyanin metabolites
have been detected in which the C6-C3-C6 structure is
494 35. Anthocyanins
maintained (i.e., C6-C3-C6 structures that are deglyco-
sylated and absorbed or conjugated with glucuronide,
sulfate, or methyl groups), and still others have been
detected in which the backbone structure has been
degraded or metabolized to simple phenolic acids due
to chemical instability or microbial metabolism in the
GIT. C6-C3-C6 metabolites appear in plasma in nM con-
centrations, whereas total phenolic acids may reach µM
concentrations. The elimination of plasma anthocyanins
is mainly through the kidney, excreted in urine follow-
ing first-order kinetics (Cao et  al., 2001; Carkeet et  al.,
2008; Hollands et al., 2008). The plethora of metabolites
generated from anthocyanins ingestion (i.e., including
unmetabolized parent compounds, aglycones, phase
I and phase II metabolites, conjugated products, and
microbial generated metabolites) suggests that total bio-
availability is much greater than previously recognized
and further supports a new framework for assessing the
“bioavailability” of anthocyanins and potentially other
plant secondary metabolites.
ANTHOCYANIN ABSORPTION,
DISTRIBUTION, METABOLISM,
AND EXCRETION
Anthocyanins are primarily absorbed in the stomach
and the small intestine. Rapid appearance of anthocya-
nins in the blood is believed to be due to gastric absorp-
tion transported into portal circulation via the organic
anion carrier, bilitranslocase (Passamonti et  al., 2002,
2003; Talavera et  al., 2005). Bilitranslocase has a higher
affinity for parent anthocyanin glycosides than for their
aglycones, which likely explains the rapid uptake and
detection of anthocyanin glycosides in plasma compared
to aglycone moieties (Passamonti et al., 2003).
In the small intestine, anthocyanin absorption appears
to be mostly in the jejunum segment (Talavera et al., 2005).
Anthocyanin glycosides are relatively polar hydrophilic
compounds; therefore, absorption requires modification
or a transport mechanism. Lactate phlorizin hydrolase, a
brush border enzyme, can hydrolyze glycosides, forming
more hydrophobic anthocyanin aglycones to allow for
passive diffusion into enterocytes (Kay, 2006). However,
passive diffusion as a mechanism for enterocyte uptake
has been challenged (Passamonti et  al., 2009). Instead,
it is expected that membrane transport mechanisms are
the primary mechanisms for anthocyanins and possibly
other flavonoid compounds. Several glucose associated
transporters (GLUT) have been suggested, including
the most often cited sodium-dependent glucose co-
transporter (SGLT1) (Fernandes et  al., 2014). Intestinal
bilitranslocase may be another mechanism for uptake
(Passamonti et  al., 2009). Once across the wall of the
enterocyte, anthocyanins are treated like xenobiotics
NUTRACEUTICALS
subject to phase I and phase II metabolism generating
oxidized or reduced products and/or sulfate, glucuro-
nide, and/or methylated metabolites through the action
of sulfotransferases (SULTs), uridine-5-diphosphate
glucuronosyltransferases (UGT), and catechol-O-meth-
yltransferases (COMTs), respectively. Compared to other
flavonoids, anthocyanins do not appear to be extensively
modified (Del Rio et al., 2013).
Efflux transporters of the ATP-binding cassette fam-
ily, including multi-drug resistance protein (MRP) and
P-glycoprotein (P-gp) at apical and basolateral surfaces,
are thought to be responsible for some of the metabolites
being transported back into the lumen of the small intes-
tine as well as facilitating efflux into portal circulation.
To what degree anthocyanins are subject to efflux back
into the intestinal lumen versus into portal circulation is
not well understood but may, at least in part, explain the
low bioavailability of anthocyanins and other flavonoids.
Once in the portal bloodstream, metabolites rapidly
reach the liver, where they can be further metabolized
(via phase I and phase II metabolism) prior to entry
into general/systemic circulation or recycled back to the
small intestine through bile excretion, where they may
be reabsorbed, degraded, or continue through to the
lower bowel (Felgines et al., 2003, 2010; Talavera et al.,
2005). Either initially or via enterohepatic circulation,
anthocyanins as parent, metabolite, or degraded prod-
ucts reaching the colon are subject to colonic metabo-
lism, generating an even broader variety of metabolites
and kinetic profiles (de Ferrars et al., 2014).
Most anthocyanins maintaining the C6-C3-C6
structure will peak in systemic circulation within 1–4 h
depending on aglycone-glycosidic chemistry as well as
gastrointestinal dynamics (i.e., gastric emptying, gut
motility, viscosity of chyme, etc.) in response to food
matrix and co-existing nutrient composition. For exam-
ple, strawberries ingested with dietary lipid in the form
of cream has been shown to shift the absorption and
clearance curve of pelargonidin-glucuronide to the right
without reducing total bioavailability compared to eat-
ing strawberries without cream (Mullen et al., 2008). In
rats fed black currants with water or with hydrated oat-
meal, producing a viscous medium, total anthocyanin
absorption was slowed and reduced in the oatmeal con-
dition (Walton et al., 2009). In human subjects, consum-
ing freeze-dried strawberry powder (12 g) prepared in a
water mixture with no other food resulted in significantly
higher peak pelargonidin-glucuronide concentrations
compared to when the same preparation was consumed
with a meal. However, for the unmetabolized compounds
(pelargondin-3-O-glycoside and pelargonidin-3-O-rutin-
soside) peak concentrations appeared approximately 1 h
earlier in the water-only condition compared to with the
meal condition. Peak plasma concentration did not dif-
fer between conditions for pelargondin-3-O-glycoside,
 Anthocyanin Safety and Toxicology
whereas the meal enhanced pelargondin-3-O-rutinoside
absorption, resulting in higher peak concentrations com-
pared to the water-only condition (personal communica-
tion, unpublished results, A. Sandhu). Whether the shifts
were due to an alteration of gastrointestinal function
in response to food/meal composition or anthocyanin
binding to food substituents (e.g., fiber, proteins) is not
clear. Lipids and viscous fibers are known to slow gastric
emptying and interfere with digestion, slowing absorp-
tion of nutrients. Aglycone chemistry and sugar moieties
impact absorption kinetics of anthocyanins, and the sug-
gested interactions with coexiting foods/nutrients fur-
ther complicate the picture. Future work will need to
determine whether these or other relevant alterations
in absorption would be significant enough to impact
anthocyanin-associated health outcomes.
The impact of dose on anthocyanin bioavailability
has not been tested extensively in humans (Kurilich
et  al., 2005; Carkeet et  al., 2008; Hollands et  al., 2008;
Banaszewski et al., 2013). Three of the four groups cited
assessed bioavailability of strawberry anthocyanins using
fresh fruit, puree, or freeze-dried powder preparations up
to maximum doses of ~440 g fresh fruit equivalent and
~368  µmol anthocyanins (pelargonidin and cyanindin
glycosides). Kurilich et  al (2005) investigated bioavail-
ability of anthocyanins from two doses of purple car-
rots providing 357 and 714 µmol anthocyanins (cyanindin
glycosides, acylated and nonacylated). Urinary excretion
increased linearly, with increasing doses of strawberry
intake providing up to ~228 µmol anthocyanins (Carkeet
et  al., 2008; Hollands et  al., 2008). Feeding strawberries
over four intake levels delivering 101–368 µmol straw-
berry anthocyanins resulted in dose-related increases in
maximum plasma concentrations (Cmax) and systemic
exposure as determined by area under the curve (AUC)
analysis. However, after adjusting for dose, these pharma-
cokinetic variables were reduced and overall bioavailabil-
ity decreased with increasing dose, suggesting possible
saturation of absorptive mechanisms as dose increases
(Banaszewski et  al., 2013). These data were confirmed
in a second larger study by the same group (personal
communication, submitted) and support findings by
Kurilich et al. (2013), who reported maximal absorption
at 350 µmol or less for cyanidin-based anthocyanins.
Anthocyanins distribution as studied in rodents indi-
cates presence in digestive organs (stomach, jejunum)
and liver, as well as in the brain and kidney, although
proportions of anthocyanins metabolites appear to dif-
fer between organs (Talavera et  al., 2005; Kalt et  al.,
2008). Radiolabeled (14C) anthocyanin administered
into animal models has also revealed deposition of the
metabolites in brain and bone tissues (Janle et al., 2010).
In humans, 13C-biolabeled cyanidin-3-glucosides were
administered and labeled metabolite concentrations
were measured using isotope ratio mass spectrometry
NUTRACEUTICALS
495
(Czank et al., 2013). Nearly half of the labeled anthocya-
nins were recovered as metabolites after elimination in
urine, feces, and breath.
In humans, anthocyanin bioavailability is regarded
as limited and appears to decrease with increasing
bolus doses beyond a certain threshold. These thresh-
olds likely differ among anthocyanins based on struc-
tural differences, attached sugars, and human variance.
Nonetheless, the current data suggest that C6-C3-C6
structures as well as their products broken down from
mammalian or microbial metabolism (phenolic metab-
olites) are managed such that excretion is rapid and
circulating concentrations are relatively low. However,
potency has not been addressed well. Several hormones
in the body circulate in very low (pM) concentrations yet
are potent biologically. Hence, evaluation of the safety
and toxicity of anthocyanins is prudent, not only because
they are used as natural colorants in foods as food addi-
tives but also because the evolving health food market
now includes a robust dietary supplements market that
is well supported by modern consumers.
ANTHOCYANIN SAFETY AND
TOXICOLOGY
The consumption of anthocyanins is generally con-
sidered safe in humans because they have been con-
sumed as part of regular diet for generations without
any adverse health effects (Brouillard, 1982). This could
be associated with their low absorption and bioavailabil-
ity (Pojer et al., 2013). However, an increasing consumer
trend toward dietary supplements to achieve health ben-
efits of anthocyanins has raised concerns. Manufacturer
recommended doses of these supplements are usually
much higher than the intake of anthocyanins from food.
Moreover, there are no regulatory standards for dietary
supplements sold in the United States, which can result
in fraudulent/adulterated products (Lee, 2014). It is
also highly likely that individuals interested in using
anthocyanins as a dietary supplement for health benefits
would also be using other supplements or conventional
drugs. As discussed in this chapter, anthocyanins are
treated as xenobiotics and, thus, can modulate activities
or compete for various drug-metabolizing enzymes and
drug transporters (Bartikova et  al., 2013). This further
enhances the risk of potential adverse effects and toxicity
due to drug–supplement interactions.
To date, there are no reports indicating adverse
health effects with ingestion of anthocyanins at usual
dietary intake levels. The safety and toxicity of antho-
cyanins have been studied, but much of the research
dates back to the 1990s and earlier. For example, bilberry
extract containing 36% anthocyanins was used to test
the tolerability and safety of anthocyanins in animals
496 35. Anthocyanins
by Morazzoni and Bombardelli (1996). No toxic effects
were observed in rats and mice at LD50 over 2,000 mg/
kg body weight (bw) and in dogs at 3,000 mg/kg bw.
However, marked discoloration of urine and feces was
observed. A 6-month study conducted on animals in
which rats (dose: 125 to 500 mg/kg bw) and dogs (dose:
80 to 320 mg/kg bw) were fed anthocyanins every day
showed no mortalities or toxic effects. Clinical safety was
confirmed in a postmarketing surveillance human study
with more than 2,000 patients suffering from venous and
retinal microcirculation disorders. Most patients (69.5%)
consumed a dose of 115 mg/day of anthocyanins from
bilberry extract for 1 to 2 months, with only 4% of people
reporting side effects that were mainly gastrointestinal
(gastric pain, pyrosis, and nausea) and related to the skin
and the nervous system (Eandi, 1987). However, high
doses of bilberry anthocyanins (>100 mg/day) should
be taken cautiously by hemorrhagic patients and those
prescribed warfarin or other antiplatelet drugs due
to interaction of anthocyanins with antiplatelet drugs
(Pulliero et al., 1989). Pourrat et al. (1967) reported that
anthocyanins can produce sedative effects in animals at
oral doses of 500 mg/kg bw. They also observed diuretic
effects in rabbits when administered a dose of 25 mg/kg
bw intravenously. No adverse effect was noted in blood
pressure of rabbits consuming an oral dose of 6 g/kg bw
of anthocyanin glycosides.
There are limited toxicological studies evaluating
anthocyanin effects on fertility/reproduction, teratoge-
nicity, and mutagenicity. A two-generation reproduction
study was conducted in rats (Sprague-Dawley) with an
18-week observation period, during which rats were fed
a grape skin extract (GSKE) preparation containing 3%
anthocyanins. The test groups received 7.5 or 15% of
the GSKE in their diet (equivalent to 112 and 225 mg
anthocyanins/kg bw per day based on an assumed 3%
anthocyanin content in GSKE). There was no differ-
ence in the reproductive performance of the control and
test groups. However, a decrease in body and organ
(liver, adrenal, and thyroid) weight of the offspring was
observed in first-generation pups after the 15% dose
and at the 7.5% dose in second-generation pups, which
could be related to decreased food intake. However, no
compound-related histological effects were observed
(Cox and Babish, 1978; EFSA, 2013) despite decreases in
organ weights of the liver, adrenal, and thyroid in the
15% group of first-generation rats. No teratogenic activ-
ity of anthocyanin glycosides from extract of currants,
blueberries, and elderberries was observed in mice, rats,
and rabbits at 1.5, 3, and 9 g/kg bw dose levels over
three successive generations (Pourrat et  al., 1967). The
same extract when administered intravenously or intra-
peritoneally at extremely high doses of 25 g/kg bw in
mice and 20 g/kg bw in rats resulted in sedation, convul-
sions, and finally death (Pourrat et al., 1967). In a study
NUTRACEUTICALS
by Morazzoni and Bombardelli (1996), bilberry extracts
showed no teratogenic effects or mutagenic activity in
different mutagenesis assays. Similarly, purified cyani-
din and delphinidin and extracts of GSKE tested neg-
ative for mutagenic activity in the Ames assay using
different strains of Salmonella with or without metabolic
activation (Brown and Dietrich, 1979; EFSA, 2013).
Genotoxic evaluation using the in vitro Comet assays in
mammalian cells with up to 10 μM delphinidin, malvidin,
pelargonidin, and peonidin did not result in increased
DNA strand breaks. However, in another study, doses
of ≥50 μM (~17.5  g/mL) delphinidin, cyanidin, mal-
vidin, pelargonidin, and peonidin did induce a small,
but significant, increase in strand breaks in HT29 cells.
Pelargonidin (doses ≤2 μM) was found to be nongeno-
toxic in a micronucleus test in HL-60 cells. Overall, in
most in vitro assays, anthocyanins tested at low doses
were not genotoxic. Some evidence of genotoxicity was
provided by a single in vitro study using pure anthocyani-
dins (aglycone). However, in a negative guideline, bone
marrow micronucleus test at a limited dose was consid-
ered to exclude in vivo genotoxicity of GSKE (EFSA, 2013).
Studies of the toxicological properties of anthocya-
nins have mainly used fruit extracts, which contain
several anthocyanins. Therefore, conclusions cannot be
drawn for specific anthocyanins, but may be made for
anthocyanins in general. From a food additive stand-
point, anthocyanin exposure is expected to be far greater
than for usual dietary intake exposure (EFSA, 2013).
However, dietary supplement usage has the potential
to increase intake to even higher levels than potentially
encountered through usual dietary intake, including
exposure of anthocyanins as a food additive. To date,
regulatory reviews of the available data indicate that it
is inadequate to establish a numerical acceptable daily
intake (ADI) for anthocyanins (for purposes of food
additive safety) (EFSA, 2013), although Joint FAO/WHO
Expert Committee on Food Additives published an ADI
of 0–2.5 mg/kg/bw per day in 1982 based on the work
of Cox and Babish (1978). Dietary reference intakes for
anthocyanins have not been established, which would
include a tolerable upper intake level (UL). Despite the
lack of evidence to make specific recommendations for
safety, the general consensus suggests that anthocyanins
are safe for human ingestion as usually consumed in
the diet. As with all dietary supplement usage, caution
should be exercised to minimize potential safety risk.
CONCLUDING REMARKS AND
FUTURE DIRECTIONS
Anthocyanins are natural plant pigments that have
beneficial effects for the plant as well as for humans and
animals. Dietary sources of anthocyanins are generally
 REFERENCES
easy to identify due to their red, blue, or purple color.
Examples include berries and red-skinned grapes,
apples, and pears and various vegetables such as rad-
ishes and red/purple cabbage. Anthocyanins may also
be ingested through their use as a food additive and as
dietary supplements, procured as anthocyanin-rich fruit
extracts, powders, and purified compounds. The bio-
availability of anthocyanins is considered to be limited;
however, recent advances in targeted and nontargeted
instrumentation have enhanced our detection capability,
indicating that anthocyanin metabolism can be exten-
sive, is complex, and that the full portfolio of antho-
cyanin metabolites are probably yet to be measured or
characterized. Nonetheless, it is clear that anthocyanin
intake is associated with various health benefits as dem-
onstrated in a number of study designs ranging from
human epidemiology and clinical trial intervention to
screening and mechanistic studies in animals and cell
culture models. Anthocyanin molecular targets include
transporters and receptors, second messenger signaling
molecules and kinase enzymes, transcription factors,
promoters, and growth factors, and a host of oxidant
defense enzymes. Despite the potential broad-spanning
biological activities of anthocyanins, safety and toxico-
logical concerns are relatively low. To date, there are
no reports indicating adverse health effects with con-
sumption of anthocyanins (in general) at usual dietary
intake levels. The collective toxicological literature sug-
gests that adverse effects occur only at extremely high
levels, although a complete toxicological assessment of
anthocyanins is lacking (EFSA, 2013). Overall, antho-
cyanin ingestion through food is unlikely to present a
safety concern and may, in fact, impart health benefits.
Currently, there is no recommended intake level of
anthocyanins to consume for optimal health or to avoid
adverse effects (UL). Future anthocyanins research and
continued consumer interest in anthocyanin-containing
foods and products with health benefits will undoubt-
edly present opportunities for pursuing dietary guid-
ance recommendations.
References
Andriambeloson, E., Magnier, C., Haan-Archipoff, G., et  al., 1998.
Natural dietary polyphenolic compounds cause endothelium-
dependent vasorelaxation in rat thoracic aorta. J. Nutr. 128 (12),
2324–2333.
Bagchi, D., Sen, C.K., Bagchi, M., et al., 2004. Anti-angiogenic, antioxi-
dant, and anti-carcinogenic properties of a novel anthocyanin-rich
berry extract formula. Biochemistry (Mosc) 69 (1), 75–80.
Banaszewski, K., Park, E., Edirisinghe, I., et al., 2013. A pilot study to
investigate bioavailability of strawberry anthocyanins and char-
acterize postprandial plasma polyphenols by Q-TOF LC/MS in
humans. J. Berry Res. 3, 113–126.
Bartikova, H., Skalova, L., Drsata, J., et al., 2013. Interaction of antho-
cyanins with drug-metabolizing and antioxidant enzymes. Curr.
Med. Chem. 20 (37), 4665–4679.
NUTRACEUTICALS
497
Basu, A., Betts, N.M., Nguyen, A., et al., 2014. Freeze-dried strawber-
ries lower serum cholesterol and lipid peroxidation in adults with
abdominal adiposity and elevated serum lipids. J. Nutr. 144 (6),
830–837.
Basu, A., Du, M., Leyva, M.J., et  al., 2010a. Blueberries decrease car-
diovascular risk factors in obese men and women with metabolic
syndrome. J. Nutr. 140 (9), 1582–1587.
Basu, A., Fu, D.X., Wilkinson, M., et al., 2010b. Strawberries decrease
atherosclerotic markers in subjects with metabolic syndrome.
Nutr. Res. 30 (7), 462–469.
Basu, A., Nguyen, A., Betts, N.M., et  al., 2014. Strawberry as a func-
tional food: an evidence-based review. Crit. Rev. Food Sci. Nutr.
54 (6), 790–806.
Bell, D.R., Gochenaur, K., 2006. Direct vasoactive and vasoprotective
properties of anthocyanin-rich extracts. J. Appl. Physiol. (1985)
100 (4), 1164–1170.
Bhagwat, S., Haytowitz, D.B., Holden, J.M. (Ret.), 2013. USDA
Database for the Flavonoid Content of Selected Foods, Release 3.1.
U.S. Department of Agriculture, Agricultural Research Service.
Nutrient Data Laboratory Home Page: http://www.ars.usda.
gov/nutrientdata/flav. Accessed on April 2014.
Blumberg, J.B., Camesano, T.A., Cassidy, A., et  al., 2013. Cranberries
and their bioactive constituents in human health. Adv. Nutr. 4 (6),
618–632.
Böhm, H., 1994. G. Mazza und E. Miniati: Anthocyanins in Fruits,
Vegetables and Grains. 362 Seiten, zahlr. Abb. und Tab. CRC Press,
Boca Raton, Ann Arbor, London, Tokyo, 1993. Preis 38 (3), 343–343.
Brouillard, R., 1982. Chemical structure of anthocyanins. In: Markakis,
P. (Ed.), Anthocyanins as Food Colors Academic Press, New York,
pp. 1–40.
Brouillard, R., Lang, J., 1990. The hemiacetal–cis-chalcone equilibrium
of malvin, a natural anthocyanin. Canad. J. Chem. 68 (5), 755–761.
Brown, E.M., McDougall, G.J., Stewart, D., et al., 2012. Persistence of
anticancer activity in berry extracts after simulated gastrointesti-
nal digestion and colonic fermentation. PLoS One 7 (11), e49740.
Brown, J.P., Dietrich, P.S., 1979. Mutagenicity of plant flavonols
in the Salmonella/mammalian microsome test. Mutat. Res. 66,
223–240.
Burton-Freeman, B., 2010. Postprandial metabolic events and fruit-
derived phenolics: a review of the science. Br. J. Nutr. 104 (Suppl.
3), S1–14.
Cao, G., Muccitelli, H.U., Sanchez-Moreno, C., et al., 2001. Anthocyanins
are absorbed in glycated forms in elderly women: a pharmacoki-
netic study. Am. J. Clin. Nutr. 73 (5), 920–926.
Cardona, F., Andres-Lacueva, C., Tulipani, S., et  al., 2013. Benefits of
polyphenols on gut microbiota and implications in human health.
J. Nutr. Biochem. 24 (8), 1415–1422.
Carkeet, C., Clevidence, B.A., Novotny, J.A., 2008. Anthocyanin excre-
tion by humans increases linearly with increasing strawberry
dose. J. Nutr. 138 (5), 897–902.
Cassidy, A., O’Reilly, E.J., Kay, C., et al., 2011. Habitual intake of flavo-
noid subclasses and incident hypertension in adults. Am. J. Clin.
Nutr. 93 (2), 338–347.
Cassidy, A., Mukamal, K.J., Liu, L., et  al., 2013. High anthocy-
anin intake is associated with a reduced risk of myocardial
infarction in young and middle-aged women. Circulation 127 (2),
188–196.
Cassidy, A., Huang, T., Rice, M.S., et al., 2014. Intake of dietary flavo-
noids and risk of epithelial ovarian cancer. Am. J. Clin. Nutr. 100
(5), 1344–1351.
Chen, T., Yan, F., Qian, J., et  al., 2012. Randomized phase II trial of
lyophilized strawberries in patients with dysplastic precancerous
lesions of the esophagus. Cancer Prev. Res. (Phila) 5 (1), 41–50.
Cooke, D., Schwarz, M., Boocock, D., et  al., 2006. Effect of cyanidin-
3-glucoside and an anthocyanin mixture from bilberry on ade-
noma development in the ApcMin mouse model of intestinal
498 35. Anthocyanins
carcinogenesis – relationship with tissue anthocyanin levels. Int.
J. Cancer 119 (9), 2213–2220.
Cox, G.E., Babish, J.C., 1978. Evaluation of the Safety of Dietary
Administration of Special Grape Color Powder (Type BW-AT) on
Reproduction, Lactation and Maturation When Fed to Sprague-
Dawley Rats. Unpublished Report No. 5417 by Food and Drug
Research Laboratories, Inc., submitted to the World Health
Organization by FDA.
Czank, C., Cassidy, A., Zhang, Q., et  al., 2013. Human metabolism
and elimination of the anthocyanin, cyanidin-3-glucoside: a (13)
C-tracer study. Am. J. Clin. Nutr. 97 (5), 995–1003.
de Ferrars, R.M., Czank, C., Saha, S., et al., 2014. Methods for isolating,
identifying, and quantifying anthocyanin metabolites in clinical
samples. Anal. Chem. 86 (20), 10052–10058.
Del Rio, D., Rodriguez-Mateos, A., Spencer, J.P., et  al., 2013. Dietary
(poly)phenolics in human health: structures, bioavailability, and
evidence of protective effects against chronic diseases. Antioxid
Redox Sign. 18 (14), 1818–1892.
Dohadwala, M.M., Holbrook, M., Hamburg, N.M., et al., 2011. Effects
of cranberry juice consumption on vascular function in patients
with coronary artery disease. Am. J. Clin. Nutr. 93 (5), 934–940.
Eandi, M., 1987. Post-marketing investigation on Tegens® preparation
with respect to side effects Cited in Morazzoni P, Bombardelli E.
Vaccinium myrtillus I. Fitoterapia 1996 67, 3–29.
Edirisinghe, I., Burton-Freeman, B., Varelis, P., et al., 2008. Strawberry
extract caused endothelium-dependent relaxation through the acti-
vation of PI3 kinase/Akt. J. Agric. Food Chem. 56 (20), 9383–9390.
Edirisinghe, I., Banaszewski, K., Cappozzo, J., et  al., 2011a. Effect of
black currant anthocyanins on the activation of endothelial nitric
oxide synthase (eNOS) in vitro in human endothelial cells. J.
Agric. Food Chem. 59 (16), 8616–8624.
Edirisinghe, I., Banaszewski, K., Cappozzo, J., et al., 2011b. Strawberry
anthocyanin and its association with postprandial inflammation
and insulin. Br. J. Nutr. 106 (6), 913–922.
Edirisinghe, I., Burton-Freeman, B., 2014. Aging associated endothelial
function: role of oxidative stress, inflammation and western diet.
J. Nutr. Aging 2 (4), 197–212.
EFSA, 2013. Scientific opinion on the re-evaluation of anthocyanins (E
163) as a food additive1. Eur. Food Saf. Author. (EFSA) 11 (4), 3145.
Erlund, I., Koli, R., Alfthan, G., et al., 2008. Favorable effects of berry
consumption on platelet function, blood pressure, and HDL cho-
lesterol. Am. J. Clin. Nutr. 87 (2), 323–331.
Felgines, C., Talavera, S., Gonthier, M.P., et al., 2003. Strawberry antho-
cyanins are recovered in urine as glucuro- and sulfoconjugates in
humans. J. Nutr. 133 (5), 1296–1301.
Felgines, C., Krisa, S., Mauray, A., et al., 2010. Radiolabelled cyanidin
3-O-glucoside is poorly absorbed in the mouse. Br. J. Nutr. 103
(12), 1738–1745.
Fernandes, I., Faria, A., Calhau, C., et al., 2014. Bioavailability of antho-
cyanins and derivatives. J. Funct. Foods 7 (0), 54–66.
Forester, S.C., Waterhouse, A.L., 2010. Gut metabolites of anthocya-
nins, gallic acid, 3-O-methylgallic acid, and 2,4,6-trihydroxybenz-
aldehyde, inhibit cell proliferation of Caco-2 cells. J. Agric. Food
Chem. 58 (9), 5320–5327.
Forester, S.C., Choy, Y.Y., Waterhouse, A.L., et al., 2014. The anthocy-
anin metabolites gallic acid, 3-O-methylgallic acid, and 2,4,6-trihy-
droxybenzaldehyde decrease human colon cancer cell viability by
regulating pro-oncogenic signals. Mol. Carcinog. 53 (6), 432–439.
Francis, F.J., 1989. Food colorants: anthocyanins. Crit. Rev. Food Sci.
Nutr. 28 (4), 273–314.
Ghosh, D., Konishi, T., 2007. Anthocyanins and anthocyanin-rich
extracts: role in diabetes and eye function. Asia Pac. J. Clin. Nutr.
16 (2), 200–208.
Giusti, M.M., Ghanadan, H., Wrolstad, R.E., 1998. Elucidation of
the structure and conformation of red radish (Raphanus sativus)
NUTRACEUTICALS
anthocyanins using one- and two-dimensional nuclear magnetic
resonance techniques. J. Agr. Food Chem. 46 (12), 4858–4863.
Gonzalez-Barrio, R., Borges, G., Mullen, W., et al., 2010. Bioavailability
of anthocyanins and ellagitannins following consumption of
raspberries by healthy humans and subjects with an ileostomy. J.
Agric. Food Chem. 58 (7), 3933–3939.
Hakimuddin, F., Paliyath, G., Meckling, K., 2004. Selective cytotoxicity
of a red grape wine flavonoid fraction against MCF-7 cells. Breast
Cancer Res. Treat. 85 (1), 65–79.
Harris, G.K., Gupta, A., Nines, R.G., et al., 2001. Effects of lyophilized
black raspberries on azoxymethane-induced colon cancer and
8-hydroxy-2'-deoxyguanosine levels in the Fischer 344 rat. Nutr.
Cancer 40 (2), 125–133.
Hassellund, S.S., Flaa, A., Sandvik, L., et al., 2012. Effects of anthocya-
nins on blood pressure and stress reactivity: a double-blind ran-
domized placebo-controlled crossover study. J. Hum. Hypertens
26 (6), 396–404.
Hassellund, S.S., Flaa, A., Kjeldsen, S.E., et al., 2013. Effects of antho-
cyanins on cardiovascular risk factors and inflammation in pre-
hypertensive men: a double-blind randomized placebo-controlled
crossover study. J. Hum. Hypertens 27 (2), 100–106.
He, J., Giusti, M.M., 2010. Anthocyanins: natural colorants with health-
promoting properties. Annu. Rev. Food Sci. Technol. 1, 163–187.
Hidalgo, J., Flores, C., Hidalgo, M.A., et  al., 2014. Delphinol(R) stan-
dardized maqui berry extract reduces postprandial blood glu-
cose increase in individuals with impaired glucose regulation
by novel mechanism of sodium glucose cotransporter inhibition.
Panminerva Med. 56 (2 Suppl. 3), 1–7.
Hollands, W., Brett, G.M., Dainty, J.R., et  al., 2008. Urinary excretion
of strawberry anthocyanins is dose dependent for physiological
oral doses of fresh fruit. Mol. Nutr. Food Res. 52 (10), 1097–1105.
Horbowicz, M., Kosson, R., Grzesiuk, A., et  al., 2008. Anthocyanins
of fruits and vegetables – their occurrence, analysis and role in
human nutrition. Veg. Crops Res. Bull. 68, 5.
Hotamisligil, G.S., 2006. Inflammation and metabolic disorders. Nature
444 (7121), 860–867.
Hou, D.X., 2003. Potential mechanisms of cancer chemoprevention by
anthocyanins. Curr. Mol. Med. 3 (2), 149–159.
Hou, D.X., Kai, K., Li, J.J., et al., 2004. Anthocyanidins inhibit activator
protein 1 activity and cell transformation: structure-activity rela-
tionship and molecular mechanisms. Carcinogenesis 25 (1), 29–36.
Hua, T., Liu, B., Chang, H., et al., 2010. Freeze-Drying of Pharmaceutical
and Food Products, Chapter  2 Fundamentals of Freeze Drying.
Woodhead Publishing, 18–67.
Huang, C., Li, J., Song, L., et al., 2006. Black raspberry extracts inhibit
benzo(a)pyrene diol-epoxide-induced activator protein 1 activa-
tion and VEGF transcription by targeting the phosphotidylinositol
3-kinase/Akt pathway. Cancer Res. 66 (1), 581–587.
Janle, E.M., Lila, M.A., Grannan, M., et  al., 2010. Pharmacokinetics
and tissue distribution of 14C-labeled grape polyphenols in the
periphery and the central nervous system following oral admin-
istration. J. Med. Food 13 (4), 926–933.
Jennings, A., Welch, A.A., Spector, T., et  al., 2014. Intakes of antho-
cyanins and flavones are associated with biomarkers of insulin
resistance and inflammation in women. J. Nutr. 144 (2), 202–208.
Joseph, S.V., Edirisinghe, I., Burton-Freeman, B.M., 2014. Berries: anti-
inflammatory effects in humans. J. Agric. Food Chem. 62 (18),
3886–3903.
Kalt, W., Blumberg, J.B., McDonald, J.E., et al., 2008. Identification of
anthocyanins in the liver, eye, and brain of blue-berry-fed pigs. J.
Agric. Food Chem. 56, 705–712.
Kamei, H., Kojima, T., Hasegawa, M., et al., 1995. Suppression of tumor
cell growth by anthocyanins in vitro. Cancer Invest. 13 (6), 590–594.
Kang, S.Y., Seeram, N.P., Nair, M.G., et  al., 2003. Tart cherry antho-
cyanins inhibit tumor development in Apc(Min) mice and reduce
 REFERENCES
proliferation of human colon cancer cells. Cancer Lett. 194 (1),
13–19.
Kay, C.D., 2006. Aspects of anthocyanin absorption, metabolism and
pharmacokinetics in humans. Nutr. Res. Rev. 19 (1), 137–146.
Koide, T., Hashimoto, Y., Kamei, H., et  al., 1997. Antitumor effect of
anthocyanin fractions extracted from red soybeans and red beans
in vitro and in vivo. Cancer Biother. Radiopharm. 12 (4), 277–280.
Kurilich, A.C., Clevidence, B.A., Britz, S.J., et  al., 2005. Plasma and
urine responses are lower for acylated vs nonacylated anthocya-
nins from raw and cooked purple carrots. J. Agric. Food Chem.
53 (16), 6537–6542.
Kurimoto, Y., Shibayama, Y., Inoue, S., et al., 2013. Black soybean seed
coat extract ameliorates hyperglycemia and insulin sensitivity via
the activation of AMP-activated protein kinase in diabetic mice. J.
Agric. Food Chem. 61 (23), 5558–5564.
Lapidot, T., Harel, S., Granit, R., et al., 1998. Bioavailability of red wine
anthocyanins as detected in human urine. J. Agr. Food Chem. 46
(10), 4297–4302.
Lee, I.T., Chan, Y.C., Lin, C.W., et al., 2008. Effect of cranberry extracts
on lipid profiles in subjects with type 2 diabetes. Diabet. Med. 25
(12), 1473–1477.
Lee, J., 2014. Marketplace analysis demonstrates quality control stan-
dards needed for black raspberry dietary supplements. Plant
Foods Hum. Nutr. 69 (2), 161–167.
Li, D., Zhang, Y., Liu, Y., et  al., 2015. Purified anthocyanin supple-
mentation reduces dyslipidemia, enhances antioxidant capacity,
and prevents insulin resistance in diabetic patients. J. Nutr. 145
(4), 742–748.
Libby, P., 2007. Inflammatory mechanisms: the molecular basis of
inflammation and disease. Nutr. Rev. 65 (12 Pt 2), S140–S146.
Lila, M.A., 2004. Anthocyanins and human health: an in vitro investiga-
tive approach. J. Biomed. Biotechnol. 2004 (5), 306–313.
Lu, H., Ouyang, W., Huang, C., 2006. Inflammation, a key event in
cancer development. Mol. Cancer Res. 4 (4), 221–233.
Manach, C., Williamson, G., Morand, C., et  al., 2005. Bioavailability
and bioefficacy of polyphenols in humans. I. Review of 97 bio-
availability studies. Am. J. Clin. Nutr. 81 (1 Suppl.), 230s–242s.
McCullough, M.L., Peterson, J.J., Patel, R., et al., 2012. Flavonoid intake
and cardiovascular disease mortality in a prospective cohort of US
adults. Am. J. Clin. Nutr. 95 (2), 454–464.
McGhie, T.K., Ainge, G.D., Barnett, L.E., et  al., 2003. Anthocyanin
glycosides from berry fruit are absorbed and excreted unmetabo-
lized by both humans and rats. J. Agric. Food Chem. 51 (16),
4539–4548.
Miguel, M.G., 2011. Anthocyanins: antioxidant and/or anti-inflamma-
tory activities. J. Appl. Pharm. Sci. 1 (6), 7–15.
Mink, P.J., Scrafford, C.G., Barraj, L.M., et  al., 2007. Flavonoid intake
and cardiovascular disease mortality: a prospective study in post-
menopausal women. Am. J. Clin. Nutr. 85 (3), 895–909.
Morazzoni, P., Bombardelli, E., 1996. Post-marketing investigation on
Tegens® preparation with respect to side effects, 1987 Cited in
Vaccinium myrtillus I. Fitoterapia 67, 3–29.
Mullen, W., Edwards, C.A., Serafini, M., et al., 2008. Bioavailability of
pelargonidin-3-O-glucoside and its metabolites in humans follow-
ing the ingestion of strawberries with and without cream. J. Agric.
Food Chem. 56 (3), 713–719.
Murphy, M.M., Barraj, L.M., Herman, D., et  al., 2012. Phytonutrient
intake by adults in the United States in relation to fruit and veg-
etable consumption. J. Acad. Nutr. Diet 112 (2), 222–229.
Nakamura, Y., Matsumoto, H., Todoki, K., 2002. Endothelium-
dependent vasorelaxation induced by black currant concentrate
in rat thoracic aorta. Jpn. J. Pharmacol. 89 (1), 29–35.
Novotny, J.A., Baer, D.J., Khoo, C., et  al., 2015. Cranberry juice
consumption lowers markers of cardiometabolic risk, includ-
ing blood pressure and circulating C-reactive protein,
NUTRACEUTICALS
499
triglyceride, and glucose concentrations in adults. J. Nutr. 145 (6),
1185–1193.
Oak, M.H., Bedoui, J.E., Madeira, S.V., et  al., 2006. Delphinidin and
cyanidin inhibit PDGF(AB)-induced VEGF release in vascular
smooth muscle cells by preventing activation of p38 MAPK and
JNK. Br. J. Pharmacol. 149 (3), 283–290.
Oliveira, J., Bras, N.F., da Silva, M.A., et al., 2014. Grape anthocyanin
oligomerization: a putative mechanism for red color stabilization?
Phytochemistry 105, 178–185.
Ovaskainen, M.L., Torronen, R., Koponen, J.M., et  al., 2008. Dietary
intake and major food sources of polyphenols in finnish adults. J.
Nutr. 138 (3), 562–566.
Park, S., Kang, S., Jeong, D.Y., et  al., 2015. Cyanidin and malvidin in
aqueous extracts of black carrots fermented with Aspergillus oryzae
prevent the impairment of energy, lipid and glucose metabolism in
estrogen-deficient rats by AMPK activation. Genes Nutr. 10 (2), 455.
Passamonti, S., Vrhovsek, U., Mattivi, F., 2002. The interaction of antho-
cyanins with bilitranslocase. Biochem. Biophys. Res. Commun.
296 (3), 631–636.
Passamonti, S., Vrhovsek, U., Vanzo, A., et al., 2003. The stomach as a site
for anthocyanins absorption from food. FEBS Lett. 544 (1-3), 210–213.
Passamonti, S., Terdoslavich, M., Franca, R., et al., 2009. Bioavailability
of flavonoids: a review of their membrane transport and the func-
tion of bilitranslocase in animal and plant organisms. Curr. Drug
Metab. 10 (4), 369–394.
Perez-Jimenez, J., Hubert, J., Hooper, L., et al., 2010. Urinary metabo-
lites as biomarkers of polyphenol intake in humans: a systematic
review. Am. J. Clin. Nutr. 92 (4), 801–809.
Pojer, E., Mattivi, F., Johnson, D., et al., 2013. The case for anthocyanin
consumption to promote human health: a review. Comprehens.
Rev. Food Sci. Food Saf. 12 (5), 483–508.
Pourrat, H., Bastide, P., Dorier, P., et  al., 1967. Préparation et activ-
ité thérapeutique de quelques glycosides d’anthocyanes. Chim.
Thérap. 2, 33–38.
Prior, R.L., Wu, X., 2006. Anthocyanins: structural characteristics that
result in unique metabolic patterns and biological activities. Free
Radic. Res. 40 (10), 1014–1028.
Pulliero, G., Montin, S., Bettini, V., et  al., 1989. Ex vivo study of the
inhibitory effects of Vaccinium myrtillus anthocyanosides on
human platelet aggregation. Fitoterapia 60, 69–75.
Qin, B., Anderson, R.A., 2012. An extract of chokeberry attenuates
weight gain and modulates insulin, adipogenic and inflammatory
signalling pathways in epididymal adipose tissue of rats fed a
fructose-rich diet. Br. J. Nutr. 108 (4), 581–587.
Qin, Y., Xia, M., Ma, J., et  al., 2009. Anthocyanin supplementation
improves serum LDL- and HDL-cholesterol concentrations asso-
ciated with the inhibition of cholesteryl ester transfer protein in
dyslipidemic subjects. Am. J. Clin. Nutr. 90 (3), 485–492.
Rechner, A.R., Kroner, C., 2005. Anthocyanins and colonic metabolites
of dietary polyphenols inhibit platelet function. Thromb. Res. 116
(4), 327–334.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure-antioxidant
activity relationships of flavonoids and phenolic acids. Free Radic.
Biol. Med. 20 (7), 933–956.
Rodrigo, K.A., Rawal, Y., Renner, R.J., et al., 2006. Suppression of the
tumorigenic phenotype in human oral squamous cell carcinoma
cells by an ethanol extract derived from freeze-dried black rasp-
berries. Nutr. Cancer 54 (1), 58–68.
Rodriguez-Fragoso, L., Martinez-Arismendi, J.L., Orozco-Bustos, D.,
et  al., 2011. Potential risks resulting from fruit/vegetable-drug
interactions: effects on drug-metabolizing enzymes and drug
transporters. J. Food Sci. 76 (4), R112–R124.
Rodriguez-Mateos, A., Heiss, C., Borges, G., et al., 2014. Berry (poly)
phenols and cardiovascular health. J. Agric. Food Chem. 62 (18),
3842–3851.
500 35. Anthocyanins
Scrivo, R., Vasile, M., Bartosiewicz, I., et  al., 2011. Inflammation as
“common soil” of the multifactorial diseases. Autoimmun. Rev.
10 (7), 369–374.
Seeram, N.P., 2008. Berry fruits: compositional elements, biochemical
activities, and the impact of their intake on human health, perfor-
mance, and disease. J. Agric. Food Chem. 56 (3), 627–629.
Sehitoglu, M.H., Farooqi, A.A., Qureshi, M.Z., et al., 2014. Anthocyanins:
targeting of signaling networks in cancer cells. Asian Pac. J. Cancer
Prev. 15 (5), 2379–2381.
Seymour, E.M., Tanone, I.I., Urcuyo-Llanes, D.E., et al., 2011. Blueberry
intake alters skeletal muscle and adipose tissue peroxisome pro-
liferator-activated receptor activity and reduces insulin resistance
in obese rats. J. Med. Food 14 (12), 1511–1518.
Shih, P.H., Yeh, C.T., Yen, G.C., 2007. Anthocyanins induce the acti-
vation of phase II enzymes through the antioxidant response
element pathway against oxidative stress-induced apoptosis. J.
Agric. Food Chem. 55 (23), 9427–9435.
Shukitt-Hale, B., Lau, F.C., Joseph, J.A., 2008. Berry fruit supplementa-
tion and the aging brain. J. Agric. Food Chem. 56 (3), 636–641.
Singletary, K.W., Jung, K.J., Giusti, M., 2007. Anthocyanin-rich grape
extract blocks breast cell DNA damage. J. Med. Food 10 (2), 244–251.
Stoner, G.D., Wang, L.S., Zikri, N., et al., 2007. Cancer prevention with
freeze-dried berries and berry components. Semin. Cancer Biol.
17 (5), 403–410.
Stull, A.J., Cash, K.C., Johnson, W.D., et al., 2010. Bioactives in blueber-
ries improve insulin sensitivity in obese, insulin-resistant men and
women. J. Nutr. 140 (10), 1764–1768.
Talavera, S., Felgines, C., Texier, O., et al., 2005. Anthocyanin metabo-
lism in rats and their distribution to digestive area, kidney, and
brain. J. Agric. Food Chem. 53 (10), 3902–3908.
Tanaka, Y., Sasaki, N., Ohmiya, A., 2008. Biosynthesis of plant pig-
ments: anthocyanins, betalains and carotenoids. Plant J. 54 (4),
733–749.
Tsuda, T., 2008. Regulation of adipocyte function by anthocyanins;
possibility of preventing the metabolic syndrome. J. Agric. Food
Chem. 56 (3), 642–646.
Tsuda, T., Ueno, Y., Aoki, H., et al., 2004. Anthocyanin enhances adi-
pocytokine secretion and adipocyte-specific gene expression in
isolated rat adipocytes. Biochem. Biophys. Res. Commun. 316 (1),
149–157.
Tulio Jr., A.Z., Chang, C., Edirisinghe, I., et  al., 2012. Berry fruits
modulated endothelial cell migration and angiogenesis via phos-
phoinositide-3 kinase/protein kinase B pathway in vitro in endo-
thelial cells. J. Agric. Food Chem. 60 (23), 5803–5812.
Walton, M.C., Hendriks, W.H., Broomfield, A.M., et  al., 2009.
Viscous food matrix influences absorption and excretion but not
NUTRACEUTICALS
metabolism of blackcurrant anthocyanins in rats. J. Food Sci. 74
(1), H22–H29.
Wang, L.S., Kuo, C.T., Cho, S.J., et  al., 2013. Black raspberry-derived
anthocyanins demethylate tumor suppressor genes through the
inhibition of DNMT1 and DNMT3B in colon cancer cells. Nutr.
Cancer 65 (1), 118–125.
Wedick, N.M., Pan, A., Cassidy, A., et  al., 2012. Dietary flavonoid
intakes and risk of type 2 diabetes in US men and women. Am. J.
Clin. Nutr. 95 (4), 925–933.
Williamson, G., Clifford, M.N., 2010. Colonic metabolites of berry poly-
phenols: the missing link to biological activity? Br. J. Nutr. 104
(Suppl. 3), S48–S66.
Wu, Q.K., Koponen, J.M., Mykkanen, H.M., et al., 2007. Berry phenolic
extracts modulate the expression of p21(WAF1) and Bax but not
Bcl-2 in HT-29 colon cancer cells. J. Agric. Food Chem. 55 (4),
1156–1163.
Wu, X., Pittman III, H.E., Prior, R.L., 2004. Pelargonidin is absorbed
and metabolized differently than cyanidin after marionberry con-
sumption in pigs. J. Nutr. 134 (10), 2603–2610.
Wu, X., Beecher, G.R., Holden, J.M., et  al., 2006. Concentrations of
anthocyanins in common foods in the United States and esti-
mation of normal consumption. J. Agric. Food Chem. 54 (11),
4069–4075.
Xu, J.W., Ikeda, K., Yamori, Y., 2004. Upregulation of endothelial nitric
oxide synthase by cyanidin-3-glucoside, a typical anthocyanin
pigment. Hypertension 44 (2), 217–222.
Zamora-Ros, R., Fedirko, V., Trichopoulou, A., et al., 2013. Dietary fla-
vonoid, lignan and antioxidant capacity and risk of hepatocellular
carcinoma in the European prospective investigation into cancer
and nutrition study. Int. J. Cancer 133 (10), 2429–2443.
Zhang, Y., Vareed, S.K., Nair, M.G., 2005. Human tumor cell growth
inhibition by nontoxic anthocyanidins, the pigments in fruits and
vegetables. Life Sci. 76 (13), 1465–1472.
Zhu, Y., Xia, M., Yang, Y., et  al., 2011. Purified anthocyanin supple-
mentation improves endothelial function via NO-cGMP activa-
tion in hypercholesterolemic individuals. Clin. Chem. 57 (11),
1524–1533.
Zhu, Y., Ling, W., Guo, H., et  al., 2013. Anti-inflammatory effect of
purified dietary anthocyanin in adults with hypercholesterolemia:
a randomized controlled trial. Nutr. Metab. Cardiovasc. Dis. 23
(9), 843–849.
Zunino, S.J., Parelman, M.A., Freytag, T.L., et al., 2012. Effects of dietary
strawberry powder on blood lipids and inflammatory markers in
obese human subjects. Br. J. Nutr. 108 (5), 900–909.