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Anthocyanins in obesity-associated
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mechanism of action

Article in Food & Function March 2016

DOI: 10.1039/C6FO00154H

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Food &
Function
REVIEW

Anthocyanins in obesity-associated
Cite this: Food Funct., 2016, 7, 2169
thrombogenesis: a review of the potential
mechanism of action
Kiara Thompson,a,b Wayne Pederickb and Abishek Bommannan Santhakumar*a

Received 5th February 2016,
Accepted 15th March 2016
DOI: 10.1039/c6fo00154h
www.rsc.org/foodfunction
Platelet dysfunction, oxidative stress and dyslipidemia are important contributors to pro-thrombotic pro-
gression particularly in obese and hyper-cholesterolemic populations. Becoming an increasingly wide-
spread endemic, obesity causes a dysfunction in the metabolic system by initiating endothelial
dysfunction; increasing free radical production; lipid peroxidation; platelet hyperactivity and aggregation;
thereby accelerating thrombogenesis. In the event of increased free radical generation under pro-throm-
botic conditions, antioxidants act as scavengers in reducing physiological oxidative stress; free radical-
mediated thrombosis and hemostatic function. Anthocyanin, a subclass of the polyphenol family avo-
noids has been shown to exhibit anti-dyslipidemic and anti-thrombotic properties by virtue of its anti-
oxidant activity. Current anti-platelet/coagulant therapeutics target specic receptor pathways to relieve
the extent of dysfunction and plaque acceleration in pro-thrombotic individuals. Though eective, they
have been associated with high bleeding risk and increased response variability. The following review
focuses on the potential role of natural dietary anthocyanins in targeting simultaneous mechanistic path-
ways in alleviating platelet activation, dyslipidemia, and oxidative stress-associated thrombus acceleration
in obese pro-thrombotic populations.

a
Introduction
School of Medical and Applied Sciences, Central Queensland University, North
Rockhampton, Queensland 4702, Australia. E-mail: a.santhakumar@cqu.edu.au, Obesity is becoming a progressively widespread endemic in
sabishekbommannan@gmail.com; Fax: +61 (0) 7 49309209; Tel: +61 (0) 7 49309626
b
wealthy nations with platelet dysfunction, oxidative stress and
QML Pathology, Rockhampton, Queensland 4702, Australia.
E-mail: kiara.thompson@cqumail.com, Wayne.Pederick@qml.com.au
dyslipidemia being important contributors to increased throm-

Miss Kiara Thompson graduated A/Prof. Wayne Pederick gradu-

with a Bachelor of Applied
Science, Medical Science at
Queensland University of
Technology, Australia in 2013.
Kiara has since worked as a
multi-disciplinary medical lab-
oratory scientist at QML Pathol-
ogy, Australia. Kiara is also a
current sessional academic and
a laboratory demonstrator at the
School of Medical and Applied
Kiara Thompson Sciences, CQ University, Austra-
lia. Her current research focusses
on evaluating the potential of anthocyanins in reducing risk
factors of thrombogenesis in obesity and metabolic syndrome. She
is an active member of the Australian Institute of Medical Scien-
tists and the Australian Society of Thrombosis and Haemostasis.
ated with a Bachelor of Applied
Science in Medical Technology in
1980 and has since gained exten-
sive experience in regional diag-
nostic pathology laboratories in
Western Australia, the Northern
Territory and Queensland. He
obtained his Masters of Public
Health and Tropical Medicine in
2008 and is a Member of the
Australian Institute of Medical
Wayne Pederick Scientists, the Australian Society
of Microbiology and the Austra-
lasian College of Tropical Medicine. Wayne was appointed as an
Adjunct Associate Professor in the School of Medical and Applied
Sciences of CQ University in 2012.

This journal is The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 21692178 | 2169
Review
bus formation. According to recent statistics from the World
Health Organization, 39% of adults aged 18 years and over
were overweight (Body Mass Index BMI between
25.029.9 kg m2) and 13% obese (BMI > 30.0 kg m2) world- The endothelium of the blood vessel plays a major role in regu-
wide. Though the current categorization of adiposity is based
on BMI, studies have shown that visceral fat accumulation par-
ticularly abdominal fat is a key risk factor for obesity-related
chronic conditions such as cardiovascular disease (CVD) and
type 2 diabetes mellitus (T2DM).1 Obesity causes a dysfunction
in the metabolic system by initiating endothelial vessel wall
damage; increasing free radical production, lipid peroxidation,
platelet hyperactivity/aggregation; thereby accelerating throm-
bus growth.24 Current therapeutics target specific thrombo-
genic pathways to alleviate the magnitude of dysfunction, for
example, aspirin targets the cyclooxygenase-1 (COX-1) pathway
resulting in irreversible inhibition of platelet-dependent
thromboxane (TxA2) formation. Nevertheless, antiplatelet
drugs have been associated with high bleeding risk and
increased response variability in target populations.5,6
Polyphenols are micronutrients common in todays diet,
abundant in fruit, and most commonly known for their rich
color. Anthocyanins are a subclass of the polyphenol family,
flavonoids, known to exhibit anti-inflammatory and anti-
thrombogenic properties by virtue of their antioxidant
activity.6,7 During excess free radical production under meta-
bolic conditions such as obesity, antioxidants act as scavengers
hence reducing physiological oxidative stress, free-radical
mediated thrombosis and managing hemostatic function. This
review aims to focus on the anti-dyslipidemic, anti-atherogenic
and antiplatelet mechanisms of action by which anthocyanins
could potentially reduce thrombogenesis under high oxidative
stress conditions such as obesity/hypercholesterolemia as a
consequence of their antioxidant capacity.
Dr Abishek Santhakumar is cur-
rently an academic and
researcher at the School of
Medical and Applied Sciences,
CQ University, Australia. Abishek
obtained his PhD in Haematol-
ogy from Grith University, Aus-
tralia in 2014. His current
research focusses on investi-
gating the role of natural dietary
antioxidants in reducing the risk
factors of thrombosis in clinical
Abishek Bommannan populations and its potential as
Santhakumar anti-platelet therapy. He is a
fellow of the Institute of Bio-
medical Science, UK and an active member in a number of scienti-
fic societies such as AIMS, ASTH and HSANZ. He is also an edi-
torial board member and active reviewer for a number of scientific
journals.
2170 | Food Funct., 2016, 7, 21692178
Food & Function
Atherogenesis, inammation and
dyslipidemia: the role of anthocyanins
lating haemostasis.8 Impairment of endothelial function due
to dyslipidemia, hypertension, systemic inflammation, oxi-
dative stress or insulin resistance each contribute to the devel-
opment and risk of vessel occlusion.8 An elevated circulating
level of low density lipoprotein (LDL) in the form of pro-athero-
genic apolipoprotein (apo) B-containing lipoproteins is a
common form of atherogenic dyslipidemia in obesity.9 An
imbalance between the anti-atherogenic apo-AI containing
lipoprotein, high density lipoprotein (HDL), and apo-B in
obesity is a highly prominent CVD risk factor.9 One of the
mechanisms of plaque acceleration in dyslipidemia is due to
the ionic interaction between the positively charged apo-B
domains and the negatively charged endothelial proteins such
as collagen, fibronectin and proteoglycans.10 In addition to
increased LDL in circulation, a low level of HDL is character-
istic of atherogenic dyslipidemia in metabolic disorders.11
Free radical mediated oxidant damage is also considered a
major contributor to atherogenic plaque development.12 Hei-
necke et al. analysis of plaque composition demonstrated the
involvement of lipid peroxidation by-products such as oxidized
phospholipids.13 There has been recent comprehensive investi-
gations on the involvement of the antioxidant action of poly-
phenols especially anthocyanins in reducing circulating lipid
levels. The proposed mechanisms by which anthocyanins
target dyslipidemia are by means of (a) improving lipid
haemostasis,1416 (b) increasing energy expenditure,14 (c) redu-
cing mass of fat,16 (d) triggering favorable changes to genes
associated with lipid metabolism, and (e) delaying fat absorp-
tion and chylomicron secretion.17 Table 1 elucidates controlled
dietary intervention trials demonstrating the anti-dyslipidemic
properties of anthocyanin supplementation.
Oxidative stress and anthocyanins in obesity
One of the important precursors to accelerated thrombus for-
mation and/or atherogenesis in obese populations is due to
excess free radical production (reactive oxygen species ROS
and reactive nitrogen species RNS) and oxidative stress
(redox imbalance).18,19 Free radicals play an important role in
oxidative stress due to platelet hyperactivity, lipid peroxidation
and impaired muscle glucose uptake.20 Some of the character-
istic pathways of systemic oxidant stress in lipid-rich diets cause
altered oxygen metabolism and decreased antioxidant
enzymes.12 Free radicals have an important biological impact on
their ability to damage the majority of classes of macromolecules
at high concentrations, thus playing a key factor in thrombosis
and its involvement in nitric oxide (NO) mediated vascular func-
tion impairment.21,22 ROS production is induced by adipokines
as a result of leptins supporting the development of atherosclero-
sis due to the proliferation and migration of the endothelial cell
to the smooth muscle cells.12 The oxidatively modified form of
LDL (ox-LDL) additionally induces platelet activation dependent
This journal is The Royal Society of Chemistry 2016
Food & Function Review

Table 1 Eect of dietary anthocyanin supplementation in modulating dyslipidemia

Population N Study design Supplementation Result Ref.

T2DM 58 (M = 29, Randomized, placebo- 320 mg anthocyanins per day LDL (7.9%) 62
F = 29) controlled, double-blind 24 weeks Triglycerides (23%)
Apolipoprotein-B-48 (16.5%)
Apolipoprotein C-III (11%)
HDL (19.4%)
Dyslipidemia 120 (M = 42, Randomized, placebo- 320 mg anthocyanins per day LDL (13.6%) 3
F = 78) controlled, double-blind 12 weeks HDL (13.7%)
Metabolic syndrome 48 (M = 4, Randomized controlled Freeze-dried blueberry beverage ox-LDL (28%) 16
F = 44) (50 g freeze-dried blueberries
350 g fresh blueberries) 8 weeks
Metabolic syndrome 16F Controlled intervention Freeze-dried strawberry powder Total cholesterol (5%) 59
25 g per day 4 weeks
LDL (6%)
Lipid peroxidation (14%)
Healthy 69 (M = 31, Randomized, parallel, Red wine. M = 300 ml per day, HDL (1116%) 63
F = 38) four-armed intervention 38.3 g alcohol per day; F = 200 ml
per day, 25.5 g alcohol
per day 4 weeks
Middle aged 71 (M = 25, Randomized, single-blind, Two portions of berries daily HDL (5.2%) 64
F = 46) placebo-controlled (100 g whole bilberries and
intervention 50 g crushed lingonberries)
8 weeks
Hypercholesterolemia 73 (M = 31, Randomized, placebo 320 mg per day anthocyanins LDL (10.4%) 33
F = 42) controlled, double-blind 24 weeks HDL (14.0%)

HDL, high density lipoprotein; LDL, low density lipoprotein; ox-LDL, oxidatively modified low density lipoprotein.

de-granulation (P-selectin expression), monocyte chemotactic
factors and damage to the endothelium.23
Antioxidants have the ability to inhibit or delay lipid oxi-
dation, making them an important dietary requirement to
combat persistent lipid peroxidation in obesity.24 Plant pheno-
lics have been shown to play a vital role in inhibition of oxi-
dative stress.8,25,26 Anthocyanins demonstrate a free radical
scavenging eect, by inhibiting ROS and release of NO improv-
ing inflammatory markers and resulting in cardioprotection.27
Anthocyanins recently have been shown to decrease multiple
oxidative stress biomarkers such as serum malondialde-
hyde,16,28,29 urinary F2-isoprostanes30 and ox-LDL31 (Table 2).
Inflammation and thrombogenesis
In addition to acting as a storage depot for lipid energy, adipo-
cytes secrete metabolically active inflammatory cytokines
(interleukin-6 IL-6 and tumor necrosis factor- TNF-). IL-6
is responsible for regulating the hepatic synthesis of the acute

Table 2 Eect of anthocyanin supplementation in reducing biomarkers of oxidative stress in humans

Population N Study design Supplement Result Ref.

T2DM 58 (M = 29, Randomized, placebo-controlled, 320 mg anthocyanin per day Serum concentration of 8-iso- 62
F = 29) double blind dietary intervention 24 weeks prostaglandin F2 (23.4%)
Obese with 48 (M = 4, Randomized, placebo Freeze-dried blueberry beverage ox-LDL (28%); serum MDA 16
metabolic F = 44) controlled, single blinded (50 g freeze-dried blueberries and hydroxynonenal
syndrome 350 g fresh blueberries) concentrations (17%)
8 weeks
Hemodialysis 21 (M = 14, Pilot intervention study 200 mL per day of red fruit juice NF-B binding activity (19%) 44
patients F = 7) high in anthocyanin/polyphenol GSH (7%)
content 4 weeks
Healthy 26 (M = 18, Randomized, double-blind, 162 mg anthocyanins Urinary 8-iso-PGF2 30
F = 8) placebo-controlled dietary (maqui berry) per day 4 weeks
intervention
Healthy 23 (M = 11, Controlled dietary 500 g strawberries 30 days Serum MDA (31.4%) 65
F = 12) intervention Urinary 8-OHdG (29.67%)
Isoprostane (27.9%)
Healthy 120 (M = 59, Parallel-designed, placebo- 300 mg anthocyanin 3 weeks IFN (inducer of NF-B 32
F = 61) controlled dietary intervention activation) (40%)
Healthy 21 (M = 10, Randomized, double-blind, 200 mL per day of anthocyanin- Plasma MDA (38%) 6
F = 11) cross-over design, placebo- rich QGPJ 4 weeks
controlled dietary intervention

GSH, glutathione; IFN, interferon ; MDA, malondialdehyde; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; ox-LDL,
oxidatively modified low density lipoprotein; QGPJ, Queen Garnet plum juice; 8-iso-PGF2, 8-iso-prostaglandin F2.

This journal is The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 21692178 | 2171
Review Food & Function

phase protein C-reactive protein (CRP), consistently associated
with vascular dysfunction.12 Excessive fat accumulation in
obesity causes cellular damage leading to increased pro-
duction of inflammatory cytokines hence accelerating ROS pro-
duction, lipid peroxidation12 and consequently leading to
foam cell formation (Fig. 1).
Furthermore, increased TNF- production leads to an
increased release of free fatty acids in the adipocytes, thereby
activating NF-B responsible for amplified expression of endo-
thelial cell surface adhesion molecules.12 Anthocyanins have
been recently shown to inhibit NF-B secretion and increase
NO production (Fig. 1).27 In a parallel-designed placebo-con-
trolled trial with 59 men and 61 women, anthocyanin-rich
extract capsule supplementation for 3 weeks exhibited favor-
able changes to plasma NF-B related chemokines, cytokines
and inflammatory mediators.32 A dietary intervention trial con-
ducted consisting of 150 hypercholesterolemic subjects
showed a 21.6% decrease in high sensitivity C-reactive protein
(hs-CRP) after 320 mg per day consumption of anthocyanins
for 24 weeks.33 Correspondingly, after consumption of an
anthocyanin-rich strawberry beverage in a placebo-controlled
cross-over design trial, hs-CRP was significantly attenuated.34
Upon endothelial injury or in the event of atherosclerotic
plaque acceleration, immune cells adhere to the site via
adhesion molecules and growth factors are expressed on the
endothelial cells. Anthocyanins have the ability to regulate this
adhesion demonstrated by a decrease in the expression of vas-
cular endothelial growth factor (VEGF) receptor and intracellu-
lar adhesion molecule (ICAM)-1 in endothelial cells.27
Anthocyanins have also been shown to reduce oxidative stress
(H2O2) and TNF- induced VEGF expression in keratinocytes.35
Favorable eects of anthocyanins on the overall vascular
thrombogenic risk were also demonstrated by a decrease in
systolic and diastolic blood pressure after blueberry consump-
tion in a dietary intervention trial consisting of forty-eight
obese subjects.16
Platelet activation and aggregation related thrombogenesis in
obesity
Platelets are extremely multifunctional cells that play a vital
role in normal hemostasis, and in particular for arresting
bleeding from arterioles during endothelial vessel wall
damage and increased shear stress.36 The adherence of plate-
lets to damaged and exposed sub endothelium initiates impor-
tant regulated functional responses including activation,
adhesion, aggregation, exposure of a pro-coagulant surface
and ultimately fibrin clot formation resulting in a hemostatic
plug.37 In the case of high shear stress; oxidant damage; endo-
thelial dysfunction/damage; atherosclerotic plaque occlusion;
inflammation and dyslipidemia; platelets get hyper-activated
and accelerate thrombus formation.38 Activation-dependent
platelet surface receptors such as P2Y1/P2Y12 (ADP receptors),
thromboxane A2 (TxA2), 2 epinephrine, PAR-1, PAR-4, throm-
bin, and GPVI/21 (collagen receptors) are triggered by con-
stant stimuli due to the damaged epithelium, oxidative stress
and the existing thrombi.39 These activation-dependent plate-
let receptors are key targets for current anti-platelet thera-
peutics. In addition to the functional properties of the
currently administered antiplatelet drugs, there has been a
debate on the ecacy and side-eects associated in pro-throm-
botic populations. Bordeaux et al. assessed platelet responsive-
ness in obesity before and after two weeks of aspirin (81 mg

Fig. 1 Anthocyanins mode of action in obesity associated dyslipidemia and atherogenesis. Anthocyanins or their active metabolites have been
demonstrated to (i) reduce oxidatively modied low density lipoprotein (ox-LDL) and circulating LDL in dyslipidemia, (ii) increase high density lipo-
protein (HDL), (iii) decrease production of reactive oxygen species via their antioxidant properties hence increasing NO bioavailability (a potent vaso-
dilator and endothelial function modulator), and (iv) target NF-B and inammatory cytokines by moderating vascular and endothelial adhesion
molecules and H2O2 production. ACN, anthocyanins; CRP, C-reactive protein; EC, endothelial cells; ED, endothelial dysfunction; eNOS, endothelial
nitric oxide synthase; HDL, high density lipoprotein; LDL, low density lipoprotein; NF-B, nuclear factor kappa-light-chain-enhancer of activated B
cells; ox-LDL, oxidatively modied low density lipoprotein; ROS, reactive oxygen species; SMC, smooth muscle cells.

2172 | Food Funct., 2016, 7, 21692178 This journal is The Royal Society of Chemistry 2016
Food & Function Review

per day) supplementation. It was concluded that due to their
pro-thrombotic state, obese individuals have higher native
platelet reactivity and maintain this state post aspirin sup-
plementation.40 Similarly obesity and T2DM have been associ-
ated with a reduced response to aspirin due to decreased
insulin sensitivity and increased oxidative stress.41 Studies
have shown a high risk of inducing gastrointestinal irritation/
bleeding particularly in high dose aspirin subjects.42,43
The benefits of naturally occurring antioxidants has
sparked great interest as a potential complementary thera-
peutic.24 In particular plant phenolics have been recently
demonstrated to blunt specific pathways of platelet activation,
adhesion and aggregation.44 In a randomized, double-blind,
crossover design, dietary intervention trial, twenty-one healthy
volunteers consumed anthocyanin-rich Queen Garnet plum
juice (QGPJ) and a commercial prune juice with no anthocya-
nins for 4 weeks.6 Post anthocyanin-rich QGPJ supplemen-
tation, findings demonstrated the inhibition of platelet
aggregation induced by the ADP-P2Y1/P2Y12 pathway, GPVI/
21-collagen, and arachidonic acid-COX-1 pathway; reduced
plasma fibrinogen concentration and P-selectin expression
stimulated by the ADP-P2Y1/P2Y12 receptor. In a follow-up trial,
QGPJ consumption inhibited platelet aggregation induced by
ADP by 10.7% and 12.7% in healthy volunteers both with and
without exercise-induced oxidative stress respectively;6
decreased arachidonic acid induced aggregation by 28.8%
under oxidative stress; reduced platelet activation related
P-selectin expression by 32.9% and 38.7% both with and

Table 3 Eect of anthocyanin intervention on platelet function in humans

Population N Study design Result Ref.

Healthy 23 (M = 11, 500 g strawberries Central clustered platelets (activation) 65

F = 12) per day 30 days
Healthy 3 In vitro treatment with ADP, collagen, TRAP stimulated platelet aggregation 46
delphinidin-3-glucoside Ex vivo thrombus formation
P-selectin expression (platelet -granules)
CD63 (-granules), CD40L (platelet cytoplasmic
inflammatory factor), PAC-1 (platelet activation-related
conformational change) expression
Fibrinogen binding to platelets
Hypercholesterolemia 146 (M = 61, 320 mg per day Plasma levels of P-selectin, platelet factor 4, 61
F = 85) anthocyanins 24 weeks transforming growth factor 1
Healthy 18 (M = 10, 1l blood orange juice No change in ADP induced P-selectin or 66
F = 8) per day 4 weeks PAC-1 expression
Healthy NA In vitro treatment with 1 M Threshold concentration of TRAP induced platelet 48
delphinidin-3-rutinoside aggregation
P-selectin expression
TRAP (4 M) induced platelet activation
Oxidative stress induced platelet activation
Healthy NA In vitro treatment with anthocyanin Arachidonic acid metabolites in thrombin 67
extracts (10 M) from red cabbage stimulated platelets
leaves Superoxide formation in platelets treated with LPS
(induced oxidative stress)
Protection of platelets against H2O2 induced protein
carbonylation
Metabolic syndrome 48 In vitro treatment with Spontaneous (10 g ml1) and ADP-induced (1 g mL1) 47
chokeberry extract platelet adhesion
10 and 100 g ml1 extracts ADP-induced platelet
aggregation
Amidolytic activity of thrombin and plasmin
Middle aged participants 71 (M = 25, Two portions of berries daily ADP and collagen induced platelet function 64
at risk of CVD F = 46) (100 g whole bilberries and No change to platelet activation, coagulation or fibrinolysis
50 g crushed lingonberries)
8 weeks
Healthy 20 (M = 12, 7 ml per kg per day of purple ADP, collagen stimulated platelet aggregation 51
F = 8) grape juice 2 weeks Platelet-derived NO release
Platelet superoxide production
Healthy 13M 50 ml per day of pomegranate Collagen stimulated platelet aggregation 68
juice 2 weeks
Healthy males NA In vitro treatment with black Collagen induced platelet aggregation, P-selectin 69
soybean extracts (10, 25, 50, expression (50 and 100 g mL1)
100 g mL1) TxA2 formation (25, 50, 100 g mL1)
Healthy NA In vitro treatment with grape seed TRAP induced platelet aggregation 70
and grape skin extracts
Healthy 10 (M = 5, 5-7.5 mL per kg per day of purple Collagen induced platelet aggregation 71
F = 5) grape juice for 1 week

ADP, adenosine diphosphate; LPS, lipopolysaccharide; NA, not available; NO, nitric oxide; PAC-1, procaspase activating compound-1; TRAP,
thrombin receptor-activating peptide; TxA2, thromboxane A2.

This journal is The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 21692178 | 2173
Review Food & Function

without oxidative stress; and prolonged clotting times and
reduced plasma fibrinogen concentration.45 Supplementation
with commercial prune juice with no anthocyanin compo-
sition did not alter the risk factors of thrombogenesis.6 Yang
et al. demonstrated the anti-thrombotic benefits of delphini-
din-3-glucoside (Dp-3-g), a major bioactive compound of
anthocyanin, via the inhibition of P-selectin expression,
GPIIbIIIa receptor, CD40L, CD63, reduction in phosphoryl-
ation of adenosine monophosphate-activated protein kinase
and attenuation of fibrinogen binding.46 Dp-3-g has also been
demonstrated to target the ADP and thrombin receptor of
platelet activation related thrombogenesis.46 Anthocyanin-rich
chokeberry extracts reduced ADP-activated platelet adhesion
and targeted the PAR1/PAR4 thrombin receptor by inhibiting
the amidolytic activity of thrombin.47 Rechner et al. validated
the eects of anthocyanins and their colonic metabolites in
vitro in reducing platelet activation and aggregation induced
by thrombin receptor-activating peptide (TRAP).48 The above
studies confirm the anti-thrombogenic and anti-platelet poten-
tial of anthocyanins by eectively blunting specific pathways of
thrombus generation and acceleration. Table 3 shows interven-
tion trials demonstrating the eects of anthocyanin sup-
plementation on platelet aggregation and activation related
thrombotic risk. In addition to the anti-thrombotic properties
demonstrated by anthocyanin-rich food supplementation
(Table 3), it should also be noted that other polyphenols
present in the food or their active metabolites could potentially
contribute to the observed vascular eects.20,49 Polyphenols
such as catechins, procyanidins, isoflavones and quercetin
have been shown to blunt specific thrombotic pathways and
might confer some of the anti-thrombotic benefits in combi-
nation with the predominant phytochemical, anthocyanin.50
A systematic literature search was performed using
PubMed, EMBASE, and Google Scholar databases to investigate
the relationship between anthocyanin-rich supplement inter-
vention and platelet aggregation. The primary outcome was to
determine the eect of intervention on platelet aggregation
exhibited in percentage and change in resistance (ohms) in
human subjects (Fig. 2). Nine original studies were identified
reflecting the eect of supplementation in dierent popu-
lations.6,45,47,51 Overall, the meta-analysis revealed a platelet
inhibitory eect ( p < 0.00001) by anthocyanin intervention
induced by ADP, collagen, arachidonic acid and phorbol ester
(PMA). The standardized mean dierence in aggregation inhi-
bition between the control group and anthocyanin-rich sup-
plementation group was 4.02 and 9.06 represented as
percentage aggregation and ohms respectively. The hetero-
geneity in data for percentage aggregation ( p < 0.00001) and

Fig. 2 Eect of anthocyanin supplementation on platelet aggregation expressed as percentage (A) and change in resistance-ohms (B). The meta-
analysis utilized the weighted mean dierences between the intervention and control groups. There was a signicant heterogeneity observed on the
extent of platelet aggregation inhibition by anthocyanin intervention between the studies. An overall inhibitory eect on platelet aggregation
induced by the dierent agonists ADP, collagen, arachidonic acid and PMA was observed ( p < 0.00001).

2174 | Food Funct., 2016, 7, 21692178 This journal is The Royal Society of Chemistry 2016
Food & Function
resistance to aggregation ( p = 0.0007) is most likely a result of
varied responses in platelet aggregation inhibition, study
population, total anthocyanin content or the influence of
additional phenolics/active metabolites present in the inter-
vention. Thus the observed eect is believed to be associated
with the structure of the phenolic compound and its func-
tional groups in mediating oxidative stress related platelet
activation.
Structure activity relationship and
bioavailability
Anthocyanins are one of the most abundant polyphenol (flavo-
noid) subclasses in fruit and vegetables with an estimated
mean intake in Europe of about 65 mg per day (ref. 6) and
have been acclaimed for their free-radical scavenging pro-
perties. Anthocyanins are usually glycosylated, polymethoxy or
polyhydroxy derivatives of 2-phenylbenzopyrylium and are
made up of two benzoyl rings (A and B) separated by a hetero-
cyclic ring (C) (Fig. 3). Structural variations in anthocyanins
arise due to the dierences in the number and degree of
methylation of hydroxyl groups; the number and nature of the
sugar moiety attached to the phenolic molecule; and the ali-
phatic or aromatic acids attached to the sugars.52 Recently four
distinct molecular structures of anthocyanins have been
recorded in aqueous solutions, with the red flavylium cation
being the most abundant when the pH is <2.53 The relative
composition of the other molecular structures (quinonoids,
hemiketal, chalcone) is dependent on pH, temperature, phy-
siochemical conditions and time. Hence anthocyanins after
oral consumption absorbed in their intact glycosylated form
from the gastro-intestinal tract are subjected to dierent con-
ditions, thereby varying their bioactive properties.53 The bio-
availability of anthocyanins has been extensively reviewed in
the past decade.53 Urinary excretion has been variable across
many studies but consistently low with less than 0.1% of the
ingested dose being excreted. Plasma tmax is known to be
between 0.754 h with the ingested dose eliminated within
68 hours.54 In spite of this challenge in extracting their finest
bioavailable functionality, regular doses of anthocyanin
extracts orally to target specific anti-thrombotic pathways
could be eective. Oliveira and Pintado have recently suggested
Fig. 3 Structure of anthocyanin.
This journal is The Royal Society of Chemistry 2016
Review
that despite the decrease in the bioavailability of polyphenols
due to gastrointestinal digestion, the levels of bioactive com-
pounds released are still relevant to exert their functional pro-
perties.45,55 Irreversible changes to platelet activity post
anthocyanin-rich supplementation have been successful in the
past therefore supporting the possibility of potent mechanistic
actions.20 It is believed that hydroxylation, methylation and
the O-diphenyl structure in the B-ring is responsible for blunt-
ing activation dependent ADP receptors (P2Y1 and P2Y12) in
addition to blocking the IIb3 integrin.46
In spite of the apparent low bioavailability of anthocyanins,
many potential active metabolites can be generated in vivo.53
Hippuric acid, a potential active metabolite of polyphenol
intake has been demonstrated to block platelet activation-
related conformational change and de-granulation hence redu-
cing thrombogenesis.49 Hippuric acid is also a metabolite of
acetylsalicylic acid (aspirin), which might be partly responsible
for the demonstrated antiplatelet activity by targeting the
COX-1 pathway resulting in reduced TxA2 synthesis.56 It is
possible that the inhibition of platelet activation could be
regulated due to platelet enzymes capable of altering mole-
cules by the addition of hydroxyl, methyl, sulphate or a glucur-
onide group to existing functional groups consequently
enhancing the antioxidant activity.57 In addition to the direct
inhibition of activation pathways, metabolites are also capable
of production of increased levels of antioxidants (tryptophan
and nicotinamide) through the shikimate pathway.49
Current therapeutic strategy
Anti-thrombogenic/coagulant/platelet therapy works by redu-
cing platelet hyper-activation or aggregation; blocking specific
platelet receptors or associated coagulation pathways.6 Most
common anti-platelet drugs administered individually or in
combination are aspirin, clopidogrel and warfarin, conse-
quently when used as long term treatment, studies have
shown resistance and potential life threatening side eects.42
Current dual antiplatelet therapy aims to target both the P2Y12
receptor and inhibits the COX-1 pathway of platelet activation/
aggregation.42,58 Another avenue of testing antithrombotic
therapies has been focused to target the GPIIbIIIa inhibitors
but most have resulted in poor outcomes, serious side eects,
high bleeding risk and increased chance of secondary throm-
botic episode.41 Similarly while the P2Y12 receptor may be an
obvious target with drugs such as clopidogrel and ticlopidine,
they can also cause high bleeding risk.58 In spite of the func-
tional benefits of antiplatelet therapeutics, side-eects and
resistance in specific population groups need consideration,
thereby justifying the necessity for alternative natural thera-
peutic agents to complement or replace the current strategy.
Anthocyanins through their antioxidant properties have
recently been successful in targeting simultaneous pathways in
obesity related thrombogenesis,59 by inhibiting platelet acti-
vation/aggregation receptors,47,60,61 coagulation proteins,
inflammatory markers59 and lipid profiles,3,16,62 with limited
Food Funct., 2016, 7, 21692178 | 2175
Review
Fig. 4 Anti-thrombogenic activity of anthocyanins vs. anti-platelet therapy. Anthocyanins target specic pathways of platelet activation-related
thrombus formation by simultaneously blunting specic receptors of activation and aggregation hence mimicking currently used drug therapeutic
strategies in pro-thrombotic populations. COX-1, cyclooxygenase-1; TxA2, thromboxane-A2; vWF, von-Willebrand factor; PAR-1, protease activated
receptor-1; PAR-4, protease activated receptor-4.
evidence suggesting any risk or side eects hence mimicking
current therapeutic strategies (Fig. 4).6,46
Conclusion
Anthocyanins have been demonstrated to reduce bio-
markers of inflammation and oxidative stress; influence
dyslipidemia and reduce platelet aggregation/hyperactivity
by blunting specific pathways in diverse populations. It is
possible that oral anthocyanin supplementation in at-risk
obese individuals or those with increased response variabil-
ity to current intervention could potentially be an eective
complementary therapeutic strategy in reducing thrombotic
progression.
Conict of interest and funding
disclosure
The authors of this manuscript have no conflicts of interest to
declare. This work was not financially supported by any exter-
nal funding or grants.
2176 | Food Funct., 2016, 7, 21692178
Food & Function
Note added after rst publication
This article replaces the version published on 17 March 2016,
which contained errors in Fig. 3, Fig. 4, and the graphical
abstract.
References
1 I. Savini, M. V. Catani, D. Evangelista, V. Gasperi and
L. Avigliano, Int. J. Mol. Sci., 2013, 14, 1049710538.
2 Y. Zhu, X. Huang, Y. Zhang, Y. Wang, Y. Liu, R. Sun and
M. Xia, J. Clin. Endocrinol. Metab., 2014, 99, 561569.
3 Y. Qin, M. Xia, J. Ma, Y. Hao, J. Liu, H. Mou, L. Cao and
W. Ling, Am. J. Clin. Nutr., 2009, 0, 18.
4 A. Ayala, M. F. Munoz and S. Arguelles, Oxid. Med. Cell.
Longevity, 2014, 2014, 360438.
5 F. Catella-Lawson, M. P. Reilly, S. C. Kapoor, A. Cucchiara,
S. DeMarco, B. Tournier, S. N. Vyas and G. Fitzgerals, N.
Engl. J. Med., 2001, 345, 18091817.
6 A. B. Santhakumar, A. R. Kundur, K. Fanning, M. Netzel,
R. Stanley and I. Singh, J. Funct. Foods, 2015, 12, 1122.
7 F. Giampieri, T. Y. Forbes-Hernandez, M. Gasparrini,
J. M. Alvarez-Suarez, S. Afrin, S. Bompadre, J. L. Quiles,
This journal is The Royal Society of Chemistry 2016
Food & Function Review

B. Mezzetti and M. Battino, Food Funct., 2015, 6, 1386 34 I. Edirisinghe, K. Banaszewski, J. Cappozzo, K. Sandhya,
1398. C. L. Ellis, R. Tadapaneni, C. T. Kappagoda and
8 M. F. Chong, R. Macdonald and J. A. Lovegrove, Br. J. Nutr., B. M. Burton-Freeman, Br. J. Nutr., 2011, 106, 913
2010, 104(Suppl 3), S28S39. 922.
9 M. J. Chapman, F. Nigon, P. Lesnik and M. Rouis, J. Lipid 35 L. S. Wang and G. D. Stoner, Cancer Lett., 2008, 269, 281
Res., 1991, 32, 17411753. 290.
10 M. F. Khalil, W. D. Wagner and I. J. Goldberg, Arterioscler., 36 Z. M. Ruggeri and G. L. Mendolicchio, Circ. Res., 2007, 100,
Thromb., Vasc. Biol., 2004, 24, 22112218. 16731685.
11 S. Rashid and J. Genest, Obesity, 2007, 15, 28752888. 37 P. Harrison, Blood Rev., 2005, 19, 111123.
12 A. Fernandez-Sanchez, E. Madrigal-Santillan, M. Bautista, 38 G. Boden, Endocrinol. Metab. Clin. North Am., 2008, 37,
J. Esquivel-Soto, A. Morales-Gonzalez, C. Esquivel-Chirino, 635646.
I. Durante-Montiel, G. Sanchez-Rivera, C. Valadez-Vega and 39 M. Bojic, Z. Debeljak, M. Tomicic, M. Medic-Saric and
J. A. Morales-Gonzalez, Int. J. Mol. Sci., 2011, 12, 3117 S. Tomic, Nutr. J., 2011, 10, 73.
3132. 40 B. C. Bordeaux, R. Qayyum, L. R. Yanek, D. Vaidya,
13 J. W. Heinecke, Atherosclerosis, 1998, 141, 115. L. C. Becker, N. Faraday and D. M. Becker, Prev. Cardiol.,
14 R. Sunkara and M. Verghese, Food Nutr. Sci., 2014, 05, 2010, 13, 5662.
13591369. 41 M. Pignone and C. D. Williams, Nat. Rev. Endocrinol., 2010,
15 S. L. Yao, Y. Xu, Y. Y. Zhang and Y. H. Lu, Food Funct., 2013, 6, 619628.
4, 16021608. 42 G. Roth and D. Calverley, Blood, 1994, 83, 885898.
16 A. Basu, M. Du, M. J. Leyva, K. Sanchez, N. M. Betts, 43 D. J. Angiolillo, A. Fernandez-Ortiz, E. Bernardo,
M. Wu, C. E. Aston and T. J. Lyons, Nutr. J., 2010, 140, C. Ramrez, M. Sabat, P. Jimenez-Quevedo, R. Hernndez,
15821587. R. Moreno, J. Escaned, F. Alfonso, C. Banuelos,
17 C. Blade, L. Arola and M. J. Salvado, Mol. Nutr. Food Res., M. A. Costa, T. A. Bass and C. Macaya, Diabetes, 2005, 54,
2010, 54, 3759. 24302435.
18 F. Perticone, R. Ceravolo, M. Candigliota, G. Ventura, 44 T. M. Spormann, F. W. Albert, T. Rath, H. Dietrich, F. Will,
S. Iacopino, F. Sinopoli and P. L. Mattioli, Diabetes, 2001, J. P. Stockis, G. Eisenbrand and C. Janzowski, Cancer Epide-
50, 159165. miol. Biomarkers Prev., 2008, 17, 33723380.
19 C. K. Roberts and K. K. Sindhu, Life Sci., 2009, 84, 705712. 45 A. B. Santhakumar, A. R. Kundur, S. Sabapathy, R. Stanley
20 A. B. Santhakumar, A. C. Bulmer and I. Singh, J. Hum. and I. Singh, J. Funct. Foods, 2015, 14, 747757.
Nutr. Diet, 2014, 27, 121. 46 Y. Yang, Z. Shi, A. Reheman, J. W. Jin, C. Li, Y. Wang,
21 D. R. Bell and K. Gochenaur, J. Appl. Physiol., 2006, 100, M. C. Andrews, P. Chen, G. Zhu, W. Ling and H. Ni, PLoS
11641170. One, 2012, 7, e37323.
22 I. Perrotta and S. Aquila, Oxid. Med. Cell. Longevity, 2015, 47 J. Sikora, M. Markowicz-Piasecka, M. Broncel and
2015, 130315. E. Mikiciuk-Olasik, Eur. J. Nutr., 2014, 53, 14931502.
23 N. R. Madamanchi, A. Vendrov and M. S. Runge, Arterios- 48 A. R. Rechner and C. Kroner, Thromb. Res., 2005, 116, 327
cler., Thromb., Vasc. Biol., 2005, 25, 2938. 334.
24 L. R. Fukumoto and G. Mazza, J. Agric. Food Chem., 2000, 49 A. B. Santhakumar, R. Stanley and I. Singh, Food Funct.,
48, 35973604. 2015, 6, 26792683.
25 Y. S. Lai, W. H. Hsu, J. J. Huang and S. C. Wu, Food Funct., 50 S. Rajaram, Am. J. Clin. Nutr., 2003, 78, 552S558S.
2012, 3, 12941301. 51 J. Freedman, C. Parker, L. Li, J. Perlman, B. Frei, V. Ivanov,
26 C. Del Bo, D. Martini, M. Porrini, D. Klimis-Zacas and L. Deak, M. Iafrati and J. Folts, Circ. Res., 2001, 103, 2792
P. Riso, Food Funct., 2015, 6, 28902917. 2798.
27 M. J. Kruger, N. Davies, K. H. Myburgh and S. Lecour, Food 52 G. Mazza and E. Miniati, Anthocyanins in fruits, vegetables,
Res. Int., 2014, 59, 4152. and grains, CRC press, 1993.
28 H. K. Vincent and A. G. Taylor, Int. J. Obes., 2006, 30, 400 53 T. K. McGhie and M. C. Walton, Mol. Nutr. Food Res., 2007,
418. 51, 702713.
29 M. L. Urso and P. M. Clarkson, Toxicology, 2003, 189, 4154. 54 I. Fernandes, A. Faria, C. Calhau, V. de Freitas and
30 S. Davinelli, J. C. Bertoglio, A. Zarrelli, R. Pina and N. Mateus, J. Funct. Foods, 2014, 7, 5466.
G. Scapagnini, J. Am. Coll. Nutr., 2015, 34(Suppl 1), 2833. 55 A. Oliveira and M. Pintado, Food Funct., 2015, 6, 1611
31 J. E. Brown and M. F. Kelly, Eur. J. Lipid Sci. Technol., 2007, 1619.
109, 6671. 56 H. Deng and Y. Fang, NaunynSchmiedebergs Arch. Pharma-
32 A. Karlsen, L. Retterstol, P. Laake, I. Paur, S. K. Bohn, col., 2012, 385, 729737.
L. Sandvik and R. Blomho, J. Nutr., 2007, 137, 19511954. 57 N. P. Seeram and M. G. Nair, J. Agric. Food Chem., 2002, 50,
33 Y. Zhu, W. Ling, H. Guo, F. Song, Q. Ye, T. Zou, D. Li, 53085312.
Y. Zhang, G. Li, Y. Xiao, F. Liu, Z. Li, Z. Shi and Y. Yang, 58 C. Gachet, J. Thromb. Haemostasis., 2015, 13(Suppl 1),
Nutr. Metab. Cardiovasc. Dis., 2013, 23, 843849. S313S322.

This journal is The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 21692178 | 2177
Review
59 A. Basu, M. Wilkinson, K. Penugonda, B. Simmons,
N. M. Betts and T. J. Lyons, Nutr. J., 2009, 8, 1.
60 I. Erlund, R. Koli, G. Alfthan, J. Marniemi, P. Puukka,
P. Mustonen, P. Mattila and A. Jula, Am. J. Clin. Nutr., 2008,
87, 323331.
61 F. Song, Y. Zhu, Z. Shi, J. Tian, X. Deng, J. Ren,
M. C. Andrews, H. Ni, W. Ling and Y. Yang, Thromb. Hae-
mostasis., 2014, 112, 981991.
62 D. Li, Y. Zhang, Y. Liu, R. Sun and M. Xia, J. Nutr., 2015,
145, 742748.
63 A. S. Hansen, P. Marckmann, L. O. Dragsted, I. L. Finne
Nielsen, S. E. Nielsen and M. Gronbaek, Eur. J. Nutr., 2005,
59, 449455.
64 I. Erlund, R. Koli, G. Alfthan, J. Marniemi, P. Puukka,
P. Mustonen, P. Mattila and A. Jula, Am. J. Clin. Nutr., 2008,
87, 323331.
65 J. M. Alvarez-Suarez, F. Giampieri, S. Tulipani, T. Casoli,
G. Di Stefano, A. M. Gonzalez-Paramas, C. Santos-Buelga,
F. Busco, J. L. Quiles, M. D. Cordero, S. Bompadre,
2178 | Food Funct., 2016, 7, 21692178
View publication stats
Food & Function
B. Mezzetti and M. Battino, J. Nutr. Biochem., 2014, 25,
289294.
66 L. Giordano, W. Coletta, C. Tamburrelli, M. DImperio,
M. Crescente, C. Silvestri, P. Rapisarda, G. Reforgiato
Recupero, A. De Curtis, L. Iacoviello, G. de Gaetano,
D. Rotilio, C. Cerletti and M. B. Donati, Eur. J. Nutr., 2012,
51, 541548.
67 J. Kolodziejczyk, J. Saluk-Juszczak, M. Posmyk, K. Janas
and B. Wachowicz, Open Life Sci., 2011, 6, 565574.
68 M. Aviram, L. Dornfeld, M. Rosenblat, N. Volkova,
M. Kaplan, R. Coleman, T. Hayek, D. Presser and
B. Fuhrman, Am. J. Clin. Nutr., 2000, 71, 10621076.
69 K. Kim, K. M. Lim, C. W. Kim, H. J. Shin, D. B. Seo,
S. J. Lee, J. Y. Noh, O. N. Bae, S. Shin and J. H. Chung, J.
Nutr. Biochem., 2011, 22, 964970.
70 O. Vitseva, S. Varghese, S. Chakrabarti, J. Folts and
J. Freedman, J. Cardiovasc. Pharmacol., 2005, 46, 445451.
71 J. Keevil, H. Osman, J. Reed and J. Folts, J. Nutr., 2000, 130,
5356.
This journal is The Royal Society of Chemistry 2016