-
REVIEW
Impact of dietary polyphenols on human platelet
function – A critical review of controlled dietary
intervention studies
Luisa M. Ostertag1, Niamh O’Kennedy2, Paul A. Kroon3, Garry G.
Duthie1 and Baukje de Roos1
1 Rowett Institute of Nutrition and Health, University of
Aberdeen, Aberdeen, UK2 Provexis plc at the Rowett Institute,
Aberdeen, UK3 Institute of Food Research, Norwich, UK
Received: April 14, 2009
Revised: June 5, 2009
Accepted: June 9, 2009
Cardiovascular disease is a chronic disease influenced by many
factors, with activated blood
platelets being one of them. Platelets play a central role in
the formation of plaques within
blood vessels, contributing to early inflammatory events.
Consumption of diets rich in plant-
based products protects against the development of
cardiovascular disease. Polyphenols,
which are secondary plant metabolites found in a wide range of
foodstuffs and beverages,
may be partially responsible for these effects. Their protective
properties include inhibitory
effects on platelet function in vitro and in vivo. However, the
bioavailability of many poly-phenols is poor and it is unclear
whether sufficient quantities can be obtained by dietary
means to exert protective effects. Consequently, this review
summarizes 25 well-controlled
human intervention studies examining the effect of
polyphenol-rich diets on platelet func-
tion. These studies report a huge variety of research methods,
study designs, and study
subjects, resulting in controversial assertions. One consistent
finding is that cocoa-related
products, however, have platelet-inhibiting effects when
consumed in moderate amounts. To
assess whether other classes of dietary polyphenols, or their
metabolites, also beneficially
affect platelet function requires more well-controlled
intervention studies as well as the
adoption of more uniform methods to assess platelet aggregation
and activation.
Keywords:
Cardiovascular disease / Critical review / Dietary polyphenols /
Human intervention
studies / Platelet function
1 Introduction
Cardiovascular disease (CVD) is a widespread human
epidemic in industrialized countries [1]. In the US alone it
accounts annually for 36% of all deaths, which is more than
the combined mortalities from cancer, chronic lower
respiratory diseases, accidents, and diabetes mellitus. The
estimated cost of CVD to the US economy in 2007 was 432
billion US dollars [2]. Similarly in Europe the annual 4.3
million deaths (equals 48% of all deaths) and associated
health care ramifications from CVD cost the EU economy
192 billion Euros a year [3]. Moreover, the observed decline
in CVD since the 1980s has slowed due to the increasing
incidence of major risk factors like obesity and diabetes
mellitus [2, 3].
CVD has a multi-factorial etiology and it has been
recognized for a long time that platelet function is related
to
the risk of developing atherosclerosis [1], the principal
cause
of heart attack and stroke. Activated blood platelets play a
central role as risk factors, comparable to hypertension or
Abbreviations: ADP, adenosine diphosphate; CAD, coronary
artery disease; CVD, cardiovascular disease; LTA, light
transmis-
sion aggregometry; PFA-100, Platelet Function Analyzer-100;
PRP, platelet-rich plasma; vWF, von Willebrand factor
Correspondence: Dr. Baukje de Roos, University of Aberdeen,
Rowett Institute of Nutrition and Health, Greenburn Road,
Bucksburn, Aberdeen AB21 9SB, UK
E-mail: [email protected]
Fax: 144-1224-716629
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
60 Mol. Nutr. Food Res 2010, 54, 60–81DOI
10.1002/mnfr.200900172
-
diabetes [4], in this chronic inflammatory condition as they
contribute to plaque formation within blood vessels in the
early stages of atherogenesis. During this process,
platelets
become activated upon binding to collagen and von
Willebrand factor (vWF) multimers, which are secreted in
response to inflammatory stimuli from damaged endothelial
cells [5]. Collagen and vWF bind to receptors (glycoprotein
VI and integrin a2b1, and the receptor complex
glycoproteinIb/IX/V, respectively) on the platelet surface [6]. The
acti-
vated platelets then secrete a range of adhesion molecules,
such as P-selectin and CD40 ligand, and bind fibrinogen
from plasma. Additionally they synthesize and secrete
agonists such as adenosine diphosphate (ADP) and throm-
boxane A2, which also induce platelet aggregation and
thus amplify and maintain the initial platelet response [6].
Activated platelets further intensify atherogenesis by
contributing to recruitment and binding of leukocytes [5].
Despite extensive research on the pathways involved in
platelet activation, understanding their functional role in
the
development of atherosclerosis and CVD in humans
remains problematic [1, 5, 7]. This is in part due to the
lack
of a specific clinical test to evaluate the complex network
of signaling pathways involved in platelet function and
atherosclerosis [8]. Furthermore, mechanistic studies using
animal models are often difficult to interpret as non-human
platelets not only differ in morphology and numbers from
human ones, but also vary in their responses to agonists and
in the expression of platelet surface receptors [9–12].
Consumption of diets rich in plant-based products
protects against the development of CVD. Such effects have
been ascribed in part to non-nutritive but potentially
bioac-
tive secondary metabolites in fruits, vegetables, herbs,
spices, teas, and wines. For example, simple phenolic
compounds (hydroxybenzoic acid and hydroxycinnamic acid
derivatives) (Fig. 1A) and polyphenolic flavonoids (Fig. 1B)
are ubiquitous in plant-based foods (Table 1). Many have
numerous potential anti-atherogenic properties including
the modification of lipid profiles, vasodilatory effects and
the
ability to prevent the oxidation of low-density lipoproteins
[13]. Some dietary phenolic compounds also affect platelet
aggregation and function in vitro and in vivo afterconsumption
of supplements [14–17]. However, whether
such effects can be achieved from diet alone will depend on
the amount of the phenolic compounds in foods and their
subsequent bioavailability. Consequently, the aim of this
review is to critically evaluate controlled human
intervention
trials in order to assess whether intake of polyphenol-rich
diets or extracts impacts on platelet function. The main
food
sources, sub-classes of polyphenols, and nutritional rele-
vance of polyphenol-rich diets are discussed.
2 Materials and methods
We searched PubMed, Scopus and Ovid MEDLINEs for
human intervention studies where polyphenol-rich diets or
supplements were provided and their influence on platelet
activation and function was examined. The last search was
performed on April 7, 2009. The used search term is
included as Supporting Information.
2.1 Exclusion criteria
With the search term cited in Supporting Information a
total of 2576 English-written references were obtained
(Fig. 2). The search was then restricted to articles that
had
been published from 1980 onward, resulting in 2362
publications. As only original research articles were used
for
this review, all reviews, notes, and conference papers were
excluded and the number of references was decreased
further to 2091. We excluded all studies that were not
performed within humans or human platelets and obtained
1153 articles that still matched our criteria. The next step
was to exclude all trials examining compounds and
supplements that were not derived from a diet or that did
not have any dietary background, resulting in 136 remaining
publications. Studies that did not discuss measurements
related to platelet activation and function were also exclu-
ded. Of the remaining 115 references, 70 abstracts were
excluded, as they did not describe an intervention trial.
Finally, we excluded all studies that did not use valid
controls, ending up with 25 controlled human intervention
trials (Fig. 2).
2.2 Data evaluation
A meta-analytical approach was not possible due to a paucity
of randomized controlled trials (rather than non-rando-
mized trials) and a lack of uniformity in pre- and post-
treatment outcome measures between studies. Conse-
quently observational assessment was made of the 25
studies which survived the original systematically applied
exclusion criteria (Table 2).
3 Results
Controlled intervention studies were categorized based on
class of polyphenol (phenolic acids or flavonoids) and their
relevant food sources: chocolate and cocoa products, grape
seed extract, quercetin-rich diets/supplements, a soy
protein
supplement, black tea, wine and Armagnac, berries, purple
grape juice, and sea buckthorn juice. Table 2 summarizes
an overview of all studies including design, intervention
compounds and food matrices, doses, parameters of the
study populations, duration of intervention, platelet func-
tion tests, and outcomes. This summary of 25 intervention
studies reveals a wide range between studies in polyphenol
intake (18.6 mg to 2.20 g/day), study population (healthy,
increased risk factors, and/or clinical disease), study size
Mol. Nutr. Food Res 2010, 54, 60–81 61
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
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(between 6 and 1535 subjects), and length of supple-
mentation (acute intake for 1 day to chronic intake for over
90 days).
3.1 Cocoa studies and flavanols
Dietary flavanols, mainly found in cocoa and grape seed
extract, are the compounds most frequently tested in the
studies included in this review. Ten studies [18–27] focused
on the in vivo effects of food sources containing
(–)-epica-techin and (1)-catechin, as well as procyanidin
oligomers
and polymers. Of these studies, five measured platelet
function using the platelet function analyzer (PFA-100) [18,
24–27], allowing a straightforward comparison of the results
obtained. This device tests shear stress-induced platelet
aggregation by measuring the time taken by aggregating
platelets to occlude a collagen-epinephrine or a collagen-
ADP coated membrane (closure time). Data from these five
studies show evidence of a dose-response relationship up to
900 mg flavanol intake (maximum daily dose tested),
although linearity is not evident from the data available.
We
calculated that acute intake of flavanols led to
approximately
3–11% inhibition of collagen-epinephrine-induced closure
time per 100 mg flavanols consumed, 2–6 h after ingestion,and
also to approximately 11% inhibition of collagen-ADP-
induced closure time (Table 2). Conversely, chronic intake
of flavanols led to inhibition of collagen-ADP-induced
Phenolic acids
O
RR
1 Hydroxybenzoic acids
R1
2 Hydroxycinnamic acids
Hydroxybenzoic acid derivatives
R R R R
Salicylic acid OH H H H
p-Hydroxybenzoic acid H H OH H
Hydroxycinnamic acid derivatives R R R
p-Coumaricd acid H OH H
Caffeic acid OH OH H
OHR
R
R O
OHR
2
3
p Hydroxybenzoic acid H H OH H
Protocatechuic acid H OH OH H
Gentisic acid OH H H OH
Gallic acid H OH OH OH
Vanillic acid H MeO OH H
Syringic acid H MeO OH MeO
Caffeic acid OH OH H
HHOOeMdica cilureF
OeMHOOeMdica cipaniS
1 Flavonols
OOH
OH
R
R
1
2
3 Flavanones
OH O
R
R2
1
Flavonoids
12 Flavanols
OH
R
OH O R2
O
OH
OH OH O
Flavonols R R
Kaempferol H H
Quercetin OH H
Flavanols R R
(+)-Catechein/(-)-Epicatechin
OH H
Flavanones R R
Naringenin H OH
Hesperetin OH MeO
OH
OH
5 Flavones
OH O
R
R1
2
6 Isoflavones
OH O
4 Anthocyanins
O
OH
OH
R
R+
1
2
Quercetin OH H
Myricetin OH OHGallocatechin/Epigallocatechin
OH OH
Hesperetin OH MeO
Eriodictyol OH OH
OH O OHOR
R
1
2
OH
OH
2
Anthocyanins R R
Pelargonidin H H
Peonidin MeO H
Flavones R R
Chrysin H H
Apigenin H OH
Isoflavones R R
Daidzein H H
Genistein OH HPeonidin MeO H
Malvidin MeO MeO
Cyanidin OH H
Petunidin MeO OH
Delphinidin OH OH
Apigenin H OH
Luteolin OH OH
Genistein OH H
Glycitein H MeO
A
B
Figure 1. Chemical structures of the
two classes of phenolic acids (A), and
of the various classes of flavonoids
(B), and their respective compounds.
62 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
closure time of 3–11% per 100 mg flavanols consumed daily,but
did not affect the collagen-epinephrine-induced closure
time (Fig. 3). Such effects may be nutritionally relevant as
100 mg flavanols can be obtained from 11 g dark chocolate
with a cocoa content 70% w/w [28, 29], from 52 g milk
chocolate [28–31], or from 50 to 100 mL cocoa drink
containing 8% w/v pure cocoa [27–29]. Five other studies
also found a significant inhibition of platelet aggregation
and activation upon acute or chronic intake of dietary
flavanols from cocoa [19–23] using alternative methodolo-
gies, namely the Impact Cone and Plate(let) Analyzer, light
transmission aggregometry (LTA) in platelet-rich plasma
(PRP), or whole blood platelet aggregation by impedance or
platelet counting.
3.2 Flavonols and isoflavones
Four studies examined the effects of quercetin-rich foods
and
supplements on platelet function in healthy subjects
[32–35].
Only one of these [34] observed a significant inhibition
(21–65%) of platelet aggregation upon acute intake of 150 mg
of quercetin-40-O-b-D-glucoside, which is equivalent to
theconsumption of 400–700 g of raw yellow onions [29, 30]. Only
one study examined the effects of isoflavones on platelet
aggregation [36]. In this study consumption of a soy protein
isolate beverage powder rich in genistein, daidzein, and
glycitein did not affect LTA in PRP of healthy volunteers.
3.3 Polyphenol-rich beverages – studies using black
tea, coffee, and alcoholic beverages
We found seven controlled studies testing black tea [37–39],
coffee [40], Armagnac [41], or wines [42, 43] for their
potential
effects on platelet aggregation. Consumption of one liter of
black tea rich in gallic acid, flavanols, tannins,
theaflavins,
and thearubigins per day inhibited platelet activation by4–10%
[39]. In this study platelet activation was measured by
the very sensitive technique of flow cytometry scanning for
circulating leukocyte–platelet aggregates [44]. Two other
studies on tea consumption did not find significant effects
on
platelet function [37, 38]. Acute intake of a strong cup of
coffee, mainly containing chlorogenic acid as a phenolic
compound, also had significant anti-aggregatory effects
[40].
Table 1. Main dietary sources of phenolic acids and
flavonoids
Class of polyphenols Main dietary sources References
Phenolic acids
Hydroxybenzoic acidderivatives
Berries (blackberries, blackcurrants, raspberries, redcurrants,
strawberries,white currants), black and green tea,
condiments/herbs
[52, 56]
Hydroxycinnamic acidderivatives
Blueberries, coffee, kiwi fruits, cherries, plums, aubergines,
apples, pears,chicory, cider, spinach, cereal brans, broccoli,
kale
[52, 57]
Flavonoids
Flavonols Broccoli, curly kale, yellow onions, leek, capers,
parsley, lovage, beans,cherry tomatoes, blueberries, blackcurrants,
black and green tea, apples, apricots
[29, 52, 58]
Flavanols andproanthocyanidins
Black and green tea, cocoa, dark chocolate, cinnamon, red wine,
cider, sorghum,beans, nuts, chokeberries, apricots, plums, peaches,
cherries, grapes, cranberries,blueberries, blackberries,
blackcurrants, apples
[28, 29, 31,52, 59, 60]
Flavones Parsley, celery, sweet pepper [29, 52, 58]Isoflavones
Soy (soy flour, soy milk, soy bean, tofu, miso, tempeh), black
beans, red clover [52, 61–63]Flavanones Citrus fruits and their
juices (orange, grapefruit, lemon, lime, mandarin) [29, 52, 64]
2,576 potentially relevant abstracts identified
214 abstracts excluded:Published before 1980.
2,362 abstracts produced in 1980 or later
2,091 original research articles
938 abstracts excluded:Animal studies.
271 abstracts excluded:No original research articles
(reviews,notes, conference papers)., p p )
1,153 studies in humans or human platelets
1,017 abstracts excluded:No diets or diet-derived compounds.
136 publications where dietary compounds or diets were
studied
21 abstracts excluded:No platelet function studies.
115 promising abstracts (matched all inclusion criteria so
far)
70 abstracts excluded:No human intervention trials.
45 final articles with usable information
20 abstracts excluded:Not adequately controlled
ΣΣ = 25 controlled human intervention trials
Figure 2. Organization of the literature search.
Mol. Nutr. Food Res 2010, 54, 60–81 63
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Overv
iew
of
con
tro
lled
hu
man
inte
rven
tio
nst
ud
ies
exam
inin
gth
eeff
ect
of
po
lyp
hen
ol-
rich
die
tso
rsu
pp
lem
en
tati
on
so
np
late
let
ag
gre
gati
on
an
dfu
nct
ion
,ca
teg
ori
zed
acc
ord
ing
toth
eg
rou
ps
of
die
tary
po
lyp
hen
ols
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Fla
vo
no
ids:
Fla
van
ols
((�
)-ep
icate
ch
in,
(1)-
cate
ch
in,
pro
cyan
idin
oli
go
mers
)
Co
ntr
olled
,Fla
van
ols
446.4
mg
32
mil
dly
8-w
kP
FA
-100
–clo
su
reti
me
b)
[27]
ran
do
miz
ed
,h
yp
erc
ho
lest
ero
-ch
ron
icC
oll
ag
en
-AD
Pin
du
ced
–N
S13
NS
do
ub
leb
lin
dC
oco
ab
evera
ge
240
mL
lem
icp
ost
men
o-
inta
keC
oll
ag
en
-ep
inep
hri
ne
ind
uce
d–
NS
–N
S
pau
sal
wo
men
So
lub
leP
-sele
cti
nb
)3
NS
14
NS
57.77
2.2
years
So
lub
leE
-sele
cti
nb
)5
NS
�7
NS
So
lub
lein
terc
ell
ula
rad
hesio
nm
ole
cu
le1
�2
NS
0N
S
(IC
AM
-1)b
)
So
lub
levascu
lar
cell
ad
hesio
nm
ole
cu
le1
9N
S�
12
0.0
1
(VC
AM
-1)b
)
Co
ntr
olled
,Fla
vo
no
ids
�82
mg
c)
1535
healt
hy
1-d
ay
PFA
-100
–clo
su
reti
me
[18]
ran
do
miz
ed
,m
ale
san
dca
sual
Co
llag
en
-ep
inep
hri
ne
ind
uce
d–
NS
–0.0
1
no
base
lin
eC
ho
cola
te/c
oco
a5.9
gco
coa
fem
ale
sw
ith
inta
keU
rin
ary
11-d
eh
yd
roth
rom
bo
xan
eB
2–
NS
–0.0
3
valu
es
incr
ease
dri
skP
lasm
afi
bri
no
gen
–N
S–
NS
for
CV
D
437
13
years
Co
ntr
olled
Fla
van
ols
(to
tal
ep
icate
chin
an
dp
rocy
an
idin
s)
897.0
mg
30
healt
hy
1-d
ay
PFA
-100
–clo
su
reti
me
b)
[25]
male
san
dacu
teC
oll
ag
en
-AD
Pin
du
ced
fem
ale
s
inta
keA
fter
2h
10.0
016
NS
24–5
0years
Aft
er
6h
60.0
013
NS
Co
coa
bevera
ge
300
mL
Co
llag
en
-ep
inep
hri
ne
ind
uce
d
Aft
er
2h
9N
S8
0.0
0
Aft
er
6h
10
NS
31
0.0
0
Pla
tele
tsu
rface
P-s
ele
cti
nb
)
Wit
ho
ut
stim
ula
tio
n10
NS
�56
0.0
5
Ag
on
ist
1:
20mm
ol/
LA
DP
�3
NS
�19
0.0
1
100mm
ol/
LA
DP
7N
S�
25
0.0
3
Ag
on
ist
2:
20mm
ol/
Lep
inep
hri
ne
37
NS
�11
NS
Acti
vate
dg
lyco
pro
tein
IIb
-III
ab
)
Wit
ho
ut
stim
ula
tio
n�
10
NS
�67
0.0
4
Ag
on
ist
1:
20mm
ol/
LA
DP
�3
NS
�34
0.0
0
100mm
ol/
LA
DP
0N
S�
25
NS
Ag
on
ist
2:
20mm
ol/
Lep
inep
hri
ne
42
NS
�66
0.0
1
Pla
tele
tm
icro
part
icle
sb
)50
0.0
1�
68
0.0
0
Co
ntr
olled
,Fla
van
ols
:23
healt
hy
male
1-d
ay
PFA
-100
–clo
su
reti
me
[24]
ran
do
miz
ed
,M
on
om
ers
70.0
mg
smo
kers
acu
teC
oll
ag
en
-AD
Pin
du
ced
do
ub
leb
lin
d,
Dim
mers
an
dtr
imers
60.0
mg
237
2years
inta
keA
fter
1h
4N
S14
0.0
1
no
base
lin
eT
ota
lfl
avan
ols
130.0
mg
Aft
er
2h
6N
S13
0.0
3
valu
es
Aft
er
6h
0N
S16
0.0
0
Fla
van
ol-
rich
gra
pe
seed
extr
act
400
mg
Co
llag
en
-ep
inep
hri
ne
ind
uce
d
Aft
er
1h
9N
S8
NS
Aft
er
2h
9N
S15
0.0
3
Aft
er
6h
6N
S10
NS
64 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Co
ntr
olled
,Fla
van
ols
:17
healt
hy,
8-w
kP
FA
-100
–clo
su
reti
me
[26]
ran
do
miz
ed
,M
on
om
ers
70.0
mg
po
stm
en
op
au
sal
chro
nic
Co
llag
en
-AD
Pin
du
ced
do
ub
leb
lin
d,
Dim
mers
an
dtr
imers
60.0
mg
wo
men
inta
ke1
haft
er
inta
keo
n1st
day
3N
S8
NS
no
base
lin
eT
ota
lfl
avan
ols
130.0
mg
567
1.5
years
2h
aft
er
inta
keo
n1st
day
1N
S24
0.0
1
valu
es
Data
take
nfr
om
6h
aft
er
inta
keo
n1st
day
6N
S14
0.0
5
(23)
on
56th
day
of
tria
l–
NS
–0.0
5
Gra
pe
seed
extr
act
400
mg
Co
llag
en
-ep
inep
hri
ne
ind
uce
d–
NS
–N
S
Co
ntr
olled
,C
ate
chin
10.8
mg
22
male
an
dfe
–1-d
ay
Sh
ear
str
ess-d
ep
en
den
tp
late
let
fun
cti
on
by�
7N
S�
22
0.0
4[1
9]
ran
do
miz
ed
,E
pic
ate
chin
36.0
mg
male
heart
acu
teIm
pact
Co
ne
an
dP
late
(let)
An
aly
zerb
)
do
ub
leb
lin
dT
ota
lp
oly
ph
en
ols
624.0
mg
tran
spla
nt
inta
ke(%
of
surf
ace
covere
db
yp
late
lets
)
reci
pie
nts
Sh
ear
str
ess-d
ep
en
den
tp
late
let
fun
cti
on
by
1N
S�
3N
S
Dark
cho
cola
te40
g527
15
years
Imp
act
Co
ne
an
dP
late
(let)
An
aly
zerb
)
(siz
eo
fp
late
let
ag
gre
gate
sin
arb
itra
ryu
nit
s)
Co
ntr
olled
,C
ate
chin
10.8
mg
20
healt
hy
male
1-d
ay
Sh
ear
str
ess-d
ep
en
den
tp
late
let
fun
cti
on
b)
–N
S�
36
0.0
3[2
1]
ran
do
miz
ed
Ep
icate
chin
36.0
mg
smo
kers
acu
te(%
of
surf
ace
covere
db
yp
late
lets
)
To
tal
po
lyp
hen
ols
624.0
mg
Ag
era
ng
en
ot
inta
ke
giv
en
Dark
cho
cola
te40
g
Co
ntr
olled
,Fla
vo
no
ids
�192–2
79
mg
c)
30
healt
hy
1-d
ay
LT
Ain
PR
Pb
)[2
2]
ran
do
miz
ed
,m
ale
san
dacu
teA
go
nis
t1:
2.0mm
ol/
LA
DP
–N
S–
NS
sin
gle
bli
nd
Mil
kch
oco
late
(20%
coco
a)
100
gfe
male
sin
take
1.0mm
ol/
LA
DP
–N
S–
NS
few
data
20–5
8years
0.5mm
ol/
LA
DP
–N
S–
NS
Ag
on
ist
2:
2.0
mg
/Lco
llag
en-
ag
gre
gati
on
–N
S–
NS
-sl
op
e–
NS
–N
S
1.0
mg
/Lco
llag
en-
ag
gre
gati
on
–N
S–
NS
-sl
op
e–
NS
–N
S
0.5
mg
/Lco
llag
en-
ag
gre
gati
on
�7
NS
�82
NS
-sl
op
e�
5N
S�
48
NS
Fla
vo
no
ids
�379–1
045
mg
LT
Ain
PR
Pb
)
Ag
on
ist
1:
2.0mm
ol/
LA
DP
�N
S�
NS
Dark
cho
cola
te(7
5%
coco
a)
100
g
1.0mm
ol/
LA
DP
�N
S�
NS
0.5mm
ol/
LA
DP
�N
S�
NS
Ag
on
ist
2:
2.0
mg
/Lco
llag
en-
ag
gre
gati
on
�N
S�
NS
-sl
op
e�
NS
�N
S
1.0
mg
/Lco
llag
en-
ag
gre
gati
on
�N
S�
12
0.0
3
-sl
op
e–
NS
�24
0.0
4
0.5
mg
/Lco
llag
en-
ag
gre
gati
on
�7
NS
�93
0.0
3
-sl
op
e�
5N
S�
48
0.0
1
Co
ntr
olled
,Fla
van
ols
an
dp
rocy
an
idin
s234
mg
32
healt
hy
4-w
kP
late
let
ag
gre
gati
on
inw
ho
leb
loo
db
)[2
3]
ran
do
miz
ed
,m
ale
san
dch
ron
icA
go
nis
t1:
1.0
mm
ol/L
ara
chid
on
icaci
d–
NS
–N
Sd
ou
ble
bli
nd
fem
ale
sin
take
Ag
on
ist
2:
8.0mm
ol/
LA
DP
0N
S�
29
0.0
5
Co
coa
tab
lets
6ta
ble
ts447
7years
Ag
on
ist
3:
2.0
mg
/Lco
llag
en
�8
NS
�17
0.0
5
Mol. Nutr. Food Res 2010, 54, 60–81 65
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
(�50
gd
ark
cho
cola
te)
AT
Pre
lease
of
pla
tele
tsb
)
Ag
on
ist
2:
8.0mm
ol/
LA
DP
�14
NS
�4
NS
Ag
on
ist
3:
2.0
mg
/Lco
llag
en
�20
NS
�11
0.0
1P
late
let
su
rface
P-s
ele
cti
nb
)
Ag
on
ist
2:
3.0mm
ol/
LA
DP
�3
NS
�8
0.0
110.0mm
ol/
LA
DP
�3
NS
�3
NS
Mean
pla
tele
tco
un
tb)
5N
S�
3N
S
Mean
pla
tele
tvo
lum
eb
)1
NS
�1
NS
Co
ntr
olled
,Fla
van
ols
:380.0
mg
12
healt
hy
1-d
ay
Pla
tele
tag
gre
gati
on
inw
ho
leb
loo
dd
)[2
0]
ran
do
miz
ed
,(�
)-ep
icate
chin
,(1
)-ca
tech
in,
pro
cyan
idin
oli
go
mers
up
tod
eca
mers
male
san
dacu
teA
go
nis
t:
0.1
25
mg
/Lco
llag
en
�14
to�
23
NS
�12
to�
20
NS
do
ub
leb
lin
dfe
male
sin
take
0.2
50
mg
/Lco
llag
en
2to�
15
NS
�15
to�
17
NS
23–5
0years
0.5
00
mg
/Lco
llag
en
�2
to�
5N
S�
1to�
3N
S
Pla
tele
t–m
on
ocyte
co
nju
gate
sd
)
Co
coa
bevera
ge
300
mL
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�4
to�
8N
S0
to�
14
NS
0.2
50
mg
/Lco
llag
en
34
to20
NS
36
to14
0.0
2
0.5
00
mg
/Lco
llag
en
15
to4
NS
24
to16
NS
Pla
tele
t–n
eu
tro
ph
ilco
nju
gate
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�6
to�
20
NS
�7
to�
12
NS
0.2
50
mg
/Lco
llag
en
17
to�
13
NS
�6
to�
12
NS
0.5
00
mg
/Lco
llag
en
25
to2
NS
8to�
5N
S
Pla
tele
tacti
vati
on
d)
P-s
ele
cti
no
nm
on
ocyte
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
0to�
17
NS
�13
to�
15
NS
0.2
50
mg
/Lco
llag
en
10
to�
6N
S�
6to�
8N
S
0.5
00
mg
/Lco
llag
en
�10
to�
19
0.0
2�
9to�
10
NS
P-s
ele
cti
no
nn
eu
tro
ph
ils
d)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�8
to�
22
NS
�9
to�
16
0.0
40.2
50
mg
/Lco
llag
en
11
to�
15
NS
�11
to�
19
0.0
3
0.5
00
mg
/Lco
llag
en
�8
to�
21
0.0
1�
5to�
15
0.0
4
Fla
van
ols
:680.0
mg
Pla
tele
tag
gre
gati
on
inw
ho
leb
loo
dd
)
(�)-
ep
icate
chin
,(1
)-ca
tech
in,
pro
cyan
idin
oli
go
mers
up
tod
eca
mers
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�14
to�
23
NS
�25
to�
30
0.0
30.2
50
mg
/Lco
llag
en
2to�
15
NS
�12
to�
14
NS
0.5
00
mg
/Lco
llag
en
�2
to�
5N
S�
3to�
7N
SP
late
let–
mo
no
cyte
co
nju
gate
sd
)
Co
coa
bevera
ge
300
mL
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�4
to�
8N
S�
22
to�
39
0.0
40.2
50
mg
/Lco
llag
en
34
to20
NS
0to�
11
NS
0.5
00
mg
/Lco
llag
en
15
to4
NS
36
to17
0.0
5
66 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Pla
tele
t–n
eu
tro
ph
ilco
nju
gate
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�6
to�
20
NS
�19
to�
28
0.0
30.2
50
mg
/Lco
llag
en
17
to�
13
NS
�11
to�
24
0.0
3
0.5
00
mg
/Lco
llag
en
25
to2
NS
2to�
6N
S
Pla
tele
tacti
vati
on
d)
P-s
ele
cti
no
nm
on
ocyte
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
0to�
17
NS
�33
to�
37
0.0
10.2
50
mg
/Lco
llag
en
10
to�
6N
S�
20
to�
28
0.0
2
0.5
00
mg
/Lco
llag
en
�10
to�
19
0.0
2�
7to�
15
0.0
1
P-s
ele
cti
no
nn
eu
tro
ph
ils
d)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�8
to�
22
NS
�31
to�
36
0.0
10.2
50
mg
/Lco
llag
en
11
to�
15
NS
�22
to�
31
0.0
2
0.5
00
mg
/Lco
llag
en
�8
to�
21
0.0
1�
15
to�
20
0.0
2
Fla
van
ols
:980.0
mg
Pla
tele
tag
gre
gati
on
inw
ho
leb
loo
dd
)
(�)-
ep
icate
chin
,(1
)-ca
tech
in,
pro
cyan
idin
oli
go
mers
up
tod
eca
mers
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�14
to�
23
NS
�11
to�
20
0.0
10.2
50
mg
/Lco
llag
en
2to�
15
NS
1to�
12
NS
0.5
00
mg
/Lco
llag
en
�2
to�
5N
S0
to�
1N
S
Pla
tele
t–m
on
ocyte
co
nju
gate
sd
)
Co
coa
bevera
ge
300
mL
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�4
to�
8N
S�
16
to�
41
0.0
00.2
50
mg
/Lco
llag
en
34
to20
NS
34
to14
0.0
5
0.5
00
mg
/Lco
llag
en
15
to4
NS
26
to6
0.0
2
Pla
tele
t–n
eu
tro
ph
ilco
nju
gate
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�6
to�
20
NS
�19
to�
21
0.0
10.2
50
mg
/Lco
llag
en
17
to�
13
NS
4to�
4N
S
0.5
00
mg
/Lco
llag
en
25
to2
NS
15
to�
3N
S
Pla
tele
tacti
vati
on
d)
P-s
ele
cti
no
nm
on
ocyte
sd
)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
0to�
17
NS
�23
to�
31
0.0
30.2
50
mg
/Lco
llag
en
10
to�
6N
S�
1to�
7N
S
0.5
00
mg
/Lco
llag
en
�10
to�
19
0.0
2�
3to�
15
NS
P-s
ele
cti
no
nn
eu
tro
ph
ils
d)
Ag
on
ist:
0.1
25
mg
/Lco
llag
en
�8
to�
22
NS
�16
to�
23
0.0
10.2
50
mg
/Lco
llag
en
11
to�
15
NS
�7
to�
9N
S
0.5
00
mg
/Lco
llag
en
�8
to�
21
0.0
1�
1to�
9N
S
Mol. Nutr. Food Res 2010, 54, 60–81 67
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Fla
vo
no
ids:
qu
erc
eti
n(5
flavo
no
l)
Co
ntr
olled
,Q
uerc
eti
nan
hyd
rid
e1000.0
mg
27
healt
hy
4-w
kLT
Ain
PR
Pb
)[3
2]
ran
do
miz
ed
,O
ther
mix
ed
1000.0
mg
male
san
dA
go
nis
t:
10
mg
/Lco
llag
en
�9
NS
�11
NS
do
ub
leb
lin
db
iofl
avo
no
ids
fem
ale
s
chro
nic
Th
rom
bo
xan
eB
2p
rod
ucti
on
b)
112
NS
79
NS
Ru
tin
200.0
mg
427
3years
inta
ke
To
tal
flavo
no
ids
2200.0
mg
Su
pp
lem
en
tin
gca
psu
les
4
Co
ntr
olled
,Q
uerc
eti
n114.0
mg
18
healt
hy
6-w
kM
axim
um
wh
ole
blo
od
ag
gre
gati
on
[35]
ran
do
miz
ed
,m
ale
san
dA
go
nis
t:
2.0
mg
/Lco
llag
en
–N
S�
NS
no
base
lin
eB
ou
illo
np
ow
der,
6.6
gfe
male
s
chro
nic
LT
Ain
PR
Pvalu
es
bo
ilin
gw
ate
r1
393.4
mL
257
8years
inta
ke
Ag
on
ist
1:
2.0
mg
/Lco
llag
en
�N
S�
NS
coo
ked
yell
ow
on
ion
s220
g
Ag
on
ist
2:
1.5mm
ol/
LA
DP
�N
S�
NS
Ap
igen
in84.0
mg
3.0mm
ol/
LA
DP
�N
S�
NS
Pla
sm
aco
ncen
trati
on
of
fib
rin
og
en
�N
S�
NS
Bo
uil
lon
po
wd
er,
6.6
g
Th
rom
bo
xan
eB
2p
rod
ucti
on
inP
RP
�N
S�
NS
bo
ilin
gw
ate
r1
393.4
mL
6h
ealt
hy
male
s
Pla
tele
tn
um
ber
�N
S�
NS
dri
ed
pars
ley
4.9
g
an
dfe
male
s
Co
ntr
olled
,Q
uerc
eti
n69.0
mg
(pil
ot
stu
dy)
347
7years
1-d
ay
LT
Ain
wash
ed
pla
tele
tsaft
er
180
min
[33]
ran
do
miz
ed
,acu
teA
go
nis
t:
0.5
mg
/Lco
llag
en
–N
S�
38
NS
do
ub
leb
lin
d,
Qu
erc
eti
n-r
ich
on
ion
sou
p600
mL
inta
ke
1.0
mg
/Lco
llag
en
�N
S�
10
NS
no
base
lin
e
2.0
mg
/Lco
llag
en
�N
S�
3N
Svalu
es
3.0
mg
/Lco
llag
en
�N
S�
2N
S
Syk
tyro
sin
ep
ho
sp
ho
ryla
tio
n
Aft
er
60
min
–N
S�
28
0.0
0
Aft
er
180
min
–N
S�
60
NS
Ph
osp
ho
lip
ase
Cg
am
ma
2(P
LCc2
)ty
rosin
e
ph
osp
ho
ryla
tio
n
Aft
er
60
min
–N
S�
43
NS
Aft
er
180
min
–N
S�
23
NS
Co
ntr
olled
,Q
uerc
eti
n150.0
mg
6h
ealt
hy
male
s1-d
ay
LT
Ain
wash
ed
pla
tele
ts[3
4]
ran
do
miz
ed
,an
dfe
male
sacu
teA
go
nis
t:
0.5
mg
/Lco
llag
en
no
base
lin
e
valu
es
Qu
erc
eti
n-40 -
O-b
-D-g
luco
sid
esu
pp
lem
en
tD
ata
no
tg
iven
(pil
ot
stu
dy)
inta
ke
Aft
er
30
min
–N
S�
26
0.0
0A
fter
120
min
–N
S�
65
0.0
0
in5%
v/v
eth
an
ol
Ag
en
ot
giv
en
1.0
mg
/Lco
llag
en
Aft
er
30
min
–N
S�
2N
S
Aft
er
120
min
–N
S�
36
0.0
0
2.0
mg
/Lco
llag
en
Aft
er
30
min
–N
S�
14
NS
Aft
er
120
min
–N
S�
21
0.0
0
3.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
13
NS
4.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
15
NS
5.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
13
NS
68 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Syk
tyro
sin
ep
ho
sp
ho
ryla
tio
n
Aft
er
30
min
–N
S�
27
0.0
5
Aft
er
120
min
–N
S�
45
0.0
5
PLCc2
tyro
sin
ep
ho
sp
ho
ryla
tio
n
Aft
er
30
min
–N
S4
NS
Aft
er
120
min
–N
S�
42
0.0
5
Wh
ole
pla
tele
tp
rote
inty
rosin
e
ph
osp
ho
ryla
tio
n
Aft
er
30
min
–N
S�
12
0.0
0
Aft
er
120
min
–N
S�
60
0.0
0
Qu
erc
eti
n300.0
mg
LT
Ain
wash
ed
pla
tele
ts
Ag
on
ist:
0.5
mg
/Lco
llag
en
Qu
erc
eti
n-40 -
O-b
-D-
glu
cosi
de
sup
ple
men
t
in5%
v/v
eth
an
ol
Data
no
tg
iven
Aft
er
30
min
–N
S�
44
0.0
0A
fter
120
min
–N
S�
53
0.0
0
1.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
27
NS
2.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
24
NS
3.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
11
NS
4.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
9N
S
5.0
mg
/Lco
llag
en
,aft
er
120
min
–N
S�
14
NS
Syk
tyro
sin
ep
ho
sp
ho
ryla
tio
n
Aft
er
30
min
–N
S�
24
0.0
5
Aft
er
120
min
–N
S�
40
0.0
5
PLCc2
tyro
sin
ep
ho
sp
ho
ryla
tio
n
Aft
er
30
min
–N
S3
NS
Aft
er
120
min
–N
S�
37
0.0
5
Wh
ole
pla
tele
tp
rote
inty
rosin
e
ph
osp
ho
ryla
tio
n
Aft
er
30
min
–N
S�
90.0
0
Aft
er
120
min
–N
S�
62
0.0
0
Fla
vo
no
ids:
iso
flavo
nes
Co
ntr
olled
,G
en
iste
in80.3
mg
20
healt
hy
4-w
k
LT
Ain
PR
P[3
6]
ran
do
miz
ed
,D
aid
zein
35.6
mg
male
s
257
1years
Ag
on
ist
1:
1.5
mg
/Lco
llag
en
–N
S–
NS
no
base
lin
eG
lyci
tein
15.1
mg
chro
nic
3.0
mg
/Lco
llag
en
–N
S–
NS
valu
es
To
tal
iso
flavo
nes
131.0
mg
inta
ke
10.0
mg
/Lco
llag
en
–N
S–
NS
Ag
on
ist
2:
0.6mm
ol/
L9,1
1-d
ideo
xy-1
1a-
–N
S–
NS
So
yp
rote
inis
ola
te60
g
9a-
ep
oxy-m
eth
an
op
rost
a-
bevera
ge
po
wd
er
gla
nd
inF
2a(U
46619)
1.0mm
ol/
LU
46619
–N
S–
NS
Po
lyp
hen
ols
:g
all
icacid
,fl
avo
no
ls,
an
dell
ag
itan
nin
s
Co
ntr
olled
,Fla
vo
no
ls67.2
mg
75
healt
hy
6-w
k
Pla
tele
tacti
vati
on
b)
[39]
ran
do
miz
ed
,G
all
icaci
d11.3
mg
male
s
Mo
no
cyte
–pla
tele
tag
gre
gate
s9
NS
10.0
3
do
ub
leb
lin
dT
ota
lp
oly
ph
en
ols
78.5
mg
chro
nic
Neu
tro
ph
il–p
late
let
ag
gre
gate
s4
NS
�4
0.0
2
18–5
5years
inta
keLeu
kocy
te–p
late
let
ag
gre
gate
s3
NS
�4
0.0
3
Bla
ckte
a1000
mL
So
lub
leP
-sele
cti
nb
)�
3N
S2
NS
(tea
extr
act
)(4�
250
mL)
Pla
tele
tco
un
tb)
6N
S8
NS
Mol. Nutr. Food Res 2010, 54, 60–81 69
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Co
ntr
olled
,P
oly
ph
en
ols
1.4
65–2
.257
g22
healt
hy
12-w
kLT
Ain
PR
Pb
)[3
8]
ran
do
miz
ed
male
san
dA
go
nis
t1:
2.0mm
ol/
LA
DP
36
NS
45
NS
Bla
ckte
a1250
mL
fem
ale
sch
ron
ic
4.0mm
ol/
LA
DP
5N
S5
NS
inta
ke
8.0mm
ol/
LA
DP
�1
NS
5N
S
597
2years
Ag
on
ist
2:
0.2
mg
/Lco
llag
en
5N
S2
NS
0.6
mg
/Lco
llag
en
1N
S2
NS
2.0
mg
/Lco
llag
en
0N
S�
1N
S
Fib
rin
og
en
b)
3N
S4
NS
So
lub
leP
-sele
cti
nb
)10
NS
�7
0.0
1
So
lub
leE
-sele
cti
nb
)4
NS
�1
NS
So
lub
leIC
AM
-1b
)�
1N
S3
NS
So
lub
leV
CA
M-1
b)
3N
S5
NS
Co
ntr
olled
,G
all
icaci
d16.2
mg
49
male
an
d1-d
ay
LT
Ain
PR
P[3
7]
ran
do
miz
ed
Ep
igallo
cate
chin
9.0
mg
fem
ale
pati
en
tsacu
teA
go
nis
t1:
1.0mm
ol/
LA
DP
–N
S–
NS
Ep
igallo
cate
chin
gall
ate
17.6
mg
wit
hC
AD
,ta
kin
gin
take
2.5mm
ol/
LA
DP
–N
S–
NS
325
mg
/d5.0mm
ol/
LA
DP
–N
S–
NS
Ep
icate
chin
6.3
mg
asp
irin
10.0mm
ol/
LA
DP
–N
S–
NS
Ep
icate
chin
gall
ate
27.0
mg
Ag
on
ist
2:
5.0mm
ol/
LT
ho
mb
inre
cep
tor-
To
tal
cate
chin
s59.9
mg
557
9years
act
ivati
ng
pep
tid
e(T
RA
P)
–N
S–
NS
To
tal
theo
flavin
s27.0
mg
10.0mm
ol/
LT
RA
P–
NS
–N
ST
ota
lp
oly
ph
en
ols
To
tal
flavo
no
ids
733.5
mg
477.0
mg
20.0mm
ol/
LT
RA
P–
NS
–N
S
Bla
ckte
a(f
resh
tea
leaves)
450
mL
50.0mm
ol/
LT
RA
P–
NS
–N
S
Gall
icaci
d62.1
mg
4-w
kLT
Ain
PR
P
Ep
igallo
cate
chin
20.7
mg
Ag
on
ist
1:
1.0mm
ol/
LA
DP
–N
S–
NS
Ep
igallo
cate
chin
gall
ate
43.2
mg
chro
nic
2.5mm
ol/
LA
DP
–N
S–
NS
Ep
icate
chin
inta
ke
5.0mm
ol/
LA
DP
–N
S–
NS
19.8
mg
10.0mm
ol/
LA
DP
–N
S–
NS
Ep
icate
chin
gall
ate
32.4
mg
Ag
on
ist
2:
5.0mm
ol/
LT
RA
P–
NS
–N
ST
ota
lca
tech
ins
116.1
mg
10.0mm
ol/
LT
RA
P–
NS
–N
S
To
tal
theo
flavin
s
22.5
mg
20.0mm
ol/
LT
RA
P–
NS
–N
S
To
tal
po
lyp
hen
ols
1350.0
mg
50.0mm
ol/
LT
RA
P–
NS
–N
S
To
tal
flavo
no
ids
873.0
mg
Bla
ckte
a(f
reeze
-dri
ed
tea
leaves)
900
mL
Co
ntr
olled
2P
oly
ph
en
ols
70.8
–352.6
mg
10
healt
hy
1-d
ay
LT
Ain
PR
P[4
0]
Caff
ein
e180
mg
male
san
dacu
teA
go
nis
t1:
2.0mm
ol/
LA
DP
aft
er
60
min
29
NS�
15
NS
fem
ale
sin
take
Ag
on
ist
2:
3.0
mg
/Lco
llag
en
Co
ffee
200
ml
(6%
w/v
)A
fter
30
min
1N
S�
20.0
524–3
5years
Aft
er
60
min
0N
S�
1N
S
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
70 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Ag
on
ist
3:
0.5mm
ol/
Lara
chid
on
icaci
d
Aft
er
30
min
�15
NS
�20
NS
Aft
er
60
min
0N
S�
33
0.0
5
Pla
tele
tT
hro
mb
oxan
eB
2fo
rmati
on
Ag
on
ist:
3.0
mg
/Lco
llag
en
Aft
er
30
min
–N
S�
29
NS
Aft
er
60
min
–N
S�
34
0.0
5
Co
ntr
olled
,P
oly
ph
en
ols
(mo
stly
18.6
mg
20
healt
hy
2-w
kLT
Ain
PR
Pb
)[4
1]
ran
do
miz
ed
,ell
ag
itan
nin
s)su
bje
cts
Ag
on
ist
1:
2.0
mm
ol/
LA
DP
sin
gle
chro
nic
Aft
er
1w
k�
11
NS
�29
0.0
5(i
nvest
igato
r)A
rmag
nac
30
mL
20–5
4years
inta
ke
Aft
er
2w
k�
11
NS
�31
0.0
5-b
lin
d(4
0%
alc
oh
ol)
12
galc
oh
ol
(th
en
2
Aft
er
3w
k(f
oll
ow
up
)�
0N
S�
10
0.0
5
Gen
der
no
tw
k
Aft
er
4w
k(f
oll
ow
up
)15
NS
�3
0.0
5
giv
en
foll
ow
Ag
on
ist
2:
2.0
mg
/Lco
llag
en
up
)
Aft
er
1w
k�
9N
S�
14
NS
Aft
er
2w
k�
11
NS
�21
NS
Aft
er
3w
k(f
oll
ow
up
)�
5N
S�
11
NS
Aft
er
4w
k(f
oll
ow
up
)9
NS
�5
NS
Ag
on
ist
3:
0.3
U/m
Lth
rom
bin
Aft
er
1w
k�
13
NS
�23
0.0
5
Aft
er
2w
k�
10
NS
�23
0.0
5
Aft
er
3w
k(f
oll
ow
up
)�
5N
S�
80.0
5
Aft
er
4w
k(f
oll
ow
up
)4
NS
�6
0.0
5
Ble
ed
ing
tim
eb
)18
NS
1N
S
Co
ntr
olled
Po
lyp
hen
ols
�13.8
–21.1
mg
c)
16
healt
hy
90-d
ay
LT
Ain
PR
Pb
)[4
3]
Tan
nin
s(p
rocy
an
idin
s)�
7.5
5m
gc)
male
sch
ron
icA
go
nis
t1:
2.0mm
ol/
LA
DP
22
0.0
5�
6N
S357
5years
inta
ke
Ag
on
ist
2:
500
mg
/Lara
chid
on
icaci
d6
NS
�10
NS
Ag
on
ist
3:
750
mg
/Lad
ren
ali
n29
0.0
5�
3N
S
Wh
ite
win
e500
mL
Cyclo
oxyg
en
ase
acti
vit
yb
)8
NS
�7
NS
Th
rom
bo
xan
eA
2syn
thesis
b)
4N
S�
7N
S
Po
lyp
hen
ols
�185–5
36
mg
c)
LT
Ain
PR
Pb
)
Tan
nin
s(p
rocy
an
idin
s)�
105–2
79
mg
c)
500
mL
Ag
on
ist
1:
2.0mm
ol/
LA
DP
22
0.0
5�
16
0.0
5R
ed
win
eA
go
nis
t2:
500
mg
/Lara
chid
on
icaci
d6
NS
6N
SA
go
nis
t3:
750
mg
/Lad
ren
ali
n29
0.0
5�
16
NS
Cyclo
oxyg
en
ase
acti
vit
yb
)8
NS
�8
NS
Th
rom
bo
xan
eA
2syn
thesis
b)
4N
S�
33
NS
Mol. Nutr. Food Res 2010, 54, 60–81 71
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le2.
Co
nti
nu
ed
Do
sep
er
day
(co
mp
ou
nd
)
%C
han
ge
com
pare
d
wit
hb
ase
lin
ea)
Stu
dy
desi
gn
Co
mp
ou
nd
an
d
Fo
od
matr
ix
Am
ou
nt
per
day
(fo
od
matr
ix)
Stu
dy
po
pu
lati
on
Du
rati
on
of
stu
dy
Test
s
Co
ntr
ol
PIn
terv
en
tio
nP
Refe
ren
ce
Co
ntr
olled
Po
lyp
hen
ols
2.0
mm
ol
40
healt
hy
1-d
ay
Pla
tele
tsu
rface
P-s
ele
cti
naft
er
6h
b)
[42]
gall
icaci
dm
ale
san
dacu
teW
ith
ou
tst
imu
lati
on
36
NS
�19
NS
eq
uiv
ale
nts
fem
ale
sin
take
Ag
on
ist
1:
20mm
ol/
LA
DP
�5
NS
4N
S100mm
ol/
LA
DP
�2
NS
10
NS
Dealc
oh
oli
zed
red
win
e300
mL
21–4
9years
Ag
on
ist
2:
20mm
ol/
Lep
inep
hri
ne
�6
NS
15
NS
Acti
vate
dg
lyco
pro
tein
IIb
-III
aaft
er
6h
b)
Wit
ho
ut
stim
ula
tio
n57
NS
50
NS
Ag
on
ist
1:
20mm
ol/
LA
DP
�4
NS
�7
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100mm
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U46619,
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um
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gn
ifica
nt
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ease
vers
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base
lin
e;
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ata
no
tg
iven
.a)
Resu
lts
sho
wp
erc
en
tag
ech
an
ges
com
pare
dw
ith
base
lin
ew
ith
intr
eatm
en
tg
rou
ps.
b)
Perc
en
tag
ech
an
ge
was
calc
ula
ted
base
do
nb
ase
lin
ean
dp
ost
-tre
atm
en
tvalu
es
pro
vid
ed
inth
em
an
usc
rip
t.c)
Data
were
est
imati
on
sb
ase
do
nth
eli
tera
ture
an
dd
ata
base
s[2
8–3
1,
52,
56,
65–7
0].
d)
Perc
en
tag
ech
an
ge
was
calc
ula
ted
base
do
nb
ase
lin
ean
dp
ost
-tre
atm
en
tvalu
es
ob
tain
ed
by
pers
on
al
com
mu
nic
ati
on
wit
hth
eau
tho
rs.
72 L. M. Ostertag et al. Mol. Nutr. Food Res 2010, 54, 60–81
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Furthermore, a daily intake of 30 mL of Armagnac, which is
especially rich in ellagitannins, for 2 wk, inhibited
platelet
aggregation by 20–30% [41]. However, a study published by
the same group 2 years later questions whether these effects
are caused by the polyphenols present in Armagnac, as only
polyphenol-free fractions of freeze-dried Armagnac extracts
generated inhibition of platelet aggregation ex vivo
[45].Consumption of wine rich in proanthocyanidins, anthocya-
nins, and tannins has produced inconsistent effects on
platelet function, showing inhibition of ADP-induced
platelet
aggregation [43], no effect on platelet aggregation [42], or
even
an increase in ADP-induced platelet aggregation by almost
10% [42].
3.4 Studies testing other phenolic-rich foods
Three studies assessed the effects of polyphenols from
berries [46], purple grape juice [47], and sea buckthorn
juice
[48] on platelet function. Only the daily consumption of an
average amount of 160 g fresh berries and berry juices for
8 wk caused a significant inhibition of collagen-ADP-
induced platelet aggregation in subjects with higher risk
for
CVD [46].
4 Discussion
This review of controlled human intervention studies
assessing the impact of dietary polyphenols on human
platelet function revealed a plethora of research methods
and study designs. Findings were markedly inconsistent.
Chronic intake of polyphenols from berries, or juices
derived from berries, may result in only a relatively low
inhibition of collagen-ADP-induced platelet aggregation
under shear stress. Due to the inconsistency in data gene-
rated in well-controlled studies, it is currently not possible
to
conclude whether polyphenols from black tea, coffee and
alcoholic beverages have beneficial effects on platelet
func-
tion when consumed in nutritionally relevant quantities.
However, cocoa-related products consistently showed inhi-
bition of platelet activation and aggregation when consumed
in moderate amounts, either on an acute basis, or on a
chronic basis.
4.1 Heterogeneity of experimental approaches
4.1.1 Assessment of platelet function
The complexity of platelet interactions with their environ-
ment results in a wide variety of experimental approaches
for measuring platelet function, as illustrated by the
studies included in this review (Table 2). Various tests and
agonists have been used to assess the effect of a dietary
intervention on platelet activation and function (Table 3).
All established methods for platelet function monitoring
have been reviewed and their advantages and disadvantages
discussed [8, 49, 50]. Many older methods are unreliable
and not platelet-specific (e.g. bleeding time) or very
labor-intensive and therefore expensive, like LTA. In addition,
LTA is, albeit considered a gold standard method for
testing platelet function, relatively non-physiological as
platelets are taken out of their natural environment (i.e.blood)
and are not undergoing high-shear conditions.
Modern equipment that measures platelet function is
expensive and therefore not widely used, although the use
of the PFA-100 for human intervention trials appears to be
increasing (Table 2). The PFA-100 method uses whole
blood, and platelets will become activated under high
shear force, thereby adhering to a membrane and occluding
an aperture. Such processes mimic the conditions in
small arteries within the human body. Although this test
appears to simulate in vivo conditions more accurately,
theoutcome is dependent on vWF and hematocrit values.
Inhibition of shear-stress induced plateletaggregation (PFA-100)
in 5 studies
30
35
20
25
30
5
10
15
Inh
ibit
ion
[%
]
0
0 100 200 300 400 500 600 700 800 900 1000
Amount of polyphenols [mg/ day]
Collagen-epinephrine- 3 out of 5: significant inhibition
(acute)- 2 out of 5: no significant inhibition (chronic)
Collagen-ADP- 3 out of 4: significant inhibition (chronic +
acute)- 1 out of 4: no significant inhibition (acute)
Figure 3. Overview of the inhibition of shear stress-induced
platelet aggregation in healthy humans measured by the
PFA-100 upon daily consumption of flavanols. The PFA-100
tests shear stress-induced platelet aggregation by measuring
the
time the aggregating platelets need to occlude a collagen-
epinephrine-coated (black squares) or a collagen-ADP-coated
(gray circles) membrane. The data were obtained from n 5 5
studies where three were acute intake studies [18, 24, 25]
and
two were chronic intake studies [26, 27].
Mol. Nutr. Food Res 2010, 54, 60–81 73
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
-
Tab
le3.
Meth
od
s,p
late
let
ag
on
ists
an
dan
tib
od
ies
use
dfo
rp
late
let
fun
ctio
nass
ess
men
tin
con
tro
lled
hu
man
inte
rven
tio
nst
ud
ies,
incl
ud
ing
the
invo
lved
path
ways/
rece
pto
rs
Test
/measu
rem
en
tP
ath
ways/
rece
pto
rsin
vo
lved
Desc
rip
tio
nR
efe
ren
ces
Pri
mary
,p
late
let-
sp
ecifi
cm
easu
rem
en
ts
LT
Ain
PR
P
-A
go
nis
ts:
0.5mm
ol/L–2
.0m
mo
l/L
AD
PP
2Y
1,
P2Y
12a)
Sta
nd
ard
meth
od
toexam
ine
ag
on
ist
ind
uce
d[7
1–7
3]
0.2
–10.0
mg
/Lco
llag
en
a 2b 1
,G
PV
Ib)
pla
tele
tag
gre
gati
on
by
measu
rin
gd
ecr
easi
ng
500
mg
/Lara
chid
on
icaci
dC
OX-
TxA
2c)
turb
idit
yo
fP
RP
an
dth
ere
fore
incr
easi
ng
lig
ht
5.0
–50.0mm
ol/L
TR
AP
PA
R1-
thro
mb
ind
)
tran
smis
sio
n.
0.3
U/L
thro
mb
inP
AR
1-
thro
mb
ind
)
750
mg
/Lad
ren
ali
na 2
A-a
dre
nerg
icre
cep
tor
0.6
–1.0mm
ol/
L9,1
1-d
ideo
xy-1
1a-
9a-
ep
oxy-
Th
rom
bo
xan
ere
cep
tor
meth
an
op
rost
ag
lan
din
F2a
(U46619)
LT
Ain
wash
ed
pla
tele
ts
-A
go
nis
ts:
5.0mm
ol/L
AD
PP
2Y
1,
P2Y
12a)
Sim
ilar
toag
gre
gati
on
inP
RP
bu
tp
late
lets
are
[72,
74,
75]
0.5
–5m
g/L
collag
en
a 2b 1
,G
PV
Ib)
iso
late
d,
wash
ed
an
dre
susp
en
ded
ina
mo
difi
ed
Tyro
des–
Hep
es
bu
ffer
Pla
tele
tag
gre
gati
on
inw
ho
leb
loo
d
–b
yim
ped
an
ce
-A
go
nis
ts:
8.0
–10.0mm
ol/L
AD
PP
2Y
1,
P2Y
12a)
Measu
rem
en
to
fin
creasi
ng
ele
ctri
cal
imp
ed
an
ce[7
2,
73]
1.5
–5.0
mg
/Lco
llag
en
a 2b 1
,G
PV
Ib)
betw
een
ele
ctro
des
up
on
ag
on
ist-
ind
uce
d
1m
mo
l/L–0
.75
mo
l/L
ara
chid
on
icaci
dC
OX-
TxA
2c)
pla
tele
tact
ivati
on
an
dacc
um
ula
tio
no
n
–b
yp
late
let
co
un
tin
g
ele
ctro
des.
-A
go
nis
t:0.1
25–0
.5m
g/L
