Flavonoids and Cardiovascular Health – What
Progress Has Been Made Towards Public Health
Recommendations for Flavonoids?
Carl L. Keen
Department of Nutrition
University of California, Davis
clkeen@ucdavis.edu
ICPH 2013
Buenos Aires, Argentina
October 19th, 2013
Disclosures for: Carl L. Keen
AFFILIATION/FINANCIAL
INTERESTS
CORPORATE ORGANIZATION
Grants/Research Support:
Research Support From Mars
Incorporated; California Walnut
Board & California Strawberry Board
Scientific Advisory
Board/Consultant:
Mars/Waltham Scientific Advisory
Boards
McCormick Spice Advisory Board
Speakers Bureau:
None
Stock Shareholder:
None
Other Financial or Material
Support/Honorarium:
None
Why address this issue?
Accepted Fact: Diets rich in plant foods are associated with
an overall positive health status and a decreased risk for
certain diseases (e.g. vascular disease, certain cancers)
Fruits, vegetables and CHD
“ There are very few studies to date examining
provision of, or advice to increase the
consumption of, fruits and vegetables in the
absence of additional dietary advice or other
lifestyle interventions for the primary prevention of
CVD. The limited evidence suggests advice to
increase fruit and vegetables as a single has
favorable effects on CVD risk factors but more
trials are needed to confirm this.”
Hartley et al., The Cochrane Collaboration, 2013.
(similar points made in Dauchet et al. Nat Rev Cardiol. 2009)
The inverse association seen between the
consumption of plant food-rich diets and the risk
for CVD is due to multiple nutritional variables:
favourable sodium/potassium profile
low in saturated fat
high essential nutrient content
high fibre content
high in select “non-essential” phytochemicals
Flavonoid Intake and Risk of CHD Mortality
– Seven Countries Study
Limited Analysis:
Two Classes:
• Flavonol
quercetin
kaempferol
myricetin
• Flavone
luteolin
apigenin
Hertog et al. Arch Intern Med 1995; 155: 381-6
Flavanols & Vascular Health
Speculation:
The inverse association often reported between
the consumption of plant food-rich diets and the
risk for CVD is due to numerous factors
including flavonoid-induced changes in: oxidant
production and defence; the immune system;
platelet reactivity, vascular reactivity, tissue
repair and blood pressure.
Biomarkers are needed for the
assessing an individuals
status for the phytochemical(s)
in question, as well as for
following the acute, and
chronic impact of these
phytochemicals on select
health parameters
Evaluation of Biomarkers 2010The Biomarker Evaluation Process Recommendations
Recommendation 3:
The FDA should use the same degree of scientific rigor for
evaluation of biomarkers across regulatory areas, whether
they are proposed for use in the arenas of drugs, medical
devices, biologics, or foods and dietary supplements.
Recommendation 4:
The FDA should take into account a nutrient’s or food’s source as
well as any modifying effects of the food or supplement that serves
as the delivery vehicle and the dietary patterns associated with
consumption of the nutrient or food when reviewing health related
label claims and the safety of food and supplements.
Recommendation 5b:
Congress should grant the FDA authority to request studies and
sufficient authority to act on the results of studies on consumer
understanding of claims on foods and supplements.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic Disease; IOM, 2010
Evaluation of Biomarkers 2010Case Studies
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Consumption of Flavanol-containing Beverage Products
- Uptake of Flavanols into the Circulation-
6 hours
2 hours
1 hours
0 hours
Epicatechin
Response (nA)
300
200
Catechin
100
0
Blood Plasma Flavanols/Metabolites after eating a
high-flavanol cocoa product
Circulating Flavanols/Metabolites [nM]
3000
epicatechin
catechin
4'MEC
EC-7Gluc
4'MEC-Gluc
Sum
2500
2000
1500
1000
500
250
0
-1
0
1
2
3
4
5
6
7
time [h]
Schroeter et al. PNAS 103:1024-29,2006
It is unlikely that the antioxidant properties of the flavanols
being studied trigger the observed reduction in platelet
reactivity
Human Plasma [ORAC]
2.5e+5
Flavanol Metabolites
(−)-epicatechin
3.7
(+)-catechin
15
Procyanidin Dimer B2
0.7
O-methyl-epicatechin
7.5
Epicatechin-glucuronide
9.4
Trolox equivalent [mol/L]
Flavanol Metabolites Mixture Profile % of Total
2.0e+5
*
1.5e+5
Range of
Dietary Relevance
1.0e+5
*
5.0e+4
O-methyl-epicatechin-glucuronide
63.7
0.0
0.01 0.1
1
10
[mol/L]
100 1000 10000
Acceptable Biomarkers – Antioxidant activity?
Like Aspirin, CF (897 mg total flavonoids) can influence blood
clotting (PFA-100) times in healthy adults
Closure Time (s)
Cocoa
Aspirin
200
*
*
150
Aspirin + Cocoa
*
*
*
Epi-collagen
100
50
0
0
2
0
6
2
0
Aspirin
Cocoa
140
120
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
*
120
0
0
2
6
0
0
2
2
6
Aspirin + Cocoa
140
140
Closure Time (s)
6
6
0
*
0
2
*
ADP-collagen
6
Time (hr)
Pearson et al. Thromb Res 2002; 106: 191-7
Effects of Flavanols and Flavanol-Rich Food
Consumption on Platelet Function
Reference
Type
Dose
Duration
Subjects
n
Platelet Test
Pace-Asciak ‘96
Red wine
White wine
Purple grape juice
375 ml/day
375 ml/day
500 ml/day
4 wks
Healthy adults
24
 Aggregation
-- Aggregation
 Aggregation
Duffy ‘01
Black Tea
900 ml/day
4 wks
CAD patients
49
-- Aggregation
Freedman ‘01
Purple grape juice
7 ml/kg/day 2 wks
Healthy adults
20
 Aggregation
Rein ‘00
Cocoa beverage
1 dose
Healthy adults
10
 Platelet Reactivity
Heptinstall ‘06
Cocoa Beverage
600-900 mg 1 dose
Healthy adults
12
Flammmer ‘07
Chocolate
40 g
1 dose
Heart
transplant
Hamed ‘08
Chocolate
100 g / day
1 week
Healthy adults
28
Erlund ’08
“Berries”
100 g
8 wks
Health Adults
72
Ostertag ’10
Cocoa/Chocolate
“Moderate
amount”
Acute
Meta Analysis
10
Studies
Carnevale ’12
Chocolate
40 g
1 dose
Healthy Adults
Smokers
40
 Isoprostanes, NOX2
300 mg
Acute
Healthy Adults
42
 Platelet Reactivity
Ostertag ‘13
Chocolate
300 ml
 Collagen stimulated
Platelet Aggregation
 Platelet adhesion
 Platelet Reactivity
 Platelet Reactivity
 Platelet Reactivity
Acceptable Biomarkers - Platelet Function ?
Water-soluble tomato concentrate (WSTC I and II) and platelet
aggregation.
Scientific substantiation of a health claim related to water-soluble
tomato concentrate (WSTC I and II) and platelet aggregation
pursuant to Article 13(5) of Regulation (EC) No 1924/2006.
The EFSA Journal (2009) 1101, 1-15.
Scientific opinion on the modification of the authorization of a
health claim related to water-soluble tomato concentrate and helps
to maintain a healthy blood flow and benefits circulation pursuant
to Article 13(5) of Regulation (EC) No 1924/2006 following a request
in accordance with Article 19 of the Regulation (EC) No 1924/2006.
The EFSA Journal (2010) 8(7), 1689.
Vascular Endpoints - FMD/PAT
Ischemia
(arterial occlusion)
Hyperemia
(transient increase in
organ blood flow)
Shear Stress
(arterial occlusion)
NO-Production
(endothelial cells)
Arterial Dilation
(relaxation of smooth
muscle)
FMD – Nitric Oxide – Flavanol/Metabolites
Cocoa-mediated increases
in FMD are inhibited by L-NMMA
infusion and NOS inhibition
b
12
4
*#
6
*#
*#
*#
4
2
3
Increase RXNO (nM)
Flow-mediated dilation (%)
10
8
c
2
0
*#
3,000
*#
1
#
0
-1
* *
2,500
*#
*#
2,000
1,500
1,000
*#
*#
*#
500
*
-2
0 1 2 3 4 5 6
hFCD
lFCD
3,500
Sum of plasma flavanols (nM)
a
0
0 1 2 3 4 5 6
*
**
0 1 2 3 4 5 6
Time after cocoa drink (h)
Schroeter et al. PNAS 2006
Epicatechin - FMD
b
d
10
*#
*#
PAT index
3
*
*
*
2
1
Water
1 mg/kg Epicatechin
0
Flow-mediated dilation (%)
4
*#
8
6
4
2
Water
1 mg/kg Epicatechin
0
0
0
1
2
3
*#
4
1
2
3
4
Time after Ingestion (h)
Time after Ingestion (h)
Schroeter et al. PNAS 103:1024-29,2006
Effects of Flavanols and Flavanol-Rich Food
Consumption on Vascular Function
Reference
Type
Dose
Duration
Subjects
n
11
Vascular Response
↑FMD, RXNO
Heiss 2005
Cocoa
88 -370 mg
1-day
Smokers
Schroeter 2006
Cocoa
917 mg
1-day
Healthy Adults 16
↑FMD, RHI, RXNO
Heiss 2007
Cocoa
918 mg
1-week
Smokers
11
↑FMD
Balzer 2008
Cocoa
963 mg
30-days
Diabetics
41
↑FMD
Heiss 2010
Cocoa
375 mg
30-days
CAD patients
16
↑FMD, CAC, Nitrite
Monohan 2011
Cocoa
69 – 1095 mg
1-day
Older Healthy
Adults
23
↑FMD
Loffredo 2012
Chocolate
40 g
1-day
Smokers
40
Healthy Adults
↑FMD, ↓NOX2
Nogueira 2012
Chocolate
50 g
4 wks
Hypertensive
20
↑RHI
Mellor 2013
Chocolate
13.5 g
1-day
Diabetics
10
↑RHI
How Does FMD/PAT Compare to Blood
Pressure as An Accepted Marker?
• Predictive value of noninvasively determined endothelial
dysfunction for long-term cardiovascular events and
restenosis in patients undergoing coronary stent
implantation: a prospective study.
Akcakoyun M, et. al. Coron Artery Dis, 2008; 19:337-343
• Persistent impairment of endothelial vasomotor function
has a negative impact on outcome in patients with coronary
artery disease.
Kitta Y, et. al., J Am Coll Cardiol 2009;53:323-30
Rubinshtein, R et al. Eur Heart J 31:1142-8, 2010
Acceptable Biomarkers - FMD?
Scientific Opinion on the substantiation of a health related to cocoa
flavanols and maintenance of normal endothelium-dependent
vasodilation pursuant to article 13(5) of regulation (EC) No
1924/2006.
The EFSA Journal (2012) 10(7), 2809.
Scientific opinion on the substantiation of a health claim related to
vitamin K2 and contribution to the normal function of the heart and
blood vessels (ID 125, further assessment) pursuant to Article 13(1)
of Regulation (EC) No 1924/2006.
The EFSA Journal (2012) 10(6), 2714.
Effects of Flavanols and Flavanol-Rich Food
Consumption on Blood Pressure
Reference
Type
Dose
Grassi 2005
Chocolate
~ 500 mg
2 wks
Healthy adults
15
↓ Systolic
Taubert 2007
Chocolate
30 mg /day
18 wks
Pre Stage 1
hypertensive
44
↓ Systolic, Diastolic
Davison 2008
Cocoa
902 mg
12 wks
Overweight/
Obese
49
↓ Diastolic, MAP
Balzer 2008
Cocoa
960 mg
30 dys
Medicated
Diabetics
41
No Change
Manuiyappa 08
Cocoa
~ 900 mg
2 wk
Hypertensive
20
No Change
Shiina 2009
Chocolate
550 mg
2 wks
Healthy Adults
39
No Change
Davison 2010
Cocoa
33, 372, 712,
1052 mg /day
6 wks
Hypertensive
52
45, 520, 990
mg /day
8 wks
416 mg
4 wks
Desideri 2012
Sarria 2013
Cocoa
Cocoa
Duration
Subjects
n
Blood Pressure
↓ 24 hr ambulatory
SBP, DBP, MAP at
high flavanol intake
Cognitively
Impaired
90
Healthy Adults
44
↓ Systolic, Diastolic (520 and
990 mg)
No Change
Fruit and Vegetable Intake in Childhood is Inversely Associated
with Pulse Wave Velocity in Adulthood
Vegetable consumption in childhood inversely associated with
Adulthood PWV (p = 0.02)
Persistently high consumption of both fruits and vegetables from childhood
To adulthood was also associated with a lower PWV.
↑ PWV = ↑ Arterial Stiffness
Aatola et al, Circulation 122:2521-8, 2010
Acceptable Biomarkers - Arterial Stiffness?
Evolus® and reduce arterial stiffness. Scientific substantiation of
a health to Lacotbacillus helveticus fermented Evolus® low-fat
milk products and reduction of arterial stiffness pursuant to Article
14 of the Regulation (EC) No 1924/2006.
The EFSA Journal (2009) 1101, 1-15.
Statement on the safety of the “conjugated linoleic acid (CLA)-rich
oils” Clarinol® and Tonalin® TG 80 as Novel Food ingredients.
The EFSA Journal (2012) 10(5), 2700.
New Biomarkers - circulating EPC levels?
CAD patients (n=519)
with
Highest
Medium
Lowest
numbers of EPCs.
Werner et al., NEJM, 2005
Circulating Endothelial Progenitor Cells,
Vascular Function, and Cardiovascular Risk
Hill, JM et al. NEJM 348:593-600, 2003
CF effects in medicated CAD Patients
• n=16
• medicated
CAD patients
Readouts
Time (days)
FMD
CAC
BP
NOx
Control
Control
9 mg of flavanols
twice a day
9 mg of flavanols
twice a day
Treatment
375 mg of flavanols
twice a day
Treatment
375 mg of flavanols
twice a day
30
1
Wash-out
1
30
Day 1
Day 30
Day 1
Day 30
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Heiss et al., JACC, 2010
CF Has Vascular Effects in CAD Patients
*#
a
1.5
CD34 /KDR (%MNC)
*
0
b
1.0
*
0.5
0.0
b
140
130
*#
+
*#
1.0
120
0.5
+
Systolic BP (mmHg)
*#
+
5
+
FMD (%)
10
CD133 /KDR (%MNC)
a
20
10
0
pre
post
pre
C
FI
Heiss et al., JACC, 2010
post
*#
0.0
pre
post
C
pre
post
FI
Contribution of 30 Biomarkers to 10-Year Cardiovascular Risk
Estimation in 2 Population Cohorts: The MONICA, Risk, Genetics,
Archiving, and Monograph (MORGAM) Biomarker Project
Blackenberg, S et al. Circulation 121:2388-97, 2010
Conclusions/Summary:
The results obtained from numerous
intervention trials support the concept that the
consumption of flavanol-rich foods can result
in acute (1 – 30 days) improvements in
vascular health parameters that are
suggestive of a decreased risk for select
chronic diseases.
Conclusions/Summary:
With respect to dietary recommendations for
“flavonoids”, the emphasis should be on
defined members of the class, rather than the
entire class
Conclusions/Summary:
When considering the development of
biomarkers for following the effects of a food
or a non-essential nutrient on the risk for
select diseases, there may be value in aiming
for a model that takes into consideration the
effects of the food/nutrient on multiple risk
factors for the disease of interest.