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Polyphenol Bioactivity: Antioxidants?
Prof Kevin D Croft
University of Western Australia
Flavonoid
structures
Riemersma RA et al
QJM 2001; 94:277-82
Other dietary polyphenols with
biological activity
• Phenolic acids, eg caffeic acid (fruits , coffee
etc)
• Lignans, eg. Sesamin (sesame seed),
isolariciresinol etc (flax seed, bran)
• Stilbenes, eg. resveratrol (grapes)
• Phenylpropanoids, eg Curcumin (turmeric),
zingerone (ginger)
• Terpenoids, eg. Oleuropein (olive oil)
•low concentrations in vasculature
•metabolic transformation
•specific targets, key enzymes
Dietary polyphenols and
cardiovascular disease:
more than antioxidants
Flavonoid Metabolism
OHO
OH O
OH
OH
OH
OHO
OH O
OH
O
OH
CH3
OHO
OH O
OH
O
OH
S OH
O
O
OHO
OH O
OH
OH
O
O
COOHHO
HO
OH
glucuronidation
0 1 2 3 4 5 6 7 8 9 100
25
50
75
100
Q
MQ
QS
QG
MQG
*
#+
++
[Polyphenol] ( M)
LT
B4 I
nhib
itio
n (
%)
Human neutrophils stimulated with Ca ionophore
Loke et.al.Biochem Pharmacol 2008
LDL Q MQ QS QG MQG L K T0
2
4
6
8
10
12
14 *
##
#
#
F2-i
sopro
stan
es (
ng/
mL
)
AAPH induced oxidation of LDL, all compounds 10 uM.
F2-isoprostanes measured at 120 min
Antioxidant activity
Loke et.al.Biochem Pharmacol 2008
Atherosclerosis (animal models)
Apo E deficient mouse
Faulty uptake of lipoproteins in the liver
Very high circulating cholesterol
C57
BL/ c
ontro
l
Apo
E/ con
trol
Apo
E/ Que
rcet
in
Apo
E/ (-)
-epi
cate
chin
Apo
E/ the
afla
vin
Apo
E/ ses
amin
Apo
E/ chl
orog
enic
aci
d
0
10
20
30
40
**
*
(A)
Aort
ic s
inus
lesi
on a
rea
(% o
f to
tal
aort
ic
cross
-sec
tional
are
a)
Effect of pure polyphenols incorporated into the
diet, apoE-/- , ~1 mg/day, 20 weeks
Loke et.al. Arterioscl Throm Vascl Biol. 2010
Table 1. Effects of specific polyphenols on tested pathways at week 26 (expressed as % change compared to the ApoE-/- mice fed on
control diet).
Quercetin Epicatechin Theaflavin Sesamin Chlorogenic acid
Aortic sinus lesion
formation
-79* -14 -56* -41 -24
Thoracic aorta lesion
formation
-57* -15 -56* -24 -43
Plasma cholesterol +0.1 -24 -2 -19 +9
Aortic F2-isoprostanes -60* -77* -39 -27 +9
HO-1 protein +190* +4 ND ND ND
Aortic superoxide -42* -41* -10 -24 -15
Aortic LTB4 -53* -34 -47* -18 +8
Plasma soluble P-selectin -29* -33* -26* -7 -7
Urinary nitrate +90* +44 +65* +41 +21
Vascular eNOS activity +1446* +631 +923* +305 +466
Urinary ET-1 -52* -51* -1 -4 -40
* p< 0.05 vs ApoE-/- control mice. ND = not determined
Loke et.al. Arterioscl Throm Vascl Biol 2010
All data; lesion absence assigned 0 value
C57
Bl/
contr
olA
poE/ contr
olA
poE/ Quer
cetin
ApoE/ E
picat
echin
0
2
4
6
8
10 *
su
m p
ixels
of
HO
-1 s
tain
/ lesio
n a
rea
Loke et.al. Arterioscl Throm Vascl Biol 2010
HO-1 exerts an anti-inflammatory and antioxidant action within the
vasculature and can protect against oxidative damage.
Heme oxygenase-1 is the inducible form of the enzyme involved in heme
degradation and the release of iron, carbon monoxide and the bile pigment
biliverdin that is then converted to bilirubin.
Evidence for effects of dietary
flavonoids on major CVD risk
factors in humans
Endothelial Function
Blood Pressure
Vasodilatory mediators - NO (80%) / EDHF / PG
Increased blood
flow
Shear stress
forearm ischaemia
Endothelium-dependent FMD
Endothelial
cells
Comparison of quercetin, epicatechin,
epigallocatechin gallate on plasma NO
• Cross-over study, 12 healthy subjects
• 200mg of each flavonoid or water control
• Blood collected at baseline and after 2 hours,
urine at baseline and 5 hours.
• Plasma nitrosothiols measured by gas phase
chemiluminescence. Nitrate/nitrite measured by
GCMS
• Plasma and urinary flavonoids measured by
GCMS
Loke et.al. Am J Clin Nutr 2008
Total plasma NO
Q EC EGCG W0
5
10
15
20
25
30
35 *
**
Treatment
Pla
sma
[SN
O]
(nM
equiv
. of
NO
) * P<0.001, ** P<0.05
Loke et.al. Am J Clin Nutr 2008
Plasma and urinary nitrate
and nitrite support results
Q EC EGCG W0
102030405060708090
100110120
*
Treatment
Uri
nar
y [
ET
-1]
(pg/
mm
ol
crea
tinin
e)
Endothelin -1 was decreased by quercetin treatment
No effects on oxidative stress (plasma or urinary F2-isoprostanes)
Bioavailability of flavonoids -plasma
Q 3'-MQ Total Q0
1
2
3
4
*
*,**
#,##
(A)
Treatment
Pla
sma
[Fla
vonoid
s](
M)
EC 3'-MEC Total EC0
1
2
3
4
*
*,**
#
Treatment
Pla
sma
[Fla
vonoid
s](
M)
(B)
quercetin epicatechin How do flavonoids effect NO ?
•Increase eNOS enzyme activity, produce
more NO
•Prevent the breakdown of NO
•Recent in vitro evidence that methylated
metabolites of epicatechin can inhibit
NADPH oxidase (Steffan, Sies, FRBM 2007)
•Reduction of NO3- to NO2
- and NO
Cathy Bondonno
• 30 healthy men and women (18 to 65 years of age)
recruited from the general population
• 4 visits in random order
• 1 week washout between testing days
• Same dinner night before each visit
• Same low flavonoid / low nitrate breakfast on morning
of each visit
Bondonno et.al. Free Rad Biol Med. 2012
Visit Pre and during
visit:
During visit:
A FLAVONOID CONTROL NITRATE CONTROL
B FLAVONOID ACTIVE NITRATE CONTROL
C FLAVONOID CONTROL NITRATE ACTIVE
D FLAVONOID ACTIVE NITRATE ACTIVE
Breakfast and
flavonoid treatment
Lunch and flavonoid /
nitrate treatment
FMD
Blood sample
4 hr 2 hr
150 mg quercetin & epicatechin
<5 mg quercetin & epicatechin
0
50
100
150
200
250
NO
x n
mo
l/l
Flavonoid control/
Nitrate control
Flavonoid active/
Nitrate control
Flavonoid active/
Nitrate active
Flavonoid control/
Nitrate active
Treatment
P<0.004
P<0.001
P<0.001
NOx BY TREATMENT:
% FMD BY TIME AFTER CUFF INFLATION
0
1
2
3
4
5
6
7
8
0 30 60 90 120 150 180 210 240
% F
MD
Time (seconds)
Flavonoid control / Nitrate control
Flavonoid active / Nitrate control
Flavonoid control / Nitrate active
Flavonoid active / Nitrate Active
P<0.05 vs.
control
Bondonno et.al. Free Rad Biol Med. 2012
(-)-Epicatechin mediates beneficial effects of
flavanol-rich cocoa on vascular function in humans
H Schroeter et.al PNAS 2006
High flav cocoa; 917 mg flavanols
Pure –epicatechin, 1 or 2 mg/kg
n=3
Mechanisms of action
• Activation of key signalling pathways or enzymes
– AMPK activation, enzymes involved in lipid and glucose
metabolism, eNOS. Resveratrol, quercetin etc
• Estrogenic effects, isoflavones, some lignans
• Redox sensitive transcription factors
– NF-kB inflammation
– Nrf2 binds antioxidant response elements of genes
encoding for antioxidant enzymes eg HO-1
• Cardiovascular health effects
– vascular function, BP
– Thrombosis
– Lipids
– Inflammation
– Glucose metabolism
Bioactivity overview
• Polyphenols are a diverse range of compounds.
• Global measures such as Total Antioxidant
Capacity (TAC) may be of little value.
• Bioactive polyphenols may be beneficial either
as food components or isolated compounds.
Food matrix effects on bioavailability may
influence absorption and metabolism.
• Different polyphenols may have very different
and specific activity, eg grapefruit flavonoids and
drug metabolising enzymes.
Acknowledgements
National Health & Medical Research Council,
National Heart Foundation of Australia
Australian Research Council
Collaborators
UWA
Jonathan Hodgson
Natalie Ward
Trevor Mori
Anne Barden
Ian Puddey
Julie Proudfoot
Adeline Indrawan
PhD students:
Jason Wu
Wai Mun Loke
Cathy Bondonno
Helen Yu Shen
Aidilla Mubarak
University of Sydney
Roland Stocker
Inst of Food Research, UK
Paul Kroon