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